BCMA-targeted chimeric antigen receptor

Abstract

The present disclosure provides BCMA-targeting chimeric antigen receptors (CARs), as well as methods for preparing and using the same. The CARs of the present disclosure target BCMA-positive cells and can be used to treat BCMA-positive B-cell lymphoma, multiple myeloma, and plasma cell leukemia.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application Nos. 63 / 120,692 (filed December 2, 2020), 63 / 153,666 (filed February 25, 2021), and 63 / 212,289 (filed June 18, 2021), and U.S. Patent Application No. 17 / 476,661 (filed September 16, 2021), each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically filed in ASCII format, which is hereby incorporated by reference in its entirety. The name of the ASCII copy created on December 1, 2021 is 11299 - 008884 - WO1_ST25.txt, and its size is 41 KB.

[0003] [[ID=ID=17]]The present invention relates to the field of biomedicine, and more particularly, to chimeric antigen receptors targeting BCMA, and methods for their preparation and uses.

Background Art

[0004] BCMA is a B - cell maturation antigen, also known as CD269 or TNFRSF17, and is a member of the tumor necrosis factor receptor superfamily. Its ligands are B - cell activating factor (BAFF) and a proliferation - inducing ligand (APRIL).

[0005] The binding of BCMA to BAFF and APRIL activates NF - kB and induces up - regulation of anti - apoptotic Bcl - 2 members such as Bcl - xL or Bcl - 2 and Mcl - 1. The interaction between BCMA and its ligands regulates humoral immunity and the growth and differentiation of B cells in various ways to maintain a stable and balanced environment in the human body.

[0006] BCMA expression is limited to B cell lines. It is expressed in plasmablasts, plasma cells, and some mature B cells, and increases during terminal B cell differentiation. On the other hand, BCMA is not expressed in most B cells, such as naive B cells, memory B cells, germinal centers, and other organs. BCMA expression has been reported to be important for long-lived, fixed plasma cells in the bone marrow. Therefore, plasma cells in the bone marrow are reduced in BCMA-deficient mice, but plasma cell levels in the spleen are unaffected. Mature B cells can normally differentiate into plasma cells in BCMA knockout mice. BCMA knockout mice appeared normal and healthy, and had normal B cell counts, but plasma cells could not survive for long periods.

[0007] BCMA is also highly expressed in malignant plasma cells, such as those in multiple myeloma and plasma cell leukemia. BCMA is also detected in HRS cells from Hodgkin lymphoma patients. In the United States, hematological malignancies account for approximately 10% of all malignancies, and myeloma accounts for 15% of all malignant hematological malignancies. According to the literature, BCMA expression is associated with the progression of multiple myeloma disease. The BCMA gene is highly expressed in myeloma samples but less expressed in chronic lymphocytic leukemia, acute lymphoblastic leukemia, and acute T-cell lymphocytic leukemia. B-cell lymphoma was significantly increased in mouse models overexpressing BCMA ligands BAFF and APRIL. Ligands that bind to BCMA have been shown to regulate the growth and survival of BCMA-expressing multiple myeloma cells. Combinations of BCMA with BAFF and APRIL can enable the survival of malignant plasma cells. Therefore, the loss of tumor cells expressing BCMA and the distribution of interactions between BCMA ligands and receptors can improve outcomes in the treatment of multiple myeloma or other BCMA-positive B-cell line malignancies.

[0008] Multiple myeloma, also known as plasmacytoma or Keller's disease, is a refractory B-cell malignancy characterized by the abnormal proliferation of plasma cells. Plasma cells are a type of white blood cell responsible for antibody production. According to data published by the National Cancer Institute in 2017, myeloma accounts for 1.8% of all tumor cases, with a mortality rate of 2.1%. Statistics from 2010-2014 show an incidence rate of approximately 6.6 cases per 100,000 cases per year, with a mortality rate of approximately 50%. Multiple myeloma is a disease of middle age. The median age of onset in Europe and the United States is 68 years. It is more common in men than women. In China, the peak age of onset is 55-65 years, with a male-to-female ratio of 2.35:1. There is no confirmed epidemiological data on multiple myeloma in China. The incidence rate is estimated to be approximately 1 in 100,000 cases, similar to neighboring Southeast Asia and Japan. Traditional treatments for multiple myeloma include chemotherapy and hematopoietic stem cell transplantation, but these methods have high relapse rates. Bortezomib (PS-341) was the first proteasome inhibitor and was approved by the FDA in 2003 for the treatment of relapsed / refractory multiple myeloma alone or in combination with existing drugs. The results were satisfactory. This drug was also marketed in China in 2005 and has become one of the treatment options for multiple myeloma, alongside thalidomide and dexamethasone. Treatment for multiple myeloma is usually done in combination. However, the simultaneous use of multiple drugs can be costly and lead to cumulative side effects. There remains a clinical need to develop new methods for treating multiple myeloma.

[0009] In recent years, immunotherapy, particularly adoptive T-cell therapy, has shown strong efficacy and promising prospects in clinical trials for the treatment of hematological malignancies. T cells can be genetically modified to express chimeric antigen receptors (CARs) containing an antigen-recognition region and a T-cell activation region. Using the antigen-binding properties of monoclonal antibodies, CARs can redirect T cells and target specificity and reactivity in a non-MHC-restricted manner. This non-MHC-restricted antigen recognition allows CAR-expressing T cells to recognize antigens without antigen processing, thus circumventing the primary mechanism of tumor escape. Furthermore, CARs do not produce dimers with alpha and beta chains of the endogenous TCR.

[0010] Currently, two CD19-targeting chimeric antigen receptor T-cell therapy (CAR-T) products are approved for the treatment of acute lymphoblastic leukemia in pediatric and young adult patients, as well as for second-line or multi-line system therapy in adults with relapsed or refractory large B-cell lymphoma. However, CD19 is hardly expressed in malignant plasma cells of multiple myeloma. There is an urgent need to develop BCMA-targeting CAR-T products for the treatment of multiple myeloma. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The purpose of this disclosure is to provide a chimeric antigen receptor targeting BCMA, as well as a method for preparing the same and its uses. [Means for solving the problem]

[0012] This disclosure provides a chimeric antigen receptor (CAR). A CAR has a light chain variable region (V L ) and heavy chain variable region (V H It may include an anti-BCMA antigen binding region containing ).

[0013] V L This may include three complementarity determination regions (CDRs), LCDR1, LCDR2, and LCDR3, V HThis may include three CD-Rs: HCDR1, HCDR2, and HCDR3.

[0014] In certain embodiments, LCDR1, LCDR2, and LCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 17, 19, and 21, respectively. HCDR1, HCDR2, and HCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 24, 26, and 28, respectively.

[0015] In certain embodiments, LCDR1, LCDR2, and LCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 31, 33, and 35, respectively. HCDR1, HCDR2, and HCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 38, 40, and 42, respectively.

[0016] In certain embodiments, LCDR1, LCDR2, and LCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 45, 47, and 49, respectively. HCDR1, HCDR2, and HCDR3 may have amino acid sequences that are approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NOs. 52, 54, and 56, respectively.

[0017] CAR's V L and V H (a) each may have an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, (a) each may have an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, or (a) each may have an amino acid sequence that is approximately 80% to 100% identical to the amino acid sequences shown in S

[0018] In a particular embodiment, V L V H It is located at the N-terminus of [the node].

[0019] The anti-BCMA antigen-binding region may be a single-stranded variable fragment (scFv) that specifically binds to BCMA.

[0020] CAR may further comprise one or more of the following: (a) a signal peptide, (b) a hinge region, (c) a transmembrane domain, (d) a co-stimulatory region, and (e) a cytoplasmic signaling domain.

[0021] The co-stimulatory regions may include co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof (or may be derived from them).

[0022] The cytoplasmic signaling domain may include the cytoplasmic signaling domain of CD3ζ (or a domain that may be derived therefrom).

[0023] The hinge region may include hinge regions of CD8, CD28, CD137, Ig4, or combinations thereof (or may be derived therefrom).

[0024] The transmembrane domain may include transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof (or may be derived therefrom).

[0025] This disclosure provides immune cells expressing the CAR. The immune cells may be T cells or natural killer (NK) cells. The immune cells may be allogeneic or autologous.

[0026] The nucleic acid encoding this CAR is also included in this disclosure.

[0027] This disclosure further provides a vector containing the nucleic acid.

[0028] The present disclosure provides a method for treating cancer. The method may include administering immune cells to a subject who needs them.

[0029] The cancer can be a blood cancer. The cancer can be a plasma cell malignancy.

[0030] The cancer can be a BCMA-positive malignancy. The cancer can be multiple myeloma (MM) or plasma cell leukemia.

[0031] The immune cells can be administered by infusion, injection, transfusion, implantation, and / or transplantation.

[0032] The immune cells can be administered intravenously, subcutaneously, intradermally, intra-articularly, intratumorally, intramedullarily, intramuscularly, or intraperitoneally.

[0033] The immune cells can be administered by intravenous infusion.

[0034] The subject can be a human.

[0035] The present disclosure provides a method for treating cancer. The method may include administering the immune cells to a subject who needs them.

[0036] The chimeric antigen receptor (CAR) can generate an area under the curve (AUC) in the subject's blood within about 28 days after administration in the range of about 5.0e+05 copies / μg genomic DNA (copies / gDNA) to about 1.3e+07 copies / gDNA, about 5.0e+06 copies / μg genomic DNA (copies / gDNA) to about 1.0e+07 copies / gDNA, about 5.0e+06 copies / μg genomic DNA (copies / gDNA) to about 1.3e+07 copies / gDNA, or about 7.0e+06 copies / μg genomic DNA (copies / gDNA) to about 1.0e+07 copies / gDNA.

[0037] The chimeric antigen receptor (CAR) is in the subject's blood at about 5×10 4 copies / μg genomic DNA (copies / gDNA) to about 1.3×10 6Copy / gDNA, approximately 5 x 10⁻⁶ 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.3 × 10⁻¹⁶ 6 Copy / gDNA, or approximately 7.5 × 10⁻⁶ 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1 × 10⁻¹⁶ 6 Maximum plasma concentration within the copy / gDNA range (C max ) can be generated.

[0038] CAR is a type of T (Time-Based Care) that lasts approximately 12 to 25 days, 14 to 20 days, or 6 to 22 days. max It may have.

[0039] In certain embodiments, the anti-BCMA antigen-binding region is a light chain variable region (V) containing at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 L ) includes.

[0040] In certain embodiments, the anti-BCMA antigen binding region is a heavy chain variable region (V) containing at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical amino acid sequence to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. H ) includes.

[0041] The light chain variable regions of the anti-BCMA antigen-binding region are the CDRs of the light chain variable regions of the BCMA-20 antibody (CDR1, CDR2, and CDR3 shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 1, respectively), or the CDRs of the light chain variable regions of the BCMA-CA8 antibody (CDR1, CDR2, and CDR3 shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 3, respectively), or the CDRs of the light chain variable regions of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3 shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 5, respectively), and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or It may include one, two, or three complementary determination regions (CDRs) that are identical by approximately 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%.

[0042] The heavy chain variable region of the anti-BCMA antigen-binding region is the CDR of the heavy chain variable region of the BCMA-20 antibody (CDR1, CDR2, and CDR3 shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 2, respectively), or the CDR of the heavy chain variable region of the BCMA-CA8 antibody (CDR1, CDR2, and CDR3 shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 4, respectively), or the CDR of the heavy chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3 shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 6, respectively), and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or This may include one, two, or three complementary determination regions (CDRs) that are identical by approximately 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%.

[0043] The light chain variable regions of the anti-BCMA antigen-binding region are at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, and at least It may include one, two, or three complementary determination regions (CDRs) that are identical by or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%.

[0044] The light chain variable region of the anti-BCMA antigen-binding region is composed of the CDRs of the heavy chain variable region of the BCMA-20 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 24, 26, and 28, respectively), or the CDRs of the heavy chain variable region of the BCMA-CA8 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 38, 40, and 42, respectively), or the CDRs of the heavy chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 52, 54, and 56, respectively), and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, and at least It may include one, two, or three complementary determination regions (CDRs) that are identical by or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100%.

[0045] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3, shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 1) that are identical (e.g., 80-100% identical) to the CDRs of the heavy chain variable region of the BCMA-20 antibody, and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3, shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 2) that are identical (e.g., 80-100% identical) to the CDRs of the heavy chain variable region of the BCMA-20 antibody.

[0046] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the light chain variable region of the BCMA-20 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 17, 19, and 21, respectively), and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the heavy chain variable region of the BCMA-20 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 24, 26, and 28, respectively).

[0047] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3, shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 3, respectively) that are identical (e.g., 80-100% identical) to the CDRs of the light chain variable region of the BCMA-CA8 antibody, and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs (CDR1, CDR2, and CDR3, shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 4, respectively) that are identical (e.g., 80-100% identical) to the CDRs of the heavy chain variable region of the BCMA-CA8 antibody.

[0048] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the light chain variable region of the BCMA-CA8 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 31, 33, and 35, respectively), and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the heavy chain variable region of the BCMA-CA8 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 38, 40, and 42, respectively).

[0049] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs identical to those of the light chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3, respectively, shown at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 5), and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs identical to those of the heavy chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3, respectively, shown at positions 31-35, 50-66, and 99-110 of SEQ ID NO: 6).

[0050] In certain embodiments, the light chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the light chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 45, 47, and 49, respectively), and the heavy chain variable region of the anti-BCMA antigen-binding region includes three CDRs that are identical (e.g., 80% to 100% identical) to the CDRs of the heavy chain variable region of the BCMA-MO6 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 52, 54, and 56, respectively).

[0051] In certain embodiments, the CAR is an amino acid sequence shown in SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, and is an amino acid sequence of at least 80% to 100%, at least 85% to 100%, at least 90% to 100%, at least 95% to 100%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 81%, at least 82%, at least 83%, and at least Alternatively, they could contain approximately 84%, at least or approximately 85%, at least or approximately 86%, at least or approximately 87%, at least or approximately 88%, at least or approximately 89%, at least or approximately 90%, at least or approximately 91%, at least or approximately 92%, at least or approximately 93%, at least or approximately 94%, at least or approximately 95%, at least or approximately 96%, at least or approximately 97%, at least or approximately 98%, at least or approximately 99%, or approximately 100% identical amino acid sequences.

[0052] In certain embodiments, the nucleic acid encoding the CAR comprises the amino acid sequence shown in SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62, and approximately 80% to approximately 100%, approximately 85% to approximately 100%, approximately 90% to approximately 100%, approximately 95% to approximately 100%, at least or approximately 70%, at least or approximately 75%, at least or approximately 80%, at least or approximately 85%, at least or approximately 90%, at least or approximately 95%, at least or approximately 99%, at least or approximately 81%, at least or approximately 82%, at least or approximately 83%, It may contain at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical nucleic acid sequences.

[0053] In certain embodiments, the CAR is present in the subject's blood for approximately 28 days after administration of the CAR, at concentrations of approximately 0.5e+06 copies / μg genomic DNA (copies / gDNA) to approximately 2e+07 copies / gDNA, approximately 5.0e+05 copies / μg genomic DNA (copies / gDNA) to approximately 1.3e+07 copies / gDNA, approximately 5.0e+05 copies / μg genomic DNA (copies / gDNA) to approximately 2e+07 copies / gDNA, approximately 5.0e+05 copies / μg genomic DNA (copies / gDNA) to approximately 1.5e+07 copies / gDNA, and approximately 5 0.0e+06 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.0e+07 copies / gDNA, approximately 5.0e+06 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.3e+07 copies / gDNA, approximately 7.0e+06 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.0e+07 copies / gDNA, approximately 8.0e+06 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.0e+07 copies / gDNA, approximately 0.5e+06 copies / μg genomic DNA (copy / gDNA) ~ approximately 4e+06 copies / gDNA, Approximately 0.5e+06 copies / μg genomic DNA (copies / gDNA) to approximately 3.5e+06 copies / gDNA, approximately 1e+06 copies / μg genomic DNA (copies / gDNA) to approximately 3.5e+06 copies / gDNA, approximately 1.2e+06 copies / μg genomic DNA (copies / gDNA) to approximately 3.2e+06 copies / gDNA, approximately 0.8e+06 copies / μg genomic DNA (copies / gDNA) to approximately 3.2e+06 copies / gDNA, approximately 1.6e+06 copies / μg genomic DNA (copies / gDNA) to approximately 3.2e+06 copies / gDNA A. Approximately 1e+06 copies / μg genomic DNA (copy / gDNA) to approximately 2e+06 copies / gDNA, approximately 0.6e+06 copies / μg genomic DNA (copy / gDNA) to approximately 1.8e+06 copies / gDNA, approximately 3e+06 copies / μg genomic DNA (copy / gDNA) to approximately 3.2e+06 copies / gDNA, approximately 0.5e+06 copies / μg genomic DNA (copy / gDNA) to approximately 1.7e+06 copies / gDNA, approximately 2e+06 copies / μg genomic DNA (copy / gDNA) to approximately 3.2e+06 copies / gDNA, approximately 1.The area under the curve (AUC) can be generated for a range of approximately 5e+06 copies / μg genomic DNA (copies / gDNA) to about 2e+06 copies / gDNA, or approximately 1e+06 copies / μg genomic DNA (copies / gDNA) to about 3.2e+06 copies / gDNA. The AUC may also be the median AUC.

[0054] In a particular embodiment, the CAR is present in the blood of the subject after administration of the CAR at a concentration of approximately 5 × 10⁻¹⁶. 4 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.3 × 10⁻¹⁶ 6 Copy / gDNA, approximately 5 x 10⁻⁶ 4 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.5 × 10⁻¹⁶ 6 Copy / gDNA, approximately 5 x 10⁻⁶ 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.3 × 10⁻¹⁶ 6 Copy / gDNA, approximately 7.5 × 10⁻⁶ 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1 × 10⁻¹⁶ 6 Copy / gDNA, approximately 7 x 10 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1 × 10⁻¹⁶ 6 Copy / gDNA, approximately 8 x 10 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1 × 10⁻¹⁶ 6 Copy / gDNA, approximately 7.5 × 10⁻⁶ 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.5 × 10⁻¹⁶ 6 Copy / gDNA, approximately 7 x 10 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.5 × 10⁻¹⁶ 6 Copy / gDNA, approximately 8 x 10 5 Copy / μg genomic DNA (copy / gDNA) ~ approximately 1.5 × 10⁻¹⁶ 6copies / gDNA, approximately 0.8e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3.5e+05 copies / gDNA, approximately 1e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3.5e+05 copies / gDNA, approximately 1e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.6e+05 copies / gDNA, approximately 1e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3.3e+05 copies / gDNA, approximately 0.8e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 1.5e+05 copies / gDNA, approximately 0.8e+05 copies / μg genomic DNA Maximum plasma concentration (C) in the range of (copy / gDNA) ~ approximately 2e+05 copies / gDNA, approximately 1e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 2e+05 copies / gDNA, approximately 2e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3e+05 copies / gDNA, approximately 2e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3.5e+05 copies / gDNA, approximately 2e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 2.5e+05 copies / gDNA, or approximately 1e+05 copies / μg genomic DNA (copy / gDNA) ~ approximately 3e+05 copies / gDNA max ) generates C max is the median C max That's fine.

[0055] In a particular embodiment, the CAR is a T in the range of approximately 12 to 25 days, approximately 14 to 20 days, approximately 6 to 22 days, approximately 3 to 20 days, approximately 4 to 18 days, approximately 5 to 17 days, approximately 6 to 16 days, approximately 7 to 15 days, approximately 9 to 15 days, approximately 10 to 15 days, approximately 10 to 14 days, approximately 8 to 12 days, approximately 6 to 14 days, approximately 12 to 14 days, approximately 8 to 11 days, approximately 8 to 15 days, or approximately 10 to 14 days. max (CAR is C max It has the time it takes to reach T max is the median T max That's fine.

[0056] In a particular embodiment, the CAR is a T in the range of approximately 30 to 200 days, approximately 50 to 150 days, approximately 50 to 100 days, approximately 60 to 80 days, approximately 60 to 150 days, approximately 80 to 150 days, approximately 50 to 200 days, approximately 50 to 60 days, approximately 50 to 80 days, approximately 50 to 100 days, approximately 60 to 100 days, approximately 80 to 100 days, approximately 60 to 200 days, approximately 80 to 200 days, approximately 50 to 140 days, approximately 60 to 140 days, or approximately 80 to 140 days. last (Time corresponding to the last quantifiable CAR level) last is the median T last That's fine.

[0057] Specifically, the purpose of this disclosure is to provide the sequence of a BCMA-targeted chimeric antigen receptor, as well as a method for preparing and identifying the activity of its modified T cells (CART-BCMA).

[0058] This disclosure provides a chimeric antigen receptor structure for use in the treatment of BCMA-positive B-cell lymphoma.

[0059] In the first embodiment, a chimeric antigen receptor (CAR) (sequence) is provided, the antigen-binding domain of which is an antibody single-chain variable region sequence that targets the extracellular region of BCMA.

[0060] In another embodiment, the antigen-binding domain is a single-strand variable region sequence of an antibody that targets amino acid residues at positions 24-41 of the BCMA sequence.

[0061] In another embodiment, the NCBI accession number for the BCMA sequence is AY684975.1.

[0062] In another embodiment, the structure of the antigen-binding domain is shown in the following formula I: V L -V H (I) (In the formula, V H This is the antibody heavy chain variable region, V L The variable region of the antibody light chain is represented by "-", and the linker peptide or peptide bond is represented by "-". V L The amino acid sequence is as shown in Sequence ID No. 1, V H The amino acid sequence is as shown in Sequence ID No. 2. Or, V L The amino acid sequence is as shown in SEQ ID NO: 3, V H The amino acid sequence is as shown in SEQ ID NO: 4. Or, V L The amino acid sequence is as shown in SEQ ID NO: 5, V H The amino acid sequence is as shown in SEQ ID NO: 6.

[0063] In another embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0064] In another embodiment, the antibody single-chain variable region includes human, mouse, and human-mouse chimeric antibody single-chain variable regions.

[0065] In another embodiment, the structure of the chimeric antigen receptor is shown in formula II below: SV L -V H -H-TM-C-CD3ζ (II) During the ceremony, S is any signal peptide, H is the hinge region, TM is a transmembrane domain, C is a co-stimulus signaling molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ, V H and V L This is as stated above.

[0066] In another embodiment, S is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0067] In another embodiment, S is a signal peptide derived from CD8.

[0068] In another embodiment, the amino acid sequence of S is as shown in SEQ ID NO: 9.

[0069] In another embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0070] In another embodiment, H is a hinge region derived from CD8.

[0071] In another embodiment, the amino acid sequence of H is as shown in SEQ ID NO: 12.

[0072] In another embodiment, TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

[0073] In another embodiment, TM is a transmembrane region derived from CD8.

[0074] In another embodiment, the sequence of TM is as shown in Sequence ID No. 13.

[0075] In another embodiment, the co-stimulatory signaling molecule is a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0076] In another embodiment, C is a co-stimulatory signaling molecule derived from 4-1BB.

[0077] In another embodiment, the amino acid sequence of C is as shown in SEQ ID NO: 14.

[0078] In another embodiment, the amino acid sequence of CD3ζ is as shown in SEQ ID NO: 15.

[0079] In a second embodiment, a nucleic acid molecule encoding a chimeric antigen receptor (CAR) according to the first embodiment is provided.

[0080] In another embodiment, nucleic acid molecules are isolated.

[0081] In a third embodiment, a vector comprising a nucleic acid molecule according to the second embodiment is provided.

[0082] In another embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0083] In another embodiment, the vector is a lentiviral vector.

[0084] In a fourth embodiment, a host cell is provided that contains a vector according to the third embodiment, or an exogenous nucleic acid molecule according to the second embodiment is integrated into the chromosome, or that expresses a CAR according to the first embodiment.

[0085] In another embodiment, the cells are isolated cells and / or the cells are genetically modified cells.

[0086] In another embodiment, the cells are mammalian cells.

[0087] In another embodiment, the cell is a T cell.

[0088] In a fifth embodiment, a method for preparing CAR-T cells expressing a CAR according to the first embodiment is provided, comprising the step of transducing a nucleic acid molecule according to the second embodiment or a vector according to the third embodiment into T cells to obtain CAR-T cells.

[0089] In a sixth aspect, the present invention provides a preparation comprising a chimeric antigen receptor according to the first aspect, a nucleic acid molecule according to the second aspect, a vector according to the third aspect, or a cell according to the fourth aspect, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0090] In another embodiment, the preparation is a liquid preparation.

[0091] In another embodiment, the dosage form of the preparation is injection.

[0092] In another embodiment, the concentration of CAR-T cells in the preparation is 1 × 10⁻⁶ 3 ~1 × 10 8 cells / ml, or 1 × 10⁶ 4 ~1 × 10 7 The value is cells / ml.

[0093] In a seventh aspect, the present invention provides the use of a chimeric antigen receptor in a first aspect, a nucleic acid molecule in a second aspect, a vector in a third aspect, or a cell in a fourth aspect for the preparation of a pharmaceutical or preparation for the prevention and / or treatment of tumors or cancer.

[0094] In another embodiment, the tumor is selected from the group consisting of hematological malignancies, solid tumors, or combinations thereof.

[0095] In one embodiment, the cancer is B-cell lymphoma.

[0096] In another embodiment, the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0097] In another embodiment, the solid tumor is selected from the group consisting of gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.

[0098] In another embodiment, the tumor is a BCMA-positive tumor such as BCMA-positive B-cell lymphoma, multiple myeloma, or plasma cell leukemia.

[0099] In an eighth embodiment, a kit for preparing cells of the fourth embodiment is provided, the kit comprising a container in which a nucleic acid molecule of the second embodiment or a vector of the third embodiment is placed.

[0100] In a ninth embodiment, the use of cells according to the fourth embodiment or preparations according to the sixth embodiment for the prevention and / or treatment of cancer or tumors is provided.

[0101] In a tenth embodiment, a method for treating a disease is provided, comprising administering an appropriate amount of cells of the fourth embodiment or a preparation of the sixth embodiment to a subject in need of treatment.

[0102] In another embodiment, the disease is cancer or a tumor.

[0103] It should be understood that the various technical features of the present disclosure described above and the various technical features described below specifically (such as the examples) may be combined within the scope of the present disclosure to form new or preferred technical solutions that do not need to be described individually due to space limitations. [Brief explanation of the drawing]

[0104] [Figure 1A]The screening results for CART-BCMA comparative examples are shown. Figure 1A shows the detection of the transfection efficiency of engineered T cells using a chimeric antigen receptor targeting BCMA. The expression level of the CAR gene coding protein on the T cell membrane surface in CAR-BCMA cells cultured on day 6 was identified by Fc fragment staining of recombinant human BCMA protein. 1 × 10⁵ CART-BCMA cells cultured on day 10 were cultured in 200 μl of GT-551 medium for 18 hours in a 1:1 ratio with BCMA-positive K562-BCMA-B9 tumor cell line, naturally BCMA-expressing MM.1S and RPMI8226 tumor cell line, and BCMA-negative K562 tumor cell line, or without tumor cells. [Figure 1B] The screening results for CART-BCMA comparative examples are shown. In Figure 1B, the expression level of CD137 on the surface of the T cell membrane was detected. [Figure 1C] The screening results for CART-BCMA comparative examples are shown. In Figure 1C, the secretion level of IFNγ in the culture supernatant was detected. [Figure 2] The structure of a chimeric antigen receptor targeting BCMA is shown. The CAR structure includes a leader sequence, antigen recognition sequence, linker region, transmembrane region, costimulator signaling region, and CD3ζ signaling region. [Figure 3A] This study demonstrates the detection of transfection efficiency of engineered T cells using a chimeric antigen receptor targeting BCMA. The expression levels of CAR gene-coding proteins on the surface of the T cell membrane in CAR-BCMA cells cultured on day 7 were identified by Fc fragment staining of recombinant human BCMA protein. [Figure 3B] This study demonstrates the detection of transfection efficiency of engineered T cells using a chimeric antigen receptor targeting BCMA. The expression levels of CAR gene-coding proteins on the surface of the T cell membrane in CAR-BCMA cells cultured on day 21 were identified by Fc fragment staining of recombinant human BCMA protein. [Figure 3C]This study demonstrates the detection of transfection efficiency of engineered T cells using a chimeric antigen receptor targeting BCMA. The expression levels of CAR gene-coding proteins on the surface of the T cell membrane in CAR-BCMA cells cultured on day 29 were identified by Fc fragment staining of recombinant human BCMA protein. [Figure 4A] This shows the expression level of CD137 on the surface of the T cell membrane. Specifically, 1 × 10⁵ CART-BCMA cells cultured on day 7 were cultured in 200 μl of GT-551 medium for 18 hours in a 1:1 ratio, either with BCMA-positive K562-BCMA-E7 tumor cell lines and BCMA-negative K562 tumor cell lines, or without tumor cells. The expression level of CD137 on the surface of the T cell membrane was then detected. [Figure 4B] This shows the secretion level of IFNγ in the culture supernatant. Specifically, 1 × 10⁵ CART-BCMA cells cultured on day 7 were cultured in 200 μl of GT-551 medium for 18 hours in a 1:1 ratio, either together with BCMA-positive K562-BCMA-E7 tumor cell lines and BCMA-negative K562 tumor cell lines, or without tumor cells. Subsequently, the secretion level of IFNγ in the culture supernatant was detected. [Figure 5] This shows the detection of advanced apoptosis levels in tumor cells induced by CART-BCMA. Specifically, 1 × 10⁴ CFSE-labeled BCMA-negative (NH929) or BCMA-positive (NH929-BCMA) tumor cell lines were co-cultured with corresponding T cells in 100 μl of GT-551 medium for 4 hours at the ratios shown in the figure. The proportion of PI-positive cells among CFSE-positive cells was analyzed by flow cytometry after staining with 100 μl of 25% PI dye for 15 minutes. The figure shows the statistical analysis of PI-positive cells in the corresponding co-culture samples. [Figure 6A]This shows the detection of advanced apoptosis levels in tumor cells induced by CART-BCMA. Figure 6A shows the ratio of CART-positive cells in the analyzed samples, with NT and BCMA-MO6 calculated at 60%. 1 × 10⁴ CFSE-labeled BCMA-negative (NH929) or BCMA-positive (NH929-BCMA, MM.1S) tumor cell lines were co-cultured with corresponding T cells in 100 μl of GT-551 medium for 4 hours at the ratios shown in the figure. The proportion of PI-positive cells among CFSE-positive cells was analyzed by flow cytometry after staining with 100 μl of 25% PI dye for 15 minutes. [Figure 6B] This shows the detection of advanced apoptosis levels in tumor cells induced by CART-BCMA. Figure 6B shows a statistical analysis of PI-positive cells in the corresponding co-culture samples. [Figure 7A] This study demonstrates the inhibitory effect of CART-BCMA on the in vivo proliferation of the myeloma cell line RPMI-8226 in B-NDG mice. RPMI-8226 cells were collected during the logarithmic growth phase, and 4.0 × 10⁶ tumor cells were inoculated subcutaneously into the right dorsal region of mice. When the tumor volume reached approximately 120 mm³, the animals were randomly divided into four groups according to tumor volume. Subsequently, 7.5 × 10⁶ NT (T cells only) and 7.5 × 10⁶ CART-BCMA cells, which served as solvent controls, were injected via the tail vein. Figure 7A shows that single injections of CART-BCMA-1 and CART-BCMA-20 via the tail vein effectively inhibit the growth of human myeloma RPMI-8226 cells. [Figure 7B] Figure 7B shows the inhibitory effect of CART-BCMA on the in vivo proliferation of the myeloma cell line RPMI-8226 in B-NDG mice. CART-BCMA-1 and CART-BCMA-20 can significantly extend the survival of tumor-bearing mice carrying human myeloma RPMI-8226 cells. [Figure 8] The results of BCMA, CR2, CXADR, DDR2, and MAG expression in 293T cells after transfection with related plasmids, as determined by flow cytometry, are shown. [Figure 9A] The cytokine detection results are shown. [Figure 9B] The cytokine detection results are shown. [Figure 9C] The cytokine detection results are shown. [Figure 10] This chart shows the cell-killing ability of each cell type against target cells. Triangle: BCMA-20 (C-CAR088); Square: Positive control; Circle: NT control (untransfected (NT) T cells). [Figure 11] This demonstrates the effect of soluble BCMA on cell death activity. [Figure 12] This shows the survival rate of mice in each group. [Figure 13] This shows the experimental process of the Phase I clinical study. [Figure 14] This shows the clinical response from a Phase I clinical study. [Figure 15] This shows the treatment status of patient ID Z0203-00801 C008. [Figure 16A] This shows the treatment status of patient ID Z0203-00701C001. [Figure 16B] This shows the treatment status of patient ID Z0203-00701C001. [Figure 17] This shows an increase in C-CAR088 levels and a decrease in M ​​protein / sFLC levels in the blood. [Figure 18A] The results of evaluating the binding specificity of scFv of C-CAR088 are shown. Figure 18A shows the experimental scheme. [Figure 18B] The results of evaluating the binding specificity of scFv to C-CAR088 are shown. Figure 18B shows the structures of anti-BCMA CAR and scFv rabFc. [Figure 18C] The results of evaluating the binding specificity of scFv to C-CAR088 are shown. Figure 18C shows the experimental results. [Figure 19A] This document presents research and validation of tissue-crossreactivity IHC GLP. HAdCC (human primary adrenal cortical cells), HPTEC (human primary thyroid epithelial cells), and A549 BCMA OE (a stable strain overexpressing BCMA) are used. [Figure 19B]This document presents research and validation of tissue-crossreactivity IHC GLP. HAdCC (human primary adrenal cortical cells), HPTEC (human primary thyroid epithelial cells), and A549 BCMA OE (a stable strain overexpressing BCMA) are used. [Figure 20] An example of the CAR (C-CAR088) manufacturing process is shown. This process involves the use of serum-free media, a closed, modular, integrated semi-automated system, and digital monitoring. An asterisk indicates an improved process. The median intervening time is approximately 17 days (range: 13–83 days). The median manufacturing time is approximately 7 days (range: 5–10 days). [Figure 21] This figure shows the Phase I clinical study design for C-CAR088 (in the treatment of relapsed or refractory multiple myeloma). A Phase I open-label dose-escalation and expansion study was conducted at four medical centers. C-CAR088 is one embodiment of this CAR based on the BCMA-20 antibody. [Figure 22] The C-CAR088 clinical response includes SD, MR, PR, CR, VGPR, MRD, and PD. SD: stable disease; PR: partial response; CR: complete response; PD: progressive disease; MR: minimal response; VGPR: very good partial response; MRD: minimal residual disease. [Figure 23] This study demonstrates C-CAR088 clinical responses, including complete response (CR / sCR), gross vegetative response (VGPR), and partial response (PR). [Figure 24] The Kaplan-Meier estimates of progression-free survival (PFS) for the medium-dose and high-dose groups are shown. [Figure 25A] This shows an increase in C-CAR088 in the blood of patients after CAR administration. [Figure 25B] This shows an increase in C-CAR088 levels in the patient's blood up to their most recent visit. [Figure 25C] This shows the Cmax level in the blood of patients after CAR administration. [Figure 25D] This shows the AUC level in the patient's blood after CAR administration. [Figure 25E] This shows the Tmax level in the blood of patients after CAR administration. [Figure 25F] This shows the Tlast level in the blood of patients after CAR administration. [Modes for carrying out the invention]

[0105] This disclosure provides chimeric antigen receptors (CARs) that target BCMA. In certain embodiments, the CARs are based on four monoclonal antibodies: BCMA-1, BCMA-20, BCMA-CA8, and BCMA-MO6. This disclosure also provides analysis and identification of CAR expression levels in primary T cells, the in vitro activating ability of these chimeric antigen receptors, and tumor cell killing efficacy. Studies have shown that the chimeric antigen receptors of this disclosure target BCMA-positive cells and can be used to treat BCMA-positive B-cell lymphoma, multiple myeloma, plasma cell leukemia, or other diseases.

[0106] Specifically, this disclosure identifies the correlation between the expression time and expression intensity of different CAR structures on the cell membrane surface after viral infection, and further identifies the differences in the expression of different CAR structure proteins. These findings suggest that different CAR structures differ in the expression levels of CAR proteins on the membrane surface and the persistence of CART in vivo activity under the same infection conditions. The CAR structures of this disclosure were obtained after extensive screening. The results demonstrate that the proteins encoded by the CAR structures of this disclosure can be fully expressed and membrane-localized.

[0107] This disclosure describes an improved process for preparing CAR-modified T cells targeting BCMA antigens. In one embodiment, lymphocytes are cultured in vitro using GT-551 serum-free medium supplemented with 1% human albumin.

[0108] term The term "approximately" can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, which depends in part on how the value or composition is measured or determined. In relation to a numerical value, "approximately" can refer to ±10% of the stated value. In other words, the value can be within the range of 90% to 110% of the stated value.

[0109] The term “administer / dosage” refers to the physical delivery of the Products of this Disclosure to a subject using any one of the various methods and delivery systems known to those skilled in the art, including, but not limited to, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration by injection or infusion.

[0110] The term “antibody” (Ab) may include, but is not limited to, an immunoglobulin or its antigen-binding moiety, which specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) scattered within more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0111] Chimeric antigen receptor (CAR) The chimeric antigen receptors (CARs) of this disclosure may comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also known as an antigen-binding domain). The intracellular domain comprises a costimulatory signaling region and a ζ chain. The costimulatory signaling region refers to a portion of the intracellular domain containing a costimulatory molecule. The costimulatory molecule is not the antigen receptor or its ligand, but a cell surface molecule necessary for the efficient response of lymphocytes to the antigen.

[0112] Linkers can be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that plays a role in linking the transmembrane domain to the extracellular domain or cytoplasmic domain in a polypeptide chain. Linkers may contain 0 to 300 amino acids, 2 to 100 amino acids, or 3 to 50 amino acids.

[0113] In one embodiment, the extracellular domain of the CAR includes an antigen-binding domain that targets BCMA. When the CAR of this disclosure is expressed in T cells, antigen recognition can be performed based on antigen-binding specificity. When the CAR binds to its associated antigen, the CAR affects the tumor cell, preventing it from growing, leading to death, or affecting it in other ways, thereby reducing or eliminating the tumor burden in the patient. The antigen-binding domain may be fused to the intracellular domain from one or more costimulatory molecules and a ζ chain. The antigen-binding domain may be fused to the intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0114] As used herein, “antigen-binding domain” and “single-chain antibody fragment” may refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment having antigen-binding activity. An Fv antibody includes a heavy-chain variable region and a light-chain variable region, but does not include a constant region. An Fv antibody has the smallest antibody fragment containing all antigen-binding sites. Generally, an Fv antibody also includes a polypeptide linker between the VH and VL domains to form the structure necessary for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). The size of an scFv is typically 1 / 6 that of a complete antibody. A single-chain antibody may be an amino acid chain sequence encoded by a nucleotide chain. In certain embodiments, the scFv may include an antibody that specifically recognizes the extracellular region of BCMA, for example, amino acid residues between positions 24 and 41 of the BCMA sequence. The antibody may also be a single-chain antibody.

[0115] With respect to the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains within the CAR is used. In some embodiments, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0116] The intracellular domain of CAR includes the 4-1BB signaling domain and the CD3ζ signaling domain.

[0117] In certain embodiments, the CAR structure of this disclosure comprises a signal peptide, an antigen recognition sequence (antigen-binding domain), a linker region, a transmembrane region, a costimulator signaling region, and a CD3 zeta signaling region (ζ chain portion). The linking sequence is as follows: [CD8 S]-[VL-Linker-VH]-[Hinge-CD8TM]-[4-1BB]-[CD3 Zeta] In a particular embodiment, the sequences selected in this disclosure are as follows: (1) The signal peptide is a signal peptide sequence derived from CD8: MALPVTALLLPLALLLHAARP (Sequence ID 9) (2) Light chain (VL) sequence of the single-strand variable region derived from BCMA-1 antibody: DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGS GSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK (Sequence ID 7) (3) Heavy chain (VH) sequence of the single-strand variable region derived from BCMA-1 antibody: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS(Sequence ID 8) Of these, BCMA-1 is an antibody sequence included in publicly available Car-T sequences and is used as a control in this application.

[0118] (4) Light chain of the single-strand variable region derived from BCMA-20 antibody (V L )array: DIQMTQSPSSLSASVGDRVTITC RASQGISNYLN WYQQKPGKAPKPLIY YTSNLQS GVPSRFSGSGSGT DYTLTISSLQPEDFATYYC MGQTISSYT FGQGTKLEI(Sequence ID 1) [Table 1]

[0119] (5) Heavy chain (VH) sequence of the single-strand variable region derived from BCMA-20 antibody: EVQLVESGGGLVQPGGSLRLSCAASGFTFS NFDMA WVRQAPGKGLVWVS SITTGADHAIYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCVR HGYYDGYHLFDY WGQGTLVTVSS(Sequence ID 2) [Table 2]

[0120] (6) Light chain (VL) sequence of the single-stranded variable region derived from the BCMA-CA8 antibody: DIQLTQTTSSLSASLGDRVTISC SASTTTSNYLN WYQQKPDGTVELVIY YTSNLHG GGPSRFSGSGTGTDYSLTIGYLEPEDVATYYC QQYRKLPWT FGGGSKLEIKR (Sequence ID 3) [Table 3]

[0121] (7) Heavy chain (VH) sequence of the single-strand variable region derived from the BCMA-CA8 antibody: EVQLQQSGAVLARPGASVKMSCKGSGYTFT NYWMH WVKQRPGQGLEWIG ATYRGHSDTYYNQKFKG KAKLTAVTSTSSTAYMELSSLTNEDSA VYYCTR GAIYNGYDVLDN WGQGTLVTVSS(Sequence ID 4) [Table 4]

[0122] (8) Light chain (VL) sequence of the single-stranded variable region derived from the BCMA-MO6 antibody: DIQMTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKLLIY YTSNLHS GVPSRFSGSGSGTDFLTISSLQPEDFATYYC QQYRKLPWT FGQGTKLEIKR(Sequence ID 5) [Table 5]

[0123] (9) Heavy chain (VH) sequence of the single-strand variable region derived from BCMA-MO6 antibody: QVQLVQSGAEVKKPGSSVKVSCKASGGTFS NYWMH WVRQAPGQGLEWMG ATYRGHSDTYYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR GAIYDGYDVLDN WGQGTLVTVSS (Sequence ID 6) [Table 6] (10) The linker sequence between the heavy and light chains in the BCMA-1 single-strand variable region is as follows: GSTSGSGKPGSGEGSTKG (Sequence No. 10) (11) The linker sequences between the heavy and light chains in the single-stranded variable regions of BCMA-20, BCMA-CA8, and BCMA-MO6 are as follows: GGGGSGGGGSGGGGS (Sequence No. 11) (12) Arrangement of hinge region and linker region: FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 12) (13) The transmembrane region is the transmembrane region sequence of the CD8 (CD8™) antigen: IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 13) (14) The co-stimulatory factor signaling region is derived from the sequence of the 4-1BB cytoplasmic signaling motif: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 14) (15) The signaling region of CD3 zeta originates from the sequence of the immune receptor tyrosine activation motif (ITAM) of CD3 zeta in the TCR complex: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 15) In certain embodiments, the nucleic acid encoding the CAR (derived from the BCMA-20 antibody) may have the following sequence (SEQ ID NO: 58): In certain embodiments, the CAR (derived from the BCMA-20 antibody) may have the following amino acid sequence (SEQ ID NO: 59): MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPPKLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCMGQTISSYTFGQGTK LEIKGGGGSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQAPGKGLVWVSSITTGADHAIYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRHGYYDGY HLFDYWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR In certain embodiments, the nucleic acid encoding the CAR (derived from the BCMA-CA8 antibody) may have the following sequence (SEQ ID NO: 60): In certain embodiments, CAR (derived from BCMA-CA8 antibody) may have the following amino acid sequence (SEQ ID NO: 61): MALPVTALLLPLALLLHAARPDIQLTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVELVIYYTSNLHSGVPSRFSGSGSGTDYSLTIGYLEPEDVATYYCQQYRKLPWTFGGGSK LEIKRGGGGSGGGGSGGGGSEVQLQQSGAVLARPGASVKMSCKGSGYTFTNYWMHWVKQRPGQGLEWIGATYRGHSDTYYNQKFKGKAKLTAVTSTSTAYMELSSLTNEDSAVYYCTRGAIYNGY DVLDNWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR In certain embodiments, the nucleic acid encoding the CAR (derived from the BCMA-MO6 antibody) may have the following sequence (SEQ ID NO: 62): In certain embodiments, CAR (derived from BCMA-BCMA-MO6 antibody) may have the following amino acid sequence (SEQ ID NO: 63): MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTK LEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGY DVLDNWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Chimeric antigen receptor T cells (CAR-T cells) As used herein, the terms “CAR-T cells,” “CAR-T,” and “CART” may be used interchangeably.

[0124] This disclosure relates to the construction of a chimeric antigen receptor structure targeting BCMA, a method for preparing chimeric antigen receptor-modified T cells targeting BCMA, and the identification of their activity.

[0125] vector Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from cells expressing the gene, by inducing the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be synthesized.

[0126] This disclosure also provides vectors into which the expression cassette of this disclosure is inserted. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term and stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have advantages over vectors derived from onchoretroviruses, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

[0127] In summary, an expression cassette or nucleic acid sequence is typically operably ligated to a promoter and incorporated into an expression vector. The vector may be suitable for replication and incorporation in eukaryotes. A typical cloning vector includes transcriptional and translational terminators, an initiation sequence, and a promoter useful for regulating the expression of the desired nucleic acid sequence.

[0128] The expression constructs disclosed herein may also be used for nucleic acid immunotherapy and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. For example, they are described in U.S. Patents 5,399,346, 5,580,859 and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the disclosure provides gene therapy vectors.

[0129] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors for specific purposes include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0130] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), as well as in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes a functional origin of replication in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., International Publication No. 01 / 96584; International Publication No. 01 / 29058; and U.S. Patent Application Publication No. 6,326,193).

[0131] Several virus-based systems have been developed for gene delivery into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Numerous retroviral systems are known in the art. Adenovirus vectors are used in some embodiments. Numerous retroviral vectors are known in the art. Lentiviral vectors are used in one embodiment.

[0132] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to contain functional elements downstream of the initiation site as well. The spacing between promoter elements is often flexible, resulting in conserved promoter function if the elements are reversed or moved relative to one another. In thymidine kinase (TK) promoters, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0133] One example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, either a constitutive or inductive promoter can be used. The use of an inductive promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably ligated to it when such expression is desired, or turn off its expression when it is not desired. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0134] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either or both a selection marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selection marker may be supported on a separate DNA fragment and used in a co-transfection procedure. Both the selection marker and the reporter gene can be positioned adjacent to appropriate regulatory sequences to enable expression in host cells. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0135] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is revealed by some readily detectable characteristic, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include those encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with a minimum 5' flanking region exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated to the reporter gene and used to evaluate the action of the agent for its ability to regulate promoter-driven transcription.

[0136] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0137] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a method for introducing polynucleotides into host cells.

[0138] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0139] Chemical means for introducing polynucleotides into host cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0140] When nonviral delivery systems are used, an exemplary delivery vehicle is a liposome. Lipid formulations are intended for the delivery of nucleic acids to host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, encapsulated within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as bilayer structures, micelles, or “disintegrated” structures. They may also simply be dispersed in solution, or in some cases form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0141] In one embodiment, the vector is a lentiviral vector.

[0142] composition This disclosure provides compositions comprising immune cells (e.g., CAR-T cells) and pharmaceutically acceptable carriers, diluents and / or excipients. In one embodiment, the composition is a liquid composition. For example, the composition is an injectable composition. In a particular embodiment, the concentration of CAR-T cells in the composition is 1 × 10⁻⁶. 3 ~1 × 10 8 cells / ml or 1 × 10⁶ 4 ~1 × 10 7 The value is cells / ml.

[0143] In one embodiment, the composition may include a buffer such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition may be formulated for intravenous administration.

[0144] therapeutic use This disclosure includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this disclosure. Transduced T cells can target the tumor cell marker BCMA, synergistically activate T cells, trigger a T cell immune response, and thereby significantly increase the efficiency of tumor cell death.

[0145] Accordingly, the Disclosure also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, the method comprising the step of administering the CAR-T cells of the Disclosure to the mammal.

[0146] In one embodiment, the disclosure includes a class of cell therapies in which T cells are isolated from an autologous patient (or xenodontic donor), activated, genetically modified to produce CAR-T cells, and then injected into the same patient. The likelihood of such graft-versus-host disease is extremely low, and antigens are recognized by T cells in an MHC-independent manner. Furthermore, a single CAR-T can treat all cancers that express the antigen. Unlike antibody therapies, CAR-T cells can replicate in vivo, resulting in long-lasting persistence that may lead to sustained tumor control.

[0147] In one embodiment, the CAR-T cells of this disclosure can undergo robust in vivo T cell proliferation and can be sustained for a long period. Furthermore, CAR-mediated immune responses may be part of adoptive immunotherapy approaches in which CAR-modified T cells induce an immune response specific to the antigen-binding portion in the CAR. For example, anti-BCMA CAR-T cells induce an immune response specific to cells expressing BCMA.

[0148] The data disclosed herein specifically disclose a lentiviral vector comprising BCMA scFv, hinge and transmembrane domains, and 4-1BB and CD3ζ signaling domains; however, this disclosure should be interpreted as including any number of variations for each component of the construct, as described elsewhere in this specification.

[0149] Cancers that can be treated include non-angiogenic or mostly non-angiogenic tumors, and angiogenic tumors. Cancers may include non-solid tumors (hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. Types of cancer treated with CAR include, but are not limited to, carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, e.g., sarcomas, carcinomas and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0150] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, as well as myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), leukemia including chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (slowly progressive and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal cord dysplasia.

[0151] Solid tumors are typically abnormal masses of tissue that do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0152] CAR-modified T cells may also serve as a type of vaccine for ex vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0153] Regarding ex vivo immunization, at least one of the following is performed in vitro before administering the cells to a mammal: i) augmenting the cells, ii) introducing CAR-encoding nucleic acids into the cells, and / or iii) cryopreserving the cells.

[0154] Ex vivo procedures are well known in the art and will be discussed in more detail below. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient may be human, and the CAR-modified cells may be self to the recipient. Alternatively, the cells may be homogeneous, syngeneic, or heterogeneous to the recipient.

[0155] In addition to using cell-based vaccines with respect to ex vivo immunization, this disclosure also provides compositions and methods for in vivo immunization to induce an immune response to an antigen in a patient.

[0156] This disclosure provides a method for treating a tumor, comprising administering a therapeutically effective dose of CAR-modified T cells to a subject in need of tumor treatment.

[0157] The CAR-modified T cells of this disclosure may be administered alone or as part of a pharmaceutical composition in combination with diluents and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of this disclosure may include, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, the target cell populations described herein. Such compositions may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for intravenous administration.

[0158] The pharmaceutical compositions of this disclosure may be administered in a manner appropriate to the disease being treated (or prevented). The dosage 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 the appropriate dosage may be determined by clinical trials.

[0159] Where “immunologically effective dose,” “antitumor effective dose,” “tumor inhibitory effective dose,” or “therapeutic dose” is indicated, the exact amount of the composition of this disclosure to be administered may be determined by a physician taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The T cell-containing pharmaceutical compositions described herein include all integer values ​​within their ranges, up to 10 4 cells / kg body weight ~10 9 cells / kg body weight, or 10 5 cells / kg body weight ~10 6It can generally be stated that T cell compositions may be administered in doses of cells / kg body weight. These doses may also be administered multiple times. Cells can be administered using infusion techniques commonly known in immunotherapy (e.g., Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment plan for a particular patient can be readily determined by those skilled in the art of medicine by monitoring the patient for signs of the disease and adjusting the treatment accordingly.

[0160] The subject composition may be administered in any convenient manner, including aerosol inhalation, injection, oral ingestion, blood transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodulely, intramedullarily, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell composition of the Disclosure is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the Disclosure may be administered by intravenous injection. The T cell composition may be injected directly into a tumor, lymph node, or site of infection.

[0161] In certain embodiments of this disclosure, cells activated and enlarged using the methods described herein, or other methods known in the art in which T cells are enlarged to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously, or after) any number of relevant modes of treatment, including, but not limited to, antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab therapy for MS patients, ephalizumab therapy for psoriasis patients, or other therapies for PML patients. In further embodiments, the T cells of this disclosure may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of this disclosure is administered to a patient in conjunction with (e.g., before, simultaneously, or after) bone marrow transplantation or the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject may receive standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In a specific embodiment, after transplantation, the subject receives an injection of the enlarged immune cells of this disclosure. In a further embodiment, the enlarged cells are administered before or after surgery.

[0162] The dosage of the above treatment administered to a patient varies depending on the condition being treated and the exact characteristics of the treatment recipient. Dosage scaling for patient administration can be carried out according to established practices in the art. Generally, 1 × 10⁻⁶ 6 pieces~1×10 10 Individual modified T cells of this disclosure (e.g., CAR-T-BCMA cells) can be applied to a patient, for example, by intravenous infusion or by each treatment or treatment process.

[0163] The main benefits of this disclosure include: (a) With respect to the chimeric antigen receptor of this disclosure, the extracellular antigen-binding domain is specific anti-BCMA scFv, and the CAR exhibits great ability to kill tumor cells with low cytotoxicity and low side effects.

[0164] (b) The chimeric antigen receptors provided herein can achieve stable expression and membrane localization of the CAR protein after T cells are infected with a viral vector (e.g., a lentivirus) that carries the CAR gene.

[0165] (c) The CAR-modified T cells of this disclosure have a longer survival time and stronger antitumor effect in vivo. The scFv used in this disclosure may be a humanized antibody or a human-derived antibody and is less likely to cause specific immune rejection.

[0166] The present disclosure will be further illustrated below with specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following examples, where specific conditions are not explicitly stated, are carried out under normal conditions, for example, as described in Sambrook et al., in Molecule Clone: ​​A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturer. Percentages and parts are by weight unless otherwise specified.

[0167] [Example 1] Construction of lentiviral expression vectors Full-length DNA synthesis and cloning were commissioned to Shanghai Boyi Biotechnology Co., Ltd. to achieve the construction of the encoding plasmid. The pWPT lentiviral vector was selected as the cloning vector, and the cloning sites were the BamH I and Sal I sites. The specific sequences are as shown above.

[0168] [Example 2] Preparation of CAR-T cells (1) Mononuclear cells (PBMCs) were isolated from the venous blood of healthy individuals by density gradient centrifugation.

[0169] (2) On day 0, PBMCs were seeded into cell culture flasks pre-coated with a final concentration of 5 μg / mL of CD3 monoclonal antibody (OKT3) and a final concentration of 10 μg / mL of retronectin (purchased from TAKARA). The culture medium was GT-551 cell culture medium containing 1% human albumin. Recombinant human interleukin-2 (IL-2) was added to the medium at a final concentration of 1000 U / mL. The cells were cultured in a CO2 incubator at 37°C and 5% saturated humidity.

[0170] (3) On day 1, the supernatant of the cultured PBMCs was slowly aspirated and discarded. A new GT-551 cell culture medium containing 1% human albumin was added, and recombinant human interleukin-2 (IL-2) was added to the medium at a final concentration of 1000 U / mL. The cells were continuously cultured in a CO2 incubator at 37°C and 5% saturated humidity.

[0171] (4) On the third day, fresh culture medium, concentrated and purified CAR-BCMA lentivirus solution, protamine sulfate (12 ug / ml), and IL-2 (final concentration of 1000 U / mL) were added. After incubating for 12 hours in a 5% CO2 incubator at 37°C, the medium was discarded, fresh medium was added, and cultivation was continued in a 5% CO2 incubator at 37°C.

[0172] (5) Starting on day 6, CAR-BCMA cells can be collected for the corresponding activity assay.

[0173] [Example 3] Detection of CAR gene integration rates into the T cell genome and the expression levels of their coding proteins on the membrane surface. The CART-BCMA cell samples cultured on day 7 (Figure 3A), day 21 (Figure 3B), and day 29 (Figure 3C) of Example 2 were each measured at 0.5 × 10⁶. 6 Individual cells were collected. The expression levels of CAR-BCMA proteins on the surface of the T cell membrane were analyzed by flow cytometry after Fc fragment staining with recombinant human BCMA protein.

[0174] The results are shown in Figures 3A, 3B, and 3C, and the four CAR structures designed in this disclosure can be expressed in their corresponding modified T cells and can complete cell membrane surface localization.

[0175] [Example 4] Detection of in vitro activation ability of CAR-BCMA The cell activation level indicator proteins CD137 and IFNγ were detected using CART-BCMA cells cultured on day 7 in Example 2. 5 CART-BCMA cells were cultured in 200 μl of GT-551 medium for 18 hours in a 1:1 ratio with BCMA-positive K562-BCMA+E7 tumor cell lines and BCMA-negative K562 tumor cell lines, or without tumor cells. Subsequently, the expression level of CD137 on the surface of the T cell membrane was detected by flow cytometry, and the secretion level of IFNγ in the culture supernatant was detected by ELISA.

[0176] The results are shown in Figures 4A and 4B, where CD137 expression was detected on the surface of four CART cells, and IFNγ expression was detected in the culture supernatant. Among these, CAR-BCMA-20 showed the best levels of CD137 activation and IFNγ release. CART-BCMA-MO6, constructed based on a humanized MO6 antibody sequence, showed lower levels of CD137 activation but higher levels of IFNγ release than CART-BCMA-CA8, constructed based on a mouse antibody sequence.

[0177] Furthermore, C-CAR088 induced higher levels of IFN-γ release (Figure 4B) and CD137 expression (Figure 4A) in BCMA-positive tumor cells compared to CARs based on BCMA-1, BCMA-CA8, and BCMA-MO6.

[0178] CART-BCMA-20 induced apoptosis in larger BCMA-positive tumor cells than CART-BCMA-1 (positive control), CART-BCMA-MO6, and CART-BCMA-CA8 (Figure 5).

[0179] [Example 5] Detection of advanced apoptotic activity in tumor cells induced by CART-BCMA (1) CART-BCMA cells cultured on day 17 in Example 2 were divided into 1 × 10 4 Individual CFSE-labeled BCMA-negative cells (NH929) or BCMA-positive autoconstructive cells (NH929-BCMA overexpressing tumor cell line) were mixed with CFSE-labeled BCMA-negative cells (NH929) in ratios of 1:1, 2.5:1, 5:1, 10:1, and 20:1 (shown in Figure 5). The mixed cells were co-cultured in 100 μl of GT-551 medium for 4 hours, and then stained with 100 μl of 25% PI dye for 15 minutes. The proportion of PI-positive cells among CFSE-positive cells was analyzed by flow cytometry.

[0180] (2) CART-BCMA cells cultured on day 22 in Example 2 were divided into 1 × 10⁶ cells. 4 CFSE-labeled BCMA-negative cells (NH929), BCMA-positive autoconstructive cells (NH929-BCMA overexpressing tumor cell line), or MM.1S cell line that naturally expresses BCMA were mixed in ratios of 1:1, 5:1, 10:1, 10:1, and 40:1 (as shown in Figure 6B). The mixed cells were co-cultured in 100 μl of GT-551 medium for 4 hours, and then stained with 100 μl of 25% PI dye for 15 minutes. The proportion of PI-positive cells among CFSE-positive cells was analyzed by flow cytometry.

[0181] The results are shown in Figure 5, and Figures 6A and 6B. All four CART cells can induce apoptosis in BCMA-positive tumor cells. Among these, CART-BCMA-20 is better able to induce advanced apoptosis in BCMA-positive tumor cells than CART-BCMA-1. CART-BCMA-MO6 and CART-BCMA-CA8 have similar abilities to induce advanced apoptosis in BCMA-positive tumor cells.

[0182] [Example 6] Inhibition of CART-BCMA in the RPMI-8226 myeloma xenograft model RPMI-8226 cells were collected during the logarithmic growth phase and implanted in the subcutaneous tissue of the right dorsal region of 6-8 week old B-NDG mice (4.0 × 10⁴ cells). 6 The tumor cells were seeded. The tumor volume was approximately 120 mm². 3 Upon reaching this point, the animals were randomly divided into four groups according to tumor volume, with the difference in tumor volume between each group being less than 10% of the mean. Next, a solvent control of 7.5 × 10⁻⁶ was used. 6 Individual NTs and 7.5 × 10 6 Each CART-BCMA cell was injected via the tail vein.

[0183] The results are shown in Figures 7A and 7B. Compared to the control group, a single injection of CART-BCMA-1 and CART-BCMA-20 via the tail vein effectively inhibited the growth of human myeloma RPMI-8226 cells (relative tumor growth rate %T / CRTV ≤ 40%, P < 0.05) and significantly extended the survival time of human myeloma-bearing mice (median survival time was 23 days in the control group and over 33 days in the CART-BCMA-treated group). There were no significant differences in tumor growth rate or median survival time between mice treated with CART-BCMA-1 and CART-BCMA-20.

[0184] For in vivo studies, human myeloma RPMI-8226 cells were xenotransplanted into B-NDG mice. The tumor volume was approximately 120 mm². 3 Once the target was reached, solvent control, non-transfected (NT) T cells (negative control), or CART-BCMA cells were injected via the tail vein of the mice.

[0185] Compared to the control group, single injections of CART-BCMA-1 and CART-BCMA-20 effectively inhibited myeloma cell growth and significantly extended the survival time of human myeloma-bearing mice: the median survival time in the CART-BCMA-treated group was over 33 days, compared to 23 days in the control group. There were no significant differences in tumor growth rate or median survival time between mice treated with CART-BCMA-1 and those treated with CART-BCMA-20 (Figures 7A and 7B).

[0186] The results suggest that BCMA-20 (C-CAR088) has high in vivo antitumor efficacy.

[0187] Comparative Example In the screening process for chimeric antigen receptors described in this application, the inventors tested numerous candidate sequences, which are shown below with examples.

[0188] The antibodies screened include BCMA-1, BCMA-2, BCMA-69, BCMA-72, BCMA-2A1, BCMA-1E1, BCMA-J22.9, BCMA-20, BCMA-CA8, and BCMA-MO6. Chimeric antigen receptor structures targeting BCMA were constructed based on the above antibodies. Among these, BCMA-1 and BCMA-2 are publicly available Car-T sequences and were used as positive controls for screening. CAR-T cells were prepared in the same manner as in Example 2 and detected in the same manner as in Examples 3 and 4.

[0189] The results are shown in Figures 1A, 1B, and 1C, which represent the experimental results for two batches of CAR-T cells. Car-T expression was detected using BCMA-Fc fusion protein. As seen in Figure 1A, high expression is observed in primary T cells. In Figure 1B, it can be seen that BCMA-1, BCMA-20, BCMA-1E1, BCMA-CA8, BCMA-MO6, and BCMA-J22.9 can be activated by BCMA antigens. Figure 1C shows that activated BCMA-1, BCMA-20, BCMA-1E1, BCMA-CA8, BCMA-MO6, and BCMA-J22.9 CAR-Ts can produce higher levels of IFN-γ. The results indicate that BCMA-20, CA8, and MO6 CAR-Ts were further analyzed and studied because their CAR-T functions were similar.

[0190] [Example 7] Membrane protein array experiment Using the light chain variable region of SEQ ID NO: 1 and the heavy chain variable region of SEQ ID NO: 2, a single-chain antibody B20-scFv-rabFc was prepared and a membrane protein array experiment was conducted.

[0191] 20 ug / mL of B20-scFv-rabFc was added to HEK293 T cell arrays transiently transfected with 5344 membrane proteins each. Flow cytometry found that under this test condition, B20-scFv-rabFc could cross-recognize TNFRSF17 (Q02223), MAG (P20916), CR2 (P20023), CXADR (P78310) and DDR2 (Q16832). Here, TNFRSF17, that is, BCMA is the specific target of B20-scFv, and MAG, CR2, CXADR and DDR2 are suspected of being non-specific targets.

[0192] To further confirm whether the suspected targets can cause the activation of CAR-T, CBM.BCMA CAR-T was co-cultured with 293T cells transfected with BCMA, CR2, CXADR, DDR2 and MAG respectively. IFNγ, TNF, IL-2 and other cytokines in the co-culture supernatant were detected. 293T cells transfected with the empty vector were used as the negative control, and 293T cells transfected with BCMA were used as the positive control.

[0193] Figure 8 shows the results of the expression of BCMA, CR2, CXADR, DDR2 and MAG in 293T cells after transfection of the related plasmids by flow cytometry.

[0194] The cytokine detection results are shown in Figures 9A, 9B and 9C. Only BCMA expressed on 293T cells can induce CBM. BCMA CAR-T cells produce a large amount of IFNγ / TNF / IL-2, but the other four non-specific surface markers cannot activate CBM. BCMA CAR-T cells. Figures 8 and 9A - 9C show the whole - genome membrane proteome array and verification. TNFRSF17, MAG, CR2, CXADR, and DDR2 were identified with moderate binding to B - 20 scFv rabFc at a concentration of 20.0 μg / mL. Only cells expressing TNFRSF17 (BCMA) can induce BCMA - CAR - T cells that produce a number of cytokines (IFNγ / TNF / IL - 2).

[0195] Specific membrane staining was observed at a concentration of 20.0 μg / mL in human lymphocytes in thymus, spleen, lymph nodes, bone marrow, and scattered lymphocytes in thyroid and adrenal glands (Table 1 and Figures 19A - 19B). [Table 7]

[0196] [Example 8] In vitro antitumor activity of C - CAR088 cells The chimeric antigen receptor BCMA - 20 (hereinafter referred to as C - CAR088) was selected for subsequent experiments. C - CAR088 cells, NT cells (non - transfected T cells, used as negative control), and positive control cells (Bluebird bb2121) were co - cultured with BCMA - negative cells (NH929) or BCMA - positive cells (NH929 - BCMA) at different effector - target ratios. The killing ability of each cell against the target cells was analyzed.

[0197] The results are shown in Figure 10. C - CAR088 cells and positive control cells only have a strong killing effect on NH929 - BCMA cells and have no significant killing effect on the negative target cells NH929. The non - transfected group (NT) also has no significant killing effect on the target cells.

[0198] T cells containing C-CAR088, non-transfected (NT) T cells (negative control), and positive control cells (Bluebird bb2121) were co-cultured with BCMA-negative cells (NH929) or BCMA-positive cells (NH929-BCMA) at different effect / target ratios. Cytotoxicity was then assayed.

[0199] Figure 10 shows that C-CAR088 T cells and positive controls exhibited strong cytotoxicity against NH929-BCMA cells, rather than NH929 cells.

[0200] [Example 9] Effect of soluble BCMA on cell death activity In a co-culture system of C-CAR088 cells with BCMA-negative target cells A549 and BCMA-positive target cells A549-BCMA-2E9, the effects of adding 100 ng / ml and 500 ng / ml of soluble BCMA protein, respectively, on CD137 expression were detected.

[0201] The results are shown in Figure 11. Soluble BCMA protein did not affect the upregulation of CD137 expression, indicating that soluble BCMA does not block the specific recognition of CAR and target antigens. In in vitro cell death activity tests of IFN-γ release and ELISA, the cell death effect of C-CAR088 cells against target cells and IFN-γ release reduced soluble BCMA protein by 500 ng / ml, but the difference was not statistically significant. The above results indicate that the cell death activity of C-CAR088 cells is not fundamentally affected by soluble BCMA.

[0202] [Example 10] Dose-dependent effects of C-CAR088 cells Six-week-old B-NDG mice (half male and half female) were selected and 2.5 × 10⁶ mice were used. 6 Human multiple myeloma cells RPMI-8226 were injected intraperitoneally. Mice with similar tumor burdens were selected and divided into five groups, each receiving 2.5 × 10⁶ doses. 6 Individual C-CAR O88 cells (low-dose group), 5 × 10⁶ 6C-CAR088 cells (medium dose group), 1×10 7 C-CAR088 cells (high dose group), T cells and vehicle (a cryoprotectant (CBMG C-CFMC) was injected as the vehicle). The experiment was continued for 54 days. During the experiment, the tumor burden of the mice was evaluated every 5 days. At the end of the experiment, the survival rate of the mice was calculated.

[0203] The results are shown in Figure 12. 5×10 6 cells / mouse and 1×10 7 cells / mouse of a single administration of the C-CAR088 cell dose can effectively inhibit the growth of B-NDG mouse xenograft tumors of human myeloma RPMI-8226 cells. The relative tumor growth rates were 6.12% and 0.75%, respectively (p<0.05). At the end of the experiment, the survival rate of the tumor-bearing mice in the medium dose group was 91.7% (11 / 12). There was no death in the tumor-bearing mice in the high dose group, and the survival rate was 100.0% (12 / 12), showing a significant difference (p<0.05) compared with the vehicle group (survival rate 58.3%). The above results indicate that C-CAR088 exhibits dose-dependent in vivo antitumor activity.

[0204] [Example 11] Phase I clinical study of C-CAR088 With the approval of the ethics committee, a total of 15 volunteers carried out the Phase I clinical trial. The important eligibility criteria for the volunteers were as follows: patients aged 18 - 75 with multiple myeloma, MM cells expressing BCMA, measurable MM, having received two prior treatment lines for MM, and having received treatment with PI and IMiD, and having sufficient liver, kidney, heart and hematopoietic functions.

[0205] The experimental process is shown in Figure 13.

[0206] The results of the treatment are shown in Figure 14 and Table 2. Two patients were evaluated at the second week, one with SD and one with CR. The remaining 13 patients were evaluated at the fourth week, and the objective response rate ORR reached 100%. Among all patients, there were 3 cases of CR, 5 cases of VGPR (including 1 daratumumab-resistant patient), and 6 cases of PR.

Table 8

[0207] The adverse events occurring under treatment are shown in Table 3. Only 1 out of 15 patients developed grade 3 cytokine release syndrome. Neurotoxic events and dose-limiting toxicity (DLT) were not observed during dose escalation. Cytopenia is mainly related to Cy / Flu lymphocyte depletion. It should be noted that the occurrence of a certain degree of cytokine release syndrome after treatment also indicates the effectiveness of CART treatment from the side. None of the 15 patients had particularly severe cytokines, and C-CAR088 has better safety.

Table 9

[0208] Figure 15 shows the treatment status of the patient with ID Z0203-00801 C008. Figure 16 shows the treatment status of the patient with ID Z0203-00701C001. The PET-CT image of the cancer lesion of one patient shows that abnormal plasma cells in the bone marrow significantly decreased after C-CAR088 treatment (Figure 15). Figures 16A and 16B show that most of the PCs were abnormal (>90% were CD45lo / -) and were clones of BCMA+ and kappa light chain at the start of the experiment (baseline). Figure 16B shows that 14 or 28 days after BCMA CAR-T treatment, abnormal PCs in the BM, especially kappa+ PCs, significantly decreased, from 85.2% of the baseline level to 0%.

[0209] Figure 17 shows the increase in C-CAR088 cells and the decrease in M ​​protein / sFLC levels in the blood. The results indicated that C-CAR088 cells effectively increased after injection, and the M protein / sFLC marker levels continued to decrease. In one patient, the M marker level was reduced to 0 by day 14.

[0210] The observation results for C-CAR088 were summarized as follows:

[0211] In preclinical studies, C-CAR088 demonstrated antitumor activity both in vitro and in vivo.

[0212] Early C-CAR088 trial results in patients with r / r MM supported preclinical findings and demonstrated a promising efficacy and manageable safety profile.

[0213] • Early clinical efficacy signals at low, suboptimal doses were promising.

[0214] Compared to KarMMa data, our current dose levels from infusion patients were far below the optimal dose of bb2121. 53% (8 / 15) had recently received the medication (approximately 4 weeks prior).

[0215] Based on the PK profile, dose-dependency was observed. C-CAR088 was well-tolerated in patients.

[0216] [Example 12] Anti-BCMA CAR T-cell therapy (C-CAR088) shows a promising safety and efficacy profile in the treatment of relapsed or refractory multiple myeloma (r / r MM). Our research has demonstrated that C-CAR088 is significantly more cytotoxic to tumor cells both in vitro and in vivo compared to other anti-BCMA CARs.

[0217] The inventors conducted a clinical trial of anti-BCMA CAR (BCMA-20, also known as "C-CAR088") in the treatment of patients with relapsed / refractory multiple myeloma (R / R MM).

[0218] Clinical trials are evaluated according to several different criteria. Two key measures are the overall response rate (ORR) and the complete response rate (CR). Compared with other anti-BCMA CARs, the CAR of the method described in the claims provides better therapeutic efficacy in clinical trials, as reflected in a higher response rate (95% overall response rate or ORR and 67% complete response rate or CR) in the treatment of relapsed / refractory multiple myeloma (R / R MM).

[0219] Compared to JNJ-4528 (CARTITUDE-1), the rate of adverse events, such as neurotoxicity, was significantly lower with C-CAR 088. Specifically, with C-CAR088, only 4% of patients experienced grade 1 neurotoxicity, which resolved spontaneously. Therefore, the CAR-T cells of the method described in the claims provided a favorable safety profile.

[0220] C-CAR088 demonstrated a manageable safety profile.

[0221] Most cases of CRS were grade 1 / 2, and the median time to onset was 6 days.

[0222] Neurotoxicity (ICANS) was rare and generally low-grade, with one case of Grade 1 event.

[0223] A dose-dependent response occurred early and deepened.

[0224] ORR: 95.7%, CR rate at median time to CR: 43.5%: 1.6 months (range: 0.5-9.5). Median duration of response: 0.5 months.

[0225] • At a median follow-up of 6.2 months, 65.1% of patients were progression-free at 6 months. In preclinical studies, C-CAR088 demonstrated very favorable in vitro and in vivo antitumor activity and target specificity. Clinical trial results in 24 patients with r / r MM showed a robust therapeutic index with promising efficacy and a manageable safety profile.

[0226] The inventors conducted a clinical trial of anti-BCMA CAR (BCMA-20, also known as "C-CAR088") in the treatment of patients with relapsed / refractory multiple myeloma (R / R MM). Table 4 shows the baseline demographics and clinical characteristics of patients before the initiation of the inventors' anti-BCMA CAR treatment.

[0227] The median age of patients who received the drug was 60 years (range: 45–74 years). The median number of prior treatment lines was 4 (range: 2–12 prior treatments). All patients had received prior treatment with IMiD (immunomodulatory Drugs) and proteasome inhibitors. 25% of patients had been previously treated with anti-CD38 monoclonal antibodies, and 25% had undergone autologous hematopoietic stem cell transplantation. [Table 10]

[0228] The clinical protocol and key inclusion criteria are shown in Figure 21. Specifically, eligible subjects were enrolled, and CAR-T cells (C-CAR088) were produced using collected peripheral blood leukocytes. The CAR-T cells were then frozen and stored at below -135°C until use. For CAR-T therapy, the CAR-T cells were thawed and administered within 30–45 minutes.

[0229] 5 days and 3 days prior to CAR-T infusion, the patient received fludarabine (30 mg / m²). 2 / d, intravenously, once daily for 3 days), and cyclophosphamide (300 mg / m²) 2 They underwent lymphocyte depletion pretreatment, including intravenous administration (once daily for 3 days).

[0230] Approximately 72 hours after lymphocyte depletion, the patient was given 1.0–6.0 × 10¹⁶ doses, with a dose escalation of 3 + 3. 6 CAR-T cells / kg were administered on day 0. Patients were followed up from day 1 to 24 months after infusion.

[0231] The primary objectives included safety: evaluation of dose-limiting toxicity; incidence and severity of adverse events (CTCAEV5.0) occurring during treatment. Secondary objectives included efficacy: IMWG 2016 ORR; DOR; PFS; OS. Exploratory objectives included increasing and sustaining CAR-T receptors.

[0232] As shown in Figure 22, the overall response rate (ORR, including CR and PR) of our anti-BCMA CAR-T trial was 96%. The best overall response (BOR) included 12 stringent complete responses (sCR), 2 complete responses (CR), 8 very good partial responses (VGPR), and 1 partial response (PR). The complete response rate (CR) was 67%. (SD: stable disease; PR: partial response; CR: complete response; PD: progressive disease; MR: minimal response; VGPR: very good partial response; MRD: minimal residual disease.) Table 5 and Figure 23 show the overall CR / sCR, VGPR, and PR for each dose group. 3.0~6.0 × 10 6 In the CAR-T cell / kg dose group, 14 / 21 (66.7%) of patients achieved CR / sCR, and all (14 / 14) patients were 10% CR by flow cytometry. -4 ~10 -6 And I achieved MRD negativity. [Table 11]

[0233] Table 6 below compares the C-CAR088 trial with the trials of Munshi et al. (KarMMa: Idecabtagene Vicleucel), Mailankody et al. (EVOLVE: Orvacabtagene Autoleucel), and Madduri et al. (CARTITUDE-1: JNJ-4528). [Table 12]

[0234] Munshi et al.,Idecabtagene vicleucel(ide-cel;bb2121),a BCMA-targeted CAR T-cell therapy,in patients with relapsed and refractory multiple myeloma(RRMM):Initial KarMMa results,Journal of Clinical Oncology,2020,38(15)suppl.,Abstract 8503;Mailankody et al.,Orvacabtagene autoleucel(orva-cel),a B-cell maturation antigen(BCMA)-directed CAR T cell therapy for patients(pts)with relapsed / refractory multiple myeloma(RRMM):update of the phase 1 / 2 EVOLVE study(NCT03430011),Journal of Clinical Oncology,2020,38(15)suppl.,Abstract 8504;Madduri et al.,CARTITUDE-1:Phase 1b / 2 Study of See Ciltacabtagene Autoleucel, a B-Cell Maturation Antigen-Directed Chimeric Antigen Receptor T Cell Therapy, in Relapsed / Refractory Multiple Myeloma, 62nd ASH Annual Meeting and Exposition, December 5-8, 2020, Abstract 177.

[0235] In our clinical trials of C-CAR088, the Kaplan-Meier progression-free survival (PFS) estimates included 78.7% of 6-month PFS in both the medium-dose and high-dose groups, with a 95% confidence interval (CI) of 62.1% to 99.7%. The median duration of response (DOR) was not reached. See Table 7 and Figure 24. [Table 13]

[0236] The time course of CAR copies in the patient's blood is shown in Figures 25A and 25B. Therefore, CAR levels were maintained in the blood after administration.

[0237] C-CAR088 treatment was well-tolerated. Patient adverse reactions (adverse events, AEs) were recorded (Tables 8 and 9). Grade 3 or higher cytokine release syndrome (CRS) occurred in only one patient (4.2%). Neurotoxicity was observed in only one patient (4.2%), resolved spontaneously, and there were no grade 3 or higher neurotoxicities. Cytopenia, such as neutropenia and thrombocytopenia, was mainly associated with fludarabine / cyclophosphamide (Cy / Flu) lymphocyte depletion. No dose-limiting toxicity was observed, and all adverse events were reversible. These findings demonstrate that our anti-BCMA CAR has an excellent safety profile. [Table 14] [Table 15]

[0238] The scope of the present invention is not limited to what is specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the illustrated examples of materials, components, structures and dimensions. Numerous references, including patents and various publications, are cited and discussed in this description of the present invention. Such citations and discussions of references are provided solely to clarify the description of the present invention and do not constitute an admission that any reference is prior art to the present invention as described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Variations, modifications and other practices of those described herein will be conceivable to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matters described herein and in the accompanying drawings are provided for illustrative purposes only and not as limitations.

Claims

1. A pharmaceutical composition for treating cancer comprising immune cells expressing a chimeric antigen receptor (CAR) targeting BCMA, and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients, wherein the CAR has a light chain variable region (V Ｌ ) and heavy chain variable region (V Ｈ It includes an anti-BCMA antigen binding region, V Ｌ However, it includes three complementarity determination regions (CDRs): LCDR1, LCDR2, and LCDR3. Ｈ However, it includes three CD-Rs: HCDR1, HCDR2, and HCDR3. (a) LCDR1, LCDR2, and LCDR3 each have the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 28, (b) LCDR1, LCDR2, and LCDR3 each have the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, and SEQ ID NO: 35, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42, or (c) LCDR1, LCDR2, and LCDR3 each have the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49, respectively, and HCDR1, HCDR2, and HCDR3 each have the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56, respectively. The immune cells are T cells or natural killer (NK) cells. When the pharmaceutical composition is administered to a subject in need, the dose of immune cells is in the range of 3.0 to 6.0 × 10⁶ cells / kg body weight, and the chimeric antigen receptor generates an area under the curve (AUC) in the subject's blood for 28 days after administration, ranging from 5.0e+0.5 copies / μg genomic DNA (copies / gDNA) to 1.3e+0.7 copies / gDNA. Pharmaceutical composition.

2. The CAR is present in the target blood at 5 × 10 ４ copies / μg genomic DNA (copies / gDNA) ~ 1.3 × 10⁻¹⁶ ６ Maximum plasma concentration within the range of copies / gDNA (C ｍａｘ The pharmaceutical composition according to claim 1, which generates ).

3. The aforementioned CAR is in the range of 12 to 25 days. ｍａｘ A pharmaceutical composition according to claim 2, having the following characteristics.

4. V Ｌ is V Ｈ The pharmaceutical composition according to claim 1, which is located at the N-terminus of V.

5. V Ｌ and V Ｈ The pharmaceutical composition according to claim 1, wherein (a) each has the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, (a) each has the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, or (a) each has the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO:

6.

6. The pharmaceutical composition according to claim 1, wherein the anti-BCMA antigen-binding region is a single-stranded variable fragment (scFv) that specifically binds to BCMA.

7. The pharmaceutical composition according to claim 1, wherein the CAR further comprises one or more of the following: (a) signal peptide, (b) A hinge region, wherein the hinge region includes a hinge region of CD8, CD28, CD137, Ig4, or a combination thereof. (c) A transmembrane domain, wherein the transmembrane domain includes transmembrane domains of CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof. (d) Co-stimulatory regions, wherein the co-stimulatory regions include the co-stimulatory regions of 4-1BB (CD137), CD28, OX40, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof, and (e) A cytoplasmic signaling domain, wherein the cytoplasmic signaling domain includes a CD3ζ cytoplasmic signaling domain.

8. The pharmaceutical composition according to claim 1, wherein the cancer is a hematological cancer, a plasma cell malignancy, a BCMA-positive malignancy, multiple myeloma (MM), or plasma cell leukemia.

9. The pharmaceutical composition according to claim 1, wherein the immune cells are administered by injection, transfusion, implantation, and / or transplantation.

10. The pharmaceutical composition according to claim 1, wherein the immune cells are allogeneic or autologous.

11. The pharmaceutical composition according to claim 1, wherein the subject is a human.

12. The pharmaceutical composition according to claim 1, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63.

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