Combined chimeric antigen receptor targeting CD19 and CD20 and application thereof

Abstract

To provide a combined chimeric antigen receptor targeting CD19 and CD20 and applications thereof.SOLUTION: The present invention provides a combined chimeric antigen receptor targeting CD19 and CD20, which comprises a scFv targeting CD19 and CD20, a hinge region, a transmembrane region, and an intracellular signaling domain. The present invention provides a nucleic acid molecule encoding a chimeric antigen receptor and a corresponding expression vector, a CAR-T cell, and applications thereof. The experimental results indicates that the chimeric antigen receptor shows 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.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] Hematological malignancies account for approximately 10% of human malignant tumors, and 95% of hematological malignancies are B-cell lymphoma. Traditional chemotherapy and radiation therapy are used in the treatment of hematological malignancies. Although some patients benefit significantly, most patients are cured. It is difficult to cure. New and effective treatments are a hot topic in the field. It is.

[0003] Adoptive T cell therapy has demonstrated strong efficacy and promising prospects in the clinical treatment of malignant tumors. Currently, adoptive T cell therapy is one of the most promising methods for treating hematological malignancies. CD19 is highly expressed on the surface of most B cell malignancies. Chimeric antigen receptor (CAR)-modified T cells were independently used to inhibit the expression of CD19-expressing B cells. Multiple centers have achieved unprecedented success targeting relapsed and refractory malignancies. Both of the two CAR-T products approved by the DA target the CD19 antigen and are used to treat chronic The indications for anti-CD19 CAR-T are expanding to include lymphocytic leukemia. However, many studies have shown that CD19 chimeric antigen receptor (CAR) T cell therapy has many problems. There are still some patients who have poor treatment outcomes and are prone to relapse. Reasons for this include the tendency of tumor cells to undergo antigen escape. For example, A recent trial of CD19 CAR-cell therapy showed that 90% of patients achieved complete remission. Eleven percent of these patients eventually relapsed, primarily in patients with CD19-negative tumors. In particular, the efficacy of refractory acute B-cell lymphoma (R / R B-ALL) was demonstrated. A clinical trial conducted at the University of Pennsylvania School of Medicine using CART19 in treatment In the trial, up to 94% of patients achieved a complete remission. Early response rates in this clinical trial were high. However, almost 40% of patients relapsed one month after treatment that achieved complete remission, and 60% of patients relapsed. More than % of patients had CD19-negative tumor cell escape. Antigen escape was observed in NY-ESO Adoptive transfer of specific T cell receptors expressing 1 and a cancer vaccine found in treating melanoma Spontaneous mutation and selective expansion are the main reasons for antigenic escape.

[0004] Therefore, it is of great importance to the art to develop methods to effectively treat tumors and prevent antigen escape. There is an urgent need in the field. Summary of the Invention [Problem to be solved by the invention]

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

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

[0007] Specifically, a combination of chimeric antigen receptor sequences targeting CD19 and CD20 is The present invention provides a method for preparing and identifying the activity of modified T cells (CART-19 / 20). The present invention relates to a method for treating CD19 and CD20 positive B cell lymphoma. The present invention provides a chimeric antigen receptor structure for use in

[0008] The present disclosure provides a bispecific chimeric antigen receptor (CAR). A bispecific CAR comprises: (i) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 4 and SEQ ID NO: 3, respectively ( V L 1) and the heavy chain variable region (V H (i) an anti-CD20 antigen-binding region comprising: A light chain variable region (V) having the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6 L 2) and 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 is a single-chain variable fragment (sc 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 specifically bound to CD19. It is located at the N-terminus of the heterologous binding scFv.

[0011] Bispecific CARs consist of (a) a leader sequence, (b) a hinge region, and (c) a transmembrane domain. (d) at least one costimulatory signaling region; and (e) a cytoplasmic signaling domain. or (f) any combination thereof.

[0012] Costimulatory signaling regions include 4-1BB (CD137), CD28, OX40, and 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 is derived from Ig4, CD8, CD28, CD137, or a combination thereof. possible.

[0015] Transmembrane domains include CD8, CD28, CD3ε, CD45, CD4, CD5, and CD9. , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD13 4, CD137, CD154, or a combination thereof.

[0016] In a particular embodiment, the bispecific CAR has the amino acid sequence set forth in SEQ ID NO: 16. obtain.

[0017] The present disclosure provides an immune cell expressing a bispecific CAR of the present invention. The immune cells may be allogeneic or autologous. It is possible.

[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 includes administering immune cells to a subject in need thereof. The method may include administering to a subject.

[0021] The cancer may be a hematological cancer. The cancer may be a B-cell malignancy. The cancer may be Hodgkin's lymphoma, The cancer may be non-Hodgkin's lymphoma, leukemia, and / or multiple myeloma. Myeloma (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML) Hematologic malignancies, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL) ), or a combination thereof.

[0022] In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, The structure 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 is an antigen-binding domain that targets CD19. the other is an antigen-binding domain that targets CD20.

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

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

[0025] In another preferred embodiment, the structure of the antigen-binding domain targeting CD20 is represented by Formula B: As shown.

[0026] In another preferred embodiment, V H1 The amino acid sequence of is shown in SEQ ID NO:1, L1 No.A The amino acid sequence 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 is shown in SEQ ID NO:4. will be done.

[0027] In another preferred embodiment, V H1 and V L1 is a flexible linker (or linker peptide) and a flexible linker (or linker peptide) is linked to SEQ ID NO: 7 (GGGG S) 1 to 4, preferably 2 to 4, more preferably 3 to 4 consecutive sequences It is.

[0028] In another preferred embodiment, V L1 and V H1 is a movable nucleic acid sequence as shown in SEQ ID NO:19. The linker is linked to a carboxylic acid.

[0029] In another preferred embodiment, the structure of the antigen-binding domain targeting CD19 is As shown in formula C or formula D, 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 is an anti-CD19 antibody heavy chain variable region, and "-" is a linker peptide or a peptide bond.

[0030] In another preferred embodiment, the structure of the antigen-binding domain targeting CD19 is represented by formula D As shown.

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

[0032] In another preferred embodiment, V H2 and V L2 is a flexible linker (or linker peptide) and a flexible linker (or linker peptide) is linked to SEQ ID NO: 7 (GGGG S) 1 to 4, preferably 2 to 4, more preferably 3 to 4 consecutive sequences It is.

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

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

[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 the flexible linker I is SEQ ID NO: 7 (GGGGS). It contains 2 to 6, preferably 3 to 4, consecutive sequences as shown.

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

[0038] In another preferred embodiment, L is CD8, CD28, GM-CSF, CD4, CD1 37, and combinations thereof. be.

[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 selected from CD8, CD28, CD137, Ig4, and the like. The hinge region of the protein is selected from the group consisting of:

[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 CD28, CD3ε, CD45, CD4, CD5 , CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, C D86, CD134, CD137, CD154, and combinations thereof. The transmembrane domain of the protein to be selected.

[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 OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB(CD137), PD1, Da p10, CDS, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD 278), NKG2D, GITR, TLR2, and combinations thereof. It is a costimulatory signaling molecule that is a protein that is stimulated by

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

[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, the anti-CD20 antigen binding region is set forth in SEQ ID NO:3 or SEQ ID NO:1. and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, At least or about 99%, at least or about 81%, at least or about 82%, at least At least about 83%, at least about 84%, at least about 85%, at least 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 At least about 93%, at least about 94%, at least about 95%, at least about 96%, at least or about 97%, at least or about 98%, at least or about 99%, Alternatively, it comprises a heavy chain variable region that comprises an amino acid sequence that is about 100% identical.

[0053] In certain embodiments, the anti-CD20 antigen binding region is set forth in SEQ ID NO:4 or SEQ ID NO:2. and at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, At least or about 99%, at least or about 81%, at least or about 82%, at least At least about 83%, at least about 84%, at least about 85%, at least 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 At least about 93%, at least about 94%, at least about 95%, at least about 96%, at least or about 97%, at least or about 98%, at least or about 99%, Alternatively, it comprises a light chain variable region that comprises an amino acid sequence that is about 100% identical.

[0054] In certain embodiments, the anti-CD19 antigen binding region has the amino acid sequence set forth in SEQ ID NO:6. , at least or about 70%, at least or about 75%, at least or about 80%, At least about 85%, at least about 90%, at least about 95%, at least about 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 about 87%, at least about 88%, at least about 89%, at least about 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 about 97%, at least about 98%, at least about 99%, or at least about 10 The heavy chain variable regions contain amino acid sequences that are 0.0% identical to each other.

[0055] In certain embodiments, the anti-CD19 antigen binding region has the amino acid sequence set forth in SEQ ID NO:5. , at least or about 70%, at least or about 75%, at least or about 80%, At least about 85%, at least about 90%, at least about 95%, at least about 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 about 87%, at least about 88%, at least about 89%, at least about 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 about 97%, at least about 98%, at least about 99%, or at least about 10 The light chain variable region comprises an amino acid sequence that is 0.0% identical to the light chain variable region.

[0056] The heavy chain variable region of the anti-CD20 antigen-binding domain is complementary to the heavy chain variable region of the ofatumumab antibody. The sex determining region (CDR) (positions 30 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 3) CDR1, CDR2 and CDR3 described below), or the heavy chain variable region of the Leu16 antibody CDRs (Cs described at positions 31 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 1, respectively) DR1, CDR2 and CDR3) and at least or about 70%, at least or about 75% , at least or about 80%, at least or about 85%, at least or about 90%, At least about 95%, at least about 99%, at least about 81%, at least about 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 about 89%, at least about 90%, at least about 91%, at least about 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%, and / or a sequence that is about 99% or about 100% identical to one, two, or three CDRs. It can be seen.

[0057] The light chain variable region of the anti-CD20 antigen-binding domain is complementary to the light chain variable region of the ofatumumab antibody. The sex determining region (CDR) (positions 24-34, 50-56, and 89-97 of SEQ ID NO: 4) CDR1, CDR2 and CDR3 described respectively), or the C of the light chain variable region of the Leu16 antibody DR (CDRs described at positions 24 to 33, 49 to 55, and 88 to 96 of SEQ ID NO: 2, respectively) 1, CDR2 and CDR3) and at least or about 70%, at least or about 75%, At least about 80%, at least about 85%, at least about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 8 2%, at least or about 83%, at least or about 84%, at least or about 85%, At least about 86%, at least about 87%, at least about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 9 2%, at least or about 93%, at least or about 94%, at least or about 95%, At least about 96%, at least about 97%, at least about 98%, at least or may contain one, two, or three CDRs that are about 99%, or about 100%, identical to the sequence of the amino acid sequence of do.

[0058] The heavy chain variable region of the anti-CD20 antigen-binding domain is complementary to the heavy chain variable region of the ofatumumab antibody. Sex determining regions (CDRs) (Cs shown in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively) DR1, CDR2 and CDR3), or the CDRs of the heavy chain variable region of the Leu16 antibody (SEQ ID NO: CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 51, respectively. ) and at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, at least about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 8 3%, at least or about 84%, at least or about 85%, at least or about 86%, At least about 87%, at least about 88%, at least about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 9 3%, 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 It may contain one, two or three CDRs that are 100% identical.

[0059] The light chain variable region of the anti-CD20 antigen-binding domain is complementary to the light chain variable region of the ofatumumab antibody. Sex determining regions (CDRs) (Cs shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively) DR1, CDR2 and CDR3), or the CDRs of the light chain variable region of the Leu16 antibody (SEQ ID NO: CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:58, respectively. ) and at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, at least about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 8 3%, at least or about 84%, at least or about 85%, at least or about 86%, At least about 87%, at least about 88%, at least about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 9 3%, 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 It may contain one, two or three CDRs that are 100% identical.

[0060] In certain embodiments, the heavy chain variable region of the anti-CD20 antigen binding region is selected from the group consisting of the ofatumumab antibody. The three CDRs (positions 30 to 35 and 50 to 66 of SEQ ID NO: 3) are identical to the CDRs of the heavy chain variable region of and CDR1, CDR2 and CDR3 described at positions 99 to 111 of the sequence of the anti-CD20 antigen. The light chain variable region of the binding region has three CDRs identical to those of the light chain variable region of the ofatumumab antibody. CDRs (Cs at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 4) DR1, CDR2 and CDR3).

[0061] In certain embodiments, the heavy chain variable region of the anti-CD20 antigen binding region is selected from the group consisting of the ofatumumab antibody. The heavy chain variable region of the present invention has three CDRs identical to those of the heavy chain variable region of the present invention (SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42) and an anti-CD20 antigen-binding domain. The light chain variable region of the region has three CDRs identical to the CDRs of the light chain variable region of the ofatumumab antibody. R (CDR1, CDR2 and CDR3 shown in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively) and CDR3).

[0062] In certain embodiments, the heavy chain variable region of the anti-CD20 antigen binding region comprises the heavy chain variable region of the Leu16 antibody. The three CDRs are the same as those of the chain variable region (positions 31 to 35, 50 to 66, and 99 to 111), respectively, and The light chain variable region of the antigen-binding region has three CDRs identical to those of the light chain variable region of the Leu16 antibody. CDRs (represented at positions 24 to 33, 49 to 55, and 88 to 96 of SEQ ID NO: 2) CDR1, CDR2 and CDR3).

[0063] In certain embodiments, the heavy chain variable region of the anti-CD20 antigen binding region comprises the heavy chain variable region of the Leu16 antibody. The three CDRs (SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51) are identical to the CDRs of the chain variable region. an anti-CD20 antigen-binding region comprising CDR1, CDR2 and CDR3 as described in The light chain variable region of the antibody has three CDRs (sequences: CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:58, respectively. R3).

[0064] The heavy chain variable region of the anti-CD19 antigen binding region is a complementarity determining region of the heavy chain variable region of the FMC63 antibody. CDR (positions 31-35, 50-65, and 98-109 of SEQ ID NO:6) and at least about 70%, at least about 80%, 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 at least about 82%, at least about 83%, at least about 84%, at least about 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 at least about 92%, at least about 93%, at least about 94%, at least about 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 one, two, or three It may contain CDRs.

[0065] The light chain variable region of the anti-CD19 antigen binding domain is a complementarity determining region of the light chain variable region of the FMC63 antibody. Constant region (CDR) (positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 5, respectively) and at least about 70%, at least about 80%, 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 at least about 82%, at least about 83%, at least about 84%, at least about 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 at least about 92%, at least about 93%, at least about 94%, at least about 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 one, two, or three It may contain CDRs.

[0066] The heavy chain variable region of the anti-CD19 antigen binding region is a complementarity determining region of the heavy chain variable region of the FMC63 antibody. Constant region (CDR) (CDs shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62) R1, CDR2 and CDR3), and at least or about 70%, at least or about 75%, At least or about 80%, at least or about 85%, at least or about 90%, at least at least about 95%, at least about 99%, at least about 81%, at least about 82%, at least or about 83%, at least or about 84%, at least or about 85%, At least about 86%, at least about 87%, at least about 88%, at least at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least or about 93%, at least or about 94%, at least or about 95%, At least about 96%, at least about 97%, at least about 98%, at least or about 99%, or about 100% identical to one, two, or three CDRs. obtain.

[0067] The light chain variable region of the anti-CD19 antigen binding domain is a complementarity determining region of the light chain variable region of the FMC63 antibody. Constant region (CDR) (CDs shown in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 39) R1, CDR2 and CDR3), and at least or about 70%, at least or about 75%, At least or about 80%, at least or about 85%, at least or about 90%, at least at least about 95%, at least about 99%, at least about 81%, at least about 82%, at least or about 83%, at least or about 84%, at least or about 85%, At least about 86%, at least about 87%, at least about 88%, at least at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least or about 93%, at least or about 94%, at least or about 95%, At least about 96%, at least about 97%, at least about 98%, at least or about 99%, or about 100% identical to one, two, or three CDRs. obtain.

[0068] In a specific embodiment, the heavy chain variable region of the anti-CD19 antigen binding region comprises the heavy chain variable region of the FMC63 antibody. The three CDRs are the same as those of the chain variable region (positions 31 to 35, 50 to 65, and 98 to 109, respectively), and 9 The light chain variable region of the antigen binding region has three CDRs identical to those of the light chain variable region of the FMC63 antibody. CDRs (represented at positions 24 to 34, 50 to 56, and 89 to 97 of SEQ ID NO: 5) CDR1, CDR2 and CDR3).

[0069] In a specific embodiment, the heavy chain variable region of the anti-CD19 antigen binding region comprises the heavy chain variable region of the FMC63 antibody. The three CDRs (SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62) are identical to the CDRs of the chain variable region. an anti-CD19 antigen-binding region comprising CDR1, CDR2 and CDR3 as set forth in The light chain variable region of the antibody FMC63 has three CDRs (sequence CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 39, respectively. R3).

[0070] In a second aspect of the present invention, a nucleic acid sequence encoding a chimeric antigen receptor according to the first aspect of the present invention is provided. Provide a child.

[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 18. As shown.

[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 a plasmid, a viral vector, a transposon, and combinations thereof.

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

[0077] In another preferred embodiment, the vector is a lentiviral vector, an adenoviral vector, vector, adeno-associated virus vector, and combinations thereof. .

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

[0079] In a fourth aspect of the invention, a vector according to the third aspect of the invention or a vector according to the second aspect of the invention is provided. or the exogenous nucleic acid molecule of the first aspect of the present invention is integrated into its genome. A host cell is provided that expresses the chimeric antigen receptor.

[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 present invention, a CAR-expressing a chimeric antigen receptor according to the first aspect of the present invention is provided. The present invention provides a method for preparing T cells, the method comprising the step of: The vector of the third aspect of the invention is transduced into T cells, thereby obtaining CAR-T cells. The process includes:

[0086] In another preferred embodiment, the method detects the function and efficacy of the resulting CAR-T cells. The method further comprises the step of:

[0087] In a sixth aspect of the present invention, a chimeric antigen receptor according to the first aspect of the present invention, a nucleic acid molecule according to the third 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; 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 preparation comprises a host cell according to the fourth aspect of the invention. The concentration of is 1×10 3 ~1×10 8 cells / ml, preferably 1 x 10 4 ~1×10 7 cell / ml.

[0091] In a seventh aspect of the present invention, a drug or formulation for preventing and / or treating tumors or cancer is provided. The chimeric antigen receptor according to the first aspect of the present invention and the nucleic acid molecule according to the second aspect of the present invention for preparation The present invention provides a method for the preparation of a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention. do.

[0092] In another preferred embodiment, the tumor is selected from hematological tumors, solid tumors, and combinations thereof. Preferably, the tumor is a hematological tumor.

[0093] In another preferred embodiment, the hematological tumor is acute myeloid leukemia (AML), multiple myeloma. (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large intestine and combinations thereof. do.

[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, and kidney cancer. pancreatic cancer, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, Lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, and combinations thereof.

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

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

[0097] In a tenth aspect of the invention, a suitable amount of cells according to the fourth aspect of the invention or the sixth aspect of the invention is The present invention also provides a method for treating a disease comprising administering a formulation of the present invention to a subject in need of treatment. do.

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

[0099] The various technical features of the present invention described above and the following specific examples (such as examples) are provided. Various technical features may be combined with one another within the scope of the present invention to provide new or preferred technical features. We understand that solutions can be constructed and do not need to be explained one by one due to space limitations. I want to be understood. [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 inventors unexpectedly found that CD19 and CD20 were simultaneously Specifically, the present invention provides CAR-T cells that simultaneously target CD19 and CD20. A chimeric antigen receptor that may be targeted, comprising a signal peptide, an anti-CD20 scFv, an anti- Contains CD19scFv, hinge region, transmembrane region, and intracellular T cell signaling domain Furthermore, the anti-CD20scFv and the anti-CD19scFv are A peptide fragment (G 4S). The CAR-T cells of the present invention are linked to both the CD19 antigen and the CD20 antigen. Downregulation of antigen expression during single-target CAR-T cell therapy or reduce the risk of immune escape caused by deletions. Targeting a single antigen CAR-T cells targeting CD19 and CD20 and other dual-targeting CAR-T cells Compared with the conventional CAR-T cells, which recognize two targets simultaneously, the CAR-T cells of the present invention can bind to tumor cells. Stronger killing ability against HIV-1, less cytotoxicity, fewer side effects, and broader therapeutic range The present invention has been completed based on this.

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

[0103] The term "about" refers to an acceptable range for a particular value or composition as determined by one of ordinary skill in the art. This can refer to a value or composition that is within a reasonable margin of error, which is how the value or composition is measured or determined. will depend in part on how the IFRS 10 is determined.

[0104] The term "administer" includes administering by injection or infusion, for example, intravenously, intramuscularly, subcutaneously, intraperitoneally, Among the various methods and delivery systems known to those skilled in the art, including spinal or other parenteral administration It refers to the physical introduction of a product of the invention into a subject using any one of the methods.

[0105] The term "antibody" (Ab) refers to a molecule that specifically binds to an antigen and is linked to the antigen by disulfide bonds. an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains linked together; or Each H chain may comprise, but is not limited to, an antigen-binding portion thereof. The heavy chain constant region comprises three constant domains: Each light chain comprises a light chain variable region (abbreviated herein as VL). The light chain constant region comprises a constant domain CL. The VH and VL domains The region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are The VH and VL are interspersed within more conserved regions called framework regions (FR). It contains three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus as follows: and 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 has been remarkable, most studies have focused on CD19 chimeric antigen receptors. This shows that there are still many problems with CAR T cell therapy. There remains a small proportion of patients who experience poor response and are prone to relapse. This is due to the fact that tumor cells These include the fact that it is prone to causing loops.

[0107] To prevent CD19CAR-T antigen escape, the inventors used CD19 and A combined bispecific CAR (i.e., BICAR) that targets both CD20 and We designed a marker that allows CD20 to escape when the CD19 antigen is not expressed on tumor cells. Once recognized, tumor cells can be eliminated in vivo.

[0108] CD20 is a key player in B cell proliferation, including in some CD19-negative patients after anti-CD19 CAR-T therapy. CD20 is expressed in most patients with myeloblastic lymphocytic leukemia. CD20 is a marker that binds to B cells and is the first B cell membrane marker identified. It is known that the CD20 molecule is encoded by the MS4A gene. It has four transmembrane hydrophobic domains. It has a functional domain, with its N- and C-termini located on the cytoplasmic side and two closed loops on the outside of the cell. These form the major and minor loops, respectively. CD20 is expressed on normal and cancerous B cells. These cells are expressed specifically at the pre-B cell stage and in subsequent developmental stages. The present invention relates to a method for the production of B-cell antigens, and CD20 expression is silenced until the cells differentiate into plasma cells. CD20 is used as an alternative target for immunotherapy of 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 disease in cancer. Cellular immunotherapy is the use of biotechnology and biological agents to treat patients with The immune cells harvested from the subject are cultured and expanded in vitro, and the cells are then infused back into the patient. to stimulate and enhance the body's autoimmune function, thereby achieving the purpose of treating tumors. Those skilled in the art have developed new cellular immunotherapies to improve efficacy and reduce side effects. I've been working on releasing it.

[0110] The present invention can be effectively incorporated into primary human T cells and upon activation of the T cells. A rational and optimized single chain design capable of simultaneously targeting CD19 and CD20 The present invention provides a method and system for the preparation of a combined bispecific CAR. RT cells can recognize two antigens, CD19 and CD20. , offering a potentially highly effective method for preventing antigen escape.

[0111] The present invention uses a CAR that simultaneously targets CD19 and CD20. Compared to targeted CAR, the affinity was enhanced, T cell activity was increased, and the targeting had additive effects In addition, the heterogeneous expression of CD19 and CD20 on tumor cells Due to current levels, dual-targeted CAR-T therapy has a broader scope. On the surface of tumor cells CAR-T targeting CD19 and CD20 simultaneously may downregulate a single surface antigen or The possibility of antigen escape caused by deletions can be reduced.

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

[0113] The CD19 and CD20 bispecific CAR of the present invention has a single structure and is wherein the CAR comprises CD19scFv and CD20scFv. The amino acid sequences of CD19scFv and CD20scFv, including sequence determination and The hinge is the main factor influencing its function.

[0114] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and The extracellular domain contains a target-specific binding element (antigen binding element). The intracellular domain contains a co-stimulatory signaling region and Contains the ζ chain. Costimulatory signaling region refers to the portion of the intracellular domain that contains the costimulatory molecule Costimulatory molecules are not antigen receptors or their ligands, but rather regulate the efficient activation of lymphocytes against antigens. It is a cell surface molecule required for various responses.

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

[0116] In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention is The CAR of the present invention contains antigen-binding domains targeting CD19 and CD20. When the CAR is coupled to its associated antigen, antigen recognition can occur based on antigen binding specificity. When bound to the antigen, CAR affects tumor cells so that they cannot grow. Death or otherwise affecting the patient's tumor burden is reduced or eliminated. The 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 a 4-1BB signaling domain and a CD It is fused to the intracellular domain of a combination of 3ζ signaling domains.

[0117] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, etc., having antigen-binding activity Fv antibodies refer to a fragment, or a single Fv fragment, of an antibody that contains the heavy chain variable region and the light chain variable region of the antibody. Fv antibodies contain the entire antigen-binding site and are composed of the chain variable regions but no constant regions. Fv antibodies also generally comprise a small antibody fragment, which consists of a VH domain and a VL domain. and a polypeptide linker between them to form the structure necessary for antigen-binding. The antigen-binding domain is usually an scFv (single chain variable fragment). The length of the single chain antibody is typically 1 / 6 of that of a complete antibody. In a preferred embodiment of the present invention, the amino acid sequence encoded by scFv comprises antibodies that specifically recognize CD19 and CD20.

[0118] With respect to the hinge region and the transmembrane region (transmembrane domain), the CAR is In one embodiment, the CAR can be designed to include a transmembrane domain fused to the CAR domain. In some embodiments, a transmembrane domain that naturally associates with one of the domains in the In this paper, transmembrane domains are the transmembrane domains of surface membrane proteins that are the same or different. This avoids binding to the receptor domain and thereby interacting with other members of the receptor complex. The amino acid sequence may be selected or modified by amino acid substitutions to minimize side effects.

[0119] The intracellular domain in the CAR of the present invention is the signaling domain of 4-1BB and C Contains the signaling domain of D3ζ.

[0120] Preferably, the CAR structure of the present invention also comprises a signal peptide sequence (leader sequence). (also known as the antigen recognition sequence (antigen-binding domain)), hinge region, transmembrane region, co-intercalation region, and It contains the stimulatory factor signaling region and the CD3 zeta signaling region (the ζ chain portion). The procedure is shown in Figure 1.

[0121] In another preferred embodiment, the CAR of the present invention is TN-LEU-19. The target antigen-binding domain is the heavy chain sequence of the single variable region from the Leu16 antibody (SEQ ID NO: No. 1) and light chain (V L ) sequence (SEQ ID NO:2).

[0122] Single chain variable region heavy chain sequence (VH) from Leu16 antibody: EVQLQQSGAELVKPGASVKMSCKASGYTFT SYNMH WVKQ TPGQGLEWIG AIYPGNGDTSYNQKFKG KATLTADKSSSTA YMQLSSLTSEDSADYYCAR SNYYGSSYWFFDV WGAGTTVT VSS (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 WYQKKP GSSPKPWIY ATSNLAS GVPARFSGSGSGTSYSLTISRVEA EDAATYYC 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 present invention is TN-OF-19. The target antigen-binding domain is a heavy chain sequence of a single variable region from the ofatumumab antibody (sequence number 3) and the light chain sequence (SEQ ID NO:4).

[0125] Single chain variable region heavy chain sequence (VH) derived from the ofatumumab antibody: EVQLVESGGGLVQPGRSLRLSCAASGFTF NDYAMH WVRQ APGKGLEWVS TISWNSGSIGYADSVKG RFTISRDNAKKSL YLQMNSLRAEDTALYYCAK DIQYGNYYYGMDV WGQGTTVT VSS (SEQ ID NO: 3) OF-VH-CDR1: SEQ ID NO: 3, positions 30 to 35. The sequence of OF-VH-CDR1 is , NDYAMH (sequence number 40).

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

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

[0128] The light chain sequence of the single variable region (VL) derived from the ofatumumab antibody: EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQK PGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLE PEDFAVYYC 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 (sequence number 43).

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

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

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

[0132] The amino acid sequence of the light chain (VL) of the single variable region derived from the FMC63 antibody: DIQMTQTTSSLSASLGDRVTISC RASQDISKYLN WYQQK PDGTVKLLIY HTSRLH SGVPSRFSGSGSGTDYSLTISNLE QEDIATYFC QQGNTLPYT FGGGTKLEIT (SEQ ID NO:5) FMC63-VL-CDR1: SEQ ID NO: 5, positions 24 to 34. FMC63-VL-CD The sequence of R1 is RASQDISKYLN (SEQ ID NO:63).

[0133] FMC63-VL-CDR2: SEQ ID NO: 5, positions 50 to 56. FMC63-VL-CD The sequence of R2 is HTSRLHS (SEQ ID NO:64).

[0134] FMC63-VL-CDR3: SEQ ID NO: 5, positions 89 to 97. FMC63-VL-CD The sequence of R3 is QQGNTLPYT (sequence number 39).

[0135] Nucleotide sequence of the light chain (VL) of the single variable region derived from the FMC63 antibody: gacatccaga tgacacagac tacatcctcc ctgtct gcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatat ttaa attggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctaccat acatca agat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattct ctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaat acgc 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 WIRQ PPRKGLEWLG VIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAK HYYYGGSYAMDY WGQGTSVTVS S (SEQ ID NO: 6) FMC63-VH-CDR1: SEQ ID NO: 6, positions 31 to 35. FMC63-VH-CD The sequence of R1 is DYGVS (SEQ ID NO:60).

[0137] FMC63-VH-CDR2: SEQ ID NO: 6, positions 50 to 65. FMC63-VH-CD The sequence of R2 is VIWGSETTYYNSALKS (SEQ ID NO:61).

[0138] FMC63-VH-CDR3: SEQ ID NO: 6, positions 98 to 109. FMC63-VH-C The sequence of DR3 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 ctggtg gcgc cctcacagag cctgtccgtc 60 acatgcactg tctcaggggt ctcattaccc gactat ggtg taagctggat tcgccagcct 120 ccacgaaagg gtctggagtg gctgggagta atatgg ggta gtgaaaccac atactataat 180 tcagctctca aatccagact gaccatcatc aaggac aact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccatt tact actgtgccaa acattattac 300 tacggtggta gctatgctat ggactactgg ggccaa ggaa 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 (i.e., flexible linker I) is As shown below.

[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 of the CD8 (CD8TM) or CD28 (CD28TM) antigen. It is an array.

[0148] CD8TM: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) CD28TM:MFWVLVVVGGVLACYSLLVTVAFIIFWV (sequence no. No. 11)

[0149] The costimulatory signaling region contains the sequence of the 4-1BB or CD28 cytoplasmic signaling motif. It comes from the column.

[0150] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCEL (SEQ ID NO: 12) CD28:RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRS (SEQ ID NO: 13)

[0151] The signal transduction domain of CD3ζ is the immunoreceptor tyrosine base pair of CD3ζ in the TCR complex. It is derived from the ITAM sequence, which is an interferon-activating motif.

[0152] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14 )

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

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

[0155] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCC TTGCTGCTCCACGCCGCCAGGCCGGAAATTGTGTTGACAC AGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTAC TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGC TCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCC AGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTT ATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGG CCAAGGGACACGACTGGAGATTAAAGGCAGTACTAGCGGT GGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCG AAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTC ACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTC CAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAA TAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATC TGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTA TTACTGTGCAAAAGATATACAGTACGGCAACTACTACTAC GGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CCTCAGGAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAG CGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTG ACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCG TGAGCTGGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATG GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAAC AGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACA GCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGAC CGACGACACCGCCATCTACTACTGCGCCAAGCACTACTAC TACGGCGGCAGCTACGCCATGGACTACTGGGGCCAGGGCA CCAGCGTGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGG CAAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGACATC CAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGG GCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACAT CAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGC ACCGTCAAGCTGCTGATCTACCACACCAGCCGGCTGCACA GCGGCGTGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCAC CGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGAT ATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCT ACACCTTTGGCGGCGGAACAAAGCTGGAAATCACCGAGAG CAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGG GTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCC TGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACG GGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTT ATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTA GCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACT GCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTAC CAGCAGGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGG GCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGG CCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAAC CCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGA TGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCG GAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTG TCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGC AGGCCCTGCCCCCAAGG (SEQ ID NO: 18)

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

[0157] MALPVTALLLPLALLLHAARPEIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTRLEIKGSTSGGGSGGGSGGGGSSEVQLVESGGGLVQ PGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISW NSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAL YYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSEVKLQE SGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE WLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGSTSGS GKPGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQD ISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGGSG TDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITE SKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVK RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 16 )

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

[0159] ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTC TGGGTTCCAGGTTCCACAGGTGACATTGTGCTGACCCAAT CTCCAGCTATCCTGTCTGCATCTCCAGGGGAGAAGGTCAC AATGACTTGCAGGGCCAGCTCAAGTGTAAATTACATGGAC TGGTACCAGAAGAAGCCAGGATCCTCCCCCAAACCCTGGA TTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCG CTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACA ATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACT GCCAGCAGTGGAGTTTTAATCCACCCACGTTCGGAGGGGG GACCAAGCTGGAAATAAAAGGCAGTACTAGCGGTGGTGGC TCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAGGTGC AGCTGCAGCAGTCTGGGGCTGAGCTGGTGAAGCCTGGGGC CTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTT ACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGAC AGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGG TGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACA TTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGC TCAGCAGCCTGACATCTGAGGACTCTGCGGACTATTACTG TGCAAGATCTAATTATTACGGTAGTAGCTACTGGTTCTTC GATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG GAGGTGGTGGATCCGAGGTGAAGCTGCAGGAAAGCGGCCC TGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGC ACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCT GGATCCGGCAGCCCCCCAGGAAGGGCCTGGAATGGCTGGG CGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCC CTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGA GCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGA CACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGC GGCAGCTACGCCATGGACTACTGGGGCCAGGGCACCAGCG TGACCGTGAGCAGCGGCAGCACCTCCGGCAGCGGCAAGCC TGGCAGCGGCGAGGGCAGCACCAAGGGCGACATCCAGATG ACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACC GGGTGACCATCAGCTGCCGGGCCAGCCAGGACATCAGCAA GTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTC AAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCG TGCCCAGCCGGTTTAGCGGCAGCGGCTCCGGCACCGACTA CAGCCTGACCATCTCCAACCTGGAACAGGAAGATATCGCC ACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACCT TTGGCGGCGGAACAAAGCTGGAAATCACCGAGAGCAAGTA CGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTG GTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGG TCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAG AAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGT GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAG GGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAA GGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGA CCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCAG GAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCG AGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCG GGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACC GCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCC TGCCCCCAAGG (SEQ ID NO: 17)

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

[0161] METDTLLLWVLLLWVPGSTGDIVLTQSPAILSASPGEKV TMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPA RFSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGG GTKLEIKGSTSGGGSGGGSGGGGSS EVQLQQSGAELVKPG ASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGN GDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYY CARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSEVKLQESG PGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWL GVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS GSTSGSGK PGSGEGSTKGDIQMTQTTSSLSASLGDRVTISCRASQDIS KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTD YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITESK YGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 15)

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

[0163] First, CD19 and CD20 are expressed on most malignant B-cell neoplasms. In general, when expanding the CAR structure to enhance T cell recognition, there are some drawbacks, such as increased detrimental targeting, However, problems such as increased cytotoxicity and increased side effects are often encountered. Both CD10 and CD20 are expressed only on B cells, which have the same tumor toxicity curve. This is not the case for CD19 and CD20. Finally, the CD19 and CD20 Expression of D20 can promote B cell survival, and loss of both antigens during treatment This is a very low probability event. Therefore, targeting CD19 and CD20 This is expected to result in more effective prevention of antigen escape by malignant B cells.

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

[0165] First, the expression of B in a single T cell is significantly improved compared to expressing two independent CARs. When ICAR is expressed, the DNA footprint is significantly reduced (DNA length is reduced by 40%) The size and length of the construct influences the packaging and transduction efficiency of the viral vector. Second, two Compared with a mixture of single CARs with different concentrations, BICAR significantly reduced the treatment cost ( BICAR is fully compatible with current T cell manufacturing processes without adding any additional overhead. Finally, CD19 and CD20 are involved in many It has been tested in clinical studies and is relatively safe.

[0166] In the present invention, the sequence of the CD19 mouse-derived monoclonal antibody FMC63 and the sequence of the CD2 Based on the sequences of the mouse-derived monoclonal antibody leu-16 and ofatumumab, Two chimeric antigen receptor structures targeting D19 and CD20 (TN-LEU-19, We constructed a chimeric antigen receptor TH-OF-19 (TAR-TH-OF-19) and expressed these two antigens in primary T cells. The expression levels, in vitro activation potential, and tumor cell killing efficacy of the receptors were analyzed and identified. Finally, we developed a method to detect the expression of TN-LEU-19 or TH-OF-19 chimeric antigen receptor-modified IL-19. The T cells killed malignant tumors bearing CD19 and CD20 positive antigens in vitro. ofatumumab has a strong ability to clear leu16 in vivo, and ofatumumab is better than leu16 This has been found to be a promising therapeutic option for the treatment of CD19- and CD20-positive leukemias and lymphomas. To provide new and effective methods and preparations for the clinical application of CAR-T.

[0167] The present invention has designed and optimized monospecific and bispecific CARs. These CARs , and has potent killing ability against B cell malignancies expressing CD19 or CD20. ICAR has identified two clinically relevant cases in which a single T cell product is associated with B cell leukemia and lymphoma. Allows for targeting of validated antigens, ultimately preventing loss or escape of a single antigen The present invention further uses the new BICAR design to reduce the risk of tumor recurrence. Thus, it is possible to increase the availability of antigens and improve the efficacy of T cell therapy against cancer. Can.

[0168] Chimeric antigen receptor T cells (CAR-T cells) As used herein, "CAR-T cells," "CAR-T," "CART," The term "CAR-T cells of the invention" refers to the CD19 and CD2 0. Specifically, the CAR structure of the CAR-T cell The anti-CD19 scFv, the anti-CD20 scFv, the hinge region, the transmembrane region, and the cell and an anti-CD20 scFv and an anti-CD19 scFv, is linked to a peptide with multiple repeats (G4S) to target a single antigen Compared with CAR-T, CAR-T cells that simultaneously recognize two targets are more lethal. , the range of treatment is more extensive.

[0169] vector Nucleic acid sequences encoding the desired molecules can be synthesized using recombinant methods known in the art, e.g. For example, gene-containing by deriving the gene from a vector known to carry the gene, or by using standard techniques. The gene can be obtained by direct isolation from cells and tissues containing the gene. 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. Vectors derived from retroviruses such as rabies have been shown to mediate long-term, stable integration and expression of transgenes. and its propagation in daughter cells, thus providing an appropriate vector for achieving long-term gene transfer. Lentiviral vectors are a promising tool for transducing non-proliferating cells such as hepatocytes. Vectors derived from oncoretroviruses such as mouse leukemia viruses are Lentiviral vectors also have the advantage of being less immunogenic. has.

[0171] In summary, the expression cassette or nucleic acid sequence of the present invention typically and operably promotes The vector is ligated to a nucleic acid sequence and incorporated into an expression vector. A typical cloning vector contains transcription and translation terminators, an initiation sequence, The nucleic acid sequence includes a nucleic acid sequence, as well as a promoter useful for regulating the expression of the desired nucleic acid sequence.

[0172] The expression constructs of the present invention can also be used in nucleic acid immunization and It can be used for gene therapy. Methods for gene delivery are known in the art. For example, No. 5,399,346, the entire specification of which is incorporated herein by reference; See US Pat. Nos. 5,580,859 and 5,589,466. The present invention provides gene therapy vectors.

[0173] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into a plasmid. , phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest may be expression vectors, replication vectors, or vectors, probe generation vectors, and sequencing vectors.

[0174] Additionally, the expression vector can be provided to the cell in the form of a viral vector. The turber technique is well known in the art and is described, for example, in Sambrook et al. (2001) ,Molecular Cloning:A Laboratory Manual,C old Spring Harbor Laboratory, New York), average and other virology and molecular biology manuals. The most common viruses are retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. In general, suitable vectors include, but are not limited to, vectors for the expression of vectors of the present invention. - an origin of replication, a promoter sequence, favorable restriction sites, and a sequence that is functional in at least one organism endonuclease site, and one or more selection markers (see, e.g., WO 01 / 02366). No. 96584, WO 01 / 29058, and U.S. Pat. No. 6,326,193). .

[0175] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses are a convenient platform for gene delivery systems. The selected gene is inserted into a vector using techniques known in the art, The recombinant virus can then be isolated. and can be delivered to cells of a subject either in vivo or ex vivo. Torovirus systems are known in the art. 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 region 30-1, 10 bp upstream of the start site, but most promoters Recently, it has been shown that promoter elements also contain functional elements downstream of the initiation site. The spacing between elements is often flexible, so that elements can flip or move relative to one another. In the case of the thymidine kinase (tk) promoter, The spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may act either cooperatively or independently to activate transcription. It appears that it can function.

[0177] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence may be any polynucleotide sequence operably linked to it. A strong constitutive promoter sequence capable of driving high level expression of the appropriate promoter. Another example of a promoter is elongation growth factor-1α (EF-1α). SV40 early promoter, Mouse mammary tumor virus (MMTV), Human immunodeficiency virus HIV long terminal repeat (LTR) promoter, MoMuLV promoter, avian Leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma Viral promoters, as well as actin promoters, myosin promoters, hemoglobin promoters, Human genetic promoters, such as, but not limited to, the mitochondrial kinase promoter, and the creatine kinase promoter. Other constitutive promoter sequences may also be used, including, but not limited to, Furthermore, the present invention should not be limited to the use of constitutive promoters, but may also include inducible promoters. The use of an inducible promoter is contemplated as part of the present invention when such expression is desired. Can expression of an operably linked polynucleotide sequence be turned on when or a molecular switch that can turn off expression when expression is not desired. Examples of inducible promoters include the metallothionein promoter, the glucocorticoid promoter, promoter, progesterone promoter, and tetracycline promoter, Not limited to these.

[0178] To assess the expression of a CAR polypeptide or a portion thereof, an expression vector is introduced into a cell. The target also contains either a selection marker gene or a reporter gene, or both. Cells that are required to be transfected or infected with a virus vector This can facilitate the identification and selection of expressing cells from a population of cells. The marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the mosquito and reporter genes are flanked by appropriate regulatory sequences and are then subjected to expression in the host cell. Useful selection markers include, for example, antibiotic resistance markers such as neo. Examples include sex genes.

[0179] Reporter genes allow identification of potentially transfected cells and evaluation of the functionality of regulatory sequences. Generally, reporter genes are used to measure the expression of a gene in a recipient organism or tissue. a gene that is not present or expressed in the A gene that is manifested by some easily detectable characteristic, such as an enzymatic activity. Expression of the reporter gene was monitored at the appropriate time point after DNA was introduced into the recipient cells. Suitable reporter genes are luciferase, β-galactosidase, loramphenicol acetyltransferase, secretory alkaline phosphatase, or The gene encoding the green fluorescent protein may be included (see, for example, Ui-Tei et al. al., 2000 FEBS Letters 479:79-82). Stems are well known and can be prepared using known techniques or can be obtained commercially. In general, the reporter gene should be expressed at a minimum 5' flanking region that provides the highest level of expression. Such a promoter region is identified as a reporter gene. to evaluate agents for their ability to modulate promoter-driven transcription. can be used for.

[0180] Methods for introducing and expressing genes in cells are known in the art. In this regard, the vector can be introduced into a host cell, e.g., a mammalian cell, by any method in the art. For example, expression vectors can be easily introduced into mammalian, bacterial, yeast, or insect cells. The vector may be transferred into the host cell by chemical, or biological means.

[0181] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, Lipofection, particle gun, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. For example, Sambrook et al. (2001, Molecular Cl oning:A Laboratory Manual,Cold Spring Ha For more information, see the National Institute of Integrative Medicine, Boston, MA (Ribbon Laboratory, New York). A preferred method for introducing nucleotides is the calcium phosphate method.

[0182] Biological methods for introducing a polynucleotide of interest into a host cell include DNA and RNA. Viral vectors, particularly retroviral vectors, can be used to infect mammalian cells. , for example, has become the most widely used method for inserting genes into human cells. Other viral vectors include lentiviruses, poxviruses, and herpes simplex virus I. , adenovirus and adeno-associated virus, etc. See, e.g., U.S. Patent No. 5,355,635. See US Pat. Nos. 0,674 and 5,585,362.

[0183] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, e.g. , macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, Examples include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in the laboratory and in vivo include liposomes. (e.g., artificial membrane vesicles).

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

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

[0186] preparation The present invention relates to a CAR-T cell according to the fourth aspect of the present invention, and a pharma- ceutically acceptable carrier, diluent, In one embodiment, the formulation is a liquid formulation. In the present invention, the formulation is an injectable preparation. Preferably, the concentration of 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 comprises a buffer such as neutral buffered saline, phosphate buffered saline; glucose carbohydrates such as mannose, sucrose or dextran, mannitol; proteins; Polypeptides or amino acids such as glycine; antioxidants; cations such as EDTA or glutathione rate agents; adjuvants (e.g., aluminum hydroxide); and preservatives. The disclosed formulations are preferably formulated for intravenous administration.

[0188] therapeutic use The present invention relates to a method for the transfection of a mammalian cell line comprising the steps of: transfecting a mammalian cell line with a lentiviral vector (LV) encoding an expression cassette of the present invention; Therapeutic applications using the transduced cells (e.g., T cells). Transduced T cells can be used to , targeting tumor cell markers CD19 and CD20, synergistically activating T cells, and It is possible to induce an immune response, thereby significantly increasing the killing efficacy of tumor cells. can.

[0189] Thus, the present invention also provides a method for the production of T cell-mediated transcription factors for target cell populations or tissues in a mammal. A method for stimulating a CAR-T cell-mediated immune response comprising administering the CAR-T cell of the present invention to a mammal. The present invention provides a method comprising the steps of:

[0190] In one embodiment, the present invention provides a method for isolating T cells from an autologous patient (or a heterologous donor). The cells are then activated and genetically modified to produce CAR-T cells, which are then injected back into the same patient. This includes the class of cell therapy. With this method, the chance of graft-versus-host disease is extremely low, and the antigen is non-M. In addition, one CAR-T can target all antigen-expressing Unlike antibody therapy, CAR-T cells cannot replicate in vivo. This can result in long-term durability leading to sustained tumor control.

[0191] In one embodiment, the CAR-T cells of the present invention are capable of undergoing robust in vivo T cell expansion. Furthermore, CAR-mediated immune responses can be enhanced by CAR-modified T cells. It can be part of an adoptive immunotherapy approach to induce an immune response specific to the antigen-binding portion of the CAR. For example, anti-CD19CD20 CAR-T cells express both CD19 and CD20. Induce an immune response specifically against the cells.

[0192] The data disclosed herein demonstrate that CD19CD20 scFv, hinge and transmembrane domains In addition, lentiviral vectors containing 4-1BB and CD3ζ signaling domains Although specifically disclosed herein, the present invention also relates to the construction of It should be construed to include any number of variations of each component of the product.

[0193] Cancers that may be treated include those that are not vascularized or are largely avascularized tumors, and those that are vascularized. Cancer includes non-solid tumors (such as blood tumors, e.g., leukemia and lymphomas) Cancer types that may be treated with the CARs of the present invention include carcinomas, blastomas, and and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors Adult tumors / cancers, including but not limited to sarcomas, carcinomas, and melanomas. Also includes childhood tumors / cancer.

[0194] Hematological cancers are cancers of the blood or bone marrow. Examples of blood (or hematopoietic) cancers include acute leukemia ( For example, acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia and myeloblastic leukemia, myeloid, myelomonocytic, monocytic and erythroleukemias), chronic leukemias (e.g. chronic myeloid (granulocyte Leukemia, including chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera Lymphoma, Hodgkin's disease, Non-Hodgkin's lymphoma (indolent and aggressive), Multiple myeloma Myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome, These include hairy cell leukemia and myelodysplastic syndromes.

[0195] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors are benign. Various types of solid tumors have life-threatening consequences for the types of cells that form them. Examples of solid tumors, such as sarcomas and carcinomas, include lymphomas. These include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer and ovarian cancer.

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

[0197] For ex vivo immunization, at least one of the following is performed prior to administering the cells to the mammal: The method is carried out in vitro by: i) expanding the cells; ii) transfecting the cells with a nucleic acid encoding a CAR; and / or iii) cryopreserving the cells.

[0198] Ex vivo procedures are well known in the art and are discussed more fully below. Cells are isolated from a mammal (preferably a human) and express a CAR as disclosed herein. Genetically modify (i.e., transduce or transfect in vitro) with a vector. The AR 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 are 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 Also, compositions for in vivo immunization to induce an immune response against an antigen in a patient. and a method.

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

[0201] The CAR modified T cells of the present invention can be administered alone or in combination with diluent and / or IL-2, IL-1 7 or other cytokines or cell populations, or other components, to form pharmaceutical compositions. Briefly, the pharmaceutical compositions of the present invention can be administered in a manner that satisfies one or more pharmacological or physiological requirements. The present invention relates to a method for producing a composition comprising the target cell population described herein in combination with an acceptable carrier, diluent or excipient. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as sucrose, mannose, sucrose or dextran, mannitol; proteins ; polypeptides or amino acids such as glycine; antioxidants; EDTA or glutathione It may contain chelating agents; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the invention are preferably formulated for intravenous administration.

[0202] The pharmaceutical compositions 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 will depend on factors such as the patient's condition and the type and severity of the patient's disease. However, appropriate doses can be determined by clinical trials.

[0203] "Immunologically effective amount," "antitumor effective amount," "tumor-inhibiting effective amount," or "therapeutic amount" are indicated. If so, the exact amount of the composition of the present invention to be administered will vary depending on age, weight, tumor size, infection or This can be determined by a doctor taking into account the degree of metastasis and individual differences in the condition of the patient (subject). The pharmaceutical composition comprising the T cells described herein may be administered in a dose of 10 4 ~10 9 Cells / kg body weight, preferred 10 5 ~10 6 and all integer values ​​within those ranges. The T cell compositions may also be administered multiple times at these doses. The cells are administered by using injection techniques commonly known in immunotherapy. (e.g., Rosenberg et al., New Eng. Jo f Med. 319:1676, 1988). Dosage and treatment regimens should be determined by monitoring the patient for signs of disease and treating accordingly. can be readily determined by one of ordinary skill in the art of medicine by adjusting

[0204] Administration of the subject compositions may be by any method, including aerosol inhalation, injection, ingestion, implantation, or implantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, in lymph nodes, or in combination with other suitable methods. The drug may be administered to a patient by intravenous, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneal injection. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In embodiments, the T cell compositions of the present invention are preferably administered by iv injection. The T cell composition may be injected directly into a tumor, lymph node, or site of infection.

[0205] In certain embodiments of the invention, the methods described herein or the method for increasing T cells to therapeutic levels are The activated and expanded cells can be propagated using other methods known in the art (e.g., antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or Treatment with drugs such as efalizumab for patients with psoriasis or other treatments for patients with PML The therapeutic modalities may be administered to the patient in conjunction with any number of related therapeutic modalities, including, but not limited to, In further embodiments, the T cells of the present invention are treated with chemotherapy, radiation, cyclosporine, Immunosuppressants such as azathioprine, methotrexate, mycophenolate, and FK506 In a further embodiment, the present invention may be used in combination with a medicament, antibody, or other immunotherapeutic agent. The cell composition may be administered (e.g., prior to, simultaneously with, or following) bone marrow transplantation, or fludarabine, exogenous It is administered to patients in conjunction with chemotherapy drugs such as radiation therapy (XRT) and cyclophosphamide. For example, in one embodiment, the subject is treated with standard high-dose chemotherapy. In certain embodiments, after transplant, the subject receives a peripheral blood stem cell transplant. In a further embodiment, the expanded immune cells are administered before or after surgery. is administered.

[0206] The dosage of the above treatments administered to a patient will depend on the condition being treated and the appropriateness of the recipient of the treatment. Scaling of doses for patient administration will depend on the exact nature of the compound. This can be done according to accepted practice in the field. Generally, 1 × 10 6 ~1×10 10 The modified T cells of the invention (e.g., CAR-T-19 / 20 cells) can be administered, for example, by intravenous infusion. The composition may be administered to the patient by means of each treatment or each course of treatment.

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

[0208] (1) With respect to the chimeric antigen receptor of the present invention, the extracellular antigen-binding domain is a specific anti-CD 20 scFv and anti-CD19 scFv, and specific anti-CD20 scFv and anti-C The D19 scFv was combined with a specific hinge region and intracellular domain to form The constructed CARs showed great ability to kill tumor cells with low cytotoxicity and low side effects. vinegar.

[0209] (2) The chimeric antigen receptor provided by the present invention is a chimeric antigen receptor (CAR) that is capable of binding to T cells carrying a CAR gene. After lentivirus infection, stable expression and membrane localization of the CAR protein was achieved. This can be done.

[0210] (3) The CAR-modified T cells of the present invention have a longer survival time in vivo and a stronger anti-tumor effect. Optimized CA with efficacy and IgG4 hinge-CH2-CH3 linker region R prevents Fc receptor binding and subsequent ADCC (antibody-dependent cellular cytotoxicity) This can be done.

[0211] (4) Compared with two independent CARs, the bispecific chimeric antigen receptor of the present invention has the following advantages: It contains both CD20 scFv and anti-CD19 scFv and has a DNA footprint. The DNA length is significantly reduced (40% reduction), the size of the structure is small, and the viral vector Benefits to packaging and transduction efficiency, thus directly improving clinical efficacy Furthermore, the bispecific CAR of the present invention has lower costs, higher cure rates, and is more affordable. It is complete.

[0212] (5) Modified with the TN-LEU-19 or TH-OF-19 chimeric antigen receptor of the present invention The T cells killed malignant tumors bearing CD19 and CD20 positive antigens in vitro. It has a very strong ability to eliminate leukemia in vivo, with ofatumumab being more potent. Clinical application of CAR-T in the treatment of CD19 and CD20 positive leukemia and lymphoma The present invention provides new and effective methods and preparations for the preparation of glycerol.

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

[0214] The invention will now be further described with reference to specific examples, which are provided for illustrative purposes only. It should be understood that the following examples are not intended to limit the scope of the present invention. The experimental methods described in the Examples without specific conditions are generally carried out under conventional conditions or by the manufacturer. 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 the plasmid coding for pWPTren. The tivirus vector was selected as the cloning vector, and the cloning site was BamH. The structures of the two CARs designed in this invention are shown in Fig. The amino acid sequence of TN-LEU-19 is shown in SEQ ID NO: 15, and TN-OF-19 is shown in SEQ ID NO: 16. The amino acid sequence of (L-(OF)VL-(OF)VH-I-(FMC 63) VH-(FMC63)VL-H-TM-C-CD3ζ structure.

[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. , CD3 monoclonal antibody (OKT3) and RetroNectin (TAKARA) were used in advance. PBMCs were activated in cell culture flasks containing 1% human albumin. GT-55, which contains 300 U / mL recombinant human interleukin 2 (IL-2) On day 3, purified TN-LEU-19 or TN From day 6, TN-OF-19 and T N-LEU-19 CAR-T cells can be harvested for corresponding activity assays. Using the Protein L method, we analyzed the surface of the T cell membrane in CAR-T cells cultured for 7 days. The expression levels of the above CAR gene-encoded proteins were determined.

[0217] T cell activation markers CD137 and IFNγ release were measured in CD19 / 20 T cells cultured for 7 days. Detection was performed using bispecific CAR-T cells. 1 × 10 5 CAR-T cells were treated with CD19 , CD20 positive K562-CD19+, K562-CD20+, K562-CD19+C D20+ and Raji (naturally expressing CD19 and CD20) tumor cell lines, as well as C D19CD20-negative K562 tumor cell line, or 200 μl of GT-55 without tumor cells The T cells were co-cultured in 1:1 ratio in 1 medium for 18 hours. The expression level of D137 and the secretion level of IFNγ in the culture supernatant were detected, respectively.

[0218] The results are shown in Figure 2A and Figure 2B. The expression of CD137 on the surface of the two CART cells was examined. The expression of IFNγ in the culture supernatant was detected. -LEU-19 has higher levels of CD137 activation and IFNγ release.

[0219] [Example 3] Detection of cytotoxicity of CD19 / 20 bispecific CAR-T cells in vitro The CAR-T cells prepared in Example 2 were examined for cytotoxicity by LDH release assay. The target cells were K562, K562-CD19+, K562-CD20+, and K 562-CD19+CD20+ and Raji cells, and effector cells were NT, T N-LEU-19 and TN-OF-19 cells. Number of effector cells: Number of target cells The effector-target ratios were set at 5:1, 10:1, 20:1, and 40:1. The results are shown in Figure 3A. Both of these cells successfully induced apoptosis in CD19 / 20 positive tumor cells and suppressed LDH induction. can be released.

[0220] CD107a (T cells) during tumor cell killing induced by CAR-T19 / 20 cells The release levels of 1×10 5 Cellular Effective cells CAR-T19 / 20 (TN-OF-19 and TN-LEU-19) were cultured at 2×1 0 5 The target cells were co-cultured with K562-CD19+ , K562-CD20+, K562-CD19+CD20+, and K562 cells, Raj i cells and Romas cells (CD19+CD20+). The results are shown in Figure 3B. CART cells can successfully induce the release of CD107a during tumor cell killing. Here, TN-OF-19 cells expressed cytotoxic markers more than TN-LEU-19 cells. The release of CD107a was slightly higher in response to CD4+.

[0221] [Example 4] Inhibitory effect of CAR-T19 / 20 on transplanted tumor cells in mice The tumor cells injected into the animals were transformed with Raji cells carrying a luciferase reporter gene ( In this experiment, the tumor cells were injected into the mice and expressed luciferase (Raji). The cells were grown in mice for a week and then injected with effector T cells. The cells were divided into three groups: NT, TN-LEU-19, and TN-OF-19. Inject vector T cells into NSG mice through the tail vein, then measure the fluorescence intensity (I The body weight of the mice was recorded every 7 days. The study was discontinued and the statistical results were analyzed.

[0222] The results are shown in Figure 4. Figure 4A shows the effector T cell response in three groups of mice after injection. The weight change of CART-TN-OF-19 and CART-TN-LEU-19 was measured. Compared with CAR T cells, NT mice showed a significant reduction in body weight, and both CAR The mice in the T cell group gained a slight weight gain. Figure 4B shows the average fluorescence intensity of the three groups of mice. The results showed that the mean fluorescence intensity of the NT group mice was significantly increased, whereas the two CART cells The mean fluorescence intensity of the mice in the group was decreased, indicating that TN-OF-19 and TN-LEU Both 19CART and 19CART cells were able to inhibit tumor growth compared to NT. Here, TN-OF-19 cells were more abundant than TN-LEU-19 cells after 14 days. also had better inhibition on tumor growth, and the tumor growth curve of the TN-LEU-19 group was significantly Figure 4C shows IVIS images of the fluorescence intensity in the two CART cell groups and the NT group. TN-OF-19 CAR-T cells out-express TN-LEU-19 in vivo. The present invention is capable of inhibiting or killing tumor cells more effectively than immunosuppressive cells.

[0223] [Example 5] In vivo inhibition of TN-OF-19 CAR-T cells and CD19-specific CAR-T cells Comparison of harmful effects 1×10 6 Firefly luciferase-expressing Raji cells were cultured at 4°C for 6-8 weeks by tail vein injection. NPG mice (NOD-Cg.Prkdc SCID IL-2Rgc null / vs Seven days later, tumor engraftment was assessed by ip injection of D-luciferin and 180 s Bruker In Vivo Xtreme Imaging System (Bruker, The mice were then imaged 10-15 minutes later using a 3D Xtreme BI microscope. The patients were equally distributed into the study 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 , 2.5×10 6 , and 5 × 10 6 Each patient was treated separately with 1 × 10 CAR+ T cells. 6 of Non-transduced T cells (NT) and single target CD19-specific from the same donor CAR-T cells were used as a control. Tumor growth was assessed in all mice at days 7, 10, and 21 post-injection. The results were evaluated based on the average radiance of the whole body. T cells inhibit or kill tumor cells better than CD19-specific CAR-T cells in vivo The results show that bispecific TN-OF can suppress inflammatory bowel disease, especially in the early stages of treatment. The -19 CAR-T shows a significantly faster inhibitory effect. Figure 5B shows the results of the four CAR-T cells. IVIS imaging of fluorescence intensity in the cells and NT groups is shown.

[0224] [Example 6] Antigen specificity of CD20 / CD19 bispecific scFV in TN-OF 19CAR To investigate the affinity and specificity of bispecific scFv in TN-OF 19CAR In-frame, CD20 scFv (derived from ofatumumab mAb) and CD1 9 scFv (derived from FMC63mAb) was linked to the Fc region of rabbit IgG1. A chimeric rabbit monoclonal antibody was produced by the above procedure. The scFvs were linked alternately by G4S linkers. The order of the molecules was OF VL-VH- G4S-FMC63 VH-VL, which is the same as the scFv of TN-OF-19. Chimeric antibodies were expressed in 293T cells after transient gene transfection. The chimeric antibody was validated by flow cytometry. The cell lines (A549-CD19, A549-CD20, A549-CD19-CD20) were used as targets. CD19-CD20-A549 cells and CD19+CD20+Raji cells were used as controls. Cells were used as controls. All cells were washed, resuspended, and blocked with 2% serum for 30 min. King. 5×10 5 The cells were transferred to FACS vials, washed, and incubated with recombinant antibodies (final concentration The sections were stained with 20 μg / mL of IgG (100 μg / mL) at 4°C for 1 hour. After washing, the sections were stained with a secondary antibody (goat anti-rabbit IgG). was added for 30 min at 4°C in the dark. Finally, the cells were washed and incubated at 200°C for FACS analysis. The cells were then resuspended in 100 μL of FACS buffer. The present invention provides a chimeric antibody that binds to cells lacking both antigens but not to cells lacking both antigens, and Heteromeric binding domains require only one cognate antigen for binding, creating a new specific recognition site indicates that no formation occurred.

[0225] [Example 7] Screening for CD20-specific CARs (to generate CD20 / 19 bispecific CARs) Testing and functionality Prior to constructing the CD20 / CD19 bispecific CAR, we investigated the CD20-specific scFV candidates Extensive research was conducted to screen and narrow down the complement. Figure 7 shows six different complements. 16 CD20-specific CAs with Fv and distinct hinge / TM / signaling domains The structure of R is shown. The full-length DNA was synthesized and cloned to achieve the construction of the plasmid coding for R. We then tested the antitumor activity of these CARs on a variety of CD20-expressing target cells.

[0226] Figure 8A shows the results of CAR-T20.1, CAR-T20.5, CAR-T20.6, and CAR- Screening of CAR-T20.7, CAR-T20.8, CAR-T20.9 and CAR-T20.10 We showed the results of an IFNγ release assay for the CAR-T cells. Only CAR-T20.9(Leu16) and CAR-T20.10(Leu16) showed better The results showed a higher positive IFNγ release than the control.

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

[0228] Figure 8C shows the results of CAR-T20.9, CAR-T20.12, CAR-T20.14, and CA Screening for R-T20.17, CAR-T20.18, and CAR-T20.19 The results of an IFNγ release assay for the CAR-T 20.14(OF) and CAR-T20.19(OF) showed higher positive IFNγ release. showed.

[0229] FIG. 9A shows CAR-T20 cells tested for cytotoxicity by LDH release assay. .17 (third generation of Leu16), CAR-T20.18 (second generation of Leu16), C The results are shown for AR-T20.19 (OF second generation) cells. The target cells were CD20 positive cells. The cell lines Raji and Ramos, and the CD20-negative Molt4. 0 CAR-T cells induce apoptosis and upregulate LDH in CD20-positive tumor cells CAR-T20.17, CAR-T20.18, and CAR-T2 0.19 had a strong killing effect on target cells, CD20-positive Raji and Romas cells. This shows that the

[0230] FIG. 9B shows in vivo inhibition of tumor growth in an NSG mouse study. Animals were injected with The tumor cells are Raji, which express luciferase. In this experiment, the tumor cells Raj i and allowed to grow in the mice for a week, and then effector T cells were injected via the tail vein. The mice were then injected with IVIS fluorescent imaging and the fluorescence intensity of the mice was measured. The body weight of each animal was recorded every 7 days. The experiment was stopped on the 21st day and the statistical results were analyzed. CAR-T20.19(OF) cells were transfected with CAR-T20.17(Leu16 ) and CAR-T201.8(Leu16) cells, which showed better inhibition of tumor growth. This indicates that.

[0231] In the summary of Example 7 (FIGS. 7 to 9), through numerous experiments and comparisons, 20.1, 20.2 , 20.4, 20.5, 20.6, 20.7, 20.8 and 20.15 are essentially invalid. 20.11, 20.12, and 20.13 have specific effects, but those effects are 20.9, 20.10, 20.14, 20.16, 20.17, 20.18 and 20.1 The effect of 20.19(OF) was found to be the best, with the effect of 20.19(OF) being smaller than that of 9. Based on the structure of CD20 scFv (OF) and CD19 scFv (FMC63), were used in tandem in a new bispecific chimeric antigen receptor, TN-OF-19, were considered the best candidates for further analysis.

[0232] The amino acid sequence of the CD20-specific CAR involved in Example 7 is shown in Table 1. [Table 3] TIFF2025066770000005.tif239164TIFF2025066770000006.tif237160TIFF2025066770000007.tif227163

[0233] [Example 8] Phase I clinical trial of TN-OF-19 CAR-T cells Phase I study was conducted at Shanghai Tongji Hospital. This was performed in NHL patients and compared the efficacy and safety of C-CAR039 (i.e., TN-OF-19 CAR-T cells) The safety and efficacy of a new immunotherapy for harvesting T cells (NCT04317885) was evaluated. Following apheresis, C-CAR039 was produced and administered as a standard 3-day cyclophosphamide treatment. C-CAR039 was administered as a single intravenous injection after conditioning with fludarabine. A serum-free, semi-automated device with a median vein-to-vein time of 8 days. The device was manufactured using a digital closure system. The manufacturing success rate was 100%. Currently, 16 patients have a 1.0×10 6 ~5.0×10 6 Dose range of CAR-T cells / kg Fourteen patients received C-CAR039 at baseline and 14 received an evaluable safety profile of at least 1 month. Thirteen patients (11 with DLBCL and 2 with FL) had a survival rate of ≥1 month. Figure 10 shows the CAR039r / r NHL study design and flow -Chart shown.

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

[0235] Treatment with C-CAR039 was associated with no grade ≥3 CRS and no neurotoxic events. It was well tolerated. Reversible grade 1-2 CRS was observed in 9 (82%) of patients. Cytopenias due to conditioning were common and reversible.

[0236] At 1 month, 12 / 13 patients showed clinical improvement (ORR=92%) and no improvement in DLB 11 / 11 CL patients responded to treatment (ORR=100%). Median follow-up was 70 days. The best overall response (BOR) was 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 The proliferation and expansion of C-CAR039 in peripheral blood positively correlated with tumor regression. -Positive initial correlation between CAR039 AUC (days 0-28) and Cmax and clinical response A trend is observed.

[0240] FIG. 13 shows the growth and expansion of C-CAR039 in the blood of patients. This showed that CAR008 cells proliferated effectively after injection. [Table 6]

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

[0242] C-CAR039 showed promising efficacy and favorable results in early phase clinical trials in patients with r / r NHL. The initial clinical efficacy signals are encouraging, with anti-CD1 9 Compares favorably with CAR-T therapy. These findings are consistent with safety, efficacy, and duration of response. Evaluation in a larger number of patients with longer follow-up periods is needed to confirm this.

[0243] [Example 10] Phase I clinical trial of C-CAR066 (i.e., CAR-T20.19(OF)) Relapse due to loss of CD19 targeted epitopes represents a therapeutic limitation of CD19 CAR-T therapy. These patients generally have a poor prognosis. CD20 is a B cell non-Hodgkin It is a proven therapeutic target for B-NHL and has been previously approved and widely used. C-CAR066 is a new It is a novel second-generation chimeric antigen receptor T (CAR-T) therapy. Preclinical studies have been conducted on C-CAR 066 (derived from the scFV of ofatumumab) binds Leu16, rituximab, and Has superior antitumor activity compared to CAR-T derived from binutuzumab scFV This suggests that...

[0244] NCT04036019 is aimed at patients with r / r CR-T prior treatment with anti-CD19 CAR-T therapy To evaluate the safety and efficacy of C-CAR066 in subjects with B-cell lymphoma This is a single-arm, single-center, non-randomized Phase I clinical trial to evaluate the efficacy and safety of cyclophosphamide in patients with bronchial asthma. The secondary objective was to assess the incidence and severity of adverse events occurring under The objectives of this study include determining the ORR, PFS, and OS. C-CAR066 is manufactured in an automated digital closed system. It is administered to patients as a single intravenous dose after Famid / Fludarabine conditioning.

[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 success rate of production was 100%. All patients relapsed after anti-CD19 CAR-T therapy. Only one of the patients achieved a CR after anti-CD19 CAR-T therapy. [Table 9]

[0247] C-CAR066 treatment resulted in reversible grade 1-2 CRS in six patients and The treatment was well tolerated with grade 3 CRS in patients and no neurotoxic events. Seven patients showed clinical improvement (best overall response rate, ORR=85.7%). All patients responded to C-CAR066 treatment with different The patients showed moderate to severe tumor regression (45-100%). [Table 10]

[0248] Six out of seven patients showed clinical improvement (best overall response rate, ORR=85.7%). The overall response rate included 3CR and 3PR. All patients responded to C-CAR066 treatment. , showing 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 is associated with improved efficacy following CD19 CAR-T therapy C-CAR066 shows promising early efficacy in r / r NHL patients. CD19 has a different mechanism of action compared to CAR-T therapy. By targeting both the CD1 and CD19 tumor antigens, These results suggest that targeting CD4+ / CD20 alone may lead to clinical benefits superior to targeting CD4+ / CD20 alone. do.

[0251] All documents referred to in this application are incorporated by reference as if each were individually incorporated by reference. Further, after reading the above teachings of the present invention, Those skilled in the art may make various changes or modifications, and equivalents thereof are within the scope of the appended claims. It should also be understood that the same falls within the scope of the defined claims.

Claims

1. A chimeric antigen receptor (CAR), the structure of which is represented by the following formula I: L-scFv1-I-scFv2-H-TM-C-CD3ζ(I) During the ceremony, each "-" is independently a linker peptide or a peptide bond; L may be a signal peptide sequence; I is a flexible linker, H may be a 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 is an antigen-binding domain that targets CD19. and the other is an antigen-binding domain that targets CD20.

2. The scFv1 is an antigen-binding domain that targets CD20, and the scFv2 is The CAR of claim 1, wherein the antigen-binding domain targets CD19.

3. The structure of the chimeric antigen receptor is represented by the following formula II: L-V Ｌ１ -V Ｈ１ -I-V Ｈ２ -V Ｌ２ -H-TM-C-CD3ζ(II) In the formula, V Ｈ１ is an anti-CD20 antibody heavy chain variable region, and V Ｌ１ is an anti-CD20 antibody light chain variable is the region, and V Ｌ２ is the anti-CD19 antibody light chain variable region, and V Ｈ２ is an anti-CD19 antibody heavy chain is a variable region, and "-" is a linker peptide or a peptide bond; L, I, H, TM, C and CD3ζ are as defined in claim 1. CAR.

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

3. Ｌ１ The amino acid sequence of SEQ ID NO. The CAR according to claim 1, as shown in No.

4.

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

16.

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

7. The cell of claim 7, wherein the cell is a CAR-T cell and / or a CAR-NK cell. 。

8. A suitable amount of the cells according to claim 7 or a formulation comprising said cells is administered to a subject in need of treatment. Administering

9. The method of claim 9, wherein the disease is cancer or a tumor.

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