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.

Claims

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).sub.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.sub.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.sub.xS.sub.y(GGGGS).sub.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.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0161] FIG. 1 illustrates SEC-HPLC test results of a purified sample of bispecific antibody AP062.

[0162] FIG. 2 illustrates SDS-PAGE electrophoresis results of a purified sample of bispecific antibody AP062.

[0163] FIG. 3 illustrates SDS-PAGE results of bispecific antibody AP062 in an acceleration experiment at 5° C.

[0164] FIG. 4 illustrates SDS-PAGE results of bispecific antibody AP062 in an acceleration experiment at 25° C.

[0165] FIG. 5 illustrates an ability, detected by flow cytometry, of bispecific antibody AP062 to bind to CD20-positive tumor cells.

[0166] FIG. 6 illustrates an ability of bispecific antibody AP062 to mediate effector cells to kill Raji-luc cells.

[0167] FIG. 7 illustrates abilities, detected by a reporter gene assay, of bispecific antibodies AP062 and AB7K7 to activate Jurkat NFATRE Luc cells.

[0168] FIG. 8 illustrates an in vivo anti-tumor effect of bispecific antibody AP062 in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Raji cells.

[0169] FIG. 9 illustrates an in vivo anti-tumor effect of bispecific antibody AP062 in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Daudi cells.

[0170] FIG. 10 illustrates changes of leukocytes and lymphocytes in normal cynomolgus monkeys administered with bispecific antibody AP062 multiple times.

DETAILED DESCRIPTION

[0171] The present disclosure is further described through examples which should not be construed as further limitations. All drawings, all reference documents, and the contents of patents and published patent applications cited in the entire application are expressly incorporated herein by reference.

[0172] In the following examples, materials used in experiments may be purchased or prepared with reference to techniques disclosed in the existing art; materials whose sources and specifications are unidentified are commercially available; various processes and methods not described in detail are conventional methods well known in the art.

Example 1 Construction of an Expression Vector of a Bispecific Antibody Molecule

[0173] The bispecific antibody molecule constructed in the present disclosure is a tetravalent homodimer formed by two identical polypeptide chains binding to each other by an interchain disulfide bond in hinge regions of Fc fragments, wherein each polypeptide chain consists of an anti-CD20 scFv, a linker peptide L2, an anti-CD3 scFv and an Fc fragment in sequence from N-terminus to C-terminus, wherein VH and VL in the anti-CD20 scFv are linked by a linker peptide L1, and VH and VL in the anti-CD3 scFv are linked by a linker peptide L3. Some preferred amino acid sequences of the VH domain and its complementarity determining regions (HCDR1, HCDR2 and HCDR3) and amino acid sequences of the VL domain and its complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a first single-chain Fv against CD20 are listed in Table 1, wherein amino acid residues contained in the CDRs are defined according to a Kabat rule. The amino acid composition of the linker peptide L1 between VH and VL of the anti-CD20 scFv is (GGGGS)n, wherein n=1, 2, 3, 4 or 5.

TABLE-US-00001 TABLE 1 Amino acid sequences of the anti-CD20 scFv contained in the bispecific antibody and amino acid sequences of its CDRs CD20 SEQ ID NO: 1 HCDR1 SYNMH SEQ ID NO: 2 HCDR2 AIYPGNGDTSYNQKFKG SEQ ID NO: 3 HCDR3 STYYGGDWYFNV SEQ ID NO: 4 LCDR1 RASSSVSYIH SEQ ID NO: 5 LCDR2 ATSNLAS SEQ ID NO: 6 LCDR3 QQWTSNPPT SEQ ID NO: 25 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWY FNVWGAGTTVTVSA SEQ ID NO: 26 VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQK PGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRV EAEDAATYYCQQWTSNPPTFGGGTKLEIK SEQ ID NO: 7 HCDR1 NYYIH SEQ ID NO: 8 HCDR2 WIYPGDGNTKYNEKFKG SEQ ID NO: 9 HCDR3 DSYSNYYFDY SEQ ID NO: 10 LCDR1 RASSSVSYMH SEQ ID NO: 11 LCDR2 APSNLAS SEQ ID NO: 12 LCDR3 QQWSFNPPT SEQ ID NO: 27 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHW VRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTA DTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYW GQGTLVTVSS SEQ ID NO: 28 VL DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQ KPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQWSFNPPTFGQGTKVEIK SEQ ID NO: 13 HCDR1 YSWIN SEQ ID NO: 14 HCDR2 RIFPGDGDTDYNGKFKG SEQ ID NO: 15 HCDR3 NVFDGYWLVY SEQ ID NO: 16 LCDR1 RSSKSLLHSNGITYLY SEQ ID NO: 17 LCDR2 QMSNLVS SEQ ID NO: 18 LCDR3 AQNLELPYT SEQ ID NO: 29 VH QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINW VRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITA DKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVY WGQGTLVTVSS SEQ ID NO: 30 VL DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLY WYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDF TLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK SEQ ID NO: 19 HCDR1 DYAMH SEQ ID NO: 20 HCDR2 TISWNSGSIGYADSVKG SEQ ID NO: 21 HCDR3 DIQYGNYYYGMDV SEQ ID NO: 22 LCDR1 RASQSVSSYLA SEQ ID NO: 23 LCDR2 DASNRAT SEQ ID NO: 24 LCDR3 QQRSNWPIT SEQ ID NO: 31 VH EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHW VRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRD NAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYG MDVWGQGTTVTVSS SEQ ID NO: 32 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPITFGQGTRLEIK

[0174] The anti-CD3 scFv binds to an effector cell at an EC.sub.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 FACS binding 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.

[0175] Some preferred amino acid sequences of the VH domain and its complementarity determining regions (HCDR1, HCDR2 and HCDR3) and amino acid sequences of the VL domain and its complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the anti-CD3 scFv are listed in Table 2, wherein amino acid residues contained in the CDRs are defined according to the Kabat rule. The amino acid composition of the linker peptide L3 between VH and VL of the anti-CD3 scFv is (GGGGS)n, wherein n=1, 2, 3, 4 or 5.

TABLE-US-00002 TABLE 2 Amino acid sequences of the anti-CD3 scFv contained in the bispecific antibody and amino acid sequences of its CDRs CD3-3 SEQ ID NO: 34 HCDR1 TYAMN SEQ ID NO: 35 HCDR2 RIRSKYNNYATYYADSVKD SEQ ID NO: 36 HCDR3 HGNFGNSYVSWFAY SEQ ID NO: 37 LCDR1 RSSTGAVTTSNYAN SEQ ID NO: 38 LCDR2 GTNKRAP SEQ ID NO: 39 LCDR3 ALWYSNLWV SEQ ID NO: 46 VH EVQLLESGGGLVQPGGSLKLSCAASGFTF NTYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNTAYLQMNN LKTEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS SEQ ID NO: 47 VL ELVVTQEPSLTVSPGGTVTLTCRSSTGAV TTSNYANWVQQKPGQAPRGLIGGTNKRA PGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCALWYSNLWVFGGGTKLTVL CD3-4 SEQ ID NO: 40 HCDR1 KYAMN SEQ ID NO: 41 HCDR2 RIRSKYNNYATYYADSVKD SEQ ID NO: 42 HCDR3 HGNFGNSYISYWAY SEQ ID NO: 43 LCDR1 GSSTGAVTSGYYPN SEQ ID NO: 44 LCDR2 GTKFLAP SEQ ID NO: 45 LCDR3 ALWYSNRWV SEQ ID NO: 48 VH EVQLLESGGGLVQPGGSLKLSCAASGFTF NKYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNTAYLQMNN LKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVSS SEQ ID NO: 49 VL ELVVTQEPSLTVSPGGTVTLTCGSSTGAV TSGYYPNWVQQKPGQAPRGLIGGTKFLAP GTPARFSGSLLGGKAALTLSGVQPEDEAE YYCALWYSNRWVFGGGTKLTVL

[0176] The linker peptide that links the anti-CD20 scFv to the anti-CD3 scFv consists of a flexible peptide and a rigid peptide; preferably, an amino acid composition structure of the flexible peptide has a general formula of G.sub.xS.sub.y(GGGGS).sub.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 (as shown in SEQ ID NO: 33) consisting of amino acids 118 to 145 at the carboxyl terminus of natural human chorionic gonadotropin β-subunit or a truncated fragment thereof; preferably, the composition of the CTP rigid peptide is SSSSKAPPPS (CTP.sup.1). Some preferred amino acid sequences of the linker peptide L2 that links the anti-CD20 scFv and the anti-CD3 scFv are listed in Table 3.

TABLE-US-00003 TABLE 3 Amino acid sequences of the linker peptide that links the anti-CD20 scFv to the anti-CD3 scFv SEQ ID NO: 52 G2(GGGGS)3CTP1 GGGGGGSGGGGSGGGGSSSS SKAPPPS SEQ ID NO: 53 (GGGGS)3CTP1 GGGGSGGGGSGGGGSSSSSK APPPS SEQ ID NO: 54 GS(GGGGS)2CTP1 GSGGGGSGGGGSSSSSKAPP PS SEQ ID NO: 55 (GGGGS)1CTP4 GGGGSSSSSKAPPPSLPSPS RLPGPSDTPILPQ

[0177] The Fc fragment is linked to the anti-CD3 scFv directly or by a linker peptide, wherein the linker peptide includes 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), and most preferably, the linker peptide consists of (GGGGS)n, wherein n=1, 2, 3 or 4.

[0178] The Fc fragment is preferably selected from heavy chain constant regions of human IgG1, IgG2, IgG3 and IgG4 and more particularly selected from heavy chain constant regions of human IgG1 or IgG4; and Fc is mutated to modify the properties of the bispecific antibody molecule, e.g., reduced affinity to at least one of human FcγRs (FcγRI, FcγRIIa or FcγRIIIa) and C1q, a reduced effector cell function, or a reduced complement function. In addition, the Fc fragment may also contain amino acid substitutions that change one or more other characteristics (such as an ability of binding to an FcRn receptor, the glycosylation of the antibody or the charge heterogeneity of the antibody).

[0179] Some amino acid sequences of the Fc fragment with one or more amino acid mutations are listed in Table 4.

TABLE-US-00004 TABLE 4 Amino acid sequences of Fc from human IgG Amino acid sequence of constant region of IgG1 Fc (L234A/L235A) mutant (EU numbering) SEQ ID NO: 56 DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK Amino acid sequence of constant region of IgG1 (L234A/L235A/T250Q/N297A/P331S/M428L/K447-) mutant (EU numbering) SEQ ID NO: 57 DKTHTCPPCP APEAAGGPSV FLFPPKPKDQ LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA SIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVLHE ALHNHYTQKS LSLSP

[0180] Exemplarily, the amino acid sequence of the preferred bispecific antibody AP062 is shown by SEQ ID NO: 50, and the corresponding nucleotide sequence is shown by SEQ ID NO: 51.

[0181] A gene encoding the bispecific antibody was synthesized by a conventional molecular biology method, and cDNA encoding the obtained fusion gene was separately inserted into corresponding restriction sites of a eukaryotic expression plasmid pCMAB2M modified by PCDNA3.1. The plasmid pCMAB2M also contains a selective marker to have kanamycin resistance in bacteria and G418 resistance in mammalian cells. In addition, when host cells are deficient in the expression of DHFR genes, the expression vector pCMAB2M contains mouse dihydrofolate reductase (DHFR) genes so that target genes and the DHFR genes can be co-amplified in the presence of methotrexate (MTX) (see U.S. Pat. No. 4,399,216).

Example 2 Expression of the Bispecific Antibody Molecule

[0182] The constructed expression plasmid was transfected into a mammalian host cell line to express the bispecific antibody. To achieve stable and high-level expression, a preferred host cell line is DHFR deficient CHO-cells (see U.S. Pat. No. 4,818,679). In this example, a CHO-derived cell strain DXB11 was selected as the host cell. A preferred method of transfection is electroporation, and other methods, including calcium phosphate co-precipitation and lipofection, may also be used. During electroporation, 50 μg of DNA of the expression vector plasmid were added to 5×10.sup.7 cells in a cuvette with a Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, Calif.) set at an electric field of 300 V and a capacitance of 1500 μFd. Two days after transfection, the medium was replaced with a growth medium containing 0.6 mg/mL G418. Transfectants were subcloned by limiting dilution and the secretion rate of each cell line was measured through an ELISA. The cell strain that expressed the bispecific antibody at a high level was screened.

[0183] To achieve the higher-level expression of fusion proteins, DHFR genes inhibited by MTX should be used for co-amplification. The transfected fusion protein genes were co-amplified with the DHFR genes in growth media containing MTX with increased concentrations. Subclones with the positive expression of DHFR were subjected to limiting dilution with gradually increased pressure to screen transfectants capable of growing in media with MTX of up to 6 μM. The secretion rates of the transfectants were determined and a cell line with high foreign protein expression was screened. A cell line with a secretion rate greater than 5 (preferably about 15)μg/10.sup.6 (millions) cells/24 h was adaptively suspended using a serum-free medium. Cell supernatants were collected and the bispecific antibody was separated and purified.

Example 3 Purification Process and Stability Test of the Bispecific Antibody

[0184] 3.1 Purification of the Bispecific Antibody

[0185] The bispecific antibody was purified by three-step chromatography. The three-step chromatography was respectively affinity chromatography, hydroxyapatite chromatography and anion exchange chromatography. (The protein purifier used in this example was AKTA pure 25 M from GE in the U.S. Reagents used in this example were purchased from Sinopharm Chemical Reagent Co., Ltd and had purity at an analytical grade).

[0186] In a first step, affinity chromatography was performed. Sample capture and concentration and the removal of partial pollutants were performed using AT Protein A Diamond from Bestchrom or other commercially available affinity media (such as MabSelect Sure from GE). First, chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 150 mM NaCl, pH 7.4) at a linear flow rate of 100-200 cm/h. The clarified fermentation broth was loaded at a linear flow rate of 100-200 cm/h with a load not higher than 20 mg/mL. After loading, the chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 150 mM NaCl, pH 7.4) at a linear flow rate of 100-200 cm/h to remove unbound components. The chromatography columns were rinsed with 3-5 column volumes decontamination buffer 1 (50 mM NaAc-HAc, 1 M NaCl, pH 5.0) at a linear flow rate of 100-200 cm/h to remove partial pollutants. The chromatography columns were equilibrated with 3-5 column volumes (CVs) of decontamination buffer 2 (50 mM NaAc-HAc, pH 5.0) at a linear flow rate of 100-200 cm/h. The target product was eluted using an elution buffer (50 mM NaAc-HAc, pH 3.5) at a linear flow rate not higher than 100 cm/h and target peaks were collected.

[0187] In a second step, hydroxyapatite chromatography was performed. Intermediate purification was performed using CHT TypeII from BIO-RAD or other commercially available hydroxyapatite media to reduce the content of polymers. After the target proteins were polymerized, the polymers and monomers differed in property such as charge characteristics and calcium ion chelation. The polymers and the monomers were separated with differences between charge characteristics. First, chromatography columns were equilibrated with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, pH 7.4) at a linear flow rate of 100-200 cm/h. The target proteins separated through the affinity chromatography in the first step were loaded after its pH was adjusted to 7.0, with a load controlled to be less than 5 mg/mL. After loading, the chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, pH 7.0) at a linear flow rate of 100-200 cm/h. Finally, the target proteins were eluted using 10 column volumes (CVs) of an elution buffer (20 mM PB, 1M NaCl, pH 7.0) at a linear flow rate not higher than 100 cm/h with a gradient of 0-25%. Eluted fractions were collected and sent for SEC-HPLC, respectively. Target components with the percentage of monomers being greater than 95% were combined for chromatography in the next step.

[0188] In a third step, anion exchange chromatography was performed. Fine purification was performed by using DEAE Sepharose Fast Flow from GE or other commercially available anion exchange chromatography media (such as Q HP of Bestchrom, Toyopearl GigaCap Q-650 of TOSOH, DEAE Beads 6FF of Smart-Lifesciences, Generik MC-Q of Sepax Technologies, Inc, Fractogel EMD TMAE of Merck, and Q Ceramic HyperD F of Pall) to further remove pollutants such as HCP and DNA. First, chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 0.15 M NaCl, pH 7.0) at a linear flow rate of 100-200 cm/h. The target proteins separated through hydroxyapatite chromatography in the second stop were loaded and through-flow was collected. After loading, the chromatography columns were rinsed with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 0.15 M NaCl, pH 7.0) at a linear flow rate of 100-200 cm/h. The through-flow components were collected and sent for the detection of protein content, SEC-HPLC and electrophoresis.

[0189] The SEC-HPLC purity results and SDS-PAGE electrophoresis results of the samples are shown in FIG. 1 and FIG. 2. The SEC-HPLC results showed that the purity of the main peak of the bispecific antibody was more than 99.0% after three-step chromatography. The band pattern in the SDS-PAGE electrophoresis was as expected, where a band was shown at 160 KDa in the non-reducing electrophoresis and a clear single-chain band (80 KDa) was obtained after reduction.

[0190] 3.2 Stability Test of the Bispecific Antibody

[0191] The stability of a bispecific antibody AP062 in histidine salts (20 mM histidine, 8% sucrose, 0.02% Tween-80) at different pH (pH 5.5, 6.0 and 6.5) was investigated and the effect of shaking on the stability of the sample was explored. The bispecific antibody AP062 was stored for two weeks under accelerated conditions at 5° C. and 25° C. for the evaluation of protein stability.

[0192] AP062 was transferred to histidine buffers of pH 5.5, 6.0 and 6.5, separately. Samples were taken at different time points for detection and analysis. The detection items included SEC-HPLC and SDS-PAGE.

[0193] SEC-HPLC and SDS-PAGE test results of three preparations stored for 0-2 weeks at 5° C. and 25° C. are shown in Tables 5 and 6 and FIGS. 3 and 4. After the samples were placed still for two weeks, the proportions of the polymers, main peaks, shoulder peaks and fragments in the SEC-HPLC test results have no significant changes and differ little at different pH. There is no significant difference in SEC-HPLC results over two weeks in the histidine buffers of pH 5.5, 6.0 and 6.5.

TABLE-US-00005 TABLE 5 SEC-HPLC results of acceleration experiments at 5° C. SEC-polymer % SEC-main peak % SEC-fragment % T 0 1 W 2 W T 0 1 W 2 W T 0 1 W 2 W pH 5.5 2.5 1.1 2.7 95.6 97.4 96.6 1.8 1.6 0.7 pH 6.0 1.9 0.7 2.8 96.3 97.9 96.6 1.8 1.3 0.7 pH 6.5 2.3 0.8 2.9 95.6 97.4 96.4 2.1 1.8 0.7

TABLE-US-00006 TABLE 6 SEC-HPLC results of acceleration experiments at 25° C. SEC-polymer % SEC-main peak % SEC-fragment % T 0 1 W 2 W T 0 1 W 2 W T 0 1 W 2 W pH 5.5 2.5 0.8 2.1 95.6 97.5 97.2 1.8 1.7 0.8 pH 6.0 1.9 0.6 2.4 96.3 97.7 96.9 1.8 1.7 0.7 pH 6.5 2.3 0.6 2.2 95.6 97.6 97.0 2.1 1.8 0.9

Example 4 In Vitro Pharmacodynamic Study of the Bispecific Antibody

[0194] 4.1 Detection of the Binding Activity of AP062 to CD20-Positive Tumor Cells by Flow Cytometry

[0195] Raji cells (purchased from the cell bank of Chinese Academy of Sciences) were cultured and collected by centrifugation. The collected cells were resuspended with 1% PBSB and placed in 96-well plates, 100 μl (i.e., 2×10.sup.5 cells) per well, after the cell density was adjusted to (2×10.sup.6) cells/ml. Diluted bispecific antibodies with a series of concentrations were added and incubated for 1 hour at 4° C. The cells were centrifuged to discard the supernatant and then washed three times using a PBS solution with 1% BSA (PBSB). Diluted AF488-labeled goat anti-human IgG antibodies (Jackson Immuno Research Inc., Cat. No. 109-545-088) or mouse anti-6×His IgG antibodies (R&D Systems, Cat. No. IC050P) were added to the cells, and the cells were incubated for 1 hour at 4° C. in the dark. The obtained cells were centrifuged to discard the supernatant and then washed twice with 1% PBSB, and cells in each well were resuspended with 100 μl of 1% paraformaldehyde. The signal intensity was detected by flow cytometry. The analysis was performed with the average fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value for the binding of AP062 to Raji cells.

[0196] As shown in FIG. 5, AP062 bound well to CD20-positive cells, the signal intensity was proportional to the antibody concentration, and the EC.sub.50 value for AP062 binding to Raji cells was calculated, which was about 69.97 nM.

[0197] 4.2 AP062 Mediating Effector Cells to Target and Kill CD20-Positive Tumor Cells

[0198] Normally cultured Raji-luc cells (purchased from Beijing Biocytogen Biotechnology Co., Ltd.) were added to 96-well white plates after the cell density was adjusted to 1×10.sup.5 cells/ml, 40 μl per well. AP062 antibodies were diluted into a series of gradients and added to the 96-well white plates. After the CIK cell density was adjusted to 5×10.sup.5 cells/ml, the CIK cells were added to the 96-well white plates, 40 μl per well, to make the effector:target ratio (E:T) equal to 5:1, and cultured for 24 hours at 37° C. After 24 hours, the white plates were taken out, 100 μl of One-Glo (Promega, Cat. No. E6120) solution was added to each well, and then the white plates were placed for at least three minutes at room temperature. The luminescence value was measured by a microplate reader. The analysis was performed with the fluorescence intensity as the Y-axis and the antibody concentration as the X-axis through software GraphPad to calculate the EC.sub.50 value of AP062 killing Raji-luc cells.

[0199] As shown in FIG. 6, the EC.sub.50 for AP062 mediating effector cells to kill Raji-luc cells was only 42.8 ng/ml and AP062 had target specificity, while the EC.sub.50 of isotype antibody as a negative control was 229.5 ng/ml and isotype antibody had little killing effect on Raji-luc cells.

[0200] 4.3 Evaluation of Abilities of Bispecific Antibodies to Activate T Cells Through Reporter Gene Cell Strains

[0201] Jurkat T cells containing NFAT RE reporter genes (BPS Bioscience, Cat. No. 60621) can overexpress luciferase in the presence of bispecific antibodies and CD20-positive Raji cells, and the degree of activation of the Jurkat T cells can be quantified by detecting the activity of the luciferase. A four-parameter curve was fitted using the concentration of bispecific antibody as the X-axis and the fluorescein signal as the Y-axis.

[0202] As shown in FIG. 7, AP062 can specifically activate Jurkat NFATRE Luc cells, wherein the EC.sub.50 value was 0.2006 μg/ml and its concentration was proportional to signal intensity, while AB7K7 as a negative control had little ability to activate T cells.

Example 5 In Vivo Pharmacodynamic Study of the Bispecific Antibody

[0203] 5.1 NPG Mouse Model of Transplanted Tumor Established by Co-Inoculating Subcutaneously Human CIK Cells and Human Burkkit's Lymphoma Raji Cells

[0204] Human Burkkit's lymphoma Raji cells with positive CD20 expression were selected to observe the in vivo anti-tumor effect of the bispecific antibody in a NPG mouse model of transplanted tumor that was established by co-inoculating subcutaneously human CIK cells and human Burkkit's lymphoma Raji cells.

[0205] The peripheral blood of a normal human was taken. Human PBMCs were separated through density gradient centrifugation (Lymphoprep™, human lymphocyte isolation solution, STEMCELL), then re-suspended in a RPMI-1640 culture medium added with 10% inactivated FBS, and added with OKT3 at a final concentration of 1 μg/mL and human IL-2 at 250 IU/mL. After three days of culture, the mixture was centrifuged at 300 g for 5 min, and the medium was replaced with RPMI-1640 added with 10% inactivated FBS for cell culture and human IL-2 at 250 IU/mL. A fresh medium was then added every 2 days, and CIK cells were collected on the tenth day of culture. Female NPG mice at the age of seven to eight weeks (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) were selected and Raji cells in the logarithmic growth stage were collected. 4×10.sup.6 Raji cells and 8×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour later, the mice were randomly divided into 5 groups with 6 mice in each group according to their weight. They were intraperitoneally administered with corresponding drugs. All administration groups were administered twice per week. The positive control monoclonal antibody Rituxan (Merlot, Roche Pharmaceuticals) and the bispecific antibody AP062 were separately administered at a dose of 1 mg/kg and a dose of 0.1 mg/kg. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured twice per week. The volume of the tumor was calculated using the following formula: volume (mm.sup.3)=[Dxd.sup.2]/2. The tumor growth inhibition rate was calculated for each administration group using the following formula: TGI (%)=(1−the volume of the administration group/the volume of the control group)×100%.

[0206] As shown in FIG. 8, on Day 24 of administration, the average tumor volume of the PBS control group was 1766.84±155.62 mm.sup.3; the average tumor volume of the treated group administrated with Rituxan at a dose of 1 mg/kg was 647.92±277.11 mm.sup.3, and TGI was 63.33%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with Rituxan at a dose of 0.1 mg/kg was 1893.81±186.99 mm.sup.3, and Rituxan herein exhibited no efficacy; the average tumor volume of the treated group administrated with AP062 at a dose of 1 mg/kg was 116.18±39.50 mm.sup.3, and TGI was 93.42%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with AP062 at a dose of 0.1 mg/kg was 1226.03±340.05 mm.sup.3, and TGI was 30.61%, which was not significantly different from that of the control group. The results show that the bispecific antibody AP062 could inhibit the growth of tumor cells by activating human immune cells in animals; and at the same dose, the efficacy of the bispecific antibody was better than the efficacy of the monoclonal antibody Rituxan, and the bispecific antibody exhibited great anti-tumor effects.

[0207] 5.2 NPG Mouse Model of Transplanted Tumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Daudi Cells

[0208] CD20-positive human Burkkit's lymphoma Daudi cells were selected to study the inhibiting effect of bispecific antibodies on tumor growth in vivo in an NPG mouse model of transplanted tumor constructed by subcutaneously co-inoculating human CIK cells and human Burkkit's lymphoma Daudi cells.

[0209] CIK cells were prepared in the method as described in Example 5.1. Female NPG mice at the age of seven to eight weeks were selected, and Daudi cells in the logarithmic growth stage were collected. 4×10.sup.6 Daudi cells and 8×10.sup.5 CIK cells were mixed and inoculated subcutaneously on the right back of each NPG mouse. One hour later, the mice were randomly divided into five groups with six mice in each group according to their weights and intraperitoneally administered with corresponding drugs. All treated groups were administrated twice a week. Rituxan and bispecific antibody AP062 were both administered at doses of 1 mg/kg and 0.1 mg/kg, respectively. The day of administration was recorded as Day 0. The maximum diameter (D) and the minimum diameter (d) of the tumor were measured weekly. The volume (mm.sup.3) of the tumor of each group and the tumor growth inhibition rate (TGI) (%) of each treated group were calculated using the formulas as shown in Example 5.1.

[0210] As shown in FIG. 9, on Day 30 of administration, the average tumor volume of the PBS control group was 889.68±192.13 mm.sup.3; the average tumor volume of the treated group administrated with Rituxan at a dose of 1 mg/kg was 241.51±44.91 mm.sup.3, and TGI was 72.85%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with Rituxan at a dose of 0.1 mg/kg was 746.11±299.71 mm.sup.3, which was not significantly different from that of the control group; the average tumor volume of the treated group administrated with AP062 at a dose of 1 mg/kg was 72.05±11.89 mm.sup.3, and TGI was 91.9%, which was significantly different from that of the control group (P<0.01); the average tumor volume of the treated group administrated with AP062 at a dose of 0.1 mg/kg was 75.36±11.81 mm.sup.3, and TGI was 91.53%, which was significantly different from that of the control group (P<0.01). The results show that the bispecific antibody AP062 could inhibit the growth of tumor cells by activating human immune cells in animals; and at the same dose, the efficacy of the bispecific antibody was better than the efficacy of the monoclonal antibody Rituxan, and AP062 exhibited good anti-tumor effects even at a low dose.

Example 6 Evaluation of the Safety of Bispecific Antibodies

[0211] The toxicity of AP062 was evaluated to determine appropriate dose ranges and observation indicators for subsequent toxicity tests. Adult Female cynomolgus monkeys (purchased from Guangzhou Xiangguan Biotechnology Co., Ltd.) at the age of 3-4 years and with the weight of 3-4 kg were divided into two groups with one mouse in each group, wherein the two groups were a vehicle control group and an AP062 treated group. The groups were administrated via intravenous drip by a peristaltic pump for 1 hour. The dose amount and volume administered are shown in Table 7. The groups were administrated on Day 0 (D0), Day 7 (D7), Day 21 (D21), and Day 28 (D28), respectively, for a total of four doses, and the drug dose was gradually escalated each time. The monkeys were weighed weekly.

TABLE-US-00007 TABLE 7 Dosing schedule for cynomolgus monkey acute toxicity evaluation To-be-tested Group drugs name Dose volume Dose amount G1 Vehicle D0: 5 mL/kg N/A control group D7: 5 mL/kg D21: 10 mL/kg D28: 10 mL/kg G2 AP062 D0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg D7: 0.3 mg/kg D21: 10 mL/kg D21: 1.5 mg/kg D28: 10 mL/kg D28: 3 mg/kg

[0212] During the test, animals were periodically monitored for clinical symptoms, body weight, food consumption, body temperature, electrocardiogram, blood pressure, clinicopathological indexes (blood cell count, coagulation function measure, and blood biochemistry), lymphocyte subsets, cytokines, drug plasma concentration measure, and toxicokinetics analyses. After administration of AP062, the physical signs of cynomolgus monkeys exhibited no abnormal reaction, the body weight was relatively stable, the body temperature fluctuation was similar to the body temperature fluctuation of the vehicle control group, and no death or impending death was observed among animals during the administration period. As shown in FIG. 10, after administration, the white blood cell changes of cynomolgus monkeys in the AP062 group were similar to the white blood cell changes in the control group; the first administration of AP062 at a dose of 0.06 mg/kg had little effect on lymphocytes; 1 hour to 6 hours after the second administration, the number of lymphocytes in the animals of the treated group decreased sharply and recovered to normal after 24 hours; as the number of administrations increased, the effect of AP062 on the decrease in the number of lymphocytes was weaker and weaker despite increasing doses. In addition, after the first administration of AP062, the release of IL-2, IL-6 and TNF-α factors was promoted and the release of IL-5 was slightly stimulated, but the release of IFN-γ was not stimulated; as the number of administrations increased, the release-promoting effect of AP062 on cytokines became less and less significant, indicating that the body had already been adapted to the stimulation by bispecific antibodies.

Example 7 Pharmacokinetics Evaluation of Anti-CD20×CD3 Bispecific Antibodies

[0213] Female cynomolgus monkeys with the weight of 3-4 kg were divided into two groups with one in one monkey in each group. The first group was a blank control group, and the second group was an AP062 treated group administrated at a dose of 0.3 mg/kg. The blood sampling time points were Minute 15, Hour 1, Hour 3, Hour 6, Hour 10, Hour 24, Hour 30, Hour 48, Hour 54, Hour 72, Hour 96, and Hour 144, respectively, a total of 13 time points. Serum was collected from blood and frozen at −80° C.

[0214] The drug concentration of AP062 in serum was determined by ELISA. The pharmacokinetics parameters were calculated using software PKSolver. Specific parameters are shown in Table 8. The results show that Ti/2 of AP062 in normal cynomolgus monkeys was about 8.5 hours.

TABLE-US-00008 TABLE 8 Pharmacokinetics parameters of bispecific antibody AP062 in cynomolgus monkeys AUC Vz_obs Cl_obs t.sub.1/2 0-inf_obs (μg/kg)/ (μg/kg)/ AP062 (h) (μg/mL*h) (μg/mL) (μg/mL)/h Pharmacokinetics 8.45 168.63 21.68 1.78 parameter

[0215] Though the preferred examples of the present disclosure are illustrated and described, it should be understood that those skilled in the art may make various changes in accordance with the teachings herein within the scope of the present disclosure.

[0216] All the publications mentioned in the present disclosure are incorporated herein by reference as if each publication is separately incorporated herein by reference. In addition, it should be understood that those skilled in the art, who have read the preceding content of the present disclosure, may make various changes or modifications on the present disclosure, and these equivalent forms fall within the scope of the appended claims.