Chimeric antigen receptor and CAR-T cells that bind to 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 that comprises 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 CARs of the invention, genetically modified immune cells (preferably T cells) that express the CARs of the invention, and the use of such cells in the treatment of medical disorders associated with the presence of pathogenic B cells (e.g., plasma cell, and / or memory B cell, and / or mature B cell disorders, particularly multiple myeloma, non-Hodgkin's lymphoma, and autoantibody-dependent autoimmune diseases). [Background technology]

[0002] In cancer immunotherapy, adoptive transfer (ATT) of T cells genetically engineered to recognize tumor-specific or tumor-associated antigens is a promising approach to eradicate tumors and tumor stem cells. Therefore, unlike traditional chemotherapy, radiation therapy, and surgery, it may potentially avoid tumor recurrence. Furthermore, although novel pathway-selective drugs can often achieve excellent tumor control, the disease usually switches to a chronic phase without clear tumor elimination.

[0003] With the advent of genetically modified T cells expressing CARs, treatment of B-cell lymphoma / leukemia has proven highly successful, despite the fact that patients are heavily pretreated and have received several previous chemotherapy, antibody therapies, and even autologous / allogeneic bone marrow transplants. For example, ATT using CAR-T cells has been used successfully 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 it may contain 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+ + or CD8 + CARs can be produced "off the shelf" and their target is typically CD19. + They are widely expressed in certain tumors (>90%), as shown in B-cell leukemias and lymphomas, suggesting that CAR-T cells may act as a "living drug" that can be maintained after a single T-cell infusion.

[0005] There is a significant medical need for the chimeric antigen receptor (CAR)-T cell products 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 unique feature is that tumor cells are primarily localized in the bone marrow. This disease is most frequently found in bone and bone marrow tumors, with a 50% 10-year survival rate in young patients treated with intensive therapy and accounting for 2% of annual cancer deaths. The incidence rate is 5 / 100,000, and the median age at diagnosis is 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 agents, localized radiation therapy, or proteasome inhibitors; allogeneic stem cell transplantation is possible in a small percentage of patients. Despite these forms of intensive therapy, the disease usually relapses, resulting in secondary resistance after multiple treatments.

[0006] Second, a much larger group of classical B-NHLs includes a variety of tumors derived from B lymphocytes that typically home to secondary lymphoid organs (e.g., diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and subgroups of chronic lymphocytic leukemia (CLL)). The combined incidence of all NHLs is approximately 10–12 / 100,000 (>85% are of B-cell origin), with the majority occurring in adults, with a significant increase in older patients. Demographic trends predict that the total number of cases will increase due to the aging of Western societies. Clinically, B-NHLs are heterogeneous, distinguishable by their aggressive or indolent course. Significant advances in the treatment of B-NHL have been made over the past 15 years. Standard therapy consists of antibody / chemotherapy combinations, either alone or in combination with autologous stem cell transplantation, immunomodulatory agents, radiation, proteasome inhibitors, and signaling pathway inhibitors, with allogeneic stem cell transplantation in a small percentage of patients. In many cases of B-NHL, the median age at diagnosis is above 55-60 years, and comorbidities are present, making intensive long-term chemotherapy and even allogeneic bone marrow transplantation impossible.

[0007] The emergence of adoptive CAR T cell therapy targeting the CD19 antigen, widely expressed on the surface of lymphoma B cells, has overcome these limitations. Currently, approximately 20 studies of CD19 CAR T cells for the treatment of B-NHL and B-ALL are registered with the FDA. To the inventors' knowledge, major breakthroughs were already achieved in clinical trials for CLL in 2011 and B-ALL in 2013, but biomedical companies were only recently permitted to use the same CD19 CAR products in Germany. Clinical trials using CD19 CAR T cells are currently being conducted in other EU countries (e.g., Austria). More importantly, antigen loss has led to the development of resistance to anti-CD19 antibody or CAR T cell therapy for B-NHL. Because treatment resistance has been observed after multiple rounds of chemotherapy / immunotherapy, alternative targeting structures are urgently needed.

[0008] For multiple myeloma, two anti-BCMA CAR products have previously been reported and are in phase I clinical trials. However, these trials have not demonstrated the applicability of anti-BCMA CARs to 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 include anti-CD19 CARs, TCR-transduced T cells directed against NY-ESO1, and MAGE-A1. These strategies differ significantly from BCMA as a tumor target, offering a much smaller selection of patients because these target antigens are expressed in less than 10% of cases. Other targeted therapies include anti-CD38 and anti-SLAMF7 antibodies, but these therapies represent a completely different approach because antibodies are not self-reliant, do not form memory, and, to our knowledge, have not yet been shown to contribute to sufficient tumor eradication.

[0009] Additionally, the ability to specifically target plasma cells is believed to 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 potent immunosuppressants, such as cyclophosphamide, a cytotoxic agent that targets circulating cells.

[0010] More recently, the Food and Drug Administration (FDA) approved belimumab, an antibody that targets the cytokine BAFF (found at elevated levels in the serum of patients with autoimmune diseases), for use in SLE. However, only newly formed B cells depend on BAFF for survival 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, and tocilizumab. 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, residing in survival niches within the bone marrow, are often resistant to traditional immunosuppressants and cytotoxic drugs, as well as to therapies targeting B cells and their activation. Rituximab, in particular, appears unsuitable for such treatment because its target antigen, CD20, is not expressed on the surface of plasma cells. This therapeutic challenge could be addressed by using anti-BCMA CAR-T cell constructs, because BCMA is expressed on the surface of long-lived plasma cells.

[0012] 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, using in vitro assays and preclinical studies in mice (Carpenter et al., 2013; Clin Cancer Res; 19(8); 2048–2060). In June 2015, Bluebird Bio and Celgene announced their collaboration to focus on BCMA CAR-T cell therapy. Recruitment for a phase I clinical trial for patients with multiple myeloid leukemia 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 myeloid leukemia (ClinicalTrials.gov identifier: NCT02546167). CARs directed against BCMA are described in WO 2016 / 014789, WO 2016 / 014565, and WO 2013 / 154760. WO 2015 / 128653 also discloses CAR sequences that bind to BCMA. The epitope recognition portion of the CAR is a variant of the APRIL ligand, which exhibits 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 treatments are under development, there continues to be 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 drugs 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] Accordingly, the present invention 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 from residues 13 to 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 suitable for transplantation (preferably autologous T cells) comprising an anti-BCMA CAR for treating different stages of mature B-NHL and multiple myeloma. In a preferred embodiment of the immunotherapeutic approach of the present invention, patient-derived T cells are transduced, preferably retrovirally, to express the artificial immune receptors described herein, which consist 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 anti-tumor cytolytic capabilities to the transduced T cells.

[0019] As demonstrated with other CAR-T cell delivery in the clinic, the present invention is characterized by the predictable, tolerable, and manageable side effects of the CAR-based anti-BCMA CAR-T cells described herein. Preclinical studies of the anti-BCMA CAR-T cells described herein demonstrate 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 sparing normal hematopoietic cells. In a preferred embodiment, the transplantation of autologous T cells avoids the potential for graft-versus-host disease. The formation of memory CAR-T cells, which are important for preventing relapse, may develop.

[0020] The high affinity and avidity of the anti-BCMA CAR-T cells described herein allows them to recognize even mature B-NHLs 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 CARs 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 advantages over other BCMA CAR constructs by allowing for reduced off-target reactivity. It was surprising that the high specificity and affinity of the original antibody could be maintained in the CARs described herein, allowing for targeting even B cells with very low expression of the BCMA antigen.

[0023] The CARs of the present invention preferably bind to an epitope comprising one or more amino acids at the N-terminus of BCMA, residues 13 to 32. In alternative embodiments, they 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 populations (4-1BB signaling domain). As shown herein, the exchange of various signaling domains into many configurations allows for flexibility in the design of CARs without losing their 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 unparalleled high affinity, conferring exceptional specificity and avidity to T cells. These properties enable them to i) recognize, ii) activate against, and iii) kill tumor cells with high BCMA surface expression, and surprisingly, tumor cells with low BCMA surface expression.

[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 quantify 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 indicator of the number of BCMA molecules present on the cell surface. The fluorescence density associated with myeloma cells is typically at least 2-3 logs higher than that of low-fluorescence B-NHL cells. 10 This means that the density of BCMA antigen is at least 2–3 log 10This indicates that the value can vary within a factor of two.

[0027] It was found that none of the competing anti-BCMA CARs are reactive with B-NHLs other than multiple myeloma cells, or in very rare cases, Burkitt's lymphoma. Therefore, the anti-BCMA CAR shows reactivity against an unprecedented variety of B-NHLs. These properties are an unexpected and surprising advantage for CAR-T therapy. According to typical predictions by those skilled in the art, high expression of the target antigen is required for CAR-T targeting to be possible. CAR-T using the CAR of the present invention shows unprecedented activity against B cells with low expression levels of the 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 for CAR-bound BCMA epitopes The CAR of the present invention is preferably directed against an epitope comprising one or more amino acids from residues 13 to 32 of the N-terminus of human BCMA. The amino acid sequence of residues 13 to 32 of CD269 is set forth in SEQ ID NO: 33. The N-terminal sequence of CD269 is set forth in SEQ ID NO: 32. The extracellular domain of CD269 is set forth 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 of the present invention, an antigen comprising the extracellular domain of CD269, set forth in SEQ ID NO: 31, was used for vaccination. When generating antibodies, using the entire CD269 protein or a fragment thereof comprising the membrane-associated or intracellular domain as an antigen can result in the generation of antibodies that bind to the cryptic or intracellular domain of CD269, rendering 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 unexpectedly preferred feature of the present invention.

[0031] Fab fragments prepared from the murine or chimeric antibodies from which the CARs of the present invention are derived were crystallized in complex with purified BCMA extracellular domains, and the complex structures were resolved. Structural analysis revealed detailed information about the epitopes in the binding regions of the antibody / CARs of the present invention and their biological significance. The binding of the antibodies of the present invention to an epitope comprising one or more amino acids from residues 13-32 of BCMA in the extracellular domain is an advantageous property due to its high binding specificity and extracellular location. To the inventors' knowledge, no CARs binding to this region have been previously reported.

[0032] In one embodiment, a CAR of the invention is characterized by binding 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, a CAR of the invention is characterized by binding 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 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 N-terminal sequence of BCMA.

[0033] As previously disclosed, the affinity of the antibodies from which the CARs of the present invention are derived is surprisingly high and superior to similar approaches attempted in the prior art. Thus, the CARs of the present invention are 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 is derived binds to BCMA with high affinity.When measured by surface plasmon resonance (for example, Biacore), the antibody binds to human BCMA with an affinity of 100nM, 90, 80, 70, 60, 50, 40, 30nM or less, or 20nM or less, or 15nM or less, or 5nM or less, or 1000pM or less, or 500pM or less, or 100pM or less, or 80pM or less, or for example, about 50pM.Therefore, the CAR of the present invention exhibits 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). 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 by the ability to detect BCMA present on the cell surface 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 for 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) 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) 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) that has 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) that has at least 80% sequence identity with SEQ ID NO: 28 (KASQSVESNVA); - a light chain complementarity-determining region 2 (L-CDR2) that has 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); and 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 sequences: GFTFSRYW (H-CDR1; SEQ ID NO: 1), INPX2X3STI (H-CDR2; SEQ ID NO: 7) (wherein X2X3 are SS, NS, TS, GS, KS, RS, SD, SN, DE), and 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) 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 VL domain comprises:

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

[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 sequences: H-CDR1:RYWFS (SEQ ID NO: 25), H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO: 26), and 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 within the scope of the present invention, particularly when such sequence variants exhibit the desired BCMA binding specificity (functionally similar / equivalent).

[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 that provides at least 70%, preferably 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NOs: 11 and 12, but in which 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] Thus, the present invention relates to CARs comprising at least the CDR sequences described herein in the VH and VL domains, although sequence variations within the 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, particularly 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 binds to BCMA on the surface of non-Hodgkin's lymphoma (B-NHL) through the CAR, is activated, and thereby induces cytotoxic activity against the B-NHL.

[0053] In one 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 has the surprising property that even when the level of BCMA on the cell surface is very low, it can lead to CAR binding, T cell activation, and cell killing of the bound cells.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 has unexpected advantages compared to the CARs known in the prior art.

[0055] Derivatization of the above-described murine, chimeric, and / or human antibodies to generate the CARs described herein can, in some embodiments, provide this advantage. 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 enable the sensitivity of the CARs of the invention. However, this property was unexpectedly achieved in combination with CARs derived 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, exhibited sufficient specificity and potency to activate and target CAR-T cells to cells with minimal BCMA expression.

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

[0057] In a preferred embodiment, the isolated chimeric antibody receptor (CAR) polypeptide of the present invention, when expressed in a genetically modified immune cell (preferably a T lymphocyte), binds to BCMA on the surface of multiple myeloma (MM) cells via the CAR, activating the immune cell and 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, one skilled in the art would not have predicted that the CAR described herein would be capable of exhibiting activity against both types of cells.

[0058] Preferred embodiments for 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 preferred embodiments, these humanized sequences are incorporated into CARs of the invention. Results of binding assays performed with the corresponding antibodies demonstrate 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 the method used to determine the CDRs.

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

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

[0061] This HC mouse sequence is a heavy chain (VH) variable region originally developed for the chimeric antibody J22.9-xi, and contains a VL domain and a VH domain obtained from a mouse antibody, capable of binding to an epitope in the extracellular domain of CD269 (BCMA), with the VL domain and VH domain fused to a human CL domain and a 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 DYW 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, which have altered sequences compared to both the original chimeric sequence and the partially humanized sequences described herein.

[0067] The PTM mutations are intended to remove potentially harmful post-translational modification motifs from the protein while maintaining favorable binding properties. Positions 1, 5, 6, 19, 27, 28, 34, 39, 46, 48, 54, 69, 84, 85, 86, 88, 93, 107, and 115 of hHC01 and hHC02 are preferably mutated (substituted) relative to the original chimeric sequence. The significance of the substitution primarily relates to the resulting amino acid, not the original amino acid. Therefore, the change may occur from the corresponding amino acid in the original chimeric sequence or in 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 substituted with 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 with V; and / or - amino acids D54 and S55 of the HC (VH) sequence are substituted with 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] Potential alterations in residue positions required for direct interaction with BCMA: hHC03 - Altered amino acids involved in interaction with BCMA (SEQ ID NO: 42): NYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SX 5 X 6 X 7 DYGDX 8 MDYW 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 contains amino acid sequences that are altered relative to both the original chimeric sequence and the partially humanized sequence. These changes in sequence are intended to reflect potential changes in substitutable 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 - 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 a feature of the CDR sequence when considered independently of the overall framework sequence. Such modified CDR sequences can be considered defining features of the invention, either 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 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 potential post-translational modification sites 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 BCMA in FACS and ELISA and was crystallized in complex with BCMA. Surprisingly, 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, potentially including non-sugar derivatizations, would be tolerated at this position.

[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 potential mutation position.

[0076] Preferred embodiments for 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, such that the VL and VH binding regions have been altered relative to their sequences to make them more suitable for administration to humans.

[0080] Humanized VL sequence: hLC01 (SEQ ID NO: 49): EIVMTQSPATLSVSPGERATLSC KASQSVDSNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR Humanized VL sequence 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 sequence and the partially humanized sequences described herein.

[0082] The PTM mutations are intended to remove potentially harmful post-translational modification motifs from the protein while maintaining favorable binding properties. 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 preferably mutated (substituted) relative to the original chimeric sequence. The significance of the substitutions primarily relates to the resulting amino acid, not the original amino acid. Therefore, changes may occur from the corresponding amino acid in 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 - 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 - 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 substituted with 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 - Amino acid D70 of the LC(VL) sequence is substituted with E.

[0084] Potential alterations in the positions of residues required for direct interaction with BCMA within the CDR binding region: hLC03 - Altered 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 contains amino acid sequences that are altered relative to both the original chimeric sequence and the partially humanized sequence. These changes in sequence are intended to reflect potential changes in substitutable 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.

[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 a feature of the CDR sequence when considered independently of the overall framework sequence. Such modified CDR sequences can be considered defining features of the invention, either 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 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 potential mutation position.

[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 can be generated that exhibit the desired binding characteristics of the originally developed chimeric antibodies shown herein. The antibodies or portions thereof described herein also include sequences that are at least 80%, and preferably 90%, identical in sequence to the humanized sequences explicitly disclosed or disclosed through sequence formulas.

[0092] The present invention further relates to X1VQLX2X3SGGGLVQPGGSLX4LSCAASGX5X6FX7X8YWZ1SWVRX9AP GKGLEWX 10 GEINPZ2SSTINYAPSLKX 11 X 12 FX 13 ISRDNAKNTLYLQMX 14 X 15 X 16 RX 17 EDTAX 18 YYCASLYYDYGDAZ3DYWGQGTX 19 X1: Q,E; X2: Q,V; X3: Q,E; 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,E; X21: Q,V; 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; 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 advantageous humanizations performed in the CDRs described herein.

[0094] The present invention further relates to a method for producing a semiconductor device comprising: 10 QX 11 PKX 12 LIX 13 SX 14 X 15 LRFSGVPARFX 16 GSGSGTDFTLTISX 17 LQSEDX 18 AX 19 YX 20 X1: Q,P; X2: R,A; X3: F,T; X4: M,L; X5: T,S; X6: T,V; X7: R,E; X8: S,T; X9: V,L; X10: V,L; X11: V,P; X12: V,L; X13: V,L; X14: V,L; X15: V,L; X16: V,L; X17: V,L; X18: V,L; X19: V,L; X20: V,L; X21: V,L; X22: V,L; X23: V,L; X24: V,L; X25: V,L; X26: V,L; X27: V,L; X28: V,L; X29: V,L; X30: V,L; X31: V,L; X32: V,L; X33: V,L; 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 advantageous humanizations performed 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), or a linker having at least 80% sequence identity to 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 It is characterized by being made from

[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 LLSLVITLYC), a CD28 domain (SEQ ID NO: 21; FWVLVVVGGVLACYSLLVTVAFIIFWV), and 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 is characterized in that the intracellular domain comprises a costimulatory domain, and the costimulatory domain is preferably selected from the group consisting of the 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 that has at least 80% sequence identity with SEQ ID NO: 22 or 23.

[0100] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide of the present invention comprises a signaling domain, which signaling domain is preferably the 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 CD3ζ 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, preferably selected from the group consisting of an IgK leader (SEQ ID NO: 55; MDFQVQIFSFLLISASVIMSR), a GMCSF leader (SEQ ID NO: 56; MLLLVTSLLLCELPHPAFLLI), a leader sequence that has at least 80% sequence identity to 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 sequence 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 set forth in 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) nucleic acid molecules which, as a consequence of the degeneracy of the genetic code, result in the nucleotide sequences set forth in a) to c); e) Nucleic acid molecules according to a) to d) which have been altered 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). 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 the nucleic acid molecule described herein, preferably a viral vector, more preferably a gamma retroviral vector.

[0107] Yet another aspect of the present invention relates to genetically modified immune cells comprising a nucleic acid molecule or vector described herein and / or expressing a CAR 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 CARs described herein are CD4 + and / or CD8 + T cells (CD4 + T cells and CD8 + These populations of T cells are preferably CD4 + Transduced cells and CD8 + Compositions containing both the transformed cells exhibit particularly effective cytolytic activity against various malignant B cells (e.g., multiple myeloma and B-NHL), preferably against these cells and / or the related 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 CARs described herein are CD4 + T cells and CD8 + Preferably, the ratio of CD4 T cells is 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 1:1 ratio leads to advantageous properties while treating the diseases mentioned herein, for example, these ratios result in improved therapeutic response and reduced toxicity.

[0110] In a preferred embodiment, immune cells for administration in the treatment of the diseases referred to 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). In the context of the present invention, which aims to modify immune cells to express the CARs described herein, the Sleeping Beauty transposon system is a synthetic DNA transposon designed to introduce precisely defined DNA sequences into vertebrate chromosomes. The Sleeping Beauty transposon combines the advantages of viruses and naked DNA. Viruses have been selected in evolution for 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 important for societal and regulatory reasons. Therefore, the use of non-viral vectors such as the Sleeping Beauty system circumvents many, if not all, of the defenses that cells employ against vectors. For these reasons, the Sleeping Beauty system allows for 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, particularly 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 being treated is associated with pathogenic mature B cells. To the inventors' knowledge, no previous disclosures in the art have been shown to teach that such mature B cells can be effective targets for the BCMA CAR-T described herein. Several of the tumor cell lines tested in the Examples below involve mature B cells, but not necessarily memory types. In contrast, immature B cells are considered to be the B cells that cause acute lymphoblastic leukemia. Accordingly, the present invention also includes methods for treating the medical disorders disclosed herein, comprising administering a therapeutically effective amount of a CAR, or a therapeutic agent comprising a CAR of the present invention, to a subject in need of such treatment.

[0115] Yet another aspect of the present invention relates to a pharmaceutical composition comprising a CAR, or a therapeutic agent comprising a CAR described herein, together with a pharmaceutically acceptable carrier. DETAILED DESCRIPTION OF THE INVENTION

[0116] Multiple myeloma (also known as plasmacytoma) is a currently incurable B-cell lymphoma derived from a malignantly transformed plasma cell clone. This disease is most frequently found in bone and bone marrow, has a median life expectancy of 7 years, and accounts for 2% of annual cancer deaths. Malignant transformation is thought to occur during development in the germinal centers of secondary lymphoid organs, where B cells undergo VDJ rearrangement and isotype switching. The median age at diagnosis is 70 years. This indicates that many patients have comorbidities that preclude intensive and prolonged chemotherapy and radiation therapy. Furthermore, allogeneic bone marrow transplantation is usually excluded in this patient cohort. Clinically, the disease is characterized by osteolytic damage, hypercalcemia, hematopoietic failure, amyloid deposition, renal failure, excessive heavy and / or light chain production, hyperviscosity, infections, and bleeding disorders. Standard treatments include 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 these 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. Second, resistance to anti-CD19 antibody therapy or anti-CD19 CAR-T cell therapy for B-cell non-Hodgkin's lymphoma (B-NHL) occurs due to antigen loss. Because treatment resistance in B-NHL occurs after multiple chemotherapy / immunotherapy cycles, alternative targeting structures are warranted. Because BCMA is a suitable target for mature B-NHL, high-affinity anti-BCMA CAR-T can be used for treatment, even in B-NHL, as specifically described below.

[0118] Transplantation of BCMA CAR-T cells against the tumor-associated antigen BCMA is selective and effective in elderly patients, even after the development of multidrug resistance. This transplantation has predictable, tolerable, and manageable side effects. Autologous T cells bearing anti-BCMA CARs 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 this is an autologous T cell transplant, graft-versus-host disease cannot occur. Due to their high affinity and avidity, anti-BCMA CAR-T cells can recognize even mature B-cell NHLs with low BCMA expression, enabling T cell activation and tumor cell killing. These 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 ineligible for other therapies, more specifically, i) patients with multidrug resistance, and / or ii) patients who are ineligible 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 disease progression and failure of multiple other standard therapies, 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 be successful, and / or vii) the structure of the original antibody in complex with BCMA at near-atomic resolution demonstrates the remarkable specificity of the CAR, which is biologically safe, a feature not seen with other anti-BCMA CAR-T cells, and / or viii) the CAR can be applied as a monotherapy, which is not the case with antibodies.

[0120] Other anti-BCMA CAR-T cells described in the prior art have only been shown to be reactive with multiple myeloid cells and patients. In contrast, our anti-BCMA CAR has unexpectedly high sensitivity even against B-NHL cell lines with low BCMA expression. Our anti-BCMA CAR confers the extremely high 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. This invention demonstrates 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 lineage or its precursors. Therefore, the present invention has unprecedentedly low off-target reactivity on the surface of other hematopoietic tissues. Unlike anti-CD38 CAR-T cells, our anti-BCMA CAR does not have 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. The modifications allow for full expression on the surface of T cells while still maintaining proper binding of the antigen.

[0122] Because of the nanomolar affinity of the original FSY IgG (the antibody template for the scFv portion of the CAR-T cell construct), the present invention features, in a preferred embodiment, an anti-BCMA CAR with unexpectedly high affinity, conferring exceptional specificity and avidity to T cells. This high affinity and avidity allows the CAR-T cells to i) recognize, ii) be activated against, and iii) kill tumor target cells with high, moderate, and low surface expression of BCMA. None of the above-mentioned prior art anti-BCMA CARs have demonstrated reactivity against B-NHLs other than multiple myeloid cells. Therefore, the anti-BCMA CARs of the present invention are specific and highly active reagents for a wide variety of B-NHLs 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 a BCMA epitope. For example, the anti-BCMA CARs described herein exhibit a substantially greater biological safety profile, with no known off-target reactivity in vivo or in vitro.

[0125] Additionally, the inventors have swapped signaling elements of the CAR constructs 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 then 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) multidrug resistance, ii) ineligible for allogeneic stem cell transplant, iii) patients with comorbidities precluding further chemotherapy, iv) elderly patients who cannot tolerate chemotherapy, v) for salvage therapy after disease progression, vi) for failure of multiple other standard therapies, vii) disease progression after autologous stem cell transplant, viii) disease progression after allogeneic stem cell transplant, and ix) as bridging therapy before 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 include: i) patients with multidrug resistance, ii) patients who are not suitable for allogeneic stem cell transplantation, iii) patients with comorbidities that preclude further chemotherapy, iv) elderly patients who cannot 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, viiii) patients for whom it is applied as bridging therapy before allogeneic stem cell transplantation, and ix) patients who exhibit escape variants or escape mutations of CD19 and / or CD20 on the surface of tumor cells, causing loss / downregulation of their target structure and making current antibody therapy (anti-CD20, rituximab, anti-CD19, oretuzumab, BITE CD19 / CD2, brimatsumonab) or anti-CD19 CAR therapy ineffective.

[0130] An additional surprising aspect of the present invention is the enhanced stability of the CARs disclosed herein: CAR polypeptides can be readily 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 containing 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 to the intracellular signaling moiety through the fixed transmembrane segment. 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 present 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 activating intracellular domain to generate chimeric proteins that exhibit specific anti-BCMA cellular immune activity. As used herein, the term "chimeric" describes a composition of distinct protein or DNA segments 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 target cells, resulting in CAR clustering and delivery of an activating stimulus to the CAR-containing cell. A key feature of CARs is their ability to redirect immune effector cell specificity, thereby triggering the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of cells expressing the target antigen, independent of major histocompatibility complex (MHC), enabling the cell-specific targeting of monoclonal antibodies, soluble ligands, and cell-specific co-receptors.

[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, those skilled in the art will clearly recognize the various experimental procedures available for examining binding and binding specificity. Methods for determining equilibrium association or dissociation constants are known in the prior art. While some cross-reactivity or background binding may be unavoidable in many protein-protein interactions, this does not detract from the "specificity" of the binding between the CAR and the epitope. "Specific binding" describes that an anti-BCMA antibody or antigen-binding fragment thereof (or a CAR comprising the same) binds to BCMA with a binding affinity greater than background binding. The term "toward" can also be applied when considering the term "specificity" in understanding the interaction between an antibody and an epitope.

[0137] "Antigen (Ag)" refers to a compound, composition, or substance that can elicit the production of antibodies or stimulate 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 by the tertiary folded structure of a protein.

[0138] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, present in either orientation (e.g., VL-VH or VH-VL) in a single polypeptide chain. Generally, an scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CARs discussed herein comprise an antigen-specific binding domain called an scFv. The scFv can be a murine scFv, a human scFv, or a humanized scFv. Single-chain antibodies can be cloned to generate hybridoma V region genes 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 CARs discussed herein includes the amino acid sequence set forth in SEQ ID NO: 11. One non-limiting example of a variable light chain suitable for constructing the anti-BCMA CARs 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. Accordingly, non-limiting examples of antibodies or antibody fragments of the present 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 they retain similar binding characteristics as the CARs described herein and preferably contain corresponding CDRs, or VH and VL regions, as described herein. Miniantibodies and multivalent antibodies (e.g., bivalent, trivalent, tetravalent, and pentavalent antibodies) can also be used in the methods of the present invention. The immunoglobulin molecules of the present invention can be immunoglobulin molecules of any class (i.e., IgG, IgE, IgM, IgD, and IgA) or subclass. Thus, as used herein, the term antibody also includes antibodies and antibody fragments comprising a 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, "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 also be considered to 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 consists of two identical pairs of polypeptide chains, each pair having one "light" (L) chain (approximately 25 kD) and one "heavy" (H) chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 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 a portion of the framework regions, and possibly some of the CDR 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 the heavy or light chains, or from cDNAs encoding the heavy or light chains (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibodies used in the methods of the present invention can be human monoclonal antibodies. Human antibodies can be obtained, for example, using phage display technology (WO 91 / 17271; WO 92 / 01047).

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

[0146] As used herein, the terms "human antibody," "human antibody fragment," "humanized antibody," and "humanized antibody fragment" refer to antibodies produced by humans and / or antibodies having amino acid sequences corresponding to those of antibodies 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 for their epitope specificity to a particular mouse antibody through competitive binding experiments or other methods. Surprisingly, the humanized antibodies of the present invention 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 non-fibrillar 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, 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 by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site.

[0148] There are a number of 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 present invention provides antibodies or fragments thereof to be incorporated into CARs that contain 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, three, four, five, or six CDRs that are substantially identical to, or derived from, at least two, three, four, 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] CAR Add-ons In some embodiments, the CARs discussed herein can include linker residues between the various domains to provide appropriate spacing and conformation for the molecule. For example, the linker can include an amino acid sequence that connects the VH and VL domains and provides a spacer function to accommodate the interaction of the two sub-binding domains, so that the resulting polypeptide retains the same specific binding affinity for a target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. The CARs discussed herein can include one, two, three, four, five, or more linkers. In particular embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, 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). Because glycine and glycine-serine polymers are relatively unstructured, they may serve as neutral tethers between domains of fusion proteins (e.g., the CARs 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, allowing for 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 hinge region of a native immunoglobulin 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 a 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. A 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 native immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative 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 can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The transmembrane domain can 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 CARs discussed herein contain a transmembrane domain derived from CD8α or CD28.

[0155] In particular embodiments, the CARs discussed herein comprise an intracellular signaling domain. The term "intracellular signaling domain" refers to the portion of the CAR that is involved in transmitting a message to the inside of an immune effector cell that the anti-BCMA CAR has successfully bound to a human BCMA polypeptide, thereby inducing effector cell function (e.g., activation, cytokine production, proliferation, cytotoxic activity, including the release of cytotoxic factors toward the target cell to which the CAR is bound, and other cellular responses induced by antigen binding to the extracellular CAR domain). The term "effector function" refers to the specialized function of an immune effector cell. Effector functions of T cells can include, for example, support or activity such as cytolytic activity and cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and directs a 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 conventional 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.) and 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- 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 its in vitro cellular environment and from association with other cellular components; i.e., the peptide or polypeptide is not significantly associated with biological materials. Similarly, an "isolated cell" refers to a cell obtained from an in vivo 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 reference sequence described herein, and typically, the variant retains at least one biological activity of the reference sequence. In various illustrative examples, some aspects of the invention contemplate polynucleotides, including expression vectors, viral vectors, and delivery plasmids, as well as compositions and cells containing the same.

[0161] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of well-established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors include plasmids, autonomously replicating sequences, and transcribable elements. Additional non-limiting examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and PI-derived artificial chromosomes (PACs)), bacteriophages (e.g., λ phage, MI3 phage), and animal viruses. Non-limiting examples of categories of animal viruses useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequence for a chimeric protein disclosed herein can be ligated into such an expression vector to express the chimeric protein in mammalian cells. The "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, may 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, when immune effector cells are transduced with a vector encoding a CAR comprising a humanized anti-BCMA antibody or antigen-binding fragment that binds to a BCMA polypeptide and has a transmembrane domain and an intracellular signaling domain, these transduced cells can induce a CAR-mediated cytotoxic response.

[0163] Retroviruses are common tools for gene delivery. In particular embodiments, 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 into the host genome. Once integrated into the host genome, the virus is called a "provirus." The provirus serves as a template for RNA polymerase II, which directs the expression of RNA molecules encoding the 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 lentivirus.

[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); and Simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In a particular embodiment, a lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.

[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. A vector can contain sequences that direct autonomous replication within a cell or can contain sequences sufficient to allow integration into the DNA of a 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 skilled in the art, the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) that typically contains viral-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 nucleic acid transfer. Viral particles typically contain various viral components, and sometimes host cell components in addition to the nucleic acid.

[0168] The term viral vector can refer to a virus or viral particle capable of transferring a nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, 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 contains additional sequences (e.g., sequences that facilitate 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 CAR-encoding sequence 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-encoding 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 mRNA (e.g., mRNA comprising a cap and poly-A tail). Any transposase system can be used in embodiments of the present 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 (incorporated herein by reference). In some embodiments, the transposase is an engineered enzyme with enhanced 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, incorporated herein by reference). For example, one method can include electroporating a cell with mRNA encoding either SB 10 transposase, SB 11 transposase, or SB 100X transposase.

[0171] Sequence variants: 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 invention that maintain similar binding properties to those of the invention are also within the scope of the 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 functional mimics. Sequence identity relates to the percentage of nucleotide or amino acid identity when the sequences are aligned.

[0172] As used herein, " sequence identity " refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid in the comparison window.Therefore, " sequence identity percentage " can be calculated by: comparing two sequences that are optimally aligned in the comparison window, determining the number of positions in both sequences that have identical 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 identical positions; dividing the number of identical 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 sequence identity percentage. Included are nucleotide and polypeptide variants that have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 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, many nucleotide sequences exist that encode the polypeptides described herein. Some of these polynucleotides will 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 within the sequence that correspond to the sequence identity described herein are also encompassed by the present invention.

[0174] Potential changes in protein sequence resulting from substitutions are also within the scope of the present invention. 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. Like 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 particularly true when the change involves a "conservative" amino acid substitution, in which 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 substituted based on size, charge, polarity, or 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] Generally, the following conserved amino acid groups are represented: nonpolar amino acids Gly, Ala, Val, Ile, Leu; nonpolar 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 and 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 involve removing at least one amino acid residue in the antibody molecule and inserting a different residue 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 labeled "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 changes in the biological properties of the antibody are 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 region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, and (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 of 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 natural amino acids, this list is not exhaustive and is merely illustrative of 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 a 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 to 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 alter the expression of a gene or to express a therapeutic polypeptide (e.g., a CAR). In particular embodiments, a CAR discussed herein is introduced into 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, cytokine secretion, 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 that are genetically different from the comparison cell. As used herein, "syngeneic" refers to cells derived from a different subject but that are genetically identical to 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 CARs discussed herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are known in the art and are 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-associated complex (MHC)-restricted natural killer (NK)-like T lymphocytes. T cells can be T helper (Th) cells (e.g., T helper 1 (Th1) cells or T helper 2 (Th2) cells). T cells can be T helper cells (HT1; 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 CARs of the invention described herein can recognize and kill tumor cells when reintroduced into patients after autologous cell transplantation. CIK cells are a preferred embodiment of the immune cells of the invention because they have greater cytotoxic activity than 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 methods 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, thereby causing the immune effector cells to express one or more CARs as discussed herein. In some embodiments, the immune effector cells are isolated from the individual and then genetically modified without further in vitro manipulation. Such cells can then be readministered directly 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 comprise T cells. T cells can be obtained from numerous 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)), etc. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically comprises 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 an appropriate buffer or medium for further processing. 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, washing steps can be accomplished by methods known in the art, such as semi-automated flow-through centrifugation, such as the Cobe 2991 cell processor or the Baxter CytoMate. After washing, cells can be resuspended in a variety of biocompatible buffers or other saline solutions containing or not containing 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 a CAR using the methods discussed herein. In some embodiments, after PBMC isolation, T lymphocytes are further isolated, and in some embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations before or after genetic modification and / or expansion. CD8 + The cells can be obtained using standard methods. In some embodiments, 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 discussed herein (e.g., transduced with a viral vector comprising 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 using, 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 that express two or more different CAR proteins.

[0192] In one embodiment, the present invention provides a method for storing immune effector cells that express a genetically modified mouse CAR protein, or a human CAR protein, or a humanized CAR protein and target a BMCA protein, comprising cryopreserving the immune effector cells so that the cells remain viable when frozen. Cryopreserving a portion of the immune effector cells expressing the CAR protein using methods known in the art can 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 expand the volume when needed to produce larger quantities of such cells.

[0193] Compositions and Formulations

[0194] The compositions discussed herein can include one or more of the polypeptides, polynucleotides, vectors containing the same, genetically modified immune effector cells, etc. discussed herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated into a pharmaceutically 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 be understood that, if desired, the compositions of the present invention can be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, and various other pharmaceutically active agents. There is virtually no limit to the other components that can be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to achieve its intended therapeutic effect.

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

[0196] As used herein, non-limiting examples of "pharmaceutically 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 U.S. Food and Drug Administration as acceptable for human and veterinary use. Non-limiting examples of pharmaceutically acceptable carriers include sugars (e.g., lactose, glucose, sucrose); starches (e.g., corn starch, potato starch); cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate); gum 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; or any other compatible substance used in pharmaceutical formulations.

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

[0198] A "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 prior to the onset or at an early stage of disease, a prophylactically effective amount is typically, but not necessarily, lower than a therapeutically effective amount. The term prophylactic does not necessarily refer to complete suppression or prevention of a particular medical disorder. The term prophylactic can also refer to a reduction in the risk of developing or worsening a medical disorder.

[0199] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as an individual's disease state, age, sex, and weight, as well as 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 any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the positive effects of the treatment. The phrase "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 the condition of the patient (subject). Generally, pharmaceutical compositions comprising T cells described herein may be administered in doses of 10 cells per kg of body weight. 2 ~10 10 a dose of 10 cells per kg of body weight, preferably 10 5 ~10 6It can be said that the composition can be administered in a dose of 10 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 less than 10 cells. 6 cells / ml, generally 10 7 > 10 cells / ml 8 Clinically significant numbers of immune cells are distributed over multiple infusions, with cumulative values ​​exceeding 10 cells / ml or higher. 5 pieces, 10 6 pieces, 10 7 More than 10 8 More than 10 9 More than 10 10 More than 10 11 More than 10 12 The number of cells can be more than one. In some aspects of the invention, fewer cells can be administered, particularly since all cells introduced via 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] Generally, 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 the CAR-modified T cells discussed herein are used to treat B-cell malignancies. The CAR-modified T cells of the invention can be administered alone or in pharmaceutical compositions in combination with carriers, diluents, or excipients, and / or in combination with other components (e.g., IL-2 or other cytokines) or cell populations. In particular embodiments, the pharmaceutical compositions discussed herein comprise a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or 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) administration, intraperitoneal administration, or intramuscular administration).

[0202] Liquid pharmaceutical compositions, whether in the form of a solution, suspension, or other similar form, can contain one or more of the following: a sterile diluent (e.g., water for injection, saline (preferably physiological saline), Ringer's solution, isotonic sodium chloride); a fixed oil (e.g., synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents which can serve as solvents or suspending media); an antibacterial agent (e.g., benzyl alcohol or methylparabens); an antioxidant (e.g., ascorbic acid or sodium hydrogen sulfate); a chelating agent (e.g., ethylenediaminetetraacetic acid); a buffer (e.g., acetate, citrate, phosphate); or a tonicity adjuster (e.g., sodium chloride or dextrose). Parenteral preparations can 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 administered alone or in combination with other known cancer treatments (e.g., radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc.). The composition can also be administered in combination with antibiotics. Such therapeutic agents are accepted in the art as standard therapy for the particular disease states (e.g., particular cancers) described herein. Examples of contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, and other active adjunctive agents.

[0204] treatment method 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 disorders 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 cancer, that forms in B cells. 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 treating B cell-mediated diseases, plasma cell-mediated diseases, and 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), solitary plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström's macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, type I and II cryoglobulinemia, light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid, and epidermolysis bullosa acquisita; The disease may be any BCMA-expressing non-Hodgkin's lymphoma, B-cell leukemia, Hodgkin's lymphoma (HL), 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 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 within 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 comprise 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 present in extranodal sites, such as the central nervous system and mucosal tissues (e.g., lungs, small intestine, large intestine, and gastrointestinal tract). Many different types of non-Hodgkin's lymphoma exist. Non-Hodgkin's lymphomas can be divided into aggressive (fast-growing) and indolent (slow-growing) types, for example. Although non-Hodgkin's lymphomas can originate from B cells and T cells, the terms "non-Hodgkin's lymphoma" and "B-cell non-Hodgkin's lymphoma" are used interchangeably herein. B-cell non-Hodgkin's lymphomas (NHLs) include Burkitt's 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's 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 this 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 immune cells expressing the CAR, are contemplated for use in treating autoimmune diseases, preferably autoantibody-dependent autoimmune diseases, preferably autoimmune diseases with an inflammatory component.The autoimmune diseases of choice are 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, connective tissue disease vasculitis, Henoch-Schönlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, and tropical aortitis, sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, lepromatous arteritis, focal central nervous system vasculitis, thromboangiitis obliterans, paraneoplastic arteritis, urticaria, Degos' disease, myelodysplastic syndrome, persistent erythema elevatum, hyperimmunoglobulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Morbus Chron, ulcerative colitis, autoimmune myositis, diabetes, Guillain-Barré 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, Sjögren'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 disease is 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, with hay fever, periodontal disease, atherosclerosis, and rheumatoid arthritis being particularly preferred, and SLE being most preferred.

[0212] Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease in which the body's immune system attacks healthy tissue 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 of 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 preferred embodiments, a 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), livestock, farm 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 a B-cell malignancy.

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

[0215] As used herein, "prevent" and similar terms such as "prevented," "preventing," and "prophylactic" refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. This term also refers 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 its onset or recurrence.

[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 and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

[0217] Administration of the compositions discussed herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. 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 enteral 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 injection and infusion. 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 examples and drawings disclosed herein clarify the present invention through illustration. The drawings presented herein depict specific embodiments of the present invention and are not intended to limit the scope of the present invention. The drawings should be considered to provide further illustration of possible and possibly preferred embodiments that provide greater technical support for one or more non-limiting embodiments. [Brief explanation of the drawings]

[0219] [Figure 1] Figure 1: Schematic of preferred CAR structures. [Figure 2] Figure 2: Schematic representation of preferred CAR constructs IX, X, XI, XV, XVI, and XVII. [Figure 3] Figure 3: List of preferred constructs of CARs described herein and possible combinations of various structural elements. [Figure 4] Figure 4: Sequence comparison between the mAb binding region and the preferred humanized sequence used in the CAR of the present invention. [Figure 5] Figure 5: GeneArt™ plasmid carrying the BCMA-CAR sequence. [Figure 6] Figure 6: Gel electrophoresis of the construct and vector after restriction. [Figure 7] Figure 7: Confirmation of BCMA CAR expression in human T cells after 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 were 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, along 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, along 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, along 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, along with anti-BCMA and anti-CD19 staining. [Figure 12] Figure 12: Schematic representation of the binding interaction between the scFv of the CAR and the BCMA epitope. [Figure 13] Figure 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. To measure tumor burden, imaging was extended to 300 seconds (day -1). (B) To track treatment efficacy and reduce bioluminescence intensity for better visualization, mice as in (A) were imaged again for 30 seconds on day -1. The 30-second IVIS exposure after implantation of CAR-T cells (control SP6 CAR-T cells (n = 4) and BCMA CAR-T cells (n = 6)) allowed for better comparison between day -1 and day 17, when intensity was at its peak. White crosses: Mice were euthanized due to disease progression and animal welfare regulations. (C) The 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) Implantation of mantle cell lymphoma in a xenograft NSG mouse model. Mice were challenged with 6 x 10 JeKo-1 cells intraperitoneally. Tumor cell growth was visualized by IVIS imaging on day 7 after tumor inoculation. IVIS exposure, 120 seconds. (B) To track the therapeutic effect and reduce the bioluminescence intensity for better visualization, mice similar to (A) were imaged again on day 0 for 30 seconds. Performing IVIS exposure for 30 seconds after implantation of CAR-T cells (control SP6 CAR-T cells (n = 7) and BCMA CAR-T cells (n = 7)) allowed for better comparison between day 0 and day 16, when intensity was at its peak. (C) Mean bioluminescence signal intensity values ​​obtained from regions of interest covering the entire body of each mouse are plotted for each group at each time point. [Example]

[0220] The present invention is illustrated by the examples disclosed herein. The examples are presented as technical support and a more detailed description of potentially preferred, non-limiting embodiments of the present invention. To demonstrate the function of the CAR described herein, the inventors performed 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 was demonstrated; virtually no killing was observed in BCMA-negative cell lines or primary cells (e.g., HUVEC (endothelial cell-derived), 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 upon 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 with an established anti-CD19 CAR-T cell product.

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

[0222] The 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) at 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 back into MACH-1. A maxi-preparation of the MP71-BCMA-CAR plasmid was generated.

[0223] MP71 is a single-stranded RNA virus. Reverse transcriptase converts the retroviral RNA genome into a DNA copy, which integrates into the target genome at a random location 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 of CaCl and 150 μl of HO. Cells were incubated at 37°C for 6 hours, the medium was changed, and the cells were incubated at 37°C for an additional 48 hours.

[0226] Coat a 24-well non-tissue culture plate with anti-huCD3 and anti-huCD28 antibodies: Prepare anti-CD3 / anti-CD28 antibody solution in 0.5 ml of 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, then 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 of 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 two 50 ml Falcon tubes, the 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 white 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. The pellet was resuspended in 45 ml of RPMI (+100 IU / ml penicillin, streptomycin), centrifuged, and the pellet combined in 10–20 ml of T cell medium. One sample was stained with trypan blue, the cells were counted, and 1–1.5 × 10 cells were plated 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 The remaining viral supernatant (4°C) and the second supernatant (0.45 μl) from Hekt or GalV cells are filtered. 1 ml to 1.5 ml of supernatant is harvested from PBLs. Stimulated PBMCs are treated with 1 ml to 1.5 ml of viral supernatant and centrifuged 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 2000 rpm at 32°C. Days 7-13: Culture PBLs 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 after retroviral transduction

[0231] We obtained evidence that the CAR receptor folds and is transported in the context of human T cells; and 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-containing medium or IL-7 / IL-15-containing medium, and then BCMA-CAR expression was analyzed.

[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 compartment within the spacer region of the CAR construct. CD3 / CD8 / CD4 T cells were simultaneously stained. See Figure 7 for results.

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

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

[0236] Retrovirally transduced human T cells are generated as detailed previously; all BCMA CAR receptor variants (IX-XVII), SP6 negative control CAR, CD19 CAR, UT = untransduced T cells are used. The following human cell lines are used as target cells in co-culture:

[0237] [Table 4]

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

[0239] III. CD107a (LAMP1) staining of CAR-T cells cultured with multiple myeloma cells: Degranulating 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 with the listed cell lines at a 1:1 ratio for 18 hours.

[0241] Anti-CD107a (LAMP1) antibody is added to the cell culture medium after overnight incubation; the antibody binds to T cells continuously as secretory lysosomes fuse with the plasma membrane, releasing enzymes within 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 cytometry analysis: Greater CD107a reactivity, expressed as mean fluorescence intensity (MFI), indicates stronger T cell activation. This confirms 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 quantify cytotoxic T lymphocyte activity and measure cytolysis of target cells.

[0244] Generate retrovirally transduced human T cells as previously detailed; use 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 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 collected. The supernatant is transferred to a LUMA-scintillation plate and the released 51 Cr is measured in a gamma-scintillation counter. Highest release: target cells lysed by Triton X-100 permeabilizing agent. Lowest release: target cells only. See Figure 10 for results.

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

[0248] Example 4: In vivo experiments using a xenograft NSG mouse model to evaluate adoptively transferred CAR-T cells compared to B-NHL and multiple myeloma cell lines In vivo experiments using xenografts in NSG mice: 1) To demonstrate that CAR-T cells equipped with various anti-BCMA variants have effector activity in situ, we intravenously transfected multiple myeloma cells with various BCMA antigen densities 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), and 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's lymphoma (B-NHL): To prove the concept that the robust in vitro activity of BCMA CAR-modified T cells translates into effective antitumor activity in vivo, we used NOD.Cg-Prkdc scid Il2rg tm1 WjlCohorts of / SzJ (NSG) mice were intravenously inoculated 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 a luciferase gene tandemly linked to GFP. Because NSG mice do not develop T cells, B cells, or NK cells, they are well suited to the tolerance and growth of xenotransplanted 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 7–8 days later. CAR-T cells were intravenously injected 1 day after tumor growth was confirmed (=day 0). CAR construct IX (B IX) was used for functional in vivo experiments. A total of 6–7 × 10 CAR-T cells were administered per mouse. 6 The number of transduced, active CAR-T cells never exceeded 3 × 10, and the average transduction rate of T cells within this population was 40–60%. The transduction rates of SP6 and BCMA for each donor were consistent within ±10%. In the two experiments shown, transduced, active CAR-T cells were 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 × 10 6 ) and the observation period was extended to day 17. Virtually all mice treated with the SP6 CAR either progressed to 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 animal protection laws (Federal State of Berlin), whereas this was apparently not the case in the BCMA CAR-treated group. We conclude that even with this relatively low number of CAR-T cells, BCMA CAR-T cells possess 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, 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 Figures 11, 10, 9, and 8). To demonstrate that BCMA is a suitable targeting structure in B-NHLs, transduced CAR-T cells (total: 6–7 × 10 6 We transplanted BCMA CAR-T cells (individually 200 cells) into NSG mice previously challenged with the mantle lymphoma cell line JeKo-1. Virtually all mice treated with the SP6 CAR progressed to lymphoma (disease progression characterized by strong fluorescent signals in the liver, thoracic organs, hindlimb bone marrow, and spleen), whereas those treated with the BCMA CAR clearly 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 16A-C).

[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 phycoerythrin fluorescent detection kit (also called BD Quantibrite assay (BD Bioscience)) 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. Flow cytometry detection 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 was For NCI-H929 , DOHH-2:1 / 20, JeKo-1:1 / 250, and MEC-1:1 / 34.

Claims

1. A chimeric antigen receptor (CAR) polypeptide, wherein the CAR is: i. an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide; ii. a transmembrane domain; iii. Intracellular domain Contains wherein the antigen-binding domain comprises a variable heavy chain (VH) having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11, and said VH is: - the amino acid sequence of SEQ ID NO: 34 (RYWX 1 S) [In the sequence, X 1 is I, F, or M]; - the amino acid sequence of amino acids 50-67 of SEQ ID NO: 53 (EINPZ 2 SSTINYAPSLKX 11 X 12 ) [in the sequence, Z 2 is S, N, or D, and X 11 is D or G, and X 12 is K or R; and - amino acid sequence of SEQ ID NO: 36 (SLYX 4 DYGDAX 5 DYW) [in the sequence, X 4 is Y and X 5 is Y or M]; and the antigen-binding domain comprises a variable light chain (VL) having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12, wherein the VL is: - the amino acid sequence of SEQ ID NO: 37 (KASQSVX 1 X 2 NVA) [in the sequence, X 1 X 2 is ES or DS]; - a light chain complementarity determining region 2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 29 (SASLRFS); - light chain complementarity determining region 3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 30 (QQYNNYPLTFG) Including, wherein expression of said chimeric antigen receptor (CAR) in immune effector cells is effective in increasing the cytotoxicity of the immune effector cells against (i) multiple myeloma cells, and (ii) mantle cell lymphoma cells; and A chimeric antigen receptor (CAR) polypeptide, wherein the CAR does not comprise a variable heavy chain of the amino acid sequence of SEQ ID NO: 11 and a variable light chain of the amino acid sequence of SEQ ID NO:

12.

2. 2. The chimeric antigen receptor (CAR) polypeptide of claim 1, wherein the CAR binds to an epitope comprising one or more amino acids from residues 13 to 32 of the N-terminus of human BCMA of the amino acid sequence of SEQ ID NO:

32.

3. The following array: i. H-CDR1: RYWFS (SEQ ID NO: 25), RYWIS, or RYWMS; ii. H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO: 26), EINPNSSTINYAPSLKDK, or EINPDSSTINYAPSLKDK, iii. H-CDR3: SLYYDYGDAYDYW (SEQ ID NO: 27) or SLYYDYGDAMDYW; iv. L-CDR1: KASQSVESNVA (SEQ ID NO: 28) or KASQSVDSNVA, v. L-CDR2: SASLRFS (SEQ ID NO: 29), and vi. L-CDR3: QQYNNYPLTFG (SEQ ID NO: 30) 3. The chimeric antigen receptor (CAR) polypeptide of claim 1 or 2, comprising:

4. a VH domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11; and A VL domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

12. The chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 3, comprising:

5. - at least W36, E50, L99, Y100, Y101 and A106 of SEQ ID NO: 11, or at least the CDR sequences of SEQ ID NOs: 25 to 27, and at least S31, A34, S50, L53, Q89, Y91, Y94 and L96 of SEQ ID NO: 12, or at least the CDR sequences of SEQ ID NOs: 28 to 30 5. The chimeric antigen receptor (CAR) polypeptide of claim 3 or 4, comprising:

6. 6. The isolated chimeric antigen receptor (CAR) polypeptide according to any one of claims 1 to 5, wherein the CAR, when expressed in a genetically modified immune cell, causes the immune cell to bind to BCMA on the surface of non-Hodgkin's lymphoma (B-NHL) via the CAR, thereby being activated and inducing cytotoxic activity against the B-NHL.

7. The chimeric antigen receptor (CAR) polypeptide according to claim 6, wherein the B-NHL is a JeKo-1 cell line, a DOHH-2 cell line, a SU-DHL4 cell line, a JVM-3 cell line, and / or an MEC-1 cell line.

8. The chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 7, wherein the extracellular antigen-binding domain comprises a linker polypeptide located between the VH domain and the VL domain.

9. 9. The chimeric antigen receptor (CAR) polypeptide of claim 8, wherein the linker is selected from a Whitlow linker or a Gly-Ser linker, or a linker having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13 or 14.

10. a spacer peptide located between the extracellular antigen-binding domain and the transmembrane domain, wherein the spacer is selected by: 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), or f. A spacer having at least 80% identity with the amino acid sequence of any one of SEQ ID NOs: 15 to 19. The chimeric antigen receptor (CAR) polypeptide according to any one of claims 1 to 9,

11. The chimeric antigen receptor (CAR) polypeptide according to any one of claims 1 to 10, wherein the transmembrane domain is selected from the transmembrane domains of CD8α or CD28 having the amino acid sequence of SEQ ID NO: 20 or 21.

12. The isolated chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 11, wherein the intracellular domain comprises a costimulatory domain of 4-1BB or CD28 having an amino acid sequence of SEQ ID NO: 22 or 23.

13. The chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 12, comprising a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO:

24.

14. The chimeric antigen receptor (CAR) polypeptide according to any one of claims 1 to 13, comprising a tandem costimulatory signal domain comprising a 4-1BB costimulatory domain, a CD28 costimulatory domain, and a CD3ζ signaling / activation domain.

15. A nucleic acid molecule encoding the chimeric antigen receptor (CAR) polypeptide of any one of claims 1 to 14.

16. A cell comprising the nucleic acid molecule of claim 15 and / or expressing the CAR of any one of claims 1 to 14.

17. The cell of claim 16, wherein the cell is activated in response to BCMA binding of the CAR via signal transduction by the intracellular domain.

18. The cell of claim 16 or 17, wherein the cell is selected from the group consisting of a T lymphocyte and a NK cell.

19. The cell of claim 18, wherein the T lymphocyte is a cytotoxic T lymphocyte.

20. A medical agent comprising the cells of any one of claims 17 to 19 for use in the treatment of a medical disorder associated with the presence of pathogenic B cells.

21. 21. The medical agent of claim 20, for use as a medicament, wherein the medical disorder is associated with the presence of pathogenic B cells.

22. 21. The medical agent of claim 20 for use as a pharmaceutical, wherein the medical disorder is a disorder of plasma cells, mature B cells and / or memory B cells.

23. 21. The medical agent of claim 20 for use as a pharmaceutical, wherein the medical disorder is multiple myeloma.

24. 21. The medical agent of claim 20 for use as a pharmaceutical, wherein the medical disorder is non-Hodgkin's lymphoma.

25. 21. The medical agent of claim 20 for use as a pharmaceutical, wherein the medical disorder is an autoantibody-dependent autoimmune disease.

26. 26. The medical agent of claim 25 for use as a medicine, wherein the medical disorder is systemic lupus erythematosus (SLE) or rheumatoid arthritis.

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