Combined chimeric antigen receptor targeting CD19 and CD20 and its applications

Abstract

A combined chimeric antigen receptor targeting CD19 and CD20 and applications thereof are provided. Specifically, a combined chimeric antigen receptor targeting CD19 and CD20 is provided, comprising an scFv targeting CD19 and CD20, a hinge region, a transmembrane region, and an intracellular signaling domain. A nucleic acid molecule encoding the chimeric antigen receptor and a corresponding expression vector, CAR-T cells, and applications thereof are also provided. Experimental results show that the chimeric antigen receptor exhibits extremely high killing ability against tumor cells. The chimeric antigen receptor targets CD19- and / or CD20-positive cells and can be used to treat CD19- and / or CD20-positive B-cell lymphoma, leukemia, and other diseases.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine, and more particularly to a combined chimeric antigen receptor targeting CD19 and CD20 and uses thereof. [Background technology]

[0002] Hematological malignancies account for about 10% of human malignancies, and 95% of hematological malignancies originate from B lymphocytes. Traditional chemotherapy and radiotherapy play an important role in the treatment of hematological malignancies. Although some patients have significant efficacy, it is difficult to cure most patients. Novel and effective treatments have become a hot topic in this field.

[0003] Adoptive T cell therapy has shown its strong efficacy and bright prospects in the clinical treatment of malignant tumors. Currently, adoptive T cell therapy is regarded as one of the most promising methods for treating hematological tumors. CD19 is highly expressed on the surface of most B cell malignancies. Multiple institutions have independently used chimeric antigen receptor (CAR)-modified T cells to target relapsed and refractory malignancies of CD19-expressing B cells, achieving unprecedented success. Currently, two CAR-T products approved by the FDA both target the CD19 antigen, and their indications, such as chronic lymphocytic leukemia, are also expanding. Although the efficacy of anti-CD19 CAR-T is remarkable, many studies have shown that there are also many problems with CD19 chimeric antigen receptor (CAR) T cell therapy. There are still some patients with poor treatment outcomes and prone to relapse. The reasons for this include the susceptibility of tumor cells to antigen escape. For example, recent experiments with CD19 CAR-cell therapy showed that 90% of patients achieved complete remission, but 11% of these patients eventually relapsed, with the relapsed patients being mainly those with CD19-negative tumors. In particular, in a clinical trial conducted at the University of Pennsylvania School of Medicine using CART19 in the treatment of relapsed refractory acute B-cell lymphoma (R / R B-ALL), up to 94% of patients achieved complete remission. Although the initial response rate of this clinical trial was high, nearly 40% of patients relapsed one month after treatment achieving complete remission, and more than 60% of relapsed patients had CD19-negative tumor cell escape. Antigen escape has been found in adoptively transferred specific T cell receptors expressing NY-ESO1 and cancer vaccines treating melanoma. Spontaneous mutation and selective expansion are the main reasons for antigen escape.

[0004] Thus, there is an urgent need in the art to develop methods to effectively treat tumors and prevent antigen escape. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide a method for effectively treating tumors and preventing antigen escape.

[0006] An object of the present invention is to provide a combined chimeric antigen receptor that targets CD19 and CD20 and a method for preparing the same. [Means for solving the problem]

[0007] Specifically, it is an object of the present invention to provide a combined chimeric antigen receptor sequence targeting CD19 and CD20, as well as a method for preparing and identifying the activity of the modified T cells (CART-19 / 20).The present invention provides a chimeric antigen receptor structure for use in the treatment of CD19 and CD20 positive B cell lymphoma.

[0008] The present disclosure provides a bispecific chimeric antigen receptor (CAR). The bispecific CAR comprises: (i) a light chain variable region (V) having the amino acid sequences set forth in SEQ ID NO:4 and SEQ ID NO:3, respectively; L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen binding region comprising ... L 2) and the heavy chain variable region (V H 2), and an anti-CD19 antigen-binding region comprising

[0009] In certain embodiments, V L 1 is V H 1. In certain embodiments, H 2 is V L It is located at the N-terminus of 2.

[0010] The anti-CD20 antigen-binding region can be a single chain variable fragment (scFv) that specifically binds to CD20. The anti-CD19 antigen-binding region can be an scFv that specifically binds to CD19. In certain embodiments, the scFv that specifically binds to CD20 is located at the N-terminus of the scFv that specifically binds to CD19.

[0011] The bispecific CAR may further comprise (a) a leader sequence, (b) a hinge region, (c) a transmembrane domain, (d) at least one costimulatory signaling region, (e) a cytoplasmic signaling domain, or (f) a combination thereof.

[0012] The costimulatory signaling region can be derived from 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 a combination thereof.

[0013] The cytoplasmic signaling domain may be derived from CD3ζ.

[0014] The hinge region can be derived from Ig4, CD8, CD28, CD137, or a combination thereof.

[0015] The transmembrane domain can be derived from CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0016] In certain embodiments, the bispecific CAR may have the amino acid sequence set forth in SEQ ID NO:16.

[0017] The present disclosure provides an immune cell expressing a bispecific CAR of the present invention. The immune cell can be a T cell or a natural killer (NK) cell. The immune cell can be allogeneic or autologous.

[0018] Nucleic acids encoding the bispecific CARs of the invention are also encompassed by the disclosure.

[0019] The disclosure further provides a vector comprising a nucleic acid of the invention.

[0020] The present disclosure provides a method of treating cancer. The method may include administering immune cells to a subject in need thereof.

[0021] The cancer may be a hematological cancer. The cancer may be a B-cell malignancy. The cancer may be Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. The cancer may be acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0022] In a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR), the structure of which is shown in Formula I below: L-scFv1-I-scFv2-H-TM-C-CD3ζ(I) During the ceremony, each "-" is independently a linker peptide or a peptide bond; L is an optional signal peptide sequence, I is a flexible linker, H is an optional hinge region, TM is the transmembrane domain, C is a costimulatory signaling molecule, CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ One of scFv1 and scFv2 has an antigen-binding domain that targets CD19, and the other has an antigen-binding domain that targets CD20.

[0023] In another preferred embodiment, scFv1 is an antigen-binding domain that targets CD20 and scFv2 is an antigen-binding domain that targets CD19.

[0024] In another preferred embodiment, the structure of the antigen-binding domain targeting CD20 is shown in Formula A or Formula B below: V H1 -V L1 (A);V L1 -V H1 (B) V H1 is the anti-CD20 antibody heavy chain variable region, and V L1 represents the anti-CD20 antibody light chain variable region, and "-" represents a linker peptide or a peptide bond.

[0025] In another preferred embodiment, the structure of the antigen-binding domain that targets CD20 is shown in Formula B.

[0026] In another preferred embodiment, V H1 The amino acid sequence of is shown in SEQ ID NO:1, L1 the amino acid sequence of which is shown in SEQ ID NO:2; or V H1 The amino acid sequence of is shown in SEQ ID NO:3, L1 The amino acid sequence of is shown in SEQ ID NO:4.

[0027] In another preferred embodiment, V H1 and V L1 is linked to a flexible linker (or linker peptide), and the flexible linker (or linker peptide) is a continuous sequence of 1 to 4, preferably 2 to 4, and more preferably 3 to 4, as shown in SEQ ID NO: 7 (GGGGS).

[0028] In another preferred embodiment, V L1 and V H1 is linked to a flexible linker such as that shown in SEQ ID NO:19.

[0029] In another preferred embodiment, the structure of the antigen-binding domain targeting CD19 is shown in Formula C or Formula D below: V L2 -V H2 (C);V H2 -V L2 (D) In the formula, V L2 is the anti-CD19 antibody light chain variable region, and V H2 indicates an anti-CD19 antibody heavy chain variable region, and "-" indicates a linker peptide or a peptide bond.

[0030] In another preferred embodiment, the structure of the antigen-binding domain that targets CD19 is shown in Formula D.

[0031] In another preferred embodiment, V L2 The amino acid sequence of is shown in SEQ ID NO:5, H2 The amino acid sequence is shown in SEQ ID NO:6.

[0032] In another preferred embodiment, V H2 and V L2 is linked to a flexible linker (or linker peptide), and the flexible linker (or linker peptide) is a continuous sequence of 1 to 4, preferably 2 to 4, and more preferably 3 to 4, as shown in SEQ ID NO: 7 (GGGGS).

[0033] In another preferred embodiment, V H2 and V L1 is linked to a flexible linker as shown in SEQ ID NO:20.

[0034] In another preferred embodiment, scFv1 and / or scFv2 are murine, humanized, chimeric humanized and murine, or fully humanized single chain antibody variable region fragments.

[0035] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula II below: L.V. L1 -V H1 -IV H2 -V L2 -H-TM-C-CD3ζ(II) In the formula, each element is as defined above.

[0036] In another preferred embodiment, the sequence of flexible linker I comprises 2 to 6, preferably 3 to 4, consecutive sequences as shown in SEQ ID NO: 7 (GGGGS).

[0037] In another preferred embodiment, the sequence of flexible linker I is as shown in SEQ ID NO:7.

[0038] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, and combinations thereof.

[0039] In another preferred embodiment, L is the signal peptide derived from CD8.

[0040] In another preferred embodiment, the amino acid sequence of L is set forth in SEQ ID NO:8.

[0041] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, Ig4, and combinations thereof.

[0042] In another preferred embodiment, H is a hinge region derived from Ig4.

[0043] In another preferred embodiment, the amino acid sequence of H is set forth in SEQ ID NO:9.

[0044] In another preferred embodiment, the 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, and combinations thereof.

[0045] In another preferred embodiment, the TM is a transmembrane region derived from CD8 or CD28.

[0046] In another preferred embodiment, the sequence of the TM is shown in SEQ ID NO:10 or SEQ ID NO:11.

[0047] In another preferred embodiment, C is a costimulatory signaling molecule of 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, and combinations thereof.

[0048] In another preferred embodiment, C is a costimulatory signaling molecule derived from 4-1BB or CD28.

[0049] In another preferred embodiment, the amino acid sequence of C is set forth in SEQ ID NO:12 or 13.

[0050] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:14.

[0051] In another preferred embodiment, the amino acid sequence of CAR is set forth in SEQ ID NO: 15 or 16.

[0052] In certain embodiments, an anti-CD20 antigen-binding region comprises a heavy chain variable region comprising an amino acid sequence that is 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 to the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:1.

[0053] In certain embodiments, the anti-CD20 antigen-binding region comprises a light chain variable region comprising an amino acid sequence that is 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 to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:2.

[0054] In certain embodiments, the anti-CD19 antigen binding region comprises a heavy chain variable region comprising an amino acid sequence that is 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 to the amino acid sequence set forth in SEQ ID NO:6.

[0055] In certain embodiments, the anti-CD19 antigen binding region comprises a light chain variable region comprising an amino acid sequence that is 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 to the amino acid sequence set forth in SEQ ID NO:5.

[0056] The heavy chain variable region of the anti-CD20 antigen-binding region has a complementarity determining region (CDR) of the heavy chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 respectively depicted at positions 30 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 3) or a CDR of the heavy chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3 respectively depicted at positions 31 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 1). It may comprise one, two or three CDRs that are 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 even about 100% identical.

[0057] The light chain variable region of the anti-CD20 antigen-binding region has a complementarity determining region (CDR) of the light chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 respectively set forth at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 4) or a CDR of the light chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3 respectively set forth at positions 24 to 33, 49 to 55, and 88 to 96 of SEQ ID NO: 2). The sequence may comprise one, two or three CDRs that are 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 even about 100% identical to the sequence of the sequence of the original.

[0058] The heavy chain variable region of the anti-CD20 antigen binding region has a complementarity determining region (CDR) of the heavy chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively) or a CDR of the heavy chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 51, respectively). The sequence may comprise one, two or three CDRs that are 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.

[0059] The light chain variable region of the anti-CD20 antigen binding region has a complementarity determining region (CDR) of the light chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively) or a CDR of the light chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58, respectively). The sequence may comprise one, two or three CDRs that are 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.

[0060] In a specific embodiment, the heavy chain variable region of the anti-CD20 antigen-binding region comprises three CDRs identical to the CDRs of the heavy chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 described at positions 30 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 3), and the light chain variable region of the anti-CD20 antigen-binding region comprises three CDRs identical to the CDRs of the light chain variable region of the ofatumumab antibody (CDR1, CDR2, and CDR3 described at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 4, respectively).

[0061] In certain embodiments, the heavy chain variable region of the anti-CD20 antigen binding region comprises three CDRs identical to the CDRs of the heavy chain variable region of the ofatumumab antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, respectively), and the light chain variable region of the anti-CD20 antigen binding region comprises three CDRs identical to the CDRs of the light chain variable region of the ofatumumab antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively).

[0062] In a specific embodiment, the heavy chain variable region of the anti-CD20 antigen-binding region contains three CDRs identical to the CDRs of the heavy chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3, respectively, described at positions 31 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 1), and the light chain variable region of the anti-CD20 antigen-binding region contains three CDRs identical to the CDRs of the light chain variable region of the Leu16 antibody (CDR1, CDR2, and CDR3, respectively, described at positions 24 to 33, 49 to 55, and 88 to 96 of SEQ ID NO: 2).

[0063] In a specific embodiment, the heavy chain variable region of the anti-CD20 antigen binding region comprises three CDRs identical to the CDRs of the heavy chain variable region of the Leu16 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 51, respectively), and the light chain variable region of the anti-CD20 antigen binding region comprises three CDRs identical to the CDRs of the light chain variable region of the Leu16 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 58, respectively).

[0064] The heavy chain variable region of the anti-CD19 antigen-binding region has a complementary determining region (CDR) (CDR1, CDR2, and CDR3 at positions 31 to 35, 50 to 65, and 98 to 109, respectively, of SEQ ID NO: 6) of the heavy chain variable region of the FMC63 antibody, which is 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 The sequence may comprise one, two or three CDRs that are at least 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 even about 100% identical to the sequence of the sequence of the amino acid ...

[0065] The light chain variable region of the anti-CD19 antigen-binding region has a complementarity determining region (CDR) of the light chain variable region of the FMC63 antibody (CDR1, CDR2, and CDR3 respectively set forth at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 5), which is 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 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 97%, at least or about 9 ... The sequence may comprise one, two or three CDRs that are at least 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 even about 100% identical to the sequence of the sequence of the amino acid ...

[0066] The heavy chain variable region of the anti-CD19 antigen-binding region has a complementarity determining region (CDR) (CDR1, CDR2, and CDR3 shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62) of the heavy chain variable region of the FMC63 antibody of 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 may comprise one, two or three CDRs that are 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 even about 100% identical.

[0067] The light chain variable region of the anti-CD19 antigen binding region has a complementarity determining region (CDR) of the light chain variable region of the FMC63 antibody (CDR1, CDR2, and CDR3 shown in SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:39, respectively) that is 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 may comprise one, two or three CDRs that are 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 even about 100% identical.

[0068] In a specific embodiment, the heavy chain variable region of the anti-CD19 antigen binding region contains three CDRs identical to the CDRs of the heavy chain variable region of the FMC63 antibody (CDR1, CDR2, and CDR3, respectively, described at positions 31 to 35, 50 to 65, and 98 to 109 of SEQ ID NO: 6), and the light chain variable region of the anti-CD19 antigen binding region contains three CDRs identical to the CDRs of the light chain variable region of the FMC63 antibody (CDR1, CDR2, and CDR3, respectively, described at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 5).

[0069] In a specific embodiment, the heavy chain variable region of the anti-CD19 antigen binding region comprises three CDRs identical to the CDRs of the heavy chain variable region of the FMC63 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively), and the light chain variable region of the anti-CD19 antigen binding region comprises three CDRs identical to the CDRs of the light chain variable region of the FMC63 antibody (CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 39, respectively).

[0070] In a second aspect of the invention, there is provided a nucleic acid molecule encoding a chimeric antigen receptor of the first aspect of the invention.

[0071] In another preferred embodiment, the nucleic acid molecule is isolated.

[0072] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO:17 or SEQ ID NO:18.

[0073] In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule of the second aspect of the invention.

[0074] In another preferred embodiment, the vector comprises DNA and RNA.

[0075] In another preferred embodiment, the vector is selected from the group consisting of a plasmid, a viral vector, a transposon, and combinations thereof.

[0076] In another preferred embodiment, the vectors include DNA viruses and retroviral vectors.

[0077] In another preferred embodiment, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and combinations thereof.

[0078] In another preferred embodiment, the vector is a lentiviral vector.

[0079] In a fourth aspect of the invention, there is provided a host cell which comprises a vector of the third aspect of the invention, or has integrated into its genome an exogenous nucleic acid molecule of the second aspect of the invention, or which expresses a chimeric antigen receptor of the first aspect of the invention.

[0080] In another preferred embodiment, the cell is an isolated cell.

[0081] In another preferred embodiment, the cell is a genetically engineered cell.

[0082] In another preferred embodiment, the cell is a mammalian cell.

[0083] In another preferred embodiment, the cells are CAR-T cells and / or CAR-NK cells.

[0084] In another preferred embodiment, the cells are targeted to both CD19 and CD20.

[0085] In a fifth aspect of the invention, there is provided a method for preparing a CAR-T cell expressing a chimeric antigen receptor of the first aspect of the invention, the method comprising the step of transducing a T cell with a nucleic acid molecule of the second aspect of the invention or a vector of the third aspect of the invention, thereby obtaining the CAR-T cell.

[0086] In another preferred embodiment, the method further comprises detecting the function and efficacy of the obtained CAR-T cells.

[0087] In a sixth aspect of the invention there is provided a formulation comprising a chimeric antigen receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, a vector of the third aspect of the invention or a host cell of the fourth aspect of the invention and a pharma- ceutically acceptable carrier, diluent or excipient.

[0088] In another preferred embodiment, the formulation is a liquid formulation.

[0089] In another preferred embodiment, the pharmaceutical formulation is an injectable solution.

[0090] In another preferred embodiment, the formulation comprises host cells of the fourth aspect of the invention, the concentration of the host cells being 1×10 3 ~1×10 8 cells / ml, preferably 1 x 10 4 ~1×10 7 cells / ml.

[0091] In a seventh aspect of the invention, there is provided the use of a chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention for the preparation of a medicament or formulation for preventing and / or treating a tumour or cancer.

[0092] In another preferred embodiment, the tumor is selected from the group consisting of a hematological tumor, a solid tumor, and a combination thereof, preferably, the tumor is a hematological tumor.

[0093] In another preferred embodiment, the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), and combinations thereof.

[0094] In another preferred 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, and combinations thereof.

[0095] In an eighth aspect of the present invention, there is provided a kit for preparing a cell of the fourth aspect of the invention, the kit comprising a container and, disposed within the container, a nucleic acid molecule of the second aspect of the invention or a vector of the third aspect of the invention.

[0096] In a ninth aspect of the invention there is provided the use of a cell according to the fourth aspect of the invention, or a formulation according to the sixth aspect of the invention, for the prevention and / or treatment of cancer or tumours.

[0097] In a tenth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of cells according to the fourth aspect of the invention or a formulation according to the sixth aspect of the invention.

[0098] In another preferred embodiment, the disease is a cancer or tumor.

[0099] It should be understood that the various technical features of the present invention described above and the various technical features specifically described below (such as in the examples) can be combined with each other within the scope of the present invention to form new or preferred technical solutions, and need not be described one by one due to space limitations. [Brief description of the drawings]

[0100] [Figure 1] Figure 1 shows the structure of a combined chimeric antigen receptor targeting CD19 and CD20. The CAR structure includes a leader sequence, an antigen recognition sequence, a hinge region, a transmembrane region, a costimulatory signal region, and a CD3ζ signaling region. [Figure 2A] FIG. 2A shows the expression level of CD137 on the surface of the T cell membrane. [Figure 2B] Figure 2B shows the secretion level of IFNγ in the culture supernatant.Specifically, 1x105 CAR-T19 / 20 cells cultured for 7 days were harvested and cultured in 200μl of GT-551 medium at a ratio of 1:1 with CD19 positive K562-CD19+ tumor cell line, CD20 positive K562-CD20+ tumor cell line, CD19 and CD20 double positive K562-CD19+CD20+ tumor cell line, RAJI tumor cell line that naturally expresses CD19 and CD20, and CD19 and CD20 double negative K562 tumor cell line, or without tumor cells, respectively, for 18 hours.Then, the expression level of CD137 on the surface of T cell membrane and the secretion level of IFNγ in the culture supernatant were detected, respectively. [Figure 3A] Figure 3A shows the detection of tumoricidal activity of CAR-T19 / 20 cells mainly by detecting the secretion level of LDH in the supernatant after co-culture. Specifically, 1x104 cells of CD19-positive K562-CD19+ tumor cell line, CD20-positive K562-CD20+ tumor cell line, CD19 and CD20 double-positive K562-CD19+CD20+ tumor cell line, RAJI or RAMOS tumor cell line naturally expressing CD19 and CD20, or CD19 and CD20 double-negative K562 tumor cell line were co-cultured with corresponding T cells in 100μl of GT-551 medium at the ratio shown in the figure for 8 hours. Then, the secretion level of LDH was detected, and the figure shows the statistical analysis result of the percentage of LDH release in the corresponding co-culture sample. [Figure 3B] Figure 3B shows the detection of the tumoricidal activity of CAR-T19 / 20 cells, mainly by detecting the expression level of CD107a on the surface of T cell membrane.Specifically, 1x105 CAR-T19 / 20 cells were harvested and cultured in 200μl of GT-551 medium at a ratio of 1:2 with CD19 positive K562-CD19+ tumor cell line, CD20 positive K562-CD20+ tumor cell line, CD19 and CD20 double positive K562-CD19+CD20+ tumor cell line, RAJI or RAMOS tumor cell line that naturally expresses CD19 and CD20, and CD19 and CD20 double negative K562 tumor cell line, or without tumor cells, for 4 hours.Then, the expression level of CD137 on the surface of T cell membrane was detected, respectively. [Figure 4A] FIG. 4A shows the mean weight change and mean fluorescence intensity change of mice injected with CAR-T19 / 20 cells within 21 days, recorded every 7 days. [Figure 4B] FIG. 4B shows the mean fluorescence intensity of the three groups of mice. [Figure 4C] Figure 4C shows in vivo imaging of mice injected with CAR-T19 / 20 cells at days 0 (D0), 7 (D7), 14 (D14) and 21 (D21) post-injection. [Figure 5A] Figure 5A shows that bispecific TN-OF-19 CAR-T cells can inhibit or kill tumor cells better than CD19-specific CAR-T cells in vivo. [Figure 5B] Figure 5B shows IVIS imaging of fluorescence intensity in the four CAR-T cell groups and the NT group. [Figure 6] FIG. 6 shows chimeric antibodies that bind to cells bearing CD19 or CD20 or both, but not to cells lacking both antigens, indicating that the bispecific binding domain requires only one cognate antigen for binding and that no new specific recognition sites were formed. [Figure 7] FIG. 7 shows the structures of 16 CD20-specific CARs with 6 different scFvs and different hinge / TM / signaling domains. [Figure 8A] Figure 8A shows the results of IFNγ release assay to screen CAR-T20.1, CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10, among these CAR-Ts, only CAR-T20.9(Leu16) and CAR-T20.10(Leu16) showed higher positive IFNγ release. [Figure 8B]Figure 8B shows the results of IFNγ release assay for screening CAR-T20.1, CAR-T20.9, CAR-T20.10, CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15 and CAR-T20.16. Among these CAR-Ts, CAR-T20.10 (Leu16) and CAR-T20.14 (OF) showed higher positive IFNγ release. [Figure 8C] Figure 8C shows the results of IFNγ release assay for screening CAR-T20.9, CAR-T20.12, CAR-T20.14, CAR-T20.17, CAR-T20.18, and CAR-T20.19. Among these CAR-Ts, CAR-T20.14(OF) and CAR-T20.19(OF) showed higher positive IFNγ release. [Figure 9A] Figure 9A shows the results of CAR-T20.17 (Leu16 3rd generation), CAR-T20.18 (Leu16 2nd generation), and CAR-T20.19 (OF 2nd generation) cells tested for cytotoxicity by LDH release assay. [Figure 9B] Figure 9B shows in vivo inhibition of tumor growth in an NSG mouse study. The tumor cells injected into the animals are Raji, which express luciferase. [Figure 10] FIG. 10 shows the CAR039r / r NHL study design and flow chart. [Figure 11] Figure 11 shows a summary of C-CAR039 clinical results. [Figure 12] Figure 12 shows an example of a patient before and after C-CAR039 treatment. [Figure 13] Figure 13 shows the growth and expansion of C-CAR039 in the patient's blood. The results showed that C-CAR008 cells expanded effectively after injection. [Figure 14] FIG. 14 shows the C-CAR066-NHL study design. [Figure 15] FIG. 15 shows an example of a PET-CT of a patient before and after treatment with C-CAR066. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] After extensive and intensive research, the present inventors unexpectedly obtained CAR-T cells that simultaneously target CD19 and CD20. Specifically, the present invention provides a chimeric antigen receptor that simultaneously targets CD19 and CD20, comprising a signal peptide, an anti-CD20scFv, an anti-CD19scFv, a hinge region, a transmembrane region, and an intracellular T cell signaling region. Furthermore, the anti-CD20scFv and the anti-CD19scFv were obtained by multiple screening and linked with a peptide fragment (G4S) having a multiple repeat structure. The CAR-T cells of the present invention can simultaneously recognize both CD19 and CD20 antigens, reducing the risk of immune escape caused by downregulation or deletion of antigen expression during the treatment of single-target CAR-T cells. Compared with CAR-T cells targeting a single antigen and other dual-target CAR-T cells (targeting CD19 and CD20), the CAR-T cells of the present invention, which simultaneously recognize two targets, have stronger killing ability against tumor cells, less cytotoxicity, less side effects, a broader therapeutic range, a lower recurrence rate and better efficacy. The present invention has been completed based on this.

[0102] term In order to make this disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning indicated below. Other definitions are set forth throughout this application.

[0103] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0104] The term "administering" refers to the physical introduction of a product of the invention into a subject using any one of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration, such as by injection or infusion.

[0105] The term "antibody" (Ab) may include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. 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 can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed among more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0106] CD20 Although the efficacy of anti-CD19 CAR-T is remarkable, many studies have shown that there are still many problems with CD19 chimeric antigen receptor (CAR) T cell therapy. There are still some patients who have poor treatment outcomes and are prone to relapse. This includes the susceptibility of tumor cells to antigen escape.

[0107] To prevent CD19CAR-T antigen escape, we designed a combined bispecific CAR (i.e., BICAR) that targets both CD19 and CD20, so that when the CD19 antigen escapes and is not expressed on tumor cells, CD20 can be recognized and eliminate tumor cells in vivo.

[0108] CD20 is expressed in most patients with B-cell acute lymphoblastic leukemia, including some CD19-negative patients after anti-CD19 CAR-T therapy. CD20 is a glycosylated protein and is the first identified B-cell membrane marker. CD20 is also known as B1 and is encoded by the MS4A gene. The CD20 molecule has four transmembrane hydrophobic regions, its N-terminus and C-terminus are located on the cytoplasmic side, and form two closed loops on the outside of the cell, called the large loop and the small loop, respectively. CD20 is specifically expressed on more than 95% of normal and cancerous B cells. These cells are in the pre-B cell stage and subsequent developmental stages, and CD20 is not expressed until the cells differentiate into plasma cells. The present invention uses CD20 as another target for immunotherapy of B-cell malignancies.

[0109] A bispecific chimeric antigen receptor targeting CD19 and CD20 Cellular immunotherapy is a new and highly effective tumor treatment model and a new type of autoimmune treatment for cancer. Cellular immunotherapy is a method that uses biotechnology and biological agents to culture and expand immune cells taken from patients in vitro, and then inject the cells back into the patient to stimulate and enhance the body's autoimmune function, thereby achieving the goal of treating tumors. Those skilled in the art have been working to develop new cellular immunotherapy methods to improve efficacy and reduce side effects.

[0110] The present invention proposes a rational and optimized single chain design and system, i.e., a combined bispecific CAR, that can be effectively incorporated into primary human T cells and can simultaneously target CD19 and CD20 when T cells are activated. The CAR-T cells of the present invention can recognize two antigens (CD19 and CD20). The present invention provides a highly effective potential method to prevent antigen escape.

[0111] The present invention uses CAR that simultaneously targets CD19 and CD20. Compared with CAR that targets a single antigen, the affinity is enhanced, the activity of T cells is increased, and the target has additive or synergistic effects. In addition, due to the heterogeneous expression levels of CD19 and CD20 on tumor cells, dual-target CAR-T therapy has a broader scope. CAR-T that simultaneously targets CD19 and CD20 on the surface of tumor cells can reduce the possibility of antigen escape caused by the downregulation or deletion of a single surface antigen.

[0112] Bispecificity means that the same CAR can specifically bind and immune recognize two different antigens and that the CAR can generate an immune response by binding to either one of the antigens.

[0113] The CD19 and CD20 bispecific CAR of the present invention has a single structure and comprises an anti-CD19scFv and an anti-CD20scFv, where the CAR comprises CD19scFv and CD20scFv, and the amino acid sequences, sequencing and hinges of CD19scFv and CD20scFv are the main factors that affect its function.

[0114] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises 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 zeta chain. The costimulatory signaling region refers to the portion of the intracellular domain that comprises a costimulatory molecule. A costimulatory molecule is a cell surface molecule that is not an antigen receptor or its ligand, but is required for an efficient response of lymphocytes to antigens.

[0115] A linker may be incorporated between the extracellular and transmembrane domains of a CAR, or between the cytoplasmic and transmembrane domains of a CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to link a transmembrane domain to an extracellular or cytoplasmic domain in a polypeptide chain. A linker may comprise 0-300 amino acids, preferably 2-100 amino acids, and most preferably 3-50 amino acids.

[0116] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided herein comprises an antigen binding domain targeting CD19 and CD20. When the CAR of the present invention is expressed in a T cell, antigen recognition can be performed based on antigen binding specificity. When the CAR binds to its associated antigen, the CAR affects the tumor cell such that the tumor cell cannot grow, is killed, or is otherwise affected, reducing or eliminating the tumor burden in the patient. The antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of a 4-1BB signaling domain and a CD3 zeta signaling domain.

[0117] As used herein, an "antigen-binding domain" and a "single-chain antibody fragment" refer to a Fab fragment, Fab' fragment, F(ab') fragment, or a single-chain antibody fragment that has antigen-binding activity. 2The term "Fv" refers to a fragment, or a single Fv fragment. An Fv antibody comprises an antibody heavy chain variable region and a light chain variable region, but does not comprise a constant region. An Fv antibody comprises the smallest antibody fragment that comprises all antigen-binding sites. In general, an Fv antibody also comprises a polypeptide linker between the VH domain and the VL domain, and can form the structure required for antigen binding. The antigen-binding domain is usually an scFv (single chain variable fragment). The size of an scFv is typically 1 / 6 of that of a complete antibody. A single chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes CD19 and CD20.

[0118] With regard 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 in the CAR is used. In some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0119] The intracellular domain in the CAR of the present invention comprises the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0120] Preferably, the CAR structure of the present invention also comprises a signal peptide sequence (also known as a leader sequence), an antigen recognition sequence (antigen binding domain), a hinge region, a transmembrane region, a costimulatory signal region, and a CD3 zeta signaling region (ζ chain portion). The order of connections is shown in FIG.

[0121] In another preferred embodiment, the CAR of the present invention is TN-LEU-19. The antigen-binding domain targeting CD20 comprises the heavy chain sequence (SEQ ID NO: 1) and the light chain (V L ) sequence (SEQ ID NO:2).

[0122] Single chain variable region heavy chain sequence (VH) from Leu16 antibody: EVQLQQSGAELVKPGASVKMSCKASGYTFT SYNMH WVKQTPGQGLEWIG AIYPGNGDTSYNQKFKG KATLTADKSSSTAYMQLSSLTSEDSADYYCAR SNYYGSSYWFFDV WGAGTTVTVSS (SEQ ID NO: 1) LEU-VH-CDR1: SEQ ID NO:1, positions 31-35; LEU-VH-CDR2: SEQ ID NO:1, positions 50-66; LEU-VH-CDR3: sequence number 1, positions 99-111.

[0123] The light chain sequence (VL) of the single variable region derived from the Leu16 antibody: DIVLTQSPAILSASPGEKVTMTC RASSSVNYMD WYQKKPGSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSFNPPT FGGGTKLEIK (SEQ ID NO: 2) LEU-VL-CDR1: SEQ ID NO:2, positions 24-33; LEU-VL-CDR2: SEQ ID NO:2, positions 49-55; LEU-VL-CDR3: sequence number 2, positions 88-96. [Table 1] [Table 2]

[0124] In another preferred embodiment, the CAR of the invention is TN-OF-19. The antigen-binding domain targeting CD20 comprises the heavy chain sequence (SEQ ID NO:3) and the light chain sequence (SEQ ID NO:4) of the single chain variable region from the ofatumumab antibody.

[0125] Single variable region heavy chain sequence (VH) from the ofatumumab antibody: EVQLVESGGGLVQPGRSLRLSCAASGFTF NDYAMH WVRQAPGKGLEWVS TISWNSGSIGYADSVKG RFTISRDNAKKSLYLQMNSLRAEDTALYYCAK DIQYGNYYYGMDV WGQGTTVTVSS (SEQ ID NO: 3) OF-VH-CDR1: SEQ ID NO: 3, positions 30 to 35. The sequence of OF-VH-CDR1 is NDYAMH (SEQ ID NO: 40).

[0126] OF-VH-CDR2: SEQ ID NO: 3, positions 50 to 66. The sequence of OF-VH-CDR2 is TISWNSGSIGYADSVKG (SEQ ID NO: 41).

[0127] OF-VH-CDR3: SEQ ID NO: 3, positions 99 to 111. The sequence of OF-VH-CDR3 is DIQYGNYYYGMDV (SEQ ID NO: 42).

[0128] The light chain sequence of the single variable region (VL) derived from the ofatumumab antibody: EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPIT FGQGTRLEIK (SEQ ID NO: 4) OF-VL-CDR1: SEQ ID NO: 4, positions 24 to 34. The sequence of OF-VL-CDR1 is RASQSVSSYLA (SEQ ID NO: 43).

[0129] OF-VL-CDR2: SEQ ID NO: 4, positions 50 to 56. The sequence of OF-VL-CDR2 is DASNRAT (SEQ ID NO: 44).

[0130] OF-VL-CDR3: SEQ ID NO: 4, positions 89 to 97. The sequence of OF-VL-CDR3 is QQRSNWPIT (SEQ ID NO: 45).

[0131] In another preferred embodiment, the antigen-binding domain targeting CD19 in the CAR of the invention comprises the light chain (VL) sequence (SEQ ID NO: 5) and heavy chain sequence (SEQ ID NO: 6) of a single chain variable region derived from the FMC63 antibody.

[0132] The amino acid sequence of the light chain (VL) of the single variable region derived from the FMC63 antibody: DIQMTQTTSSLSASLGDRVTISC RASQDISKYLN WYQQKPDGTVKLLIY HTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYT FGGGTKLEIT (SEQ ID NO:5) FMC63-VL-CDR1: SEQ ID NO:5, positions 24 to 34. The sequence of FMC63-VL-CDR1 is RASQDISKYLN (SEQ ID NO:63).

[0133] FMC63-VL-CDR2: SEQ ID NO:5, positions 50 to 56. The sequence of FMC63-VL-CDR2 is HTSRLHS (SEQ ID NO:64).

[0134] FMC63-VL-CDR3: SEQ ID NO:5, positions 89 to 97. The sequence of FMC63-VL-CDR3 is QQGNTLPYT (SEQ ID NO:39).

[0135] Nucleotide sequence of the light chain (VL) of the single variable region derived from the FMC63 antibody: gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggagatcac a 321 (SEQ ID NO: 21)

[0136] The amino acid sequence of the single variable region (VH) heavy chain from the FMC63 antibody: EVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVS WIRQPPRKGLEWLG VIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAK HYYYGGSYAMDY WGQGTSVTVSS (SEQ ID NO: 6) FMC63-VH-CDR1: SEQ ID NO: 6, positions 31 to 35. The sequence of FMC63-VH-CDR1 is DYGVS (SEQ ID NO: 60).

[0137] FMC63-VH-CDR2: SEQ ID NO: 6, positions 50 to 65. The sequence of FMC63-VH-CDR2 is VIWGSETTYYNSALKS (SEQ ID NO: 61).

[0138] FMC63-VH-CDR3: SEQ ID NO: 6, positions 98 to 109. The sequence of FMC63-VH-CDR3 is HYYYGGSYAMDY (SEQ ID NO: 62).

[0139] Nucleotide sequence of the heavy chain (VH) of the single variable region derived from the FMC63 antibody: gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60 acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120 ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180 tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300 tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360 (SEQ ID NO:22)

[0140] Specifically, the sequences of other elements in the CAR of the present invention are as follows:

[0141] The leader sequence is the leader sequence of the CD8 antigen.

[0142] MALPVTALLLPLALLLHAARP (SEQ ID NO: 8)

[0143] The linker sequence between the heavy and light chains of the single-chain variable region (ie, flexible linker I) is as follows:

[0144] Amino acid sequence of the linker between VL and VH of CD20 scfv: GSTSGGGSGGGSGGGGSS (SEQ ID NO: 19) Amino acid sequence of the linker between the VH and VL of CD19 scFv: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 20)

[0145] The hinge region is selected from the sequence of the short form of the IgG4 hinge.

[0146] ESKYGPPCPPCP (SEQ ID NO: 9)

[0147] The transmembrane domain is the transmembrane domain sequence of the CD8 (CD8TM) or CD28 (CD28TM) antigen.

[0148] CD8TM: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) CD28TM: MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 11)

[0149] The costimulatory signal region is derived from the sequence of the 4-1BB or CD28 cytoplasmic signaling motif.

[0150] 4-1BB: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12) CD28: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 13)

[0151] The signal transduction domain of CD3ζ is derived from the immunoreceptor tyrosine-based activation motif (ITAM) sequence of CD3ζ in the TCR complex.

[0152] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14)

[0153] In a preferred embodiment, the complete nucleic acid and amino acid sequences of the two CARs constructed in the present invention are as follows:

[0154] The complete nucleic acid sequence of TN-OF-19 is as follows:

[0155]

[0156] The complete amino acid sequence of TN-OF-19 is as follows:

[0157] (SEQ ID NO:16)

[0158] The complete nucleic acid sequence of TN-LEU-19 is as follows:

[0159]

[0160] The complete amino acid sequence of TN-LEU-19 is as follows:

[0161] METDTLLLWVLLLWVPGSTGDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGGTKLEIKGSTSGGGSGGGSGGGGSS EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSG GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS GSTSGSGKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 15)

[0162] The BICAR design of the present invention has the following advantages:

[0163] First, CD19 and CD20 are expressed in most malignant B cell tumors. Second, in general, when expanding the CAR structure to enhance T cell recognition ability, problems such as increased harmful targeting, increased cytotoxicity, and increased side effects are often encountered. However, this is not true for CD19 and CD20, since both CD19 and CD20 are only expressed in B cells, which have the same tumor toxicity curve. Finally, the expression of CD19 and CD20 in B cells can promote the survival of B cells. Also, the loss of both antigens during treatment is a very low probability event. Therefore, it is expected that targeting CD19 and CD20 will result in effective prevention of antigen escape of malignant B cells.

[0164] Compared with single CD19 or CD20 CARs, BICARs have the following advantages:

[0165] First, the DNA footprint is significantly reduced (40% reduction in DNA length) when expressing BICAR in a single T cell compared to expressing two independent CARs. The size and length of the structure can significantly affect the packaging and transduction efficiency of the viral vector, thus directly affecting the clinical efficacy. Second, compared to a mixture of two different single CARs, BICAR can significantly reduce the cost of treatment (BICAR is fully compatible with the current T cell manufacturing process without adding any additional burden) and increase the clinical cure rate. Finally, CD19 and CD20 have been validated in numerous clinical studies and are relatively safe.

[0166] In the present invention, based on the sequence of CD19 mouse-derived monoclonal antibody FMC63 and the sequence of CD20 mouse-derived monoclonal antibodies leu-16 and ofatumumab, two chimeric antigen receptor structures (TN-LEU-19, TH-OF-19) targeting CD19 and CD20 were constructed. We completed the analysis and identification of the expression level, in vitro activation ability, and tumor cell killing efficacy of these two chimeric antigen receptors in primary T cells. Finally, it was found that T cells modified with TN-LEU-19 or TH-OF-19 chimeric antigen receptors have a strong ability to kill malignant tumors bearing CD19 and CD20 positive antigens in vitro and eliminate them in vivo, and ofatumumab is better than leu16. This provides a new and effective method and preparation for the clinical application of CAR-T in the treatment of CD19 and CD20 positive leukemia and lymphoma.

[0167] The present invention has designed and optimized monospecific and bispecific CARs. These CARs have strong killing ability against B cell malignancies expressing CD19 or CD20. BICARs allow a single T cell product to target two clinically validated antigens associated with B cell leukemia and lymphoma, ultimately lowering the risk of tumor recurrence due to loss or escape of a single antigen. The present invention can further be used in the design of new BICARs, thus increasing the applicability of antigens and enhancing the efficacy of T cell therapy against cancer.

[0168] Chimeric antigen receptor T cells (CAR-T cells) As used herein, the terms "CAR-T cell", "CAR-T", "CART", and "CAR-T cell of the present invention" all refer to the CAR-T cell targeting both CD19 and CD20 of the fourth aspect of the present invention. Specifically, the CAR structure of the CAR-T cell comprises anti-CD19 scFv, anti-CD20 scFv, hinge region, transmembrane region, and intracellular T cell signaling region in order, and the anti-CD20 scFv and anti-CD19 scFv are linked to a peptide (G4S) having multiple repeat structures. Compared with CAR-T targeting a single antigen, CAR-T cells that simultaneously recognize two targets are more lethal and have a broader therapeutic scope.

[0169] vector Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the gene, or by direct isolation from cells and tissues which contain the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0170] The present invention also provides a vector into which the expression cassette of the present invention is inserted. A vector derived from a retrovirus, such as a lentivirus, is a suitable tool for achieving long-term gene transfer, since it allows long-term and stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors have the advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, that they can transduce non-proliferating cells, such as hepatocytes. Lentivirus vectors also have the advantage of being less immunogenic.

[0171] In summary, the expression cassette or nucleic acid sequence of the present invention is typically operably linked to a promoter and incorporated into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0172] The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entire specification of which is incorporated herein by reference. In another embodiment, the present invention provides a gene therapy vector.

[0173] Nucleic acids can be cloned into many 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 of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0174] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584, WO 01 / 29058, and U.S. Patent No. 6,326,193).

[0175] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into a retroviral particle. The recombinant virus can then be isolated and delivered to cells of a subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. A number of adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0176] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-1, 10 bp upstream of the start site, but many promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0177] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the 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 leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters, and inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0178] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells can also contain either a selection marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells that are desired to be transfected or infected via a viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate control sequences to allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0179] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present or expressed in the recipient organism or tissue, that encode a polypeptide, and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-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 may be prepared using known techniques or may be obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0180] Methods for introducing and expressing genes into cells are known in the art. In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0181] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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). A preferred method for introducing polynucleotides into host cells is the calcium phosphate method.

[0182] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0183] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as 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 a liposome (e.g., an artificial membrane vesicle).

[0184] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in a suspension in lipid, contained or complexed with micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist as a bilayer structure, micelles, or in a "collapsed" structure. They may also simply be dispersed in the solution, and in some cases form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0185] In a preferred embodiment of the invention, the vector is a lentiviral vector.

[0186] preparation The present invention provides a formulation comprising the CAR-T cells of the fourth aspect of the invention and a pharma- ceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1×10 3 ~1×10 8 cells / ml, more preferably 1×10 4 ~1×10 7 cells / ml.

[0187] In one embodiment, the formulation may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol, etc.; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0188] therapeutic use The present invention includes therapeutic applications using cells (e.g., T cells) transduced with lentiviral vectors (LV) encoding the expression cassettes of the present invention. The transduced T cells can target the tumor cell markers CD19 and CD20, synergistically activating T cells and eliciting a T cell immune response, thereby significantly enhancing the killing efficacy against tumor cells.

[0189] Thus, the present invention 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 administering a CAR-T cell of the present invention to the mammal.

[0190] In one embodiment, the present invention comprises a class of cell therapy in which T cells from an autologous patient (or a xenogeneic donor) are isolated, activated, and genetically modified to generate CAR-T cells, which are then injected back into the same patient. In this way, the probability of graft-versus-host disease is extremely low, and antigens are recognized by T cells in a non-MHC-restricted manner. Furthermore, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-term persistence and leading to sustained tumor control.

[0191] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can persist for extended periods of time. Furthermore, CAR-mediated immune responses can be part of an adoptive immunotherapy approach in which CAR-modified T cells induce an immune response specific to the antigen-binding moiety in the CAR. For example, anti-CD19CD20 CAR-T cells induce an immune response specifically against cells expressing CD19 and CD20.

[0192] Although the data disclosed herein specifically discloses lentiviral vectors comprising CD19CD20 scFv, hinge and transmembrane domains, and 4-1BB and CD3ζ signaling domains, the invention should be construed to include any number of mutations in each component of the construct, as described elsewhere herein.

[0193] Cancers that can be treated include non-vascularized or mostly non-vascularized tumors, and vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or solid tumors. Types of cancers that can be treated with the CARs of the present invention 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.

[0194] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0195] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0196] The CAR-modified T cells of the present invention can also serve as a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. Preferably, the mammal is a human.

[0197] For ex vivo immunization, at least one of the following is performed in vitro prior to administering the cells to a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0198] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transduced in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0199] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0200] The present invention provides a method for treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR modified T cell of the present invention.

[0201] The CAR modified T cells of the present invention may be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include the target cell populations described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, 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 the present invention are preferably formulated for intravenous administration.

[0202] The pharmaceutical composition of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, and the appropriate dose can be determined by clinical trials.

[0203] When an "immunologically effective amount", "antitumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 6The T cell composition may be administered at a dose of cells / kg body weight, generally including all integer values ​​within these ranges. The T cell composition may also be administered multiple times at these doses. The cells may be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient may be easily determined by those skilled in the art of medicine by monitoring the patient for symptoms of disease and adjusting the treatment accordingly.

[0204] Administration of the subject compositions may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intralymph node, intramedullary, intramuscularly, intravenously (iv) injection, or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the invention are preferably administered by iv injection. The T cell compositions may be injected directly into a tumor, lymph node, or site of infection.

[0205] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art whereby T cells are expanded to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously, or after) any number of relevant therapeutic modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are 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), cyclophosphamide, etc. For example, in one embodiment, a subject may undergo standard treatment with high dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplant, the subject receives an infusion of the expanded immune cells of the invention. In further embodiments, the expanded cells are administered before or after surgery.

[0206] The dosage of the above treatments administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for patient administration can be performed according to art-accepted practices. Generally, dosages of 1×10 6 ~1×10 10 The modified T cells of the present invention (e.g., CAR-T-19 / 20 cells) can be administered to the patient by means of each treatment or each course of treatment, for example, by intravenous infusion.

[0207] The advantages of the present invention are as follows:

[0208] (1) Regarding the chimeric antigen receptor of the present invention, the extracellular antigen-binding domain is a specific anti-CD20 scFv and an anti-CD19 scFv, and the CAR formed by combining a specific anti-CD20 scFv and an anti-CD19 scFv with a specific hinge region and an intracellular domain shows a great ability to kill tumor cells with low cytotoxicity and low side effects.

[0209] (2) The chimeric antigen receptor provided by the present invention can achieve stable expression and membrane localization of the CAR protein after T cells are infected with a lentivirus carrying the CAR gene.

[0210] (3) The CAR-modified T cells of the present invention have a longer survival time and stronger anti-tumor efficacy in vivo, and the optimized CAR with an IgG4 hinge-CH2-CH3 linker region can avoid Fc receptor binding and subsequent ADCC (antibody-dependent cellular cytotoxicity).

[0211] (4) Compared with two independent CARs, the bispecific chimeric antigen receptor of the present invention contains both anti-CD20 scFv and anti-CD19 scFv, which significantly reduces the DNA footprint (DNA length is reduced by 40%) and has a small structure size, which is beneficial to the packaging and transduction efficiency of the viral vector, thus directly improving the clinical efficacy. Furthermore, the bispecific CAR of the present invention has a lower cost, a higher cure rate, and is safer.

[0212] (5) T cells modified with the TN-LEU-19 or TH-OF-19 chimeric antigen receptor of the present invention have a very strong ability to kill malignant tumors bearing CD19 and CD20 positive antigens in vitro and eliminate them in vivo, with ofatumumab being more potent, which provides a new and effective method and preparation for the clinical application of CAR-T in the treatment of CD19 and CD20 positive leukemia and lymphoma.

[0213] (6) The CAR-T cells of the present invention have a killing effect on most malignant B cell tumors, have a broader therapeutic range and greater applicable rate, and can more effectively prevent tumor cell escape.

[0214] The present invention will be further described below with reference to 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. The experimental methods without specific conditions described in the following examples are generally carried out under conventional conditions or according to the manufacturer's instructions. Percentages and parts are by weight unless otherwise stated.

[0215] [Example 1] Construction of lentiviral expression vector The full-length DNA was synthesized and cloned to construct a plasmid coding. The pWPT lentiviral vector was selected as the cloning vector, and the cloning sites were BamH I and Sal I sites. Here, the structures of the two CARs designed in the present invention are shown in Figure 1. The amino acid sequence of TN-LEU-19 is shown in SEQ ID NO: 15, and the amino acid sequence of TN-OF-19 is shown in SEQ ID NO: 16 (having the structure of L-(OF)VL-(OF)VH-I-(FMC63)VH-(FMC63)VL-H-TM-C-CD3ζ).

[0216] [Example 2] Detection of in vitro activation ability of bispecific CARs PBMCs were isolated from venous blood of healthy donors by density gradient centrifugation. On day 0, PBMCs were activated in cell culture flasks pre-coated with CD3 monoclonal antibody (OKT3) and RetroNectin (TAKARA). The medium was GT-551 cell culture medium containing 1% human albumin and 300 U / mL recombinant human interleukin 2 (IL-2). On day 3, activated PBMCs were transduced with purified TN-LEU-19 or TN-OF-19 lentivirus solution. From day 6, TN-OF-19 and TN-LEU-19 CAR-T cells can be harvested for corresponding activity assays. Protein L method was used to determine the expression levels of CAR gene-encoded proteins on the surface of T cell membrane in CAR-T cells cultured for 7 days.

[0217] T cell activation marker CD137 and IFNγ release were detected using CD19 / 20 bispecific CAR-T cells cultured for 7 days. 5 CAR-T cells were co-cultured with CD19, CD20 positive K562-CD19+, K562-CD20+, K562-CD19+CD20+ and Raji (naturally expressing CD19 and CD20) tumor cell lines, and CD19CD20 negative K562 tumor cell lines, or without tumor cells, in 200 μl of GT-551 medium at a ratio of 1:1 for 18 hours. Then, the expression level of CD137 on the surface of T cells and the secretion level of IFNγ in the culture supernatant were detected, respectively.

[0218] The results are shown in Figures 2A and 2B. The expression of CD137 on the surface of the two CART cells was detected, and the expression of IFNγ in the culture supernatant was detected. Here, TN-OF-19 cells have higher CD137 activation levels and IFNγ release levels than TN-LEU-19 cells.

[0219] [Example 3] Detection of cytotoxicity of CD19 / 20 bispecific CAR-T cells in vitro The CAR-T cells prepared in Example 2 were tested for cytotoxicity by LDH release assay. The target cells were K562, K562-CD19+, K562-CD20+, K562-CD19+CD20+, and Raji cells, and the effector cells were NT, TN-LEU-19, and TN-OF-19 cells. The effector-target ratios were set as follows: effector cell number: target cell number=5:1, 10:1, 20:1, and 40:1. The results are shown in Figure 3A. Both TN-LEU-19 CAR-T cells and TN-OF-19 CAR-T cells can successfully induce apoptosis and release LDH in CD19 / 20 positive tumor cells.

[0220] The levels of CD107a (a marker of T cell degranulation) release during tumor cell killing induced by CAR-T19 / 20 cells were analyzed by flow cytometry. 5 The effective cells of CAR-T19 / 20 (TN-OF-19 and TN-LEU-19) were added at 2 × 10 5 The TN-OF-19 cells were co-cultured with target tumor cells of each of the 19 and 20 cells. The target cells were K562-CD19+, K562-CD20+, K562-CD19+CD20+, and K562, Raji, and Romas cells (CD19+CD20+), respectively. The results are shown in Figure 3B. Both CART cells can successfully induce the release of CD107a during tumor cell killing. Here, TN-OF-19 cells have a slightly higher release of CD107a as a marker of cytotoxicity than TN-LEU-19 cells.

[0221] [Example 4] Inhibitory effect of CAR-T19 / 20 on transplanted tumor cells in mice The tumor cells injected into the animals were Raji cells carrying a luciferase reporter gene (luciferase-expressing Raji). In this experiment, tumor cells Raji were injected and allowed to grow in mice for one week, and then effector T cells were injected. The effector T cells were divided into three groups: NT, TN-LEU-19, and TN-OF-19. The expanded effector T cells were injected into NSG mice through the tail vein, and then the fluorescence intensity (by IVIS fluorescence imaging) and the weight of the mice were recorded every 7 days. The experiment was stopped on day 21, and the statistical results were analyzed.

[0222] The results are shown in Figure 4. Figure 4A shows the weight change of mice after injection of effector T cells into three groups of mice. Compared with the two types of CART cells, CART-TN-OF-19 and CART-TN-LEU-19, the mice in NT showed a significant decrease in weight, and the mice in both CART cell groups showed a slight increase in weight. Figure 4B shows the average fluorescence intensity of the three groups of mice. The results show that the average fluorescence intensity of the mice in the NT group increased significantly, while the average fluorescence intensity of the mice in the two CART cell groups decreased, indicating that both TN-OF-19 and TN-LEU-19 CART cells can inhibit tumor growth compared with NT. Here, TN-OF-19 cells have better inhibition on tumor growth than TN-LEU-19 cells after 14 days, and the tumor growth curve of the TN-LEU-19 group recurred significantly. Figure 4C shows IVIS imaging of the fluorescence intensity in the two CART cell groups and the NT group. TN-OF-19 CAR-T cells can inhibit or kill tumor cells in vivo better than TN-LEU-19 cells.

[0223] [Example 5] Comparison of the inhibitory effects of TN-OF-19 CAR-T cells and CD19-specific CAR-T cells in vivo 1×10 6of firefly luciferase-expressing Raji cells were administered to 6-8 week-old NPG mice (NOD-Cg.PrkdcSCID IL-2Rgcnull / vst) by tail vein injection. Seven days later, tumor engraftment was measured by ip injection of D-luciferin and imaging 10-15 min later with a Bruker In Vivo Xtreme Imaging System (Bruker, Xtreme BI) for 180 s. Mice were equally distributed into test groups (n=6 / group) based on tumor burden. TN-OF-19(L), TN-OF-19(M), and TN-OF-19(H) groups were administered 1×10 6-cell cultures by tail vein injection. 6 , 2.5×10 6 , and 5 × 10 6 Each patient was treated separately with 1 × 10 CAR+ T cells. 6 Non-transduced T cells (NT) and single target CD19-specific CAR-T cells from the same donor were used as controls. Tumor growth was evaluated based on mouse whole body average radiance at 7, 10, and 21 days after injection. Figure 5A shows that bispecific TN-OF-19 CAR-T cells can inhibit or kill tumor cells better than CD19-specific CAR-T cells in vivo. Especially in the early stage of treatment, bispecific TN-OF-19 CAR-T shows a significantly faster inhibitory effect. Figure 5B shows IVIS imaging of the fluorescence intensity in the four CAR-T cell groups and the NT group.

[0224] [Example 6] Antigen specificity of CD20 / CD19 bispecific scFV in TN-OF 19CAR To investigate the affinity and specificity of the bispecific scFv in TN-OF 19CAR, a chimeric rabbit monoclonal antibody was generated by linking in frame CD20 scFv (derived from ofatumumab mAb) and CD19 scFv (derived from FMC63 mAb) with the Fc region of rabbit IgG1. CD19 and CD20 scFv were alternately linked by a G4S linker. The molecular order was OF VL-VH-G4S-FMC63 VH-VL, which is the same as the scFv in TN-OF-19. The chimeric antibody was expressed in 293T cells after transient transfection. Validation of this chimeric antibody for staining specificity was performed by flow cytometry. Briefly, three stable cell lines (A549-CD19, A549-CD20, A549-CD19-CD20) were used as targets, and CD19-CD20-A549 cells and CD19+CD20+Raji cells were used as controls. All cells were washed, resuspended, and blocked with 2% serum for 30 min. 5 × 10 5 The cells were transferred to FACS vials, washed and stained with the recombinant antibodies (final concentration 20 μg / mL) for 1 h at 4° C. After washing, secondary antibodies (goat anti-rabbit IgG) were added for 30 min at 4° C. in the dark. Finally, the cells were washed and resuspended in 200 μL of FACS buffer for FACS analysis. Figure 6 shows chimeric antibodies that bind to cells bearing CD19 or CD20 or both, but not to cells lacking both antigens, indicating that the bispecific binding domain requires only one cognate antigen for binding and that no new specific recognition sites were formed.

[0225] [Example 7] Screening and functional validation of CD20-specific CARs (to generate CD20 / 19 bispecific CARs) Prior to constructing the CD20 / CD19 bispecific CAR, we performed extensive studies to screen and narrow down the CD20-specific scFv candidates. Figure 7 shows the structures of 16 CD20-specific CARs with 6 different scFvs and different hinge / TM / signaling domains. Full-length DNA was synthesized and cloned to achieve the construction of plasmid-encoded, and we tested the anti-tumor activity of these CARs on various CD20-expressing target cells.

[0226] Figure 8A shows the results of IFNγ release assay to screen CAR-T20.1, CAR-T20.5, CAR-T20.6, CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10, among these CAR-Ts, only CAR-T20.9(Leu16) and CAR-T20.10(Leu16) showed higher positive IFNγ release.

[0227] Figure 8B shows the results of IFNγ release assay for screening CAR-T20.1, CAR-T20.9, CAR-T20.10, CAR-T20.11, CAR-T20.12, CAR-T20.13, CAR-T20.14, CAR-T20.15 and CAR-T20.16. Among these CAR-Ts, CAR-T20.10 (Leu16) and CAR-T20.14 (OF) showed higher positive IFNγ release.

[0228] Figure 8C shows the results of IFNγ release assay for screening CAR-T20.9, CAR-T20.12, CAR-T20.14, CAR-T20.17, CAR-T20.18, and CAR-T20.19. Among these CAR-Ts, CAR-T20.14(OF) and CAR-T20.19(OF) showed higher positive IFNγ release.

[0229] Figure 9A shows the results of CAR-T20.17 (Leu16 3rd generation), CAR-T20.18 (Leu16 2nd generation), CAR-T20.19 (OF 2nd generation) cells tested for cytotoxicity by LDH release assay. Target cells are CD20 positive cell lines Raji and Ramos, and CD20 negative Molt4. All three CD20 CAR-T cells can induce apoptosis and release LDH in CD20 positive tumor cells, indicating that CAR-T20.17, CAR-T20.18, and CAR-T20.19 have a strong killing effect on target cells CD20 positive Raji and Romas cells.

[0230] Figure 9B shows the in vivo inhibition of tumor growth in NSG mice study. The tumor cells injected into the animals are Raji expressing luciferase. In this experiment, tumor cells Raji are injected and allowed to grow in the mice for one week, then effector T cells are injected through the tail vein, and then the fluorescence intensity (by IVIS fluorescence imaging) and the weight of the mouse are recorded every 7 days. The experiment was stopped on day 21, and the statistical results were analyzed. The results show that CAR-T20.19(OF) cells have better inhibition of tumor growth than CAR-T20.17(Leu16) and CAR-T201.8(Leu16) cells after 14 days.

[0231] In summary of Example 7 (Figures 7-9), through numerous experiments and comparisons, it was found that 20.1, 20.2, 20.4, 20.5, 20.6, 20.7, 20.8 and 20.15 were basically ineffective, 20.11, 20.12 and 20.13 had certain effects, but their effects were smaller than those of 20.9, 20.10, 20.14, 20.16, 20.17, 20.18 and 20.19, and the effect of 20.19 (OF) was the best. Based on the above structure, CD20 scFv (OF) and CD19 scFv (FMC63) were used in tandem in a new bispecific chimeric antigen receptor TN-OF-19, which was considered as the best candidate for further analysis.

[0232] The amino acid sequence of the CD20-specific CAR involved in Example 7 is shown in Table 1. [Table 3] TIFF0007624967000004.tif239164TIFF0007624967000005.tif237160TIFF0007624967000006.tif227163

[0233] [Example 8] Phase I clinical trial of TN-OF-19 CAR-T cells A phase I study was conducted in r / r NHL patients at Shanghai Tongji Hospital to evaluate the safety and efficacy of C-CAR039 (i.e., TN-OF-19CAR-T cells, NCT04317885). After apheresis to harvest T cells, C-CAR039 was manufactured and infused as a single intravenous dose after a standard 3-day cyclophosphamide / fludarabine conditioning regimen. C-CAR039 was manufactured in a serum-free, semi-automated digital closed system with a median vein-to-vein time of 18 days. The manufacturing success rate was 100%. As of August 3, 2020, 16 patients have received 1.0 × 10 6 ~5.0×10 6 Patients were infused with C-CAR039 at a dose range of 100-200 mg / kg CAR-T cells. Fourteen patients had at least 1 month of evaluable safety data, and 13 patients (11 with DLBCL and 2 with FL) had efficacy data of 1 month or more. Figure 10 shows the CAR039r / r NHL study design and flow chart.

[0234] The median age of treated patients was 58.5 years (range: 28-71 years). The median number of prior lines of therapy was 2 (range: 2-5 prior therapies). Three patients (21%) had previously undergone autologous stem cell transplantation (ASCT). [Table 4] [Table 5]

[0235] C-CAR039 treatment was well tolerated, with no grade 3 or higher CRS and no neurotoxic events. Reversible grade 1-2 CRS was observed in 9 (82%) patients. Transplant conditioning cytopenias were common and reversible.

[0236] At 1 month, 12 / 13 patients showed clinical improvement (ORR=92%) and 11 / 11 DLBCL patients responded to treatment (ORR=100%). Median follow-up was 70 days (range: 35-257 days). Best overall response (BOR) included 10 complete responses (CR) and 2 partial responses (PR).

[0237] Figure 11 shows a summary of C-CAR039 clinical results.

[0238] Figure 12 shows an example of a patient before and after C-CAR039 treatment.

[0239] [Example 9] PK profile of C-CAR039 Growth and expansion of C-CAR039 in peripheral blood was positively correlated with tumor regression. A positive initial trend of correlation between C-CAR039 AUC (days 0-28) and Cmax and clinical response is observed.

[0240] Figure 13 shows the growth and expansion of C-CAR039 in the patient's blood. The results showed that C-CAR008 cells expanded effectively after injection. [Table 6]

[0241] Growth and expansion of C-CAR039 in peripheral blood was positively correlated with tumor regression. A positive initial trend of correlation between C-CAR039 AUC (days 0-28) and Cmax and clinical response is observed. [Table 7] [Table 8]

[0242] C-CAR039 shows promising efficacy and a favorable safety profile in early clinical trials in r / r NHL patients. Early clinical efficacy signals are encouraging and compare favorably with anti-CD19 CAR-T therapy. These findings need to be evaluated in larger numbers of patients with longer follow-up to confirm safety, efficacy, and duration of response.

[0243] [Example 10] Phase I clinical trial of C-CAR066 (i.e., CAR-T20.19(OF)) Relapse due to loss of CD19-targeted epitopes presents a therapeutic challenge for CD19 CAR-T therapy. These patients generally have a poor prognosis. CD20 is a proven therapeutic target for B-cell non-Hodgkin's lymphoma (B-NHL) and is supported by previously approved and widely used monoclonal antibody therapies. C-CAR066 is a novel second-generation chimeric antigen receptor T (CAR-T) therapy. Preclinical studies suggest that C-CAR066 (derived from the scFV of ofatumumab) has superior antitumor activity compared to CAR-T derived from the scFVs of Leu16, rituximab, and obinutuzumab.

[0244] NCT04036019 is a single-arm, single-center, non-randomized Phase I clinical trial to evaluate the safety and efficacy of C-CAR066 in subjects with r / r B-cell lymphoma previously treated with anti-CD19 CAR-T therapy. The primary objective of the study is to evaluate the incidence and severity of treatment-emergent adverse events. Secondary objectives include determining the overall response rate (ORR), PFS, and OS. C-CAR066 will be manufactured in a serum-free, semi-automated, digital, closed system. C-CAR066 will be administered to patients as a single intravenous dose after a standard 3-day cyclophosphamide / fludarabine transplant conditioning regimen.

[0245] FIG. 14 shows the C-CAR066-NHL study design.

[0246] As of August 3, 2020, seven patients (all with DLBCL) have been enrolled, with a recurrence rate of 2.0 × 10 6 ~5.0×10 6 C-CAR039 with a range of doses of CAR-T cells was infused. The production success rate was 100%. All patients relapsed after anti-CD19 CAR-T treatment, and only one of the patients achieved a CR after anti-CD19 CAR-T therapy. [Table 9]

[0247] C-CAR066 treatment was well tolerated with reversible grade 1-2 CRS in six patients and grade 3 CRS in another patient, without any neurotoxic events. 6 / 7 patients showed clinical improvement (best overall response rate, ORR=85.7%). Best overall responses included 3CR and 3PR. All patients responded to C-CAR066 treatment and showed different degrees of tumor regression (45-100%). [Table 10]

[0248] Six out of seven patients showed clinical improvement (best overall response rate, ORR=85.7%). Best overall responses included 3CR and 3PR. All patients responded to C-CAR066 treatment and showed different degrees of tumor regression (45-100%). [Table 11]

[0249] FIG. 15 shows an example of a PET-CT of a patient before and after treatment with C-CAR066.

[0250] C-CAR066 has a favorable safety profile and shows promising early efficacy in r / r NHL patients after CD19 CAR-T therapy. This indicates that C-CAR066 has a different mechanism of action compared to anti-CD19 CAR-T therapy. Targeting both CD20 and CD19 tumor antigens can lead to superior clinical benefit in patients with B-cell malignancies than targeting either CD19 or CD20 alone.

[0251] All documents mentioned in this application are incorporated herein by reference as if each were individually incorporated by reference. Furthermore, it should also be understood that after reading the above teachings of the present invention, a person skilled in the art may make various changes or modifications, the equivalents of which are included within the scope of the claims defined in the appended claims.

Claims

1. A chimeric antigen receptor (CAR), the structure of which is represented by formula II: L-V L1 -V H1 -I-V H2 -V L2 -H-TM-C-CD3ζ (II) During the ceremony, each "-" is independently a linker peptide or a peptide bond; L is an optional signal peptide sequence, V L1 is an anti-CD20 antibody light chain variable region; V H1 is an anti-CD20 antibody heavy chain variable region; I is a flexible linker, VH2 is an anti-CD19 antibody heavy chain variable region; V L2 is an anti-CD19 antibody light chain variable region; H is an optional hinge region, TM is the transmembrane domain; C is a costimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ, a chimeric antigen receptor.

2. The V Ｈ１ The amino acid sequence of V is shown in SEQ ID NO:

3. Ｌ１ The CAR of claim 1, wherein the amino acid sequence is set forth in SEQ ID NO:

4.

3. A cell expressing the chimeric antigen receptor of claim 1.

4. The cell of claim 3, wherein the cell is a CAR-T cell and / or a CAR-NK cell.

5. A pharmaceutical composition for treating a disease comprising the cells described in claim 3.

6. The pharmaceutical composition according to claim 5 , wherein the disease is cancer or a tumor.

7. A bispecific chimeric antigen receptor (CAR) (i) a light chain variable region (V) having the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 3, respectively; Ｌ 1) and the heavy chain variable region (V Ｈ 1), wherein V L 1 is located N-terminal to V H 1; (ii) a light chain variable region (V Ｌ 2) and the heavy chain variable region (V Ｈ 2), wherein V H2 is positioned N-terminal to V L2; (iii) a transmembrane domain having the amino acid sequence set forth in SEQ ID NO:11; (iv) a costimulatory signaling region having the amino acid sequence set forth in SEQ ID NO: 12; and (v) a cytoplasmic signaling domain having the amino acid sequence set forth in SEQ ID NO:

14. A bispecific CAR comprising:

8. 8. The bispecific CAR of claim 7, wherein said anti-CD20 antigen binding region is a single chain variable fragment (scFv) that specifically binds to CD20 and said anti-CD19 antigen binding region is a scFv that specifically binds to CD19.

9. (a) a signal peptide having the amino acid sequence set forth in SEQ ID NO:8; and (b) a hinge region having the amino acid sequence set forth in SEQ ID NO:9; The bispecific CAR of claim 7, further comprising:

10. In the N-terminal to C-terminal direction, the signal peptide, Ｌ 1. V Ｈ 1. V Ｈ 2. V Ｌ 2. The bispecific CAR of claim 9, comprising the hinge region, the transmembrane domain, the costimulatory signaling region, and the cytoplasmic signaling domain.

11. An immune cell expressing the bispecific CAR described in claim 7.

12. The immune cell of claim 11, which is a T cell or a natural killer (NK) cell.

13. A pharmaceutical composition comprising the immune cells described in claim 11.

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