Bispecific antibody binding to cd20 and cd3 and uses thereof

Abstract

Disclosed is a bispecific antibody that specifically binds to surface antigens CD3 of immune cells and CD20 antigens on the surfaces of tumor cells, and that can bind to human CD3 with high affinity, inducing T cell proliferation, and mediating tumor cell killing. The bispecific antibody in an in vitro test was able to mediate the specific killing of target cells by T cells. The construction method thereof is simple, avoiding the possibility of mismatch between two sets of light chains and heavy chains of heterobispecific antibodies, thereby reducing the difficulty of antibody purification. The affinity of the obtained antibody is high, the side effects caused by cytokines are small, and safety is high.

1 . A bispecific antibody, which is a tetravalent homodimer formed by two identical polypeptide chains that bind to each other by a covalent bond, wherein each of the polypeptide chains comprises a first single-chain Fv that specifically binds to tumor antigen CD20, a second single-chain Fv that specifically binds to effector cell antigen CD3 and an Fc fragment in sequence from N-terminus to C-terminus; wherein the first single-chain Fv is linked to the second single-chain Fv by a linker peptide, the second single-chain Fv is linked to the Fc fragment directly or by a linker peptide, and the Fc fragment has no effector functions comprising CDC, ADCC and ADCP. 
     
     
         2 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv comprises a VH domain and a VL domain that are linked by a linker peptide which has an amino acid sequence of (GGGGX)n, wherein X comprises Ser or Ala, and n is a natural number from 1 to 5. 
     
     
         3 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv is selected from the group consisting of:
 (1) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively or having sequences that are least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 1, 2 and 3; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively or having sequences that are least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions any of SEQ ID NOs: 4, 5 and 6;   (2) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 7, 8 and 9, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 7, 8 and 9; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 10, 11 and 12;   (3) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14 and 15, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 13, 14 and 15; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 16, 17 and 18; and   (4) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 19, 20 and 21, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions any of SEQ ID NOs: 19, 20 and 21; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 22, 23 and 24.   
     
     
         4 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv is selected from the group consisting of:
 (1) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 25 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 25; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 26 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions SEQ ID NO: 26;   (2) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 27 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 27; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 28 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions SEQ ID NO: 28;   (3) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 29 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 29; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 30 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 30; and   (4) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 31 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 31; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 32 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 32.   
     
     
         5 . The bispecific antibody according to  claim 1 , wherein the second single-chain Fv comprises a VH domain and a VL domain that are linked by a linker peptide which has an amino acid sequence of (GGGGX) n , wherein X comprises Ser or Ala, and n is a natural number from 1 to 5. 
     
     
         6 . The bispecific antibody according to  claim 1 , wherein the second single-chain Fv binds to an effector cell at an EC 50  value greater than about 50 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM in an in vitro binding affinity assay; more preferably, the second single-chain Fv of the bispecific antibody is capable of binding to human CD3 and specifically binding to CD3 of a cynomolgus monkey or a rhesus monkey. 
     
     
         7 . The bispecific antibody according to  claim 6 , wherein the second single-chain Fv is selected from the group consisting of:
 (1) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 34, 35 and 36, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 34, 35 and 36; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 37, 38 and 39; and   (2) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 40, 41 and 42; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively or having sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more amino acid substitutions than any of SEQ ID NOs: 43, 44 and 45.   
     
     
         8 . The bispecific antibody according to  claim 7 , wherein the second single-chain Fv is selected from the group consisting of:
 (1) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 46 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 43; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 47 or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 47; and   (2) a second single-chain Fv that specifically binds to CD3; wherein the VH domain thereof contains an amino acid sequence as shown in SEQ ID NO: 48 or has a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 48; and the VL domain thereof contains an amino acid sequence as shown in SEQ ID NO: 49 or has a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acid substitutions than SEQ ID NO: 49.   
     
     
         9 . The bispecific antibody according to  claim 1 , wherein the linker peptide that links the first single-chain Fv to the second single-chain Fv consists of a flexible peptide and a rigid peptide; wherein the flexible peptide comprises two or more amino acids, and preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A) and Thr(T); more preferably, the flexible peptide comprises G and S residues; most preferably, an amino acid composition structure of the flexible peptide has a general formula of G x S y (GGGGS) z , wherein x, y and z are integers greater than or equal to 0 and x+y+z≥1; the rigid peptide is derived from a full-length sequence consisting of amino acids 118 to 145 at carboxyl terminus of natural human chorionic gonadotropin β-subunit or a truncated fragment thereof; preferably, the rigid peptide comprises SSSSKAPPPS. 
     
     
         10 . The bispecific antibody according to  claim 9 , wherein the linker peptide contains an amino acid sequence as shown in SEQ ID NO: 52. 
     
     
         11 . The bispecific antibody according to  claim 1 , wherein the linker peptide that links the Fc fragment to the second single-chain Fv comprises 1-20 amino acids, and preferably selected from the following amino acids: Gly(G), Ser(S), Ala(A) and Thr(T); more preferably selected from Gly (G) and Ser (S); further preferably, the linker peptide consists of (GGGGS)n, wherein n=1, 2, 3 or 4. 
     
     
         12 . The bispecific antibody according to  claim 1 , wherein the Fc fragment comprises a hinge region, a CH2 domain and a CH3 domain derived from a human immunoglobulin heavy chain constant region; preferably, the Fc fragment is selected from heavy chain constant regions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; more preferably, the Fc fragment is selected from heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4; further preferably, the Fc fragment is selected from a heavy chain constant region of human IgG1 or IgG4; and compared to a natural sequence from which the Fc fragment is derived, the Fc fragment has one or more amino acid substitutions, deletions or additions selected form the group consisting of:
 (i) amino acid substitutions L234A/L235A/P331S that are determined according to an EU numbering system;   (ii) amino acid substitutions M428L, T250Q/M428L/N434S or M252Y/S254T/T256E determined according to the EU numbering system;   (iii) an amino acid substitution N297A determined according to the EU numbering system; and   (iv) an amino acid deletion K447 determined according to the EU numbering system.   
     
     
         13 - 16 . (canceled) 
     
     
         17 . The bispecific antibody according to  claim 12 , wherein the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 57 that has six amino acid substitutions or replacements L234A/L235A/N297A/P331S/T250Q/M428L determined according to the EU numbering system and a deleted or removed K447 determined according to the EU numbering system compared to the natural sequence from which the Fc fragment is derived. 
     
     
         18 . The bispecific antibody according to  claim 1 , wherein the bispecific antibody binds to human CD20 and CD3 and has an amino acid sequence as follows:
 (1) a sequence as shown in SEQ ID NO: 50;   (2) a sequence having one or more substitutions, deletions or additions relative to the sequence as shown in SEQ ID NO: 50; or   (3) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity relative to the sequence as shown in SEQ ID NO: 50.   
     
     
         19 . A DNA molecule encoding the bispecific antibody according to  claim 1 . 
     
     
         20 . The DNA molecule according to  claim 19 , which has a nucleotide sequence as shown in SEQ ID NO: 51. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . A pharmaceutical composition, comprising the bispecific antibody according to  claim 1  and a pharmaceutically acceptable excipient, carrier or diluent. 
     
     
         24 . A method for preparing the bispecific antibody according to  claim 1 , comprising: (a) obtaining a fusion gene of the bispecific antibody, and constructing an expression vector of the bispecific antibody; (b) transfecting the expression vector into a host cell by a genetic engineering method; (c) culturing the host cell under conditions that allow the bispecific antibody to be generated; (d) separating and purifying the bispecific antibody;
 wherein the expression vector in step (a) is one or more selected from a plasmid, a bacterium and a virus; preferably, the expression vector is a pCDNA3.4 vector;   wherein the host cell into which the constructed vector is transfected by a genetic engineering method in step (b) comprises a prokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, an NS0 cell or another mammalian cell, preferably a CHO cell; and   wherein the bispecific antibody is separated and purified in step (d) by a conventional immunoglobulin purification method comprising protein A affinity chromatography and ion exchange, hydrophobic chromatography or molecular sieve chromatography.   
     
     
         25 - 27 . (canceled) 
     
     
         28 . A method for preventing/treating, delaying development of, or reducing/inhibiting recurrence of a disease including diseases or disorders comprising an immune-related disease, a tumor, an autoimmune disease, an inflammatory disease or a transplant rejection-related disease or disorder, comprising administering an effective amount of the bispecific antibody of  claim 1  to an individual suffering from the diseases or disorders,
 wherein the tumor comprises acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B acute lymphocytic leukemia (B-ALL), B chronic lymphocytic leukemia (B-CLL), B-cell lymphoma (BCL), T-cell lymphoma (TCL) (such as skin), myelodysplastic syndrome (MDS), small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL), marginal zone lymphoma (MZL) (such as extranodal or splenic), follicular lymphoma (FL) (such as pediatric or gastrointestinal), B-cell prolymphocytic leukemia (B-PLL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia (WM), lymphoblastic leukemia (ALL) (such as B cell), lymphoblastic lymphoma (LBL) (such as B cell), plasmablastic lymphoma (PBL) (such as B cell), Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL) (for example, primary or inflammation-related), Burkitt's lymphoma (BL), multiple myeloma, anaplastic large-cell lymphoma, HIV-related lymphoma and Waldenstrom's macroglobulinemia, 
 the autoimmune or inflammatory disease is selected from rheumatoid arthritis (RA), osteoarthritis, reactive arthritis, systemic lupus erythematosus (SLE), Crohn's disease, multiple sclerosis, scleroderma, psoriasis, psoriatic arthritis, ulcerative colitis (such as chronic), insulin-dependent diabetes (such as juvenile), thyroiditis (such as chronic), hyperthyroidism, asthma, allergic diseases, sarcoidosis, autoimmune hemolytic anemia, pernicious anemia, graft-versus-host disease, dermatomyositis, chronic hepatitis, microscopic renal vasculitis, chronic active hepatitis, uveitis, intestinal synovitis, autoimmune intestinal disease, idiopathic leukopenia, autoimmune glomerulonephritis, autoimmune hemolytic anemia, autoimmune hepatitis, interstitial pneumonia, chronic pemphigus, pemphigus vulgaris, arteritis, polyarteritis nodosa and ankylosing spondylitis.