Homodimer-type bispecific antibody against her2 and cd3 and use thereof

Abstract

A tetravalent, homodimer-type bispecific antibody molecule that simultaneously targets immune effector cell antigen CD3 and human epidermal growth factor receptor 2 (Her2); the bispecific antibody molecule comprises, from in sequence from N-terminus to C-terminus, a first single-chain Fv capable of specifically binding to Her2, a second single-chain Fv capable of specifically binding to CD3, and an Fc fragment; the first and second single-chain Fv are connected by means of a connection peptide, and the second single-chain Fv is connected to the Fc directly fragment or is connected by means of a connection peptide; the Fc fragment does not have effector functions such as CDC, ADCC and ADCP. The bispecific antibody may significantly inhibit or kill tumor cells, and has controlled toxic side effects that may be caused by excessive activation of effector cells. The maximum safe starting dose in preclinical toxicology evaluation tests is significantly higher than other doses having the same target, and no systemic immunotoxicity occurs, suggesting that the drug administration safety window for the bispecific antibody is wide; in addition, said bispecific antibody is a homodimer that does not experience the problem of heavy chain and light chain mismatching; the steps of purification are simple and efficient, expression is high, and the physicochemical and in vivo stability of the antibody are significantly improved.

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 human epidermal growth factor receptor 2, 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 comprises a VH domain and a VL domain selected from the group consisting of:
 (i) a VH domain that contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 9, 10 and 11, 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: 9, 10 and 11; and   a VL domain that contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 12, 13 and 14, 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: 12, 13 and 14;   (ii) a VH domain that contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 17,18 and 19, 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: 17, 18 and 19; and a VL domain that contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 20, 21 and 22, 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: 20, 21 and 22; and   (iii) a VH domain that contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 25, 26 and 27, 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: 25, 26 and 27; and a VL domain that contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 28, 29 and 30, 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: 28, 29 and 30.   
     
     
         4 . The bispecific antibody according to  claim 1 , wherein the first single-chain Fv comprises a VH domain and a VL domain selected from the group consisting of:
 (i) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 15 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: 15; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 16 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: 16;   (ii) a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 23 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: 23; and a VL domain comprising an amino acid sequence as shown in SEQ ID NO: 24 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: 24; and   (iii) 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, preferably Ser, and n is a natural number from 1 to 5, preferably 3,
 wherein the second single-chain Fv binds to an effector cell an 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; and wherein, 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.   
     
     
         6 . (canceled) 
     
     
         7 . The bispecific antibody according to  claim 1 , wherein the VH domain of the second single-chain Fv contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 33, 34 and 35, 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 SEQ ID NOs: 33, 34 and 35; and the VL domain of the second single-chain Fv contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 36, 37 and 38, 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 SEQ ID NOs: 36, 37 and 38;
 the VH of the second single-chain Fv contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 41, 42 and 43, 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 SEQ ID NOs: 41, 42 and 43; and the VL domain of the second single-chain Fv contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 44, 45 and 46, 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 subsitutions than SEQ ID NOs: 44, 45 and 46.   
     
     
         8 . (canceled) 
     
     
         9 . The bispecific antibody according to  claim 7 , wherein the second single-chain Fv specifically binds to CD3; the VH domain of the second single-chain Fv contains an amino acid sequence as shown in SEQ ID NO: 39 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: 39; and the VL domain of the second single-chain Fv contains an amino acid sequence as shown in SEQ ID NO: 40 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 SEQ ID NO: 40;
 the second single-chain FV specificallybinds to CD3; the VH domain of the second single-chain Fv contains an amino acid sequence as shown in SEQ ID NO: 47 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: 47; and the VL domain of the second single-chain Fv 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 substituions than SEQ ID NO: 48.   
     
     
         10 . (canceled) 
     
     
         11 . 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 as shown in SEQ ID NO: 49 or a truncated fragment thereof; preferably, the rigid peptide comprises SSSSKAPPPS. 
     
     
         12 . The bispecific antibody according to  claim 11 , wherein the linker peptide comprises an amino acid sequence as shown in SEQ ID NO: 50, 51, 52 or 53. 
     
     
         13 . 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); preferably Gly (G) and Ser (S); more preferably, the linker peptide consists of (GGGGS)n, wherein n=1, 2, 3 or 4. 
     
     
         14 . The bispecific antibody according to  claim 1 , wherein the Fc fragment comprises a hinge region, a CH2 domain and a CH3 domain 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 the Fc fragment has one or more amino acid substitutions, deletions or additions with reduced or eliminated effector functions comprising ADCP, ADCC and CDC effects than a natural sequence from which the Fc fragment is derived. 
     
     
         15 . (canceled) 
     
     
         16 . The bispecific antibody according to  claim 15 , wherein the Fc fragment comprises amino acid substitutions L234A/L235A/P331S that are determined according to an EU numbering system. 
     
     
         17 . The bispecific antibody according to  claim 16 , wherein the Fc fragment further comprises amino acid substitutions, deletions or additions with one or more of the following properties:
 (i) enhanced binding affinity to a neonatal receptor (FcRn);   (ii) reduced or eliminated glycosylation; and   (iii) reduced or eliminated charge heterogeneity.   
     
     
         18 . The bispecific antibody according to  claim 17 , wherein the Fc fragment further comprises one or more of amino acid substitutions, deletions or additions as follow:
 (i) amino acid substitutions M428L, T250Q/M428L, M428L/N434S or M252Y/S254T/T256E determined according to the EU numbering system;   (ii) an amino acid substitution N297A determined according to the EU numbering system; and   (iii) an amino acid deletion K447 determined according to the EU numbering system.   
     
     
         19 . The bispecific antibody according to  claim 18 , wherein the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 55 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. 
     
     
         20 . The bispecific antibody according to  claim 1 , wherein the bispecific antibody comprises an amino acid sequence as follows:
 (i) a sequence as shown in SEQ ID NO: 8;   (ii) a sequence with one or more substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) than the sequence as shown in SEQ ID NO: 8; or   (iii) a sequence with 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: 8.   
     
     
         21 . A DNA molecule encoding the bispecific antibody according to  claim 1 . 
     
     
         22 . The DNA molecule according to  claim 21 , which has a nucleotide sequence as shown in SEQ ID NO: 56. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . A pharmaceutical composition, comprising the bispecific antibody according to  claim 1  and a pharmaceutically acceptable excipient, carrier or diluent,
 preferably, the pharmaceutical composition is a solution formulation, 
 preferably, the pharmaceutical composition further comprises a pH regulator, a stabilizer, and a surfactant: preferably, the pH regulator is a citrate buffer or a histidine buffer, the stabilizer is sucrose, and the surfactant is tween˜80: more preferably, the formulation comprises 0.5 mg/mL of the bispecific antibody, 20 mM of citrate or histidine, 8% (w/v) of sucrose and 0.02% (w/v) of PS80: the formulation has a pH of 5.5. 
 
     
     
         26 . (canceled) 
     
     
         27 . 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 an pCDNA3.4 vector;   wherein the host cell into which the constructed vector is transfected by the genetic engineering method in step (b) comprises a prokaryotic cell, a yeast cell 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.   
     
     
         28 - 30 . (canceled) 
     
     
         31 . A method for treating, preventing or ameliorating a tumor or an immune disorder or disease in a patient or subject, which comprises administering a therapeutically effective amount of the bispecific antibody of  claim 1  to the patient or subject, wherein the tumor comprises breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, cervial cancer, pancreatic cancer, testicular cancer, malignant melanoma, and soft tissue cancer.