Chimeric antigen receptor (CAR) polypeptides, nucleic acid molecules encoding the same, cells comprising the same, and uses thereof

Abstract

This invention relates to a chimeric antigen receptor (CAR) polypeptide comprising i. an extracellular antigen-binding domain containing an antibody or antibody fragment binding to a B-cell maturation antigen (BCMA) polypeptide, ii. a transmembrane domain, and iii. an intracellular domain, wherein the antigen-binding domain comprises: a variable heavy chain (VH) containing heavy chain complementarity-determining regions 1 (H-CDR1), 2 (H-CDR2), and 3 (H-CDR3); and a variable light chain (VL) containing light chain complementarity-determining regions 1 (L-CDR1), 2 (L-CDR2), and 3 (L-CDR3), wherein expression of the CAR in immune effector cells increases the cytotoxicity of the immune effector cells against (i) BCMA-expressing multiple myeloma cells and (ii) BCMA-expressing mantle cell lymphoma cells. The invention also relates to nucleic acid molecules encoding CAR polypeptides, cells containing them, and uses thereof.

Description

[0001] This application is a divisional application of Chinese patent application filed on June 7, 2017, with application number 201780035831.4 and invention title "Chimeric antigen receptor and CAR-T cells bound to BCMA". Technical Field

[0002] This invention relates to isolated chimeric antigen receptor polypeptides (CARs), wherein the CAR comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a B cell maturation antigen (BCMA) polypeptide. The CAR preferably binds to an epitope comprising one or more amino acids from the 13th to 32nd residues of the N-terminus of human BCMA. The invention also relates to nucleic acid molecules encoding the CAR of the invention, genetically modified immune cells (preferably T cells) expressing the CAR of the invention, and the use of said cells in the treatment of medical conditions associated with the presence of pathogenic B cells, such as diseases of plasma cells, memory B cells, and / or mature B cells, particularly multiple myeloma, non-Hodgkin's lymphoma, or autoantibody-dependent autoimmune diseases. Background Technology

[0003] In cancer immunotherapy, adoptive transfer of genetically modified T cells (ATTs) to recognize tumor-specific or tumor-associated antigens is a promising approach for eradicating tumors and cancer stem cells. Therefore, it can potentially avoid tumor recurrence compared to traditional chemotherapy, radiotherapy, and surgery. Furthermore, novel pathway-selective drugs often allow for excellent tumor control, but the disease course typically transitions to a chronic phase without definitive tumor elimination.

[0004] The emergence of genetically modified T cells expressing CARs has proven highly successful in the treatment of B-cell lymphoma / leukemia, despite patients having undergone extensive pretreatment and prior to multiple chemotherapy therapies, antibody therapies, and even autologous / allogeneic bone marrow transplants. Therefore, ATT with CAR-T cells has been successful as a salvage therapy.

[0005] CARs are synthetic, engineered immunoglobulin-derived receptors that can recognize surface antigens in an MHC-independent manner. Unlike TCRs, CARs have a broader affinity, allowing them to bind to target antigens without necessarily exhibiting cross-reactivity. Target antigens must be located on the surface and can include tumor-associated proteins, carbohydrates, or even glycolipids. Another advantage of CAR-T cells is their rapid generation via transduction of autologous T cells, which can be CD4+ or CD8+ derived. CARs can be generated “off-the-shelf,” and their targets are typically widely expressed (>90%) in identified tumor entities, as exemplified by CD19+ B-cell leukemia and lymphoma. It has been proposed that CAR T cells can serve as a “living drug,” maintaining their activity even after a single T-cell infusion.

[0006] There is a strong medical need for the chimeric antigen receptor (CAR)-T cell products described in this article. First, multiple myeloma is an incurable B-cell non-Hodgkin lymphoma (B-NHL) originating from malignantly transformed plasma cell clones. As a characteristic feature, the tumor cells are primarily located in the bone marrow. This disease is the most common tumor in bone and bone marrow, with a 10-year survival rate of 50% in young patients treated with intensive therapy, and causes 2% of cancer deaths annually. With an incidence of 5 / 100,000 and a median age at diagnosis of 70 years, this indicates that many patients have comorbidities unsuitable for intensive and long-term chemotherapy. Standard treatment is chemotherapy alone, or a combination of chemotherapy with autologous stem cell transplantation, immunomodulatory drugs, local radiation, proteasome inhibitors, and, in rare cases, allogeneic stem cell transplantation. Despite intensive treatment with these approaches, the disease often relapses and secondary resistance develops after multiple lines of therapy.

[0007] Secondly, the much larger classic B-NHL group includes a variety of tumor entities derived from B lymphocytes that are typically located in secondary lymphoid organs, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and the chronic lymphocytic leukemia (CLL) subset. While the total incidence of all NHL is approximately 10 to 12 per 100,000 (>85% of which are B-cell derived), the majority are adult-onset diseases, with a significant increase in older adults. Population development projects that the total number will increase due to aging populations in Western societies. Clinically, B-NHL is heterogeneous and can be identified by its aggressive and indolent processes. Substantial progress has been made in the treatment of B-NHL over the past 15 years, with standard treatment being combination antibody / chemotherapy, or a combination of antibody / chemotherapy with autologous stem cell transplantation, immunomodulatory drugs, radiation, proteasome inhibitors, signaling pathway inhibitors, and, in rare cases, allogeneic stem cell transplantation. Because the median age at diagnosis in many B-NHL cases is >55 to 60 years, there are also comorbidities that make patients unsuitable for intensive and long-term chemotherapy or even allogeneic bone marrow transplantation.

[0008] The emergence of adoptive CAR-T cell therapy targeting the CD19 antigen, which is widely expressed on lymphoma B cells, has made it possible to overcome these limitations. Currently, approximately 20 CD19 CAR-T cell studies for the treatment of B-NHL and B-ALL are registered with the FDA. While significant breakthroughs were achieved in clinical trials for CLL in 2011 and B-ALL in 2013, to the inventor's knowledge, biopharmaceutical companies have only recently received approval to use the same CD19 CAR product in Germany. Clinical trials using CD19 CAR T cells are also underway in other EU countries, such as Austria. More importantly, resistance has occurred in anti-CD19 antibody or CAR-T cell therapy for B-NHL due to antigen loss. Because treatment resistance has been observed after multiple lines of chemotherapy / immunotherapy, there is an urgent need for alternative target structures.

[0009] For indications of multiple myeloma, two anti-BCMA-CAR products have been previously described and they have entered Phase I clinical trials. These studies have not demonstrated the applicability of anti-BCMA CARs for B-NHL. Regarding B-NHL, anti-BCMA targeted therapy represents a possible alternative, especially when anti-CD19 CARs fail. Other immunotherapy strategies targeting multiple myeloma and tested in clinical trials include anti-CD19 CARs, NY-ESO1, and MAGE-A1-guided TCR-transduced T cells. In stark contrast to BCMA as a tumor target, the frequency of eligibility in patients is much lower, as the expression rate of these target antigens is less than 10%. Other targeted therapies include anti-CD38 and anti-SLAMF7 antibodies; conceptually, these therapies are quite different because the antibodies are not self-sustaining, do not form memory, and, to our knowledge, have not been shown to mediate adequate tumor eradication.

[0010] Furthermore, the ability to specifically target plasma cells would be highly beneficial for treating 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) involve organ dysfunction due to active disease and are typically treated with a combination of steroids and strong immunosuppressants such as cyclophosphamide (a cytotoxic agent that targets circulating cells).

[0011] Just recently, belimumab, an antibody against the cytokine BAFF, was found to have elevated levels in the serum of patients with autoimmune diseases and has been approved by the U.S. Food and Drug Administration (FDA) for 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 licensed biologics, followed by abatacept, rituximab, and tocilizumab, among others: they inhibit key inflammatory pathways associated with joint inflammation and damage; however, at the cost of increased risk of infection due to relative immunosuppression (Chan et al. (2010) Nat Rev Immunol 10:301–316, Keyser (2011) CurrRheumatol Rev 7:77–87).

[0012] Only recently have CAR-T cells been discussed as a targeted approach for treating autoantibody-mediated diseases (Ellebrecht et al. (2016) Science 353:179-184). Long-lived and stabilized plasma cells residing in survival niches in the bone marrow are typically resistant to conventional immunosuppressive and cytotoxic drugs, as well as therapies targeting B cells and their activation. In particular, rituximab appears unsuitable for this treatment because its target antigen, CD20, is not expressed on plasma cells. This therapeutic challenge can be addressed by using anti-BCMA CAR-T cell constructs, as BCMA is expressed on long-lived plasma cells.

[0013] Currently, many other anti-BCMA CAR constructs have been described in this field. In 2013, James N. Kochenderfer's group published the first anti-BCMA CAR-transduced T-cell approach, a preclinical study using in vitro assays and mouse experiments (Carpenter et al., 2013; Clin Cancer Res; 19(8); 2048–2060). In June 2015, Bluebird Bio and Celgene announced a collaboration to develop BCMA CAR-T cell therapy. In January 2016, enrollment for a Phase I clinical trial in patients with multiple myeloma began. In early 2016, the Abramson Cancer Center at the University of Pennsylvania began recruiting for a Phase I study of treating multiple myeloma patients with anti-BCMA CAR-transduced T-cell therapy (ClinicalTrials.gov ID: NCT02546167). CARs targeting BCMA have been described in WO 2016 / 014789, WO2016 / 014565, and WO2013 / 154760. WO 2015 / 128653 also discloses a CAR sequence that binds to BCMA, wherein the CAR portion responsible for epitope recognition is a variant of the APRIL ligand, which shows improved binding to BCMA compared to wild-type APRIL. Alternative treatment strategies involve anti-CD38 CARs. BCMA-binding antibodies have been disclosed in WO 2015 / 166073 and WO 2014 / 068079.

[0014] Although many potential alternative therapies are under development, there is still a need to provide effective methods to address medical conditions associated with the presence of pathogenic B cells, particularly multiple myeloma, non-Hodgkin's lymphoma, or autoantibody-dependent autoimmune diseases. Summary of the Invention

[0015] In view of the prior art, the technical problem of the present invention is to provide a medicament suitable for treating diseases associated with pathogenic B cells.

[0016] This problem is solved by the features of the independent claims. The dependent claims provide preferred embodiments of the invention.

[0017] Therefore, this invention relates to isolated chimeric antigen receptor polypeptides (CARs), wherein the CARs comprise:

[0018] i. An extracellular antigen-binding domain containing an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide.

[0019] ii. Transmembrane domain, and

[0020] iii. Intracellular domain,

[0021] And the CAR is said to bind to an epitope of one or more amino acids from the 13th to 32nd residues of the N-terminus of BCMA.

[0022] Therefore, the present invention relates to genetically modified immune cells (preferably T cells) expressing the CAR of the present invention, and the use of said cells in the treatment of medical conditions associated with the presence of pathogenic B cells.

[0023] Therefore, this invention provides a preferred autologous T cell containing anti-BCMA CAR suitable for transplantation for the treatment of mature B-NHL and multiple myeloma at different stages. In a preferred embodiment of the immunotherapy method of this invention, patient-derived T cells, preferably retrovirally transduced, are transduced to express an artificial immune receptor as described herein, which consists of an extracellular antibody-derived antigen recognition portion fused to a transmembrane portion and an intracellular signaling domain. The construct described herein endows the transduced T cells with antitumor cell lysis capabilities.

[0024] As demonstrated in other clinical CAR-T cell transfers, this invention is characterized by predictable, tolerable, and manageable side effects of the anti-BCMA CAR-T cells based on the CAR described herein. Preclinical testing of the BCMA CAR-T cells described herein has shown selectivity for the tumor-associated antigen BCMA. T cells equipped with the anti-BCMA CAR exhibit high affinity and avidity, recognizing and destroying multiple myeloma cells while preserving normal hematopoietic cells. In a preferred embodiment, the transfer of autologous T cells prevents the possibility of graft-versus-host disease. The formation of memory CAR-T cells is important for preventing relapse and can potentially develop.

[0025] Due to the high affinity and cohesion of the anti-BCMA CAR-T cells described in this article, they can even recognize mature B-NHL cells with low BCMA expression, thereby allowing T cell activation and tumor cell killing.

[0026] In a preferred embodiment, such mature B-NHL entities include certain stages of FL (follicular lymphoma), DLBCL (diffuse large B-cell lymphoma), mantle cell lymphoma (MCL), and CLL (chronic lymphocytic leukemia).

[0027] The antigen recognition portion of the CAR described herein is preferably based on the humanized antibody described in WO / 2015 / 166073. The antibody described therein is used to construct numerous CAR constructs that retain high affinity and specificity for BCMA. This high affinity and specificity reduces off-target reactivity, thus providing an advantage over other BCMA CAR constructs. Surprisingly, as described herein, the high specificity and affinity of the original antibody can be maintained in the CAR to target B cells expressing even very low levels of BCMA antigen.

[0028] The CAR of the present invention preferably binds to an epitope containing one or more amino acid residues from the 13th to 32nd residues of the N-terminus of BCMA. In other embodiments, it may also bind to other epitopes of BCMA, particularly the N-terminus of BCMA.

[0029] This invention also includes various signal transduction domains. The exchange of signal transduction domains satisfies the need for potent and rapid-effect phases (CD28 co-stimulatory domain) or long-acting relapse control protected by T-cell memory populations (4-1BB signal transduction domain). As demonstrated herein, various signal transduction domains can be interchanged in multiple configurations, thereby providing flexibility in the design of CARs without sacrificing advantageous binding properties.

[0030] 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 possess unparalleled high affinity and confer extremely high specificity and affinity to T cells. These properties enable CAR-T cells to i) recognize tumor target cells with high and surprisingly low BCMA surface expression, ii) be activated against said tumor target cells, and iii) kill said tumor target cells.

[0031] The amount of BCMA antigen expressed on the surface of tumor cells can be quantified using an anti-BCMA antibody conjugated to a fluorescent dye and attached to Quantibrite beads (from Becton Dicksinson). The preferred method for quantifying BCMA antigen expression on the surface of tumor cells is fluorescence-activated cell sorting / cell analysis (FACS). The fluorescence intensity of the beads is directly correlated with the amount of fluorescent antibody bound to the cells, which is a measure of the number of BCMA molecules on the cells. Myeloma cells typically exhibit at least 2–3 log [log] higher fluorescence density compared to low-fluorescence B-NHL cells. 10 This indicates that the BCMA antigen density can also be at least 2-3 log [value missing]. 10 It varies within a range of times.

[0032] Aside from multiple myeloma cells or, in the very rare case of Burkitt lymphoma, no competitive anti-BCMA CAR has been shown to be responsive to B-NHL. Therefore, the anti-BCMA CAR exhibits unprecedented responsiveness to the diversity of B-NHL. These properties represent an unexpected and surprising benefit of CAR-T therapy. Typical expectations from those skilled in the art require the expression of high levels of the target antigen to achieve CAR-T targeting. CAR-T using the CAR of this invention has shown unprecedented activity against B cells with low levels of target antigen expression.

[0033] In a preferred embodiment, the combination with the MP71-vector and γ-retroviral expression system can achieve an exceptionally high transduction rate for human T cells.

[0034] Preferred implementation of BCMA epitopes bound by CAR

[0035] The CAR of the present invention preferably targets 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 shown in SEQ ID No. 33. The N-terminal sequence of CD269 is provided in SEQ ID No. 32. The extracellular domain of CD269 is provided in SEQ ID No. 31.

[0036] Vaccination using an antigen comprising the extracellular domain of CD269 according to SEQ ID No. 31 produces binding specificity of mouse antibodies and chimeric antibodies (WO / 2014 / 068079) as described herein and previously, said chimeric antibodies having been modified for the CAR form of this invention. Using the entire CD269 protein or fragments thereof comprising a membrane-binding domain or an intracellular domain as an antigen during antibody production can produce antibodies that bind to the cryptic or intracellular domains of CD269, thus rendering these agents unsuitable or detrimental to therapeutic applications. Therefore, the CAR of this invention is defined by its binding to the extracellular portion of CD269. Specific epitopes in the extracellular domain also represent a preferred novel and additional characteristic of this invention.

[0037] The Fab fragment prepared from the mouse antibody or chimeric antibody derived from the CAR of this invention was crystallized as a complex with the purified extracellular domain of BCMA, and the structure of this complex was resolved. Structural analysis revealed detailed information about the binding epitopes of the antibody / CAR of this invention and their biological relevance. Due to the high binding specificity and extracellular location of the antibody of this invention, binding of the antibody of this invention to one or more amino acid epitopes from residues 13 to 32 of BCMA containing the extracellular domain is advantageous. To the inventors' knowledge, no CAR binding to this region has been previously described.

[0038] In one embodiment, the CAR of the present invention is characterized in that the CAR binds to an epitope comprising one or more of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, or 32 of CD269 (BCMA). In another embodiment, the CAR of the present invention is characterized in that the antibody binds to an epitope consisting of amino acids 13, 15, 16, 17, 18, 19, 20, 22, 23, 26, 27, and 32 of CD269 (BCMA). These residues represent amino acids that directly interact with the antibody of the present invention, as identified by the crystal structure data provided herein. These residues have been numbered for SEQ ID No. 32, which provides the N-terminal sequence of human BCMA.

[0039] As previously disclosed, the antibody from which the CAR of this invention is derived exhibits surprisingly high affinity and is superior to similar approaches attempted in the prior art. Therefore, the CAR of this invention is characterized by high affinity not seen in other anti-BCMA CAR molecules. In conventional practice, Kd in the pM range (as shown below) is generally considered to represent unexpectedly excellent affinity.

[0040] In another aspect, the CAR of the present invention is derived from a humanized antibody or antibody fragment that binds to BCMA with high affinity, for example, when measured by surface plasmon resonance (e.g., Biacore), the antibody binds to human BCMA with an affinity of 100 nM, 90, 80, 70, 60, 50, 40, 30 nM or lower, or 20 nM or lower, or 15 nM or lower, or 5 nM or lower, or 1000 pM or lower, or 500 pM or lower, or 100 pM or lower, or 80 pM or lower, or for example, about 50 pM. Therefore, the CAR of the present invention exhibits corresponding affinity.

[0041] In another embodiment, the antibody derived from the CAR of the present invention binds to human CD269 and, when measured by surface plasmon resonance, has an affinity between about 1 pM and about 100 nM, or between about 100 pM and about 50 nM, or between about 200 pM and about 20 nM. Therefore, the CAR of the present invention exhibits the corresponding affinity.

[0042] In one embodiment, the CAR and / or CAR-T of the present invention is characterized by CAR binding to cells expressing BCMA, wherein BCMA is detectable on the cell surface, and wherein, compared to multiple myeloma cells, preferably compared to those multiple myeloma cell lines used in the embodiments described herein, BCMA is expressed at a rate 1 to 4 log lower. 10 Times, preferably 2-3 log 10 These cells exist in quantities several times greater than those present on the cell surface. Examples of such cells are non-Hodgkin lymphoma (B-NHL) cells, such as DOHH-2, SU-DHL4, JEKO-1, JVM-3, and / or MEC-1 cell lines, but are not limited thereto.

[0043] Preferred implementation methods for CAR sequences:

[0044] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises:

[0045] - Heavy chain complementarity determination region 1 (H-CDR1) with at least 80% sequence identity to SEQ ID NO 1 (GFTFSRYW).

[0046] - Heavy chain complementarity-determining region 2 (H-CDR2) with at least 80% sequence identity to SEQ ID NO 2 (INPSSSTI), and

[0047] - Heavy chain complementarity determination region 3 (H-CDR3) with at least 80% sequence identity to SEQ ID NO 3 (ASLYYDYGDAYDY).

[0048] The VL includes:

[0049] - Light chain complementarity determination region 1 (L-CDR1) with at least 80% sequence identity to SEQ ID NO 4 (QSVESN).

[0050] - Light chain complementarity-determining region 2 (L-CDR2) with at least 80% sequence identity to SEQ ID NO 5 (SAS), and

[0051] - Light chain complementarity determination region 3 (L-CDR3) with at least 80% sequence identity to SEQ ID NO 6 (QQYNNYPLT).

[0052] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises:

[0053] - Heavy chain complementarity determination region 1 (H-CDR1) with at least 80% sequence identity to SEQ ID NO 25 (RYWFS).

[0054] - Heavy chain complementarity-determining region 2 (H-CDR2) with at least 80% sequence identity to SEQ ID NO 26 (EINPSSSTINYAPSLKDK), and

[0055] - Heavy chain complementarity determination region 3 (H-CDR3) with at least 80% sequence identity to SEQ ID NO 27 (SLYYDYGDAYDYW).

[0056] The VL includes:

[0057] - Light chain complementarity determination region 1 (L-CDR1) with at least 80% sequence identity to SEQ ID NO 28 (KASQSVESNVA).

[0058] - Light chain complementarity-determining region 2 (L-CDR2) with at least 80% sequence identity to SEQ ID NO 29 (SASLRFS), and

[0059] - Light chain complementarity determination region 3 (L-CDR3) with at least 80% sequence identity to SEQ ID NO 30 (QQYNNYPLTFG).

[0060] The CDR sequences listed above with reference to SEQ ID NO 25-30 represent embodiments obtained by using alternative parameters for defining the CDR region and including, for example, additional flanking amino acids compared to SEQ ID NO 1-6.

[0061] The CDR sequences of SEQ ID NO 1-6 and 25-30 may also be specified, such that the invention covers polypeptide sequences having at least 70%, 75%, 80%, 85%, 90%, or at least 95% sequence identity with the specific sequences listed.

[0062] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises the following CDR sequence:

[0063] - GFTFSRYW (H-CDR1; SEQ ID NO.1);

[0064] - INPX2X3STI (H-CDR2; SEQ ID No. 7), where X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE; and

[0065] - ASLYX4DYGDAX5DY (H-CDR3; SEQ ID NO 8), where X4: Y, L, A, V, F, I, W, and / or X5: Y, L, F, I, V, A, C;

[0066] The VL field contains the following CDR sequences:

[0067] - QSVX1X2N (L-CDR1; SEQ ID NO 9), where X1X2: ES, SS, TS, QS, HS, DH;

[0068] - SAS (L-CDR2; SEQ ID NO 5); and

[0069] - QQYNNYPLTFG (L-CDR3; SEQ ID NO. 10).

[0070] In an alternative embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention comprises a VH domain and a VL domain, wherein the VH domain comprises the following CDR sequence:

[0071] - RYWX1S (H-CDR1; SEQ ID NO 34), where X1: I, F, L, V, Y, C, G, A, S, T;

[0072] - EINPX2X3STINYAPSLKDK(H-CDR2; SEQ ID NO 35), where X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE; and

[0073] - SLYX4DYGDAX5DYW (H-CDR3; SEQ ID NO 36), wherein X4: Y, L, A, V, F, I, W, and / or X5: Y, L, F, I, V, A, C;

[0074] The VL field contains the following CDR sequences:

[0075] - KASQSVX1X2NVA (L-CDR1; SEQ ID NO 37), where X1X2: ES, SS, TS, QS, HS, DH;

[0076] - SASLRFS(L-CDR2; SEQ ID NO 29); and

[0077] - QQYNNYPLTFG (L-CDR3; SEQ ID NO. 30).

[0078] The CDR sequences listed above, SEQ ID NO 34-37, represent embodiments obtained by using alternative parameters for defining the CDR region and, for example, including additional flanking amino acids compared to SEQ ID NO 1-6.

[0079] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR comprises the following sequence:

[0080] - H-CDR1: GFTFSRYW (SEQ ID NO.1),

[0081] - H-CDR2: INPSSSTI (SEQ ID NO.2),

[0082] - H-CDR3: ASLYYDYGDAYDY (SEQ ID NO.3),

[0083] - L-CDR1: QSVESN (SEQ ID NO.4),

[0084] - L-CDR2: SAS (SEQ ID NO 5), and

[0085] - L-CDR3: QQYNNYPLT (SEQ ID NO 6).

[0086] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention comprises the following CDR sequence:

[0087] - H-CDR1: RYWFS (SEQ ID NO.25),

[0088] - H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO.26),

[0089] - H-CDR3: SLYYDYGDAYDYW (SEQ ID NO.27),

[0090] - L-CDR1: KASQSVESNVA (SEQ ID NO.28),

[0091] - L-CDR2:SASLRFS (SEQ ID NO.29), and

[0092] - L-CDR3: QQYNNYPLTFG (SEQ ID NO.30),

[0093] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR contains a VH domain having at least 80% sequence identity with SEQ ID NO 11 (EVQLVESGGGLVQPGGSLRLSCAASGFT FSRYWFSWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS);

[0094] The VL field that has at least 80% sequence identity with SEQ ID NO 12 (EIVMTQSPATLSVSPGERATLSCKASQSVESNVAW YQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK).

[0095] SEQ ID NO 11 and 12 represent the “full-length” VH and VL domains of the preferred CAR. Sequences having at least 70%, preferably 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NO 11 and 12, particularly when such sequence variants exhibit the desired BCMA binding specificity (functionally similar / equivalent), are included within the scope of this invention.

[0096] In one embodiment, a separate chimeric antigen receptor (CAR) polypeptide comprising the VH and VL sequences of SEQ ID NO 11 and 12, or sequences having at least 80% identity with SEQ ID NO 11 and 12, comprises at least W36, E50, L99, Y100, Y101 and A106 of SEQ ID NO 11, and at least S31, A34, S50, L53, Q89, Y91, Y94 and L96 of SEQ ID NO 12.

[0097] The amino acid residues listed above represent amino acid residues known to directly interact with the target BCMA epitope. Therefore, this invention relates to CARs wherein sequence variations occur in the VH and VL domains within the range of at least 70%, preferably 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NO 11 and 12, but the VH and VL domains contain at least those residues known to interact with the target epitope.

[0098] In one embodiment, the isolated chimeric antigen receptor (CAR) polypeptide comprising the VH and VL sequences of SEQ ID NO 11 and 12, or sequences having at least 80% identity with SEQ ID NO 11 and 12, comprises at least the CDR sequences of SEQ ID NO 1, 7, 8, 9, 5 and 10, preferably the CDR sequences of SEQ NO 1 to 6, as described herein.

[0099] Therefore, the present invention relates to CAR, wherein sequence variations occur in the VH and VL domains within the range of having at least 70%, preferably 80%, 85%, 90%, or at least 95% sequence identity with SEQ ID NO 11 and 12, but the VH and VL domains at least contain the CDR sequence as described herein. The CDR can represent any sequence referred to herein as CDR, particularly the sequences of SEQ ID NO 1-6 or SEQ ID NO 25-30.

[0100] In a preferred embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that, when the CAR is expressed in genetically modified immune cells, preferably T lymphocytes, the immune cells are activated by binding BCMA to the surface of non-Hodgkin lymphoma (B-NHL) via the CAR, thereby inducing cytotoxic activity against the B-NHL.

[0101] In a preferred embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the B-cell lymphoma is a non-Hodgkin lymphoma (B-NHL) cell, such as DOHH-2, SU-DHL4, JEKO-1, JVM-3 and / or MEC-1 cell lines.

[0102] The CAR of the present invention is characterized by the surprising property that even very low levels of BCMA on the cell surface can lead to CAR binding, T cell activation, and cytotoxicity against bound cells. This represents a significant advantage compared to conventionally described CARs. Typically, CARs require large amounts of surface antigen to activate the CAR and subsequently achieve cytotoxic activity. Therefore, the CAR of the present invention is associated with unexpected benefits, given the CARs known in the art.

[0103] In some embodiments, derivatization of previously described mouse antibodies, chimeric antibodies, and / or human antibodies can provide this advantage in order to generate a CAR as described herein. In some embodiments, the characterization of the BCMA epitope preferably results in this advantage. In other embodiments, the high affinity and specificity of the VH and VL fragments described herein enable the sensitivity of this CAR. However, unexpectedly, this property will be combined with CARs previously described for antibodies. Completely unexpectedly, the specific sequences provided herein, preferably involving the CDR regions of the VL and VH regions, exhibit specific and strong binding sufficient to activate CAR-T cells against cells with minimal BCMA expression.

[0104] Surprisingly, the VH and VL fragments described in this paper can be arranged in multiple conformations in CARs as described herein, while still maintaining high specificity and high affinity for the target epitopes. See below. Figure 3 As shown, the CAR can be constructed in a VH-VL or VL-VH configuration, with variations in the junction, hinge, transmembrane domain, co-stimulatory domain, and / or activation domain, while still retaining its efficacy. This surprising feature of the invention allows for greater flexibility in the design of BCMA-targeted CARs, enabling further modification and / or optimization of the CAR structure based on the VH and VL domains described herein if further development is desired or desired.

[0105] In a preferred embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the SAR is expressed in genetically modified mutant cells, preferably T lymphocytes, which are immune cells that bind BCMA to the surface of multiple myeloma (MM) cells via the CAR and are activated, thereby inducing cytotoxic activity against the MM cells. Preferred MM cells are disclosed herein. In a preferred embodiment, the CAR described herein exhibits efficient binding and cytotoxic activity against both MM and B-NHL. Given the prior art, those skilled in the art would not anticipate that the CAR described herein would be able to exhibit activity against both cell types.

[0106] Preferred implementation methods for humanized VH and VL domains

[0107] As detailed herein and previously disclosed (WO / 2015 / 166073), the sequence of antibody J22.9-xi is humanized to provide a reagent more suitable for administration in human subjects. Various humanized sequence variants of J22.9-xi have been generated, and their binding affinity and specificity to human and cynomolgus BCMA have been tested. In a preferred embodiment, the CAR of the present invention comprises these humanized sequences. Results of binding analyses performed with the corresponding antibodies indicate that the humanized sequences retain the desired binding properties of the chimeric reagent J22.9-xi. In the sequences below, underlined regions represent CDRs or putative CDRs, depending on the method used for CDR determination.

[0108] Preferred implementation methods for humanized VH variants

[0109] The following provides further information regarding the humanized VH and VL sequences preferably incorporated via the CAR of the present invention.

[0110] Chimeric sequence:

[0111] HC mice (SEQ ID No. 38):

[0112] QVQLQQSGGGLVQPGGSLKLSCAASGIDFS RYWMS WVRRAPGKGLEWIG EINPDSSTINYAPSLKDK FIISRDNAKNTLYLQMSKVRSEDTALYYCA SLYYDYGDAMDYW GQGTSVTVSS

[0113] The HC mouse sequence represents the heavy chain (VH) variable region originally developed for the chimeric antibody J22.9-xi, which contains VL and VH domains derived from mouse antibodies. This heavy chain variable region is capable of binding epitopes of the extracellular domain of CD269 (BCMA), with the VL and VH domains fused to the human CL and CH domains, respectively. In some embodiments, the CAR may be incorporated into the HC mouse sequence or its CDR.

[0114] Partially humanized sequences:

[0115] HC partially humanized (SEQ ID No. 39):

[0116] EVQLVESGGGLVQPGGSLRLSCAASGFTFD DYWMS WVRQAPGKGLEWVG EINPDSSTINYAPSLKGR FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0117] The HC partially humanized sequence represents a modified amino acid sequence (through amino acid substitution) compared to the chimeric antibody disclosed herein, thereby modifying the VL and VH binding regions to make them more suitable for administration in humans.

[0118] Humanized VH sequence:

[0119] hHC01 (SEQ ID No. 40)

[0120] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWMS WVRQAPGKGLVWVG EINPDSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAMDYW GQGTLVTVSS

[0121] Humanized VH sequences with post-translational modified motifs removed:

[0122] hHC02 (SEQ ID No. 41)

[0123] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWX1S WVRQAPGKGLVWVG EINPX2X3STINYAPSLKD K FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYX4DYGDAX5DYW GQGTLVTVSS

[0124] in:

[0125] X1: I, F, L, V, Y, C, G, A, S, T, preferably I or F;

[0126] X2X3: SS, NS, TS, GS, KS, RS, SD, SN, DE, with SS being the preferred choice;

[0127] X4: Y, L, A, V, F, I, W, preferably Y; and / or

[0128] X5: Y, L, F, I, V, A, C, with Y being the preferred choice;

[0129] The humanized sequences “hHC01” and “hHC02” represent preferred amino acid sequences of the CAR of the present invention, which include sequence changes compared to the original chimeric sequence and the partially humanized sequence described herein.

[0130] PTM mutations aim to remove potentially harmful post-translational modification motifs from the protein while preserving favorable binding properties. Positions 1, 5, 6, 19, 27, 28, 34, 49, 46, 48, 54, 69, 84, 85, 86, 88, 93, 107, and / or 115 of hHC01 and hHC02 are preferably mutated (substituted) compared to the original chimeric sequence. The importance of substitutions lies primarily in the resulting amino acid, rather than the original amino acid. Therefore, alterations can also be made from the corresponding amino acid of the original chimeric amino acid or from the corresponding amino acid of other variants, such as partially humanized sequences.

[0131] In some implementations, the following substitutions are preferred and differ from the chimeric (SEQ ID No. 38) sequence:

[0132] - Amino acid M34 of the HC(VH) sequence is replaced by any amino acid, preferably I, L, F, V, Y, C, G, A, S, T;

[0133] - The amino acid E46 in the HC(VH) sequence is replaced by V;

[0134] - The amino acids D54 and S55 of the HC(VH) sequence are replaced by any combination of amino acids, preferably SS, TS, GS, KS, RS, SD, SN, DE;

[0135] - The amino acid Y101 in the HC(VH) sequence is replaced by any amino acid, preferably L, A, V, F, I, W; and / or

[0136] - The amino acid M107 of the HC(VH) sequence is replaced by any amino acid, preferably L, Y, F, I, V, A, or C.

[0137] Sequences that can be modified at those residues required for direct interaction with BCMA:

[0138] hHC03- Modified amino acids involved in the interaction with BCMA (SEQ ID No 42):

[0139] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYX1MX2 WVRQAPGKGLVX3VGX4 INPDSSTINYAPSLK DK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SX5X6X7DYGDX8MDYW GQGTLVTVSS

[0140] The preferred amino acids are:

[0141] X1: W, F, Y, W is preferred;

[0142] X2: S, T, N, Q, D, E, with S being the preferred option;

[0143] X3: W, F, Y, W is preferred;

[0144] X4: E, Q, E is preferred;

[0145] X5: L, I, V, G, A, with L being the preferred choice;

[0146] X6: Y, X, Y is preferred;

[0147] X7: Y, F, L, I, V, M, Y preferred; and / or

[0148] X8: A, G, V, A is preferred.

[0149] The "hHC03" humanized sequence represents a preferred amino acid sequence, comprising amino acid sequence changes compared to the original chimeric sequence and the partially humanized sequence. These sequence changes are designed to reflect potential variations in the amino acids that can be substituted for binding to the BCMA target, while maintaining favorable binding properties. The importance of substitutions lies primarily with the resulting amino acid, rather than the original amino acid. Therefore, changes can also be made from the corresponding amino acid of the original chimeric amino acid or from the corresponding amino acid of other variants.

[0150] For example:

[0151] - The amino acid W33 in the HC(VH) sequence is W, F, Y;

[0152] - The amino acid S35 of the HC(VH) sequence is S, T, N, Q, D, E;

[0153] - The amino acid W47 in the HC(VH) sequence is W, F, Y;

[0154] - The amino acid E50 of the HC(VH) sequence is E, Q;

[0155] - The amino acid sequence L99 of the HC(VH) sequence is L, I, V, G, A;

[0156] - The amino acid Y100 of the HC(VH) sequence is Y, X;

[0157] - The amino acid Y101 of the HC(VH) sequence is Y, F, L, I, V, M; and / or

[0158] - The amino acid sequence A106 of the HC(VH) sequence is A, G, V.

[0159] Generally, any changes made to the CDR region during humanization can also be considered a feature of the CDR sequence when considered independently of the frame sequence as a whole. Such modified CDR sequences can be considered to define the features of the invention within or independently of the context of the entire frame region described herein. For example, the underlined CDR sequences in hHC01 to hHC03 can be considered defining features of the invention, independent of the surrounding variable region sequences.

[0160] Specific examples of humanized HC (VH) sequences:

[0161] hHC04 (SEQ ID NO 43):

[0162] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0163] hHC05 (SEQ ID NO 44):

[0164] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWFS WVRQAPGKGLVWVG EINPNSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0165] hHC06 (SEQ ID NO 45):

[0166] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWIS WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0167] hHC07 (SEQ ID NO 46):

[0168] EVQLVESGGGLVQPGGSLRLSCAASGFTFS RYWFS WVRQAPGKGLVWVG EINPSSSTINYAPSLKDK FTISRDNAKNTLYLQMNSLRAEDTAVYYCA SLYYDYGDAYDYW GQGTLVTVSS

[0169] To remove potential post-translational modification sites in humanized J22.9, residue D54 of the heavy chain CDR2 was mutated to asparagine (N), thereby creating new potential modification sites for N-linked glycosylation (e.g., hHC04, O5). The mutant heavy chain containing N54 can be glycosylated. However, the corresponding IgG, J22.9-FNY, binds to BCMA in both FACS and ELISA and crystallizes with BCMA as a complex. Surprisingly, this large extension of the side chain does not disrupt the binding to BCMA, and these observations suggest that multiple and varied amino acid substitutions, possibly including derivatizations other than sugars, can be tolerated at this site.

[0170] Comparison:

[0171] Figure 13 In the CLUSTAL W (1.83) multiple sequence alignment of various substitution sites within the HC sequence provides appropriate sequence comparisons. "General sequence" represents the HC sequence, where each X represents a potential amino acid change for any given amino group. Preferred amino acid substitutions are those described above for each potential mutation site.

[0172] Preferred implementation methods for humanized VL variants

[0173] Chimeric sequence:

[0174] LC mice (SEQ ID No. 47):

[0175] DIVMTQSQRFMTTSVGDRVSVTC KASQSVDSNVA WYQQKPRQSPKALIF SASLRFS GVPARFTGSGSGTDFTLTISNLQSEDLAEYFC QQYNNYPLTFG AGTKLELKR

[0176] The LC mouse sequence represents the light chain (VL) variable region originally developed for the chimeric antibody J22.9-xi, which contains VL and VH domains derived from mouse antibodies and is capable of binding epitopes of the extracellular domain of CD269 (BCMA). In some embodiments, the mouse VL domain or its CDR can be used in the CAR of the present invention.

[0177] Partially humanized sequences:

[0178] LC partial humanization (SEQ ID NO 48):

[0179] DIVMTQSPATLSVSVGDEVTLTC KASQSVDSNVA WYQQKPGQAPKLLIY SDDLRFSGVPARFSGSGSGTDFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0180] The LC partially humanized sequence represents a modified sequence (through amino acid substitutions) compared to the chimeric antibody, thereby modifying the VL and VH binding regions to make them more suitable for administration in humans.

[0181] Humanized VL sequence:

[0182] hLC01 (SEQ ID NO 49):

[0183] EIVMTQSPATLSVSPGERATLSC KASQSVDSNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0184] Humanized VL sequences with post-translational modified motifs removed:

[0185] hLC02 (SEQ ID NO 50):

[0186] EIVMTQSPATLSVSPGERATLSC KASQSVX1X2NVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0187] in:

[0188] X1X2: ES, SS, TS, QS, HS, DH, with ES being the preferred choice.

[0189] The humanized sequences “hLC01” and “hLC02” represent preferred amino acid sequences, which include amino acid sequence changes compared to the original chimeric sequences and partially humanized sequences described herein.

[0190] PTM mutations aim to remove potentially harmful post-translational modification motifs from the protein while preserving favorable binding properties. Positions 1, 8, 9, 10, 13, 15, 17, 19, 20, 21, 22, 30, 41, 43, 45, 49, 58, 63, 70, 77, 83, 85, and / or 87 of hLC01 and hLC02 are preferably mutated (substituted) compared to the original chimeric sequence. The importance of substitutions lies primarily in the resulting amino acid, rather than the original amino acid. Therefore, changes can also be made from the corresponding amino acid of the original chimeric amino acid or the corresponding amino acid of other variants.

[0191] The following substitutions are preferred and differ from chimeric and partially humanized sequences:

[0192] - The amino acid D1 in the LC (VL) sequence is replaced by E;

[0193] - The amino acid V15 in the LC (VL) sequence is replaced by P;

[0194] - The amino acid D17 in the LC (VL) sequence is replaced by E;

[0195] - The amino acid V19 in the LC (VL) sequence is replaced by A;

[0196] - The amino acid T22 in the LC (VL) sequence is replaced by S;

[0197] - The amino acids D30 and S31 of the LC (VL) sequence may be replaced by any combination of amino acids, preferably ES, SS, TS, QS, HS, or DH;

[0198] - Amino acid V58 in the LC (VL) sequence is replaced by I; and / or

[0199] - The amino acid D70 in the LC (VL) sequence is replaced by E.

[0200] The CDR-binding region can contain sequences of residues that require interaction with BCMA:

[0201] hLC03 - Modified amino acid involved in the interaction with BCMA (SEQ ID NO 51):

[0202] EIVMTQSPATLSVSPGERATLSC KASQSVDX1X2VX3 WX4QQKPGQAPRALIX5 X6AX7X8RX9S GIPARFSGSX 10 X 11 GTEFTLTISSLQSEDFAVYYC X 12 QX 13 NNX 14 PX 15 TFG AGTKLELKR

[0203] The preferred amino acids are:

[0204] X1: S, H, T, N, D, Q;

[0205] X2: N, E, Q;

[0206] X3: A, G, V, S, T, L, I;

[0207] X4: Y, F, L, I, V, A, G;

[0208] X5: Y, F, L;

[0209] X6: S, T;

[0210] X7: S, T, D, N, H, E, Q;

[0211] X8: L, V, I, M;

[0212] X9: F, L, I, V, Y, M;

[0213] X 10 : G, X;

[0214] X 11 S, X;

[0215] X 12 : Q, V, L, I, M;

[0216] X 13 : Y, F, L, I, Q;

[0217] X 14 : Y, F, R, Q, K; and / or

[0218] X 15 L, I, V, F

[0219] The “hLC03 humanized sequence” represents a preferred amino acid sequence, comprising amino acid sequence changes compared to the original chimeric sequence and the partially humanized sequence. These sequence changes are designed to reflect potential variations in the amino acids that can be substituted for binding to the BCMA target, while maintaining favorable binding properties. The importance of substitutions lies primarily with the resulting amino acid, rather than the original amino acid. Therefore, changes can also be made from the corresponding amino acid of the original chimeric amino acid or from the corresponding amino acid of other variants.

[0220] For example:

[0221] - The amino acid S31 of the LC (VL) sequence is S, H, T, N, D, Q;

[0222] - The amino acid N32 in the LC (VL) sequence is N, E, Q;

[0223] - The amino acid A34 of the LC (VL) sequence is A, G, V, S, T, L, I;

[0224] - The amino acid Y36 of the LC (VL) sequence is Y, F, L, I, V, A, G;

[0225] - The amino acid Y49 in the LC (VL) sequence is Y, F, L;

[0226] - The amino acid S50 of the LC(VL) sequence is S, T;

[0227] - The amino acid S52 of the LC (VL) sequence is S, T, D, N, H, E, Q;

[0228] - The amino acid L53 in the LC(VL) sequence is L, V, I, M;

[0229] - The amino acid F55 of the LC(VL) sequence is F, L, I, V, Y, M;

[0230] - The amino acid G66 in the LC(VL) sequence is G, X;

[0231] - The amino acid S67 of the LC (VL) sequence is S, X;

[0232] - The amino acid Q89 in the LC (VL) sequence is Q, V, L, I, M;

[0233] - The amino acid sequence Y91 in the LC (VL) sequence is Y, F, L, I, Q;

[0234] - The amino acid Y94 of the LC (VL) sequence is Y, F, R, Q, K; and / or

[0235] - The amino acid L96 of the LC(VL) sequence is L, I, V, F.

[0236] Generally, any changes made to the CDR region can be considered a feature of the CDR sequence when considered independently of the frame sequence as a whole. Such modified CDR sequences can be considered to define the characteristics of the sequence used herein within or independently of the context of the entire frame region described herein. For example, the CDR sequences identified by underscores in hLC01 to hLC03 can be considered as defining features of the invention in their unmodified or unreplaced form, independent of the surrounding variable region sequences.

[0237] Examples of humanized LC sequences:

[0238] hLC04 (SEQ ID NO 52):

[0239] EIVMTQSPATLSVSPGERATLSC KASQSVESNVA WYQQKPGQAPRALIY SASLRFS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNYPLTFG AGTKLELKR

[0240] Comparison:

[0241] Figure 14In the LC sequence, CLUSTAL W (1.83) multiple sequence alignment provides appropriate sequence comparisons for various potential modification sites. "General sequence" represents the LC sequence, where each X represents a potential amino acid change. Preferred amino acid substitutions are those described above for each potential mutation site.

[0242] Therefore, the present invention relates to humanized sequences based on hHC01, hHC02, hHCO03, hHCO04, hHC05, hHC06, hHC07, hLC01, hLC02, hLC03 and / or hLC04 or any given combination thereof.

[0243] All possible combinations of potential modifications to any given potential variant residues presented herein (as identified by X in the “general” sequence) are included in this invention. By combining one or more of these various substitutions, humanized variants exhibiting the desired binding properties of the chimeric antibodies originally developed and demonstrated herein can be produced. The antibodies or portions thereof described herein also include sequences having at least 80%, preferably 90%, sequence identity with those humanized sequences explicitly disclosed or disclosed by way of sequence formula.

[0244] The present invention further relates to a CAR comprising a VH domain as described herein, wherein the VH domain comprises according to X1VQLX2X3SGGGLVQPGGSLX4LSCAASGX5X6FX7X8YWZ1SWVRX9APGKGLEWX 10 GEINPZ2SSTINYAPSLKX 11 X 12 FX13ISRDNAKNTLYLQMX 14 X 15 X 16 RX 17 EDTAX 18 YYCASLYYDYGDAZ3DYWGQGTX 19The sequence of VTVSS (SEQ ID No. 53) includes 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: I, V; X11: D, G; X12: K, R; X13: I, T; X14: S, N; X15: K, S; X16: V, L; X17: S, A; X18: L, V; X19: S, L; and at least one of Z1, Z2 and / or Z3 is: Z1: I, F, L, V, Y. C, G, A, S, T, preferably I or F; Z2: S, N, T, G, K, R, D, preferably S; Z3: Y, L, F, I, V, A, C, preferably Y; and wherein the antibody or fragment thereof specifically binds to the epitope of the extracellular domain of CD269 (BCMA).

[0245] This implementation includes various humanized sequences for the CAR used in this invention, particularly its VH sequence, all variants of which are defined by the favorable humanization method described in the CDR herein.

[0246] The present invention further relates to antibodies or antibody fragments comprising a VL domain as described herein, wherein the VL domain comprises according to DIVMTQSX1X2X3X4X5X6SVGDX7VX8X9TCKASQSVESNVAW YQQKPX 10 QX 11 PKX 12 LIX 13 SX 14 X 15 LRFSGVPARFX 16 GSGSGTDFTLTISX 17 LQSEDX 18 AX 19 YX 20 The sequence CQQYNNYPLTFGAGTKLELKR (SEQ ID No. 54), wherein 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: R, G; X11: S, A; X12: A, L; X13: F, Y; X14: A, D; X15: S, D; X16: T, S; X17: N, S; X18: L, F; X19: E, V; X20: F, Y; wherein the antibody or fragment thereof specifically binds to the epitope of the extracellular domain of CD269 (BCMA).

[0247] This implementation includes various humanized sequences for the CAR used in this invention, particularly its VL sequence, all variants of which are defined by the favorable humanization method described in the CDR herein.

[0248] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the extracellular antigen-binding domain comprises a linker polypeptide located between the VH domain and the VL domain, wherein the linker is preferably selected from the Whitlow (SEQ ID NO 13; GSTGSGSGKPGSGEGSTKG) or Gly-Ser (SEQ ID NO 14; SSGGGGSGGGGSGGGGS) linker, or a linker having at least 80% sequence identity with SEQ ID NO 13 or 14.

[0249] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR comprises a spacer region polypeptide located between an extracellular antigen-binding domain and a transmembrane domain, wherein the spacer region is preferably selected from:

[0250] - IgG1-CD28 spacer (SEQ ID NO 15; PAEPKSPDKTHTCPPCPAPPVAGPS VFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK),

[0251] - IgG1Δ-4-1BB spacer (SEQ ID NO 16; PAEPKSPDKTHTCPPCPAPPVAGP SVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK),

[0252] - IgG4 (Hi-CH2-CH3) spacer (SEQ ID NO 17; ESKYGPPCPPCPAPEFEG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK),

[0253] - IgG4(Hi-CH3) spacer (SEQ ID NO 18; ESKYGPPCPPCPGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK),

[0254] - IgG4(Hi) spacer region (SEQ ID NO 19; ESKYGPPCPPCP), or

[0255] - A spacer region having at least 80% sequence identity with any one of SEQ ID NO 15 to 19;

[0256] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the transmembrane domain is preferably selected from the CD8α domain (SEQ ID NO 20; IYIWAPLAGTCGVLLLSLVITLYC) or the CD28 domain (SEQ ID NO 21; FWVLVVVGGVLACYSLLVTVAFIIFWV) or the transmembrane domain having at least 80% sequence identity with SEQ ID NO 20 or 21.

[0257] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that its intracellular domain includes a co-stimulatory domain, said co-stimulatory domain preferably selected from the 4-1BB co-stimulatory domain (SEQ ID NO 22; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or the CD28 co-stimulatory domain (SEQ ID NO 23; RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRS), or a co-stimulatory domain having at least 80% sequence identity with SEQ ID NO 22 or 23; and / or

[0258] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR comprises a signal transduction domain, wherein the signal transduction domain is preferably selected from the CD3ζ (CD28 or 4-1BB) signal transduction domain (SEQ ID NO 24; LRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or a signal transduction domain having at least 80% sequence identity with SEQ ID NO 24.

[0259] In one embodiment, the isolated chimeric antigen receptor (CAR) of the present invention is characterized in that the CAR comprises a tandem co-stimulatory domain and a CD3ζ signaling / activation domain (SEQ ID NO 24), the tandem co-stimulatory domain comprising a 4-1BB co-stimulatory domain (SEQ ID NO 22) and a CD28 co-stimulatory domain (SEQ ID NO 23).

[0260] In one embodiment, the isolated chimeric antigen receptor polypeptide (CAR) of the present invention is characterized in that the CAR comprises a leader sequence, wherein the leader sequence is preferably selected from the IGK leader sequence (SEQ ID NO 55; MDFQVQIFSFLLISASVIMSR) or the GMCSF leader sequence (SEQ ID NO 56; MLLLVTSLLLCELPHPAFLLI) or a leader sequence having at least 80% sequence identity with SEQ ID NO 55 or 56.

[0261] Another aspect of the present invention relates to isolated nucleic acid molecules selected from the group consisting of:

[0262] a) Nucleic acid molecules containing nucleotide sequences,

[0263] - The nucleotide sequence encodes a chimeric antigen receptor (CAR) polypeptide isolated as described herein, and / or

[0264] - The nucleotide sequence comprises a sequence or sequence fragment of SEQ ID No. 66 and / or 67, or SEQ ID No. 86 to 94, or

[0265] b) Nucleic acid molecules complementary to the nucleotide sequence according to a);

[0266] c) A nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity with the nucleotide sequence of a) or b) such that it is functionally similar to / equivalent to the nucleotide sequence according to a) or b), preferably comprising a sequence having at least 80% sequence identity with the nucleotide sequence of a) or b);

[0267] d) Nucleic acid molecules, as a result of the genetic code, are degenerate into nucleotide sequences according to a) through c); and

[0268] e) Nucleic acid molecules based on the nucleotide sequences of a) to d), which are modified by deletion, addition, substitution, translocation, inversion and / or insertion, and are functionally similar to / equivalent to the nucleotide sequences of a) to d).

[0269] The preferred amino acid sequence of the present invention is as follows:

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] Preferred nucleotide sequences:

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] Another aspect of the invention relates to a vector comprising a nucleic acid molecule as described herein, wherein the vector is preferably a viral vector, more preferably a γ-retroviral vector.

[0304] Another aspect of the invention relates to genetically modified immune cells comprising nucleic acid molecules or vectors as described herein, and / or expressing CARs as described herein, wherein the immune cells are preferably selected from the group consisting of free T lymphocytes or NK cells, more preferably cytotoxic T lymphocytes.

[0305] In a preferred embodiment, the genetically modified immune cells comprise nucleic acid molecules or vectors as described herein, and / or express CARs as described herein. These immune cells are characterized as CD4+ and / or CD8+ T cells, preferably a mixture of CD4+ and CD8+ T cells. These T cell populations, preferably compositions comprising CD4+ and CD8+ transformed cells, exhibit particularly effective cytolytic activity against various malignant B cells (e.g., multiple myeloma and B-NHL), preferably those described herein, and / or related medical conditions.

[0306] In a preferred embodiment, the genetically modified immune cells comprise nucleic acid molecules or vectors as described herein, and / or express CARs as described herein. These immune cells are CD4+ and CD8+ T cells, preferably in a ratio of 1:10 to 10:1, more preferably 5:1 to 1:5, 2:1 to 1:2, or 1:1. BCMA-guided modified CAR-T cells express CARs in the aforementioned ratios, preferably 1:1 CD4 / CD8+. Administration of these CAR-T cells during treatment of the aforementioned diseases provides beneficial characteristics, such as improved treatment response and reduced toxicity.

[0307] In a preferred embodiment, the immune cells to be administered to the aforementioned diseases are genetically modified immune cells using the aforementioned nucleic acids, employing the "Sleeping Beauty" transposon system, particularly the Sleeping Beauty transposase, which encodes and expresses the anti-BCMA CAR described herein. The Sleeping Beauty transposon system is a synthetic DNA transposon designed to guide the previously defined DNA sequence into the chromosome of a vertebrate, used in the context of this invention to modify immune cells for the purpose of expressing the CAR described herein. The Sleeping Beauty transposon combines the advantages of viruses with naked DNA. Viruses have undergone evolutionary selection based on their ability to infect and replicate in new host cells. Simultaneously, cells have developed major molecular defense mechanisms to protect themselves from viral infection. Avoiding the use of viruses is also very important for social and managerial reasons. Therefore, using non-viral vectors (such as the Sleeping Beauty system) avoids many, but not all, of the cells' defenses against viruses. For this purpose, the Sleeping Beauty virus system enables particularly effective and safe genetic modification of immune cells for administration to patients.

[0308] A further aspect of the invention relates to immune cells as described herein, which comprise nucleic acid molecules or vectors as described herein and / or express CARs as described herein, and are used as medicaments for treating medical conditions associated with the presence of pathogenic B cells (e.g., diseases of plasma cells, memory B cells, and / or mature B cells, particularly multiple myeloma or non-Hodgkin's lymphoma).

[0309] In one implementation, the medical use of immune cells is characterized in that the medical condition to be treated is multiple myeloma.

[0310] In one implementation, the medical use of immune cells is characterized in that the medical condition to be treated is non-Hodgkin's lymphoma.

[0311] In one embodiment, the medical use of immune cells is characterized by the medical condition to be treated being associated with pathogenic mature B cells. To the knowledge of the inventors, no prior disclosure in the art explicitly teaches that BCMACAR-T can effectively target such mature B cells as described herein. Some of the tumor cell lines tested described in the following embodiments involve mature B cells and are not necessarily memory types. In contrast, immature B cells would be those that cause acute lymphoblastic leukemia. Therefore, the present invention also includes a method of treating the medical condition disclosed herein, comprising administering a therapeutically effective amount of a CAR or a therapeutic agent comprising the CAR of the present invention to a subject in need of such treatment.

[0312] Another aspect of the invention relates to a pharmaceutical composition comprising a CAR or a CAR-containing therapeutic agent, and a therapeutic agent with a pharmaceutically acceptable carrier. Detailed Implementation

[0313] Multiple myeloma, also known as plasmacytoma, is a currently incurable B-cell lymphoma originating from a malignantly transformed plasma cell clone. It is the most common tumor of bone and bone marrow, with a median life expectancy of 7 years and an annual cancer mortality rate of 2%. Malignant transformation is believed to occur in the germinal centers of secondary lymphoid organs during development, where B cells have completed VDJ rearrangement and allotype conversion. The median age at diagnosis is 70 years, indicating comorbidities in many patients that may preclude intensive and long-term chemotherapy or radiation therapy. Furthermore, this patient population is often unsuitable for allogeneic bone marrow transplantation. Clinical features of the disease include osteolytic lesions, hypercalcemia, hematopoietic insufficiency, amyloidosis, renal failure, excessive production of heavy and / or light chains antibodies, hyperviscosity, infections, and hemorrhagic disorders. The standard of care is chemotherapy alone, or a combination of chemotherapy with autologous stem cell transplantation, immunomodulatory agents such as immunomodulatory drugs (IMIDs), local radiation, proteasome inhibitors, and, in a minority of patients, allogeneic stem cell transplantation. Despite intensive treatment using the aforementioned methods, the disease often relapses and develops primary and secondary resistance after multiple lines of treatment.

[0314] The adoptive chimeric antigen receptor (CAR)-T cell therapy described in this article targets B-cell maturation antigen (BMCA). This therapy can overcome these limitations in multiple myeloma because BCCA is highly expressed in multiple myeloma tumor cells but not in normal B cells or precursor B cells. Secondly, in direct-targeting anti-CD19 antibody or anti-CD19 CAR-T cell therapies against B cells, resistance to non-Hodgkin lymphoma (B-NHL) occurs due to antigen loss. Because treatment resistance occurs after multiple lines of chemotherapy / immunotherapy in these B-NHL cases, alternative target structures are needed. For mature B-NHL, BCCA is a suitable target; therefore, high-affinity anti-BCMA CAR-T cells could even be used therapeutically for B-NHL, as described below.

[0315] BCMA CAR-T cell transfer is selective for the tumor-associated antigen BCMA, making it applicable and effective even in older patients and in cases of multidrug resistance. It has predictable, tolerable, and easily managed side effects. Autologous T cells equipped with anti-BCMA CARs have high affinity and compatibility, recognizing and destroying multiple myeloma cells while preserving normal hematopoietic cells (e.g., T cells, B cells, and their bone marrow progenitor cells); all bone marrow cells and NK cells are also spared. Due to the autologous transfer of T cells, graft-versus-host disease does not occur. The formation of memory T cells, which is important for preventing relapse, can develop. Due to the high affinity and compatibility of anti-BCMA CAR-T cells, they can even recognize mature B-cell NHL with low BCMA expression, allowing T cell activation and tumor cell killing. These mature B-NHL entities include follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and certain stages of chronic lymphocytic leukemia.

[0316] In some implementations, the anti-BCMA CAR-T cells described herein are indicated for patients with multiple myeloma and B-NHL who are ineligible for other therapies. More specifically: i) patients with multidrug resistance, ii) patients who are not eligible for allogeneic stem cell transplantation, iii) patients with comorbidities unsuitable for further chemotherapy, iv) elderly patients intolerant to chemotherapy, v) CARs are indicated as salvage therapy even after failure of progressive disease and multiple other standard treatments, vi) it is indicated even at very low antigen densities for target tumor cells where antibodies would fail, vii) the structure of the source antibody complexed with BCMA validates its precise specificity at near-atomic resolution, and other biosafety features not shown in anti-BCMA CAR-T cells, and / or vii) it is indicated as a monotherapy, unlike the case of antibodies.

[0317] Other anti-BCMA CAR-T cells described in this art have shown responsiveness only to multiple myeloma cells and patients; in contrast, our anti-BCMA CAR exhibits unexpectedly high sensitivity even to B-NHL cell lines with low BCMA expression. Our anti-BCMA CAR confers extremely high affinity to T cells, which is essential for antitumor efficacy. No other anti-BCMA CAR has been reported to respond to mature B-NHL, diffuse large B-cell lymphoma (DLBCL), defined-stage follicular lymphoma, mantle cell lymphoma, or chronic lymphocytic leukemia. This invention demonstrates that our anti-BCMA CAR does not confer responsiveness to physiological B cells, T cells, NK cells, endothelial cells, all bone marrow cell lineages, and their precursor cells. Therefore, this invention exhibits unprecedentedly low off-target responsiveness to other hematopoietic tissues. Unlike anti-CD38 CAR-T cells, our anti-BCMA CAR does not exhibit undesirable responsiveness to bone marrow cell precursors.

[0318] The amino acid sequences of the scFV fragments previously described in WO / 2015 / 166073 and WO / 2014 / 068079 have been modified to i) enable folding and expression in a transmembrane receptor structure; ii) reverse the order of the light and heavy chain segments; and iii) elongate the linker sequence between the heavy and light chains. These modifications enable adequate surface expression on T cells while maintaining proper antigen binding.

[0319] The original FSY IgG, used as the antibody template for the scFv portion of the CAR-T cell construct, has low nanomolar affinity. Therefore, a preferred embodiment of the present invention is characterized by an anti-BCMA CAR exhibiting unexpectedly high affinity and conferring extremely high specificity and affinity to T cells. This high affinity enables CAR-T cells to i) recognize tumor target cells with high, intermediate, and low BCMA surface expression, ii) be activated against said tumor target cells, and iii) kill said tumor target cells. Apart from multiple myeloma cells, the aforementioned anti-BCMA CARs in the prior art have not been shown to be responsive to B-NHL. Therefore, the anti-BCMA CAR of the present invention is a specific and highly active agent, with low levels / quantities of BCMA molecules combating unprecedented B-NHL diversity.

[0320] When combined with retroviral vectors, preferred MP71-vectors, and γ-retroviral expression systems, an exceptionally high transduction rate for human T cells can be achieved.

[0321] Another significant advantage of this invention is the detailed understanding of the BCMA epitope recognized by the scFv fragment of the CAR. To date, no other antibody-based inventions or publications have identified the BCMA epitope. Therefore, the anti-BCMA CAR described herein exhibits significantly higher biocompatibility and no known off-target reactivity in vivo or in vitro.

[0322] In addition, the inventors have swapped the signal transduction components of our CAR construct in a simple three-step cloning process, which enables the production of a clinically applicable anti-BCMA CAR modular composition.

[0323] In an in vitro co-culture system, the anti-BCMA CAR-T cells of this invention were activated upon exposure to BCMA-expressing human B-NHL and multiple myeloma tumor cell lines. These T cells then produced an effector phenotype with high levels of IFN-γ secretion, a phenotype that predicts cytotoxic activity.

[0324] Preclinical evaluation includes i) in vitro cytotoxicity testing of appropriate B-NHL cell lines and primary myeloma cells from patients, and ii) in vivo testing of anti-BCMA CAR activity of xenografted B-NHL and multiple myeloma cell lines.

[0325] In an in vivo human setting, a Phase I clinical trial was conducted to evaluate myeloma patients with the following characteristics: i) patients with multidrug resistance, ii) patients who are not eligible for allogeneic stem cell transplantation, iii) patients with comorbidities unsuitable for further chemotherapy, iv) elderly patients intolerant to chemotherapy, v) patients undergoing salvage therapy after the onset of progressive disease, vi) patients who have failed multiple other standard care therapies, vii) patients who have developed progressive disease after autologous stem cell transplantation, viii) patients who have developed progressive disease after allogeneic stem cell transplantation, and ix) bridging therapy prior to allogeneic stem cell transplantation.

[0326] In addition, in a human setting, patients with B-NHL who have diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, and mantle cell lymphoma were evaluated in a Phase I clinical trial with the following characteristics: i) patients with multidrug resistance; ii) patients who are not eligible for allogeneic stem cell transplantation; iii) patients with comorbidities unsuitable for further chemotherapy; iv) elderly patients intolerant to chemotherapy; v) patients undergoing salvage therapy after the onset of progressive disease and failure of multiple lines of other standard care; vi) patients with progressive disease after autologous stem cell transplantation; vii) patients with progressive disease after allogeneic stem cell transplantation; viiii) patients as bridging therapy prior to allogeneic stem cell transplantation; ix) patients exhibiting escape variants or CD19 and / or CD20 mutants on tumor cells, making current antibody therapies (anti-CD20, rituximab, anti-CD19, oletuzumab, BITE) less suitable for current therapy. CD19 / CD3, blimatumomab, or anti-CD19 CAR therapies have lost / downregulated their target structures and become ineffective.

[0327] Another surprising aspect of this invention is the improved stability of the CAR disclosed herein. The CAR peptide can be easily stored for extended periods under appropriate conditions without any loss of binding affinity.

[0328] Chimeric antigen receptor:

[0329] A CAR consists of an extracellular domain derived from an antibody and an intracellular domain containing a signal transduction module derived from T cell signaling proteins. In a preferred embodiment, the extracellular domain preferably comprises variable regions derived from the heavy and light chains of immunoglobulins, constructed as single-chain variable fragments (scFvs). The scFvs are preferably linked to a hinge region that provides flexibility to transduce signals to the intracellular signal transduction domain by anchoring to the transmembrane portion. The transmembrane domain is preferably derived from CD8α or CD28. In first-generation CARs, the signal transduction domain consists of the ζ chain of the TCR complex. The term "generation" refers to the structure of the intracellular signal transduction domain. Second-generation CARs are equipped with a single co-stimulatory domain derived from CD28 or 4-1BB. Third-generation CARs have included two co-stimulatory domains, such as CD28, 4-1BB, ICOS or OX40, CD3ζ. This invention preferably relates to second- or third-generation CARs.

[0330] In various embodiments, genetically engineered receptors are provided to redirect the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). A CAR is a molecule that combines antibody-based specificity against a desired antigen (e.g., BCMA) with an intracellular domain of an activated T-cell receptor to produce a chimeric protein exhibiting specific anti-BCMA cellular immune activity. As used herein, the term "chimera" describes a composition consisting of different proteins or portions of DNA from different sources.

[0331] The CARs considered in this article include the extracellular domain (also known as the binding domain or antigen-binding domain) that binds to BCMA, the transmembrane domain, and the intracellular domain or intracellular signaling domain. The binding of the CAR's anti-BCMA antigen-binding domain to BCMA on the surface of target cells leads to CAR aggregation and delivery of activating stimuli to CAR-containing cells. A key characteristic of CARs is their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or the production of molecules capable of mediating cell death on target antigen-expressing cells in a major histocompatibility (MHC)-independent manner, as well as the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0332] In various embodiments, the CAR includes an extracellular binding domain containing a humanized BCMA-specific binding domain; a transmembrane domain; and one or more intracellular signaling domains. In a particular embodiment, the CAR includes an extracellular binding domain containing a humanized anti-BCMA antigen binding fragment; one or more spacer regions; a transmembrane domain; and one or more intracellular signaling domains.

[0333] The terms "extracellular antigen-binding domain" and "extracellular binding domain" are used interchangeably and provide the CAR with the ability to specifically bind to the target antigen of interest, BCMA. The binding domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The scFV domain is preferred.

[0334] "Specific binding" should be understood by those skilled in the art, who will clearly know the various experimental methods that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. In many protein-protein interactions, some cross-reactions or background binding may be unavoidable; this does not diminish the "specificity" of the binding between the CAR and the epitope. "Specific binding" describes the binding of an anti-BCMA antibody or its antigen-binding fragment (or a CAR containing them) to BCMA with a binding affinity higher than background binding. The term "directed against" also applies when considering the term "specificity" in the context of the interaction between the antibody and the epitope.

[0335] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal. In a particular embodiment, the target antigen is an epitope of the BCMA polypeptide. An epitope is an antigenic region that binds to a binding agent. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed in the tertiary folding of a protein.

[0336] A single-chain Fv or scFv antibody fragment comprises a VH domain and a VL domain of an antibody, wherein these domains are present as a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Typically, the scFv polypeptide also includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR considered herein comprises an antigen-specific binding domain, which is the scFv and can be mouse, human, or humanized scFv. A single-chain antibody can be cloned from the V region gene of a hybridoma that is specific to a desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human BCMA polypeptide. Exemplary examples suitable for constructing a variable heavy chain of the anti-BCMA CAR considered herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO: 11. Exemplary examples suitable for constructing a variable light chain of the anti-BCMA CAR considered herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO: 12.

[0337] Antibodies and antibody fragments:

[0338] The CAR contains an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide. Therefore, the antibodies or antibody fragments of the present invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single-chain fragments (scFv), single variable fragments (ssFv), single-domain antibodies (e.g., VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments generated from Fab expression libraries, anti-idiotypic antibodies, and epitope-binding fragments, or any combination thereof, provided that they retain similar binding properties to the CARs described herein, preferably containing the corresponding CDR, or VH and VL regions as described herein. Microantibodies and multivalent antibodies such as 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 any class (i.e., IgG, IgE, IgM, IgD, and IgA) or subclass of immunoglobulin molecules. Therefore, as used herein, the term antibody also includes antibodies and antibody fragments contained in the CAR of the present invention, which are generated by modifying complete antibodies or resynthesized using recombinant DNA methods.

[0339] As used herein, "antibody" generally refers to a protein composed primarily of one or more polypeptides encoded by immunoglobulin genes or segments of immunoglobulin genes. When the term "antibody" is used, it can also be considered as "antibody fragment." Recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, and numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to consist of a tetramer or dimer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" (L) chain (approximately 25 kDa) and a "heavy" (H) chain (approximately 50–70 kDa). 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 these variable regions of the light and heavy chains, respectively. Optionally, the antibody or the immune portion of an antibody may be chemically conjugated with or expressed as a fusion protein containing other proteins.

[0340] The CAR of this invention is designed to target mammalian, particularly human, protein targets. The use of protein names may correspond to the mouse or human forms of the protein.

[0341] The affinity of the binding domain peptide and the CAR protein according to this disclosure can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by displacement assay using labeled ligands, or by using a surface plasmon resonance device (such as Biacore).

[0342] Humanized antibodies can be prepared using any method known in the art, which comprises one or more CDRs of the antibody of the present invention or are derived from one or more CDRs of said antibody. For example, a monoclonal antibody can be humanized using four general steps. These are: (1) determining the nucleotide and predicted amino acid sequences of the variable domains of the light and heavy chains of the starting antibody; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use in the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody. See, for example, 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.

[0343] The term humanized antibody refers to an immunoglobulin in which at least a portion of the framework region and optionally a portion of the CDR region, or other binding regions, are derived from or modified from the human immunoglobulin sequence. Humanized, chimeric, or partially humanized forms of mouse monoclonal antibodies can be prepared, for example, by recombinant DNA technology, thereby deviating from mouse and / or human genomic DNA sequences encoding the H and L chains or cDNA clones encoding the H and L chains. Humanized forms of mouse antibodies can be produced by linking the CDR region of a non-human antibody to a human constant region using recombinant DNA technology (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibody used in the methods of the present invention can be a human monoclonal antibody. Human antibodies can be obtained, for example, using phage display (WO 91 / 17271; WO 92 / 01047).

[0344] As used in this article, humanized antibodies also refer to the form of non-human (e.g., mouse, camel, llama, shark) antibodies, which are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins.

[0345] As used herein, a human or humanized antibody or antibody fragment refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of a human-produced antibody, and / or having been prepared using any technique known in the art or disclosed herein for the preparation of human antibodies. Human antibodies or fragments thereof can be selected by competitive binding experiments or other means to have the same epitope specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly share, to a large extent, the useful functional properties of mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillary peptides and / or non-fibrillary peptides or fragments thereof as affinity reagents. Monoclonal antibodies can be obtained from serum according to the techniques described in WO 99 / 60846.

[0346] Variable area and CDR

[0347] The variable region of an antibody refers to the variable region of a single antibody light chain, the variable region of an antibody heavy chain, or a combination thereof. The variable regions of both the heavy and light chains consist of four frame regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together closely by the FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site.

[0348] Many techniques are available for determining CDRs, such as those based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, National Institutes of Health, Bethesda Md)); and those based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., (1997) J. Molec. Biol. 273:927-948). Alternative methods include the IMGT International ImMunoGeneTics Information System (Marie-Paule Lefranc). Kabat's definition is based on sequence variability and is the most commonly used method. Chothia's definition is based on the location of structural loop regions, while AbM's definition is a compromise between the two used by the Oxford Molecular AbM antibody modeling software (see www.bioinf.org.uk:Dc Andrew CR Martin group). As used herein, a CDR can refer to a CDR defined by one or more methods, or by a combination of these methods.

[0349] In some embodiments, the present invention provides antibodies or fragments thereof incorporated into a CAR, wherein the antibody or fragment thereof comprises at least one, at least two, at least three or more CDRs substantially identical to at least one, at least two, at least three or more CDRs of the antibody of the present invention. Other embodiments include antibodies having at least two, three, four, five or six CDRs substantially identical to or derived from at least two, three, four, five or six CDRs of the antibody of the present invention. In some embodiments, the at least one, two, three, four, five or six CDRs are at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% identical to at least one, two or three CDRs of the antibody of the present invention. It should be understood that, for the purposes of the present invention, binding specificity and / or overall activity are generally preserved, although the degree of activity may vary (may be greater or less) compared to the antibody.

[0350] Other components of CAR

[0351] In some embodiments, the CARs considered herein may include linker residues added between the various domains for proper spacing and conformation of the molecule, such as linkers comprising amino acid sequences that connect the VH and VL domains and provide spacer region functions compatible with the interaction of the two sub-binding domains, such that the resulting peptide maintains specific binding affinity to the same target molecule as antibodies containing the same light and heavy chain variable regions. The CARs considered herein may contain one, two, three, four, or 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, or about 10 to about 20 amino acids, or any intervening length of amino acids.

[0352] Exemplary examples of connectors include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible connectors known in the art, such as the Whitlow connector. Glycine and glycine-serine polymers are relatively unstructured and can therefore serve as neutral linkers between domains of fusion proteins (such as the CAR described herein).

[0353] In a particular embodiment, the binding domain of the CAR is followed by one or more "spacer regions" or "spacer polypeptides," which refer to regions that distance the antigen-binding region from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. In some embodiments, the spacer region is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer region may include naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge region amino acid sequences. In one embodiment, the spacer region includes the CH2 and CH3 domains of IgG1 or IgG4.

[0354] In some embodiments, the binding domain of a CAR may be followed by one or more "hinge domains" that function to localize the antigen-binding domain to a location distant from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for the CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8α, CD4, CD28, PD1, CD152, and CD7), which may be wild-type hinge regions from these molecules or may be modified. In another embodiment, the hinge domain includes a PD1, CD152, or CD8α hinge region.

[0355] The transmembrane domain is part of the CAR, fusing an extracellular binding portion and an intracellular signaling domain to anchor the CAR to the plasma membrane of immune effector cells. The TM domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from the α, β, or ζ chains of the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In one embodiment, the CAR considered herein comprises a TM domain derived from CD8α or CD28.

[0356] In a particular embodiment, the CAR considered herein includes an intracellular signaling domain. An “intracellular signaling domain” refers to a domain involved in transducing information about the binding of an effective anti-BCMA CAR to a human BCMA peptide into the immune effector cell to trigger effector cell functions (e.g., activation, cytokine production, proliferation, and cytotoxic activity, said cytotoxic activity including the release of cytotoxic factors to the target cell to which the CAR is bound) or other cellular responses triggered by the binding of an antigen to an extracellular CAR domain. The term “effective function” refers to a specialized function of an immune effector cell. For example, the effector function of a T cell may be cytolytic activity or an assisting or detrimental activity including cytokine secretion. The term “intracellular signaling domain” refers to a portion of a protein that transduces effector function signals and directs the cell to perform specialized functions.

[0357] The CARs considered in this paper contain one or more co-stimulatory signaling domains to enhance the efficacy, expansion, and / or memory formation of T cells expressing CAR receptors. As used herein, the term "co-stimulatory signaling domain" refers to the intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to antigens.

[0358] polypeptide

[0359] The terms “peptide,” “polypeptide,” “polypeptide fragment,” and “protein” are used interchangeably unless otherwise stated and are used in their conventional sense as an amino acid sequence. Polypeptides are not limited to a specific length; for example, they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other modifications known in the art, including both naturally occurring and non-naturally occurring modifications.

[0360] In various embodiments, the CAR peptides considered herein comprise a signal (or leader) sequence at the N-terminus of a protein that guides protein transfer during co-translation or post-translation. The peptides can be prepared using a variety of well-known recombinant and / or synthetic techniques. Specifically, the peptides considered herein include the CARs of this disclosure, or sequences having one or more amino acid deletions, additions, and / or substitutions of the CARs disclosed herein.

[0361] As used herein, "isolated peptide" or "isolated polypeptide" refers to peptide or polypeptide molecules that have been isolated and / or purified in vitro from the cellular environment or from their binding to other components of the cell (i.e., they are not significantly associated with substances in vivo). Similarly, "isolated cell" refers to a cell obtained from an in vivo tissue or organ that is substantially free of extracellular matrix.

[0362] Nucleic acid

[0363] As used herein, the term "polynucleotide" or "nucleic acid molecule" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention comprise polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any reference sequence described herein, typically wherein the variant retains at least one biological activity of the reference sequence. In various exemplary embodiments, the present invention is partly contemplated regarding polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising them.

[0364] Polynucleotides can be prepared, manipulated, and / or expressed using any of the various well-established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Other exemplary vectors include, but are not limited to, plasmids, phagemids, granules, artificial chromosomes (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), bacteriophages (e.g., λ phage or M13 phage), and animal viruses. Examples of animal virus classes useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells. TM pLenti6 / V5-DEST TM And pLenti6.2 / V5-GW / lacZ (Invitrogen). In a particular embodiment, the coding sequence of the chimeric protein disclosed herein can be ligated into such expression vectors for expressing the chimeric protein in mammalian cells. The “control elements” or “regulatory sequences” present in the expression vector are the untranslated regions of the vector (i.e., origin of replication, selection cassette, promoter, enhancer, translation initiation signal (ShineDalgarno sequence or Kozak sequence) intron, polyadenylated sequences, 5' and 3' untranslated regions), which interact with host cell proteins for transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0365] carrier

[0366] In a particular embodiment, cells (e.g., immune effector cells, such as T cells) are transduced using a retroviral vector encoding a CAR (e.g., a lentiviral vector). For example, immune effector cells are transduced using a vector encoding a CAR, the vector containing a humanized anti-BCMA antibody or antigen-binding fragment that binds to a BCMA polypeptide, the humanized anti-BCMA antibody or antigen-binding fragment having a transmembrane domain and an intracellular signaling domain, such that these transduced cells can elicit a CAR-mediated cytotoxic response.

[0367] Retroviruses are a common tool for gene delivery. In particular implementations, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) into cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy, which is then covalently integrated 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 and directs the expression of RNA molecules that encode the structural proteins and enzymes required to produce new viral particles.

[0368] Exemplary retroviruses suitable for particular implementations include, but are not limited to: Moloney mouse leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibberish leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Fried mouse leukemia virus, mouse stem cell virus (MSCV), and Rouss sarcoma virus (RSV), and lentiviruses.

[0369] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (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, the lentivirus is used to deliver a CAR-containing polynucleotide into cells.

[0370] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, for example, inserted into, the vector nucleic acid molecule. Vectors may include sequences that guide autonomous replication within the cell, or sequences sufficient to allow integration into the host cell's DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, granules, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0371] As will be apparent to those skilled in the art, the term "viral vector" is widely used to refer to nucleic acid molecules (such as transfer plasmids) or viral particles that mediate nucleic acid transfer. Nucleic acid molecules include virus-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the cellular genome. Viral particles typically comprise a variety of viral components and sometimes also include host cell components other than nucleic acids.

[0372] The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid primarily derived from retroviruses that contains structural and functional genetic elements or portions thereof.

[0373] Therefore, in a preferred embodiment, the present invention relates to a method of transfecting cells with an expression vector encoding a CAR. For example, in some embodiments, the vector contains additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly-A signals, and / or one or more introns. In a preferred embodiment, the CAR coding sequence is flanked by transposon sequences, such that transposases are present to allow the coding sequence to be integrated into the genome of the transfected cells.

[0374] In some embodiments, the genetically transformed cells are further transfected with a transposase, the transposase promoting the integration of the CAR-coding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided as an expressible RNA or protein such that the transposase does not undergo long-term expression in the transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA containing a cap and a poly-A tail). Any transposase system can be used according to embodiments of the invention. However, in some embodiments, the transposase is a salmon-type Tel-like transposase (SB). For example, the transposase can be a so-called "Sleeping Beauty" transposase, see, for example, U.S. Patent 6,489,458, which is incorporated herein by reference. In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB 100X transposases (see, for example, Mates et al., 2009, Nat Genet. 41(6):753-61 or US9228180, which are incorporated herein by reference). For example, methods may include electroporation of cells having mRNA encoding SB 10, SB 11, or SB 100X transposases.

[0375] Sequence variants:

[0376] Sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments, and / or CARs (e.g., those defined by percentage sequence identity) are also included within the scope of this invention, maintaining similar binding properties of the invention. These variants exhibit alternative sequences but maintain substantially the same binding properties, such as target specificity, because the particular sequence provided is known to be a functional analogue or functionally similar. Sequence identity refers to the percentage of identical nucleotides or amino acids when sequence alignment is performed.

[0377] As used in this paper, “sequence identity” refers to the degree of sequence similarity based on nucleotide-nucleotide or amino acid-amino acid sequences within a comparison window. Therefore, the “percentage of sequence identity” can be calculated as follows: Compare two best-aligned sequences within the comparison window, determine the number of positions on both sequences that contain the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to generate the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the percentage of sequence identity. This includes nucleotides or polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any reference sequence described herein, wherein the polypeptide variant typically retains at least one biological activity of the reference polypeptide.

[0378] Those skilled in the art will understand that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology or sequence identity with the nucleotide sequence of any natural gene. Nevertheless, the present invention specifically considers polynucleotides that vary due to differences in codon usage. Deletions, substitutions, and other variations in sequences falling within the aforementioned sequence identity are also included in the present invention.

[0379] Protein sequence modifications that can occur through substitution are also included within the scope of this invention. Substitution, as defined herein, is a modification of the amino acid sequence of a protein, whereby one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the primary protein. Substitutions can be made, preferably without significantly altering the function of the protein. As with additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering protein function. This is especially true when the modification involves a “conserved” amino acid substitution, where one amino acid replaces another amino acid with similar properties. Such “conserved” amino acids can be natural or synthetic amino acids that can be substituted due to size, charge, polarity, and conformation without significantly affecting the structure and function of the protein. Typically, many amino acids can be substituted with conserved amino acids without adversely affecting the function of the protein.

[0380] Generally, the nonpolar amino acids Gly, Ala, Val, Ile, and Leu; the nonpolar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu represent conserved amino acid groups. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be substituted for each other, even though they belong to different groups.

[0381] Substitutional variants remove at least one amino acid residue from the antibody molecule and insert a different residue at its position. For substitutional mutagenesis to occur, the most interesting sites include hypervariable regions, but FR alterations are also considered. If such substitutions result in changes in biological activity, more extensive alterations (referred to as “exemplary substitutions” in the table below, or further described below regarding amino acid classes) can be introduced, and products can be screened.

[0382] Potential amino acid substitutions:

[0383]

[0384] Substantial modifications to the biological properties of antibodies are achieved by selecting substitutions that have significantly different effects in maintaining the following aspects: (a) the structure of the polypeptide backbone in the substituted region (e.g., sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the body of the side chain.

[0385] Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids include ω-amino acids of various chain lengths and cyclohexylalanine as neutral nonpolar analogs; citrulline and methionine sulfoxide as neutral nonpolar analogs; phenylglycine as an aromatic neutral analog; cysteine ​​as a negatively charged analog; and ornithine as a positively charged amino acid analog. As with naturally occurring amino acids, this list is not exhaustive, but merely an example of substitutions well known in the art.

[0386] Gene-modified cells and immune cells

[0387] In a particular embodiment, the present invention contemplates cells genetically modified to express the CARs contemplated herein for the treatment of B-cell-related conditions. As used herein, the terms “genetically modified” or “genetically engineered” refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of 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 of a cell, which restores, corrects, or modifies gene expression, or is used to express a therapeutic peptide (e.g., a CAR). In a particular embodiment, the CARs contemplated herein are introduced into and expressed in immune effector cells to redirect their specificity for a target antigen of interest, such as a BCMA peptide.

[0388] "Immune cells" or "immune effector cells" are any cells of the immune system that have one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC).

[0389] The immune effector cells of the present invention can be autologous / autogenetic (“own”) or non-autologous (“non-own”, such as allogeneic, syngeneic, or allogeneic). As used herein, “autologous” means cells from the same subject, representing a preferred embodiment of the invention. As used herein, “allogeneic” means cells of the same species as the comparison but genetically different. As used herein, “syngeneic” means cells from a different subject that are genetically identical to the comparison cells. As used herein, “alienate” means cells of a different species than the comparison cells. In a preferred embodiment, the cells of the present invention are autologous or allogeneic.

[0390] Exemplary immune effector cells used with the CAR considered herein include T lymphocytes. The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3- and CD56-positive non-major histocompatibility complex (MHC) cells, which are restricted natural killer (NK)-like T lymphocytes. T cells can be T helper cells (Th), such as T helper cell 1 (Th1) or T helper cell 2 (Th2). T cells can be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells, CD4CD8 T cells, or any other T cell subset. Other exemplary T cell populations suitable for particular embodiments include naive T cells and memory T cells.

[0391] For example, when reintroduced into a patient after autologous cell transplantation, the CAR-modified T cells of the present invention described herein can recognize and kill tumor cells. CIK cells, compared to other T cells, can exhibit enhanced cytotoxic activity, and therefore represent a preferred embodiment of the immune cells of the present invention.

[0392] As those skilled in the art will understand, other cells can also be used as immune effector cells with the CAR described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitor cells of effector cells, which can be induced to differentiate into immune effector cells in vivo or in vitro.

[0393] This invention provides a method for preparing immune effector cells expressing a CAR as described herein. In one embodiment, the method includes transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described herein. In some embodiments, the immune effector cells are isolated from an individual and genetically modified without further in vitro manipulation. These cells can then be directly re-administered to the individual. In a further embodiment, the immune effector cells are first activated and stimulated to proliferate in vitro, and then 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 described herein).

[0394] In a particular embodiment, the cell source is obtained from the subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In a particular embodiment, the CAR-modified immune effector cells comprise T cells. T cells can be obtained from many sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained using any number of techniques known to those skilled in the art, such as sedimentation (e.g., FICOLL). TM Separation), antibody-based conjugated bead methods (e.g., MACS) TM Cells obtained from apheresis (miltenyi) are derived from units of blood collected from a subject. In one embodiment, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected via apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing. Cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most (if not all) divalent cations. As will be understood by those skilled in the art, the washing step can be performed by methods known to those skilled in the art, for example, using a semi-automatic flow-through centrifuge (e.g., Cobe2991 cell processor, Baxter CytoMate, etc.). After washing, cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In some embodiments, unwanted components of the apheresis sample can be removed from cells resuspended directly in a culture medium.

[0395] In some implementations, this is achieved by lysing red blood cells and depleting monocytes (e.g., by PERCOLL). TM T cells are isolated from peripheral blood mononuclear cells (PBMCs) using gradient centrifugation. Specific T cell subsets can be further isolated using positive or negative selection techniques. One method used in this paper involves cell sorting and / or selection using a mixture of monoclonal antibodies targeting cell surface markers present on negatively selected cells via negative magnetic immunoadhesion or flow cytometry.

[0396] PBMCs can be directly genetically modified to express CAR using the methods considered herein. In some embodiments, T lymphocytes are further isolated after PBMC isolation, and in some embodiments, cytotoxic and helper T lymphocytes can be sorted into primary, memory, and effector T cell subsets before or after genetic modification and / or expansion. CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into primary, central memory, and effector cells by identifying each associated cell surface antigen in these types of CD8+ cells.

[0397] Immune effector cells (e.g., T cells) can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In a particular embodiment, immune effector cells (e.g., T cells) are genetically modified with chimeric antigen receptors as considered herein (e.g., transduced with a viral vector containing nucleic acid encoding a CAR), and then activated and expanded in vitro. In various embodiments, methods described, for example, 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, can be used to activate and expand T cells before or after genetic modification to express CAR.

[0398] In another implementation, for example, a mixture of one, two, three, four, five, or more different expression vectors can be used to genetically modify a donor population of immune effector cells, wherein each vector encodes a different chimeric antigen receptor protein as considered herein. The resulting modified immune effector cells form a mixed population of modified cells, a subset of which express more than one different CAR protein.

[0399] In one embodiment, the present invention provides a method for storing immune effector cells expressing genetically modified mouse, human, or humanized CAR proteins that target BCMA protein, comprising cryopreserving the immune effector cells so that the cells remain viable upon thawing. A subset of the CAR protein-expressing immune effector cells can be cryopreserved using methods known in the art to provide a permanent source of such cells for future treatment of patients with B-cell-related diseases. When needed, the cryopreserved transformed immune effector cells can be thawed, grown, and expanded to obtain more such cells.

[0400] Compositions and Formulations

[0401] The compositions considered herein may comprise one or more polypeptides, polynucleotides, carriers containing such polynucleotides, genetically modified immune effector cells, etc., as considered herein. Compositions include, but are not limited to, pharmaceutical compositions. A “pharmaceutical composition” means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration, alone or in combination with one or more other therapeutic modalities, to cells or animals. It should also be understood that, if desired, the compositions of the present invention may also be administered in combination with other pharmaceutical agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are practically no limitations on other components that may be included in the composition, provided that additional components do not adversely affect the composition’s ability to deliver the intended therapy.

[0402] The term “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissue contact with reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications and in proportion to a reasonable benefit / risk ratio.

[0403] As used herein, “pharmaceuticalally acceptable carriers, diluents, or excipients” include, but are not limited to, any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; astragalus gum; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable oils; paraffin wax; organosilicon; bentonite; silicic acid; zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and any other compatible substances used in pharmaceutical preparations.

[0404] In a particular embodiment, the composition of the present invention comprises an amount of CAR-expressing immune effector cells as conceived herein. As used herein, the term “amount” refers to an “effective amount” of genetically modified therapeutic cells (e.g., T cells) that achieve a beneficial or desired preventive or therapeutic outcome, including clinical outcomes.

[0405] "Prophylactic effective dose" refers to the amount of genetically modified therapeutic cells that effectively achieve the desired preventative outcome. It is usually, but not always, less necessary because the preventative dose is used in subjects before or in the early stages of disease, so the prophylactic effective dose is less than the therapeutic effective dose. The term "prophylaxis" does not necessarily mean the complete prohibition or prevention of a specific medical condition. Prophylaxis also refers to reducing the risk of developing a particular medical condition or worsening its symptoms.

[0406] The “therapeutic effective amount” of genetically modified therapeutic cells can vary depending on various factors, such as disease state, age, sex, and individual weight, as well as the ability of stem cells and progenitor cells to elicit a desired response in an individual. Therapeutic effective amount is also a amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the virus or transduced therapeutic cells. The term “therapeutic effective amount” includes the amount that effectively “treats” a subject (e.g., a patient). When indicating a therapeutic amount, the precise amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. It can generally be specified that pharmaceutical compositions containing T cells described herein can be administered in quantities of 10... 2 Up to 10 10 Cells / kg body weight, preferably 10 5 Up to 10 6 The dosage is administered at 1 cell / kg body weight (inclusive of all integer values ​​within these ranges). The number of cells will depend on the end use of the composition and the cell type contained therein. For the uses provided herein, cells are typically in volumes of 1 L or less, and may be 500 mL or less, or even 250 mL or 100 mL or less. Therefore, a cell density greater than 10 is generally desired. 6 cells / ml, typically greater than 10 7 Cells / ml, typically 10 8 Cells / ml or higher. A clinically relevant number of immune cells can be allocated to multiple infusions, with the cumulative number of infusions equal to or exceeding 10. 5 10 6 10 7 10 8 10 9 10 10 10 11 Or 10 12 Cells. In some aspects of the invention, particularly because all infused cells will be redirected to a specific target antigen, a lower number of cells can be administered. CAR-expressing cell compositions can be administered multiple times at doses within these ranges. For the patient receiving treatment, the cells can be allogeneic, syngeneic, allogeneic, or autologous.

[0407] Generally, compositions comprising activated and expanded cells as described herein can be used to treat and prevent diseases in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells as considered herein are used to treat B-cell malignancies. The CAR-modified T cells of the present invention can be administered alone or as pharmaceutical compositions in combination with carriers, diluents, excipients, and / or other components (e.g., IL-2) or other cytokines or cell populations. In particular embodiments, the pharmaceutical compositions considered herein comprise a quantity of genetically modified T cells, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0408] The pharmaceutical compositions of the present invention comprising a population of immune effector cells (e.g., T cells) expressing CAR may include: a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA) or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intra-arterial), intraperitoneal, or intramuscular administration.

[0409] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may include one or more of the following: sterile diluents (e.g., water for injection), saline solutions (preferably physiological saline, Ringer's solution, or isotonic sodium chloride), fixed oils (e.g., synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and tonicity modifiers, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0410] In particular embodiments, the compositions contemplated herein comprise an effective amount of CAR-expressing immune effector cells alone, or in combination with one or more therapeutic agents. Thus, CAR-expressing immune effector cell compositions can be administered alone or in combination with other known cancer treatments such as radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents are acceptable in the art as standard treatment for specific disease states (e.g., specific cancers) as described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapy agents, radiotherapy agents, therapeutic antibodies, or other active and adjuvant agents.

[0411] Treatment

[0412] This article considers genetically modified immune effector cells to provide an improved approach to adoptive immunotherapy for the treatment of B-cell-related conditions, including but not limited to immunomodulatory disorders and hematologic malignancies.

[0413] In a particular embodiment, a composition comprising immune effector cells containing the CAR considered herein is used to treat conditions associated with abnormal B cell activity, also known as “medical conditions associated with the presence of pathogenic B cells”.

[0414] As used herein, “medical condition associated with the presence of pathogenic B cells” or “B-cell malignancy” refers to a medical condition that develops in B cells, such as cancer. In a particular embodiment, the compositions comprising CAR-modified T cells contemplated herein are intended for the treatment of hematologic malignancies, including but not limited to B-cell malignancies such as multiple myeloma (MM) and non-Hodgkin lymphoma (NHL).

[0415] In another aspect of the invention, CARs and CAR-Ts according to the invention as described herein are provided for the treatment of a selection of B-cell-mediated or plasma cell-mediated diseases or antibody-mediated diseases or conditions: multiple myeloma (MM), chronic lymphocytic leukemia (CLL), non-secreting multiple myeloma, condensing multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's macroglobulinemia. Macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / sclerosing myeloma, type I and II cryoglobulinemia, light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and bullous pemphigoid, and epidermolysis bullosa; or any non-Hodgkin lymphoma B-cell leukemia or Hodgkin lymphoma (HL) with BCMA expression or any disease in which the patient develops neutralizing antibodies against recombinant protein replacement therapy, wherein the method comprises administering a therapeutically effective amount of CAR or CAR-T as described herein to the patient.

[0416] Multiple myeloma is a malignant tumor of B cells with a mature plasma cell morphology, characterized by the tumor transformation of a single clone of these cell types. These plasma cells proliferate within the bone marrow and may invade adjacent bone and sometimes the bloodstream. Variations of multiple myeloma include overt multiple myeloma, condensatory multiple myeloma, plasma cell leukemia, non-secreting myeloma, IgD myeloma, osteosclerosing myeloma, solitary plasmacytoma, and extramedullary plasmacytoma.

[0417] Non-Hodgkin lymphoma comprises a large group of lymphocytic carcinomas (white blood cells). Non-Hodgkin lymphoma can occur at any age and is typically marked by larger-than-normal lymph nodes, fever, and weight loss. Non-Hodgkin lymphoma can also be found in extranodal locations, such as the central nervous system, mucosal tissues including the lungs, intestines, colon, and internal organs. There are many different types of non-Hodgkin lymphoma. For example, non-Hodgkin lymphoma can be classified as aggressive (rapid-growing) or indolent (slow-growing) types. Although non-Hodgkin lymphoma can originate from both B cells and T cells, as used herein, the terms "non-Hodgkin lymphoma" and "B-cell non-Hodgkin lymphoma" are used interchangeably. B-cell non-Hodgkin lymphoma (NHL) includes Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that develop after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin lymphomas.

[0418] Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells called B lymphocytes or B cells. The cancer cells spread through the blood and bone marrow and can also affect lymph nodes or other organs, such as the liver and spleen. CLL eventually leads to bone marrow failure. A different presentation of the disease is called small lymphocytic lymphoma and primarily locates in secondary lymphoid organs such as lymph nodes and the spleen.

[0419] In one embodiment of the invention, the CAR or immune cells expressing the CAR are intended for the treatment of autoimmune diseases, preferably autoantibody-dependent autoimmune diseases, and more preferably autoimmune diseases with an inflammatory component, wherein the autoimmune disease is preferably selected from the following: Goran's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutaneous polyarteritis nodosa, hepatitis-associated arteritis, Behcet's syndrome, Wegener's granulomatosis, ANCA vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, connective tissue disease vasculitis, Hennoch-Schönlein purpura, cryoglobulin vasculitis, cutaneous leukocytoclastic vasculitis, tropical aortitis, sarcoidosis, Cogan's syndrome, and Wiskott-Aldrich syndrome. Syndrome, Leprosy nodular arteritis, CNS primary vasculitis, thromboangiitis obliterans, paraneoplasticateritis, urticaria, Dego's disease, myelodysplastic syndrome, persistent erythema raisedae, hyperimmune globulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Morbus Chronosis, ulcerative colitis, autoimmune myositis, diabetes, Guillain-Barré syndrome, histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, psoriatic arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease Disease), erythema nodosum, otitis media, cryoglobulinemia, Sjögren's syndromeAutoimmune syndrome, lupus erythematosus (preferably systemic lupus erythematosus (SLE)), aplastic anemia, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura disease, systemic sclerosis, chronic perivascular inflammation, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic achalasia, bleomycin-induced lung inflammation, cytarabine-induced lung inflammation, autoimmune thrombocytopenic purpura, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, Chagas disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atrophic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, pemphigus vulgaris, pemphigus foliaceus, herpetic dermatitis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiffness syndrome (Stiff-Man syndrome) SLE is the preferred treatment for conditions such as Goodpass syndrome, sympathetic ophthalmia, folliculitis, Sharp syndrome, and / or Evans syndrome, especially hay fever, periodontitis, atherosclerosis, and rheumatoid arthritis.

[0420] Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease in which the body's immune system attacks healthy tissues throughout the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, fatigue, and a red rash most commonly seen on the face.

[0421] As used herein, the terms “individual” and “subject” are generally used interchangeably and refer to any animal exhibiting symptoms of a disease, condition, or illness that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. In a preferred embodiment, a subject includes any animal exhibiting symptoms of a disease, condition, or illness of the hematopoietic system (e.g., B-cell malignancy) that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. Typical subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and livestock or pets (e.g., cats or dogs). Non-human primates are included, preferably including human patients. Typical subjects include human patients with B-cell malignancy who have been diagnosed with B-cell malignancy or are at risk of having B-cell malignancy.

[0422] As used herein, “treatment / treating” includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even the smallest reduction in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve the relief or improvement of symptoms of the disease or condition, or the delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition or its associated symptoms.

[0423] As used herein, “prevent” and similar terms, such as “prevented / preventing” or “prophylactic,” refer to methods of preventing, suppressing, or reducing the likelihood of a disease or condition occurring or recurring. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the occurrence or recurrence of its symptoms. As used herein, “prevent” and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs.

[0424] In one embodiment, a method of treating B-cell-related conditions in subjects with this need includes administering an effective amount, such as a therapeutically effective amount, of a composition comprising genetically modified immune effector cells as considered herein. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although an appropriate dose can be determined through clinical trials.

[0425] The compositions considered herein can be administered in any convenient manner, including via aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. The phrase "parenteral administration / administered parenterally" as used herein refers to administration other than enteral and local administration, typically by injection, including but not limited to intravascular, intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intratumoral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions. In one embodiment, the compositions considered herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection. Attached Figure Description

[0426] The invention is illustrated by way of example and accompanying drawings disclosed herein. The accompanying drawings provided herein represent particular embodiments of the invention and are not intended to limit the scope of the invention. These drawings should be considered as further descriptions of possible and potentially preferred embodiments that enhance the technical support for one or more non-limiting embodiments.

[0427] Figure 1 : Schematic diagram of the preferred CAR structure.

[0428] Figure 2 : Schematic diagram of the preferred CAR constructs IX, X; XI, XV, XVI, XVII.

[0429] Figure 3 A list of preferred constructs and potential combinations of various structural elements of CAR as described herein.

[0430] Figure 4 The diagram shows a sequence comparison between the mAb binding region and the preferred humanized sequence used in the CAR of this invention. The upper figure shows the J22.9 hHC FSY change, where the sequence is codon-optimized for Homo sapiens, and the optimized sequence has 81% homology with the original sequence; the lower figure shows the J22.9 hLC E change, where the sequence is codon-optimized for Homo sapiens, and the optimized sequence has 78% homology with the original sequence.

[0431] Figure 5 GeneArt™ plasmids containing the BCMA-CAR sequence.

[0432] Figure 6 : Restricted gel electrophoresis of cleaved constructs and carriers.

[0433] Figure 7 Confirmation of BCMA CAR expression on human T cells after retroviral transduction: CAR expression, construct IX-XII, CD19, SP6.

[0434] Figure 8 Co-culture of CAR-transduced human T cells with different target cell lines showed that different BCMAs + Specific T-cell activation in multiple myeloma (MM) and B-NHL cell lines. Functional in vitro co-culture and IFN-γ ELISA.

[0435] Figure 9 CD107a (LAMP1) staining of co-cultured CAR-T cells and multiple myeloma cells: Degranulated CD8+ T cells activated after antigen-specific (BCMA) stimulation were detected by flow cytometry. Functional in vitro co-culture and LAMP1 detection were determined by FACS.

[0436] Figure 10 Cytotoxicity analysis revealed 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 were also performed.

[0437] Figure 11 BCMA and CD19 expression on cell types assessed in functional analysis. Results of MACS-based B cells isolated from PBMCs are also shown, along with anti-BCMA and anti-CD19 staining.

[0438] Figure 12 : Schematic diagram of the binding interaction between CAR's scFV and BCMA epitopes.

[0439] Figure 13 Sequence alignment of the preferred humanized sequence of HC compared to J22.9-xi.

[0440] Figure 14 Sequence alignment of the preferred humanized sequence of LC compared to J22.9-xi.

[0441] Figure 15 BCMA-redirected CAR-T cells are effective against MM tumors in a xenografted NSG mouse model. (A) MM tumors were implanted in a xenografted NSG mouse model. Mice were challenged by intravenous transplantation of MM.1S cells. Tumor cell growth was visualized by IVIS imaging on day 8 post-tumor inoculation. To measure tumor burden, imaging was extended to 300 seconds (day -1). (B) Following treatment efficacy and proportionally reducing bioluminescence intensity for better presentation, mice in (A) were re-imagined for 30 seconds on day -1. Subsequent IVIS exposures of control SP6 CAR-T cells (n = 4) and BCMA-AR-T cells (n = 6) were completed within 30 seconds to better compare which day had the highest intensity between day -1 and day 17. Animals were euthanized by tear gas (White cross) based on advanced disease and animal protection laws. (C) Average bioluminescence signal intensity obtained from regions of interest covering the entire body of each mouse was plotted for each group at each time point.

[0442] Figure 16 BCMA-redirected CAR-T cells were effective against B-NHL tumors in a xenografted NSG mouse model. (A) Mantle cell lymphoma was implanted in a xenografted NSG mouse model. 6 × 10⁶ cells were transplanted intravenously. 5JeKo-1 cells were used to attack mice. Tumor cell growth was visualized by IVIS imaging on day 7 post-tumor inoculation. IVIS exposure, 120 seconds. (B) Following treatment efficacy and with bioluminescence intensity reduced proportionally for better presentation, mice in (A) were imaged again for 30 seconds on day 0. Subsequent IVIS exposures of control SP6 CAR-T cells (n = 7) and BCMA CAR-T cells (n = 7) were performed within 30 seconds after CAR-T cell transfer to better compare which day had the highest intensity between day 0 and day 16. (C) The average bioluminescence signal intensity obtained from the region of interest covering the entire body of each mouse was plotted for each group at each time point.

[0443] Example

[0444] The invention is illustrated by the embodiments disclosed herein. These embodiments provide technical support and a more detailed description of potential preferred, non-limiting embodiments of the invention. To demonstrate the functionality of the CAR described herein, the inventors conducted the following experiments:

[0445] - Co-culture of CAR-transduced human T cells with different target cell lines showed specific T cell activation via different BCMA+ MM and NHL cell lines; the readings were the release of IFN-γ from T cells as an effector cytokine;

[0446] - Cytotoxicity analysis revealed selective killing of BCMA+ cell lines; virtually no killing was observed in BCMA-negative cell lines or primary cells (e.g., HUVEC (endothelial origin), HEK293 (kidney), peripheral blood B cells, peripheral blood total leukocytes, T- and B-ALL, colon cancer).

[0447] - CD107a staining of co-cultured CAR-T cells and multiple myeloma cells, and flow cytometry detection of degranulated CD8+ T cells after antigen-specific (BCMA) stimulation.

[0448] - In vivo experiments involved using a xenograft NSG mouse model to generate data on the following: i) functionality, ii) off-target reactivity, iii) T-cell memory, and iv) biosafety of adoptive CAR-T cells against B-NHL and myeloma cell lines. For B-NHL, the cytolytic capacity of anti-BCMA CAR-T cells was compared with established anti-CD19 CAR-T cell products.

[0449] Example 1: Cloning and plasmid preparation:

[0450] Using GeneArt TM(Gene Synthesis Service) Synthesize CAR sequences. Perform restriction digestion of CAR constructs using NotI and EcoRI. Figure 5 The retroviral vector MP71 was also digested with NotI and EcoRI, followed by dephosphorylation.

[0451] CAR and vector were separated using gel electrophoresis. Figure 6 The fragment was then purified. The CAR construct was subsequently ligated into a vector (50 ng) at a 3:1 ratio. The ligation mixture was then converted into MACH-1 ( Figure 3 Control digestion was performed, and the mini-preparation was sequenced. The construct was then re-transformed into MACH-1. Large-scale preparation of the MP71-BCMA-CAR plasmid was then carried out.

[0452] MP71 is a single-stranded (+)-stranded RNA virus. Reverse transcriptase converts the retroviral RNA genome into a DNA copy. The DNA, acting as the provirus, integrates into the target genome at random locations. Through cell division, the virus stably regenerates as the provirus.

[0453] Example 2: Transfection and transduction:

[0454] Day 0: Seed HekT (293T) or GalV cells for virus production in 6-well plates.

[0455] Day 1: Transient 3-plasmid transfection (calcium phosphate transfection) to generate retroviruses. Following the standard protocol, 18 μg of DNA was used per well in 250 mM CaCl2 and 150 μl H2O. Cells were cultured at 37°C for 6 hours, the medium was changed, and the cells were cultured at 37°C for an additional 48 hours.

[0456] 24-well tissue culture plates were coated with anti-huCD3 and anti-huCD28 antibodies:

[0457] Prepare PBS solutions of anti-CD3 / anti-CD28 antibodies (5 μg / ml anti-CD3, 1 μg / ml anti-CD28), 0.5 ml per well. Incubate each well with 0.5 ml of antibody solution at 37°C for 2 hours, then replace with sterile 2% BSA solution (aqueous solution) and incubate for 30 minutes (37°C). Remove the BSA solution and wash the wells with 2 ml of PBS.

[0458] From 40 ml of blood (~2.5×10) 7 Purifying PBMCs from 1 PBMC:

[0459] Prepare 12.5 ml of sucrose gradient medium in 2 × 50 ml Falcon tubes. Dilute blood to 45 ml with RPMI (+100 IU / ml penicillin, streptomycin), mix, and spread onto 22.5 ml of the blood-medium mixture. Centrifuge (20 min, 20°C, 1800 rpm, RZB*648, G17.9). Discard 15 ml of the upper phase. Transfer the remaining upper phase and the milky white intermediate phase containing PBMCs to a new 50 ml Falcon tube. Fill to 45 ml with RPMI (+100 IU / ml penicillin, streptomycin) and centrifuge. Resuspend the globules in 45 ml of RPMI (+100 IU / ml penicillin, streptomycin), centrifuge, and transfer the globules to 10–20 ml of T cell culture medium. Stain one sample with trypan blue, count the cells, and set the concentration at 1–1.5 × 10⁻⁶. 6 Cells / ml (T cell culture medium (+100 IU / ml IL-2) corresponds to 400 U / ml clinical-IL-2) were added to wells coated with anti-CD3 and anti-CD28. The remaining PBMCs were centrifuged, resuspended in freezing medium, and stored in cryovials at -80°C.

[0460] Day 3: Transduction of PBL

[0461] Remove and filter (0.45 μm filter) the viral supernatant from Hekt or GalV cells. Treat stimulated PBMCs with 1.5 ml of viral supernatant.

[0462] Day 4: Transduction of PBL

[0463] Filter the remaining viral supernatant (4°C) and the secondary supernatant from Hekt- or GalV- cells (0.45 μm). Collect 1 to 1.5 ml of supernatant from the PBL. Treat stimulated PBMCs with 1 to 1.5 ml of viral supernatant and centrifuge in CD3 / CD28-coated wells (90 min, 32°C, 2000 rpm). The final concentrations are 100 IU / ml IL2 (1 μl von 400 U / μl) or 10 ng / μl IL7 and 10 ng / μl IL15, plus an additional 4 μg / ml (8 μl) protamine sulfate. Centrifuge at 2000 rpm, 32°C for 90 min.

[0464] Days 7 to 13: PBL cultured, T cell culture medium treated with fresh IL2 or IL7 / IL15.

[0465] Day 13: End T cell stimulation.

[0466] Rinse the PBL culture from the cell culture flask, centrifuge, and resuspend the pellets in T cell culture medium (+10 IU / ml IL2).

[0467] From Day 15: Functional Analysis

[0468] Example 3: Functional in vitro test of anti-BCMA CAR T cells

[0469] I. Confirmation of BCMA CAR expression on human T cells after retroviral transduction

[0470] Evidence was obtained regarding the folding and transport of the CAR receptor in the context of human T cells; the functionality of the retroviral transduction protocol was evaluated.

[0471] Human peripheral blood leukocytes were purified using a sucrose gradient. Cells were cultured, stimulated, and transduced with retroviruses as described above. After transduction, cells were further cultured in medium containing IL-2 or IL-7 / IL-15 before analyzing BCMA-CAR expression.

[0472] Transduction rate and viability were assessed by flow cytometry (FACS). To detect BCMA-CAR expression, cells were stained with an anti-human Ig antibody that selectively recognizes human IgG1 or IgG4 segments in the spacer region of the CAR construct. Co-staining of CD3 / CD8 / CD4 T cells was performed. Results are shown below. Figure 7 .

[0473] II. Co-culture of CAR-transduced human T cells with different target cell lines showed efficacy through different BCMA pathways. + Multiple bone Specific T cell activation in myeloma (MM) and B-NHL cell lines

[0474] The readings are the release of IFN-γ, an effector cytokine, from T cells.

[0475] As detailed previously, retrovirally transduced human T cells were generated; all BCMA CAR receptor variants (IX-XVII), SP6 negative control CAR, CD19 CAR, and UT = untransduced T cells were used. The following human cell lines were used as target cells in co-culture:

[0476]

[0477] Retrovirus-transduced T cells were co-cultured at a 1:1 ratio for 18 to 20 hours in the presence of the listed cell lines or primary cells. Afterward, cell-free culture supernatant was collected; maximal release was induced by stimulating effector T cells with PMA / iomycin; minimum release was induced by T cell stimulation alone. IFN-γ release in the supernatant was determined by ELISA. For results, please refer to [link to results]. Figure 8 .

[0478] III. CD107a (LAMP1) staining of co-cultured CAR-T cells and multiple myeloma cells: by flow cytometry Cytological detection of degranulated CD8+ T cells activated after antigen-specific (BCMA) stimulation.

[0479] As detailed previously, retroviral transduced human T cells were generated; using the BCMA CAR receptor variant (IX-XI) and SP6 negative control CAR.

[0480] Retrovirus-transduced T cells were co-cultured at a 1:1 ratio for 18 hours in the presence of the listed cell lines.

[0481] Anti-CD107a (LAMP1) antibody was added to the cell culture medium for overnight incubation; the antibody continuously bound to T cells as secretory lysosomes fused to the plasma membrane and released the enzyme contents of their vesicles. These vesicles contained cytolytic mediators such as granzymes and perforin. The next day, the T cells were co-stained with anti-CD8 and / or anti-CD3.

[0482] Flow cytometry analysis showed that higher CD107a reactivity, expressed as mean fluorescence intensity (MFI), indicated stronger T cell activation. This confirms antigen-dependent activation of T cells. For results, please refer to [link to relevant documentation]. Figure 9 .

[0483] IV. Cytotoxicity analysis revealed selective killing of BCMA-positive cell lines; and minimal damage in BCMA-negative cell lines. No casualties were observed.

[0484] use 51 Cr-release assays were used to quantify cytotoxic T lymphocyte activity. Cell lysis of target cells was measured.

[0485] As detailed previously, retroviral transduced human T cells were generated; BCMA CAR receptor variant (IX-XI), SP6 negative control CAR, and CD19 CAR were used as controls.

[0486] use 51 Cr-labeled target cells were then used. CAR-T cells and labeled target cells were then co-cultured for 4 hours. The effector-to-target ratio was measured by titration.

[0487] Effect: target 80:1 40:1 20:1 10:1 5:1 2.5:1

[0494] Harvest cell-free cell culture supernatant. Transfer the supernatant to a LUMA scintillation plate and measure the released [products / effects] using a gamma scintillation counter. 51 Cr. Maximum release: Target cells lysed via Triton X-100 permeation. Minimum release: Target cells only. See results for details. Figure 10 .

[0495] also, Figure 11 The results provided show the amounts of BCMA and CD19 expressed on the surfaces of each cell type for which cytotoxicity was assessed. Figure 12 A schematic diagram of the interaction between the scFV binding region of CAR and the BCMA epitope is provided.

[0496] Example 4: In vivo experiments using a xenograft NSG mouse model to evaluate adoptive transfer of CAR-T cells targeting B-NHL and myeloma cell lines.

[0497] In vivo experiments using xenografts into NSG mice:

[0498] 1) To demonstrate that CAR T cells equipped with different anti-BCMA variants also possess effector activity under in situ conditions, multiple myeloma cells with different BCMA antigen densities were intravenously transplanted into NSG mice (NOD.Cg-Prkdc). scid Il2rg tm1 Wjl / SzJ). The usable multiple myeloma cell lines are: RPMI-8226, low BCMA; MM1S, medium BCMA density; and NCI-H229, high BCMA density.

[0499] 2) To confirm the responsiveness of anti-BCMA CAR T cells to B-NHL cell lines in situ, luciferase-transduced cell lines, such as SU-DHL4 (DLBCL), JEKO-1 (mantle cell lymphoma), JVM3 (CLL), MEC1 (CLL), and DOHH-2 (FL), were intravenously injected into NSG mice.

[0500] In vivo antitumor activity of BCMA CAR-T cells in mouse models of multiple myeloma (MM) and B-cell non-Hodgkin lymphoma (B-NHL):

[0501] To provide a proof-of-concept for converting the strong in vitro activity of BCMA CAR-modified T cells into effective in vivo antitumor activity, we used a human MM.1S cell line transduced with the luciferase gene tandemly with GFP (… Figure 15 ) or B-NHL cell line JeKo-1 (mantle cell lymphoma) Figure 16 Intravenous infusion of one group of NOD.Cg-Prkdc scid Il2rg tm1 Wjl / SzJ (NSG) mice. NSG mice do not develop T, B, and NK cells, thus making them suitable for tolerance and growth of xenografted human cells. No graft-versus-host disease (GvHD) response (xenogeneic reactivity) was observed within the experimental timeframe presented here (data not shown). Tumor growth was monitored by IVIS imaging and fluorescein injection 7–8 days thereafter. CAR-T cells were intravenously injected one day after tumor growth was confirmed (=day 0). For functional in vivo experiments, CAR construct IX (B IX) was used. The total number never exceeded 6–7 × 10⁻⁶. 6 CAR-T cells / animal, with an average T cell transduction rate of 40-60% in this population. Transduction rates for each donor, SP6, and BCMA were matched within + / - 10%. For the two experiments shown, 3 × 10⁶ CAR-T cells / animal were used. 6 The efficacy rate of transduced CAR-T cells. Control mice receiving SP6 CAR-T cells.

[0502] In the MM1.S experiment ( Figure 15 In ), 3 × 10 6 6-7 × 10⁶ transduced CAR-T cells (as described above, total: 10⁶) 6 (and the observation interval was extended to 17 days). Although almost all animals treated with SP6 CAR had progressive MM disease characterized by strong luminescent signals in the spine, pelvis, and hind legs, or were euthanized due to disease progression under the (Berlin) animal protection law, this was clearly not the case in the BCMA CAR treatment group. We conclude that BCMA CAR-T cells already possess anti-myeloma activity at a relatively low number of CAR-T cells. Figure 15 AC).

[0503] Due to the high affinity and cohesion of anti-BCMA CAR-T cells, even mature B-cell NHL with low BCMA expression can be recognized, leading to T cell activation and tumor cell killing. These mature B-NHL entities include follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and certain stages of chronic lymphocytic leukemia (see [link to relevant documentation]). Figure 11 , 10 9, 8). To demonstrate the suitability of BCMA as a target structure in B-NHL entities, transduced CAR-T cells (total: 6-7 × 10⁸) were used. 6(1) cells were transplanted into NSG mice that had been challenged with the mantle cell lymphoma cell line JeKo-1. Although almost all animals treated with SP6 CAR had progressive lymphoma disease characterized by strong luminescent signals in the liver, thoracic organs, hind limb bone marrow, and spleen, this was clearly not the case in the BCMA CAR treatment group. Thus, we provide the first preclinical in vivo evidence that BCMA CAR-T cells possess antitumor activity not only against multiple myeloma but also against B-NHL lymphoma solids ( Figure 16 AC).

[0504] Example 5: Determining the surface density of BCMA molecules

[0505] The high affinity and cohesion of anti-BCMA CAR-T cells enable them to recognize even mature B-cell NHL entities with low BCMA expression, leading to T cell activation and tumor cell killing. These mature B-NHL entities include certain stages of follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

[0506] To quantify the surface density of BCMA molecules We used the PE phycoerythrin fluorescence detection kit, also known as the BD Quantibrite assay (BD Bioscience). The number of PE molecules per cell can be converted into antibodies per cell, providing a quantitative estimate of the amount of antigen per cell. Flow cytometry was used for detection.

[0507] Using this method, we found that the relative surface BCMA antigen density of the multiple myeloma cell line NCI-H929 was 12555, the multiple myeloma cell line OPM-2 had 3443 BCMA molecules, and the relative value of the multiple myeloma cell line MM.1S was 3181.

[0508] Compared to NCI-H929 The BCMA antigen density of the mentioned B-NHL cell line is:

[0509] DOHH-2: 1 / 20, JeKo-1: 1 / 250, MEC-1: 1 / 34.

Claims

1. A chimeric antigen receptor (CAR) polypeptide, wherein, The CAR includes: i. An extracellular antigen-binding domain containing an antibody or antibody fragment that binds to a B-cell maturation antigen (BCMA) polypeptide. ii. Transmembrane domain, and iii. Intracellular domain, Furthermore, the antigen-binding domain comprises a variable heavy chain (VH), wherein the VH comprises: - Heavy chain complementarity determination region 1 (H-CDR1), which contains SEQ ID NO 34 (RYWX1S), where X1 is I, F or M; - a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acids at positions 50-67 of SEQ ID NO 53 (EINPZ2SSTINYAPSLKX 11 X 12 ), wherein Z2 is S, N or D; X 11 is D or G; X 12 is K or R; and - Heavy chain complementarity determination region 3 (H-CDR3), which contains SEQ ID NO 36 (SLYX4DYGDAX5DYW), wherein X4 is Y and X5 is Y or M; Furthermore, the antigen-binding domain comprises a variable light chain (VL), the VL comprising: - Light chain complementarity determination region 1 (L-CDR1), which contains SEQ ID NO 37 (KASQSVX1X2NVA), where X1X2 is ES or DS; - Light chain complementarity determination region 2 (L-CDR2), which contains SEQ ID NO 29 (SASLRFS); and - Light chain complementarity determination region 3 (L-CDR3), which contains SEQ ID NO 30 (QQYNNYPLTFG), The expression of the CAR in immune effector cells increases the cytotoxicity of the immune effector cells against the following cells: (i) multiple myeloma cells expressing BCMA, and (ii) mantle cell lymphoma cells expressing BCMA.

2. The chimeric antigen receptor (CAR) polypeptide of claim 1, wherein, The VH has at least 80% sequence identity with SEQ ID NO 11, and the VL has at least 70% sequence identity with SEQ ID NO 12.

3. The chimeric antigen receptor (CAR) polypeptide according to claim 1, comprising the following sequence: - H-CDR1: RYWFS (SEQ ID NO.25) or RYWMS, - H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO.26) or EINPDSSTINYAPSLKDK, - H-CDR3: SLYYDYGDAYDYW (SEQ ID NO.27) or SLYYDYGDAMDYW, - L-CDR1: KASQSVESNVA (SEQ ID NO.28) or KASQSVDSNVA, - L-CDR2:SASLRFS (SEQ ID NO.29), and - L-CDR3: QQYNNYPLTFG (SEQ ID NO. 30).

4. The chimeric antigen receptor (CAR) polypeptide of claim 1, wherein, (a) The VH comprises the sequence of SEQ ID NO 41, and H-CDR1, H-CDR2 and H-CDR3; (b) The VL comprises the sequence of SEQ ID NO 50, and L-CDR1, L-CDR2 and L-CDR3.

5. The chimeric antigen receptor (CAR) polypeptide according to claim 1, comprising at least W36, E50, L99, Y100, Y101 and A106 of SEQ ID NO 11, and at least S31, A34, S50, L53, Q89, Y91, Y94 and L96 of SEQ ID NO 12.

6. The chimeric antigen receptor (CAR) polypeptide according to claim 1, comprising the VH domain according to SEQ ID NO 11 and the VL domain according to SEQ ID NO 12.

7. The chimeric antigen receptor (CAR) polypeptide of claim 1, wherein, The mantle cell lymphoma is the JeKo-1 cell line.

8. The chimeric antigen receptor (CAR) polypeptide of claim 1, wherein, The extracellular antigen-binding domain contains a linker polypeptide located between the VH and VL domains.

9. The chimeric antigen receptor (CAR) polypeptide according to claim 8, wherein, The connector is selected from Whitlow (SEQ ID NO 13; GSTGSGSGKPGSGEGSTKG) or Gly-Ser (SEQ ID NO 14; SSGGGGSGGGGSGGGGS) connectors, or connectors that have at least 80% sequence identity with SEQ ID NO 13 or 14.

10. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising a spacer region polypeptide located between the extracellular antigen-binding domain and the transmembrane domain, wherein, The interval region is selected from: a. IgG1-CD28 spacer region (SEQ ID NO 15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKK), b. IgG1Δ-4-1BB spacer region (SEQ ID NO 16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK), c. IgG4 (Hi-CH2-CH3) spacer region (SEQ ID NO 17; ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), d. IgG4(Hi-CH3) spacer region (SEQ ID NO 18; ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), e. IgG4(Hi) spacer region (SEQ ID NO 19; ESKYGPPCPPCP), or f. A spacer region having at least 80% sequence identity with any of SEQ ID NO 15 to 19.

11. The chimeric antigen receptor (CAR) polypeptide according to claim 1, wherein, The transmembrane domain is selected from the CD8α domain (SEQ ID NO 20; IYIWAPLAGTCGVLLLSLVITLYC) or the CD28 domain (SEQ ID NO 21; FWVLVVVGGVLACYSLLVTVAFIIFWV) or a transmembrane domain having at least 80% sequence identity with SEQ ID NO 20 or 21.

12. The chimeric antigen receptor (CAR) polypeptide according to claim 1, wherein, The intracellular domain includes a co-stimulatory domain selected from the 4-1BB co-stimulatory domain (SEQ ID NO 22; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or the CD28 co-stimulatory domain (SEQ ID NO 23; RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS), or a co-stimulatory domain having at least 80% sequence identity with SEQ ID NO 22 or 23.

13. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising a signal transduction domain, wherein the signal transduction domain is selected from the CD3ζ (CD28 or 4-1BB) signal transduction domain (SEQ ID NO 24); LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or a signal transduction domain having at least 80% sequence identity with SEQ ID NO 24.

14. The chimeric antigen receptor (CAR) polypeptide of claim 1, comprising a tandem co-stimulatory domain and a CD3ζ signaling / activation domain (SEQ ID NO 24), wherein the tandem co-stimulatory domain comprises a 4-1BB co-stimulatory domain (SEQ ID NO 22) and a CD28 co-stimulatory domain (SEQ ID NO 23).

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

16. A cell comprising the CAR as described in any one of claims 1 to 14, or a nucleic acid molecule encoding said CAR.

17. The cell according to claim 16, wherein, The cells were selected from a group consisting of T lymphocytes and NK cells.

18. The cell according to claim 17, wherein, The cells in question are cytotoxic T lymphocytes.

19. Use of the cell according to claim 16 in the preparation of a medicament for treating medical conditions associated with the presence of pathogenic B cells.

20. The use according to claim 19, wherein, The medical condition is a disease of plasma cells, mature B cells, and / or memory B cells.

21. The use according to claim 19, wherein, The medical condition described is multiple myeloma.

22. The use according to claim 19, wherein, The medical condition described is non-Hodgkin's lymphoma.

23. The use according to claim 19, wherein, The medical condition described is an autoantibody-dependent autoimmune disease.

24. The use according to claim 23, wherein, The medical condition mentioned is systemic lupus erythematosus (SLE) or rheumatoid arthritis.

Images