Construction and application of bispecific antibody HER2xCD3

• US 20160145339A1
• Filed: 07/20/2015
• Published: 05/26/2016
• Est. Priority Date: 07/21/2014
• Status: Active Grant

• Current Assignee: Wuhan YZY Biopharma Co., Ltd.
• Original Assignee: Wuhan YZY Biopharma Co., Ltd.
• Inventors: Hu, Lingli, Wang, Rui, Zhou, Xiang, Fan, Kesu, Zhou, Pengfei, Zhang, Jing
• Granted Patent: US 9,611,325 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/60 characterized by non-natural combinations of immunoglobulin fragments
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The present invention provides a bispecific antibody. The bispecific antibody provided by the present invention comprises a single-chain unit and a monovalent unit, wherein the single-chain unit has a specific binding capability against a surface antigen CD3 of an immune cell; the monovalent unit has a specific binding capability against a surface antigen HER2 of a tumor cell; the single-chain unit comprises a single-chain variable fragment ScFv fused with an Fc fragment; and the monovalent unit comprises a light chain and heavy chain pair. The present invention also provides a preparation method of the bispecific antibody and pharmaceutical use of these antibodies.

Claims

1. A bispecific antibody, characterized in that it comprises:

(a) a monovalent unit which is a light chain-heavy chain pair having a specific binding capability against surface antigens of tumor cells, comprising HER2, CD20, CD30 or CD133; and

(b) a single-chain unit which is a fusion peptide containing a single-chain variable fragment (ScFv) and an FC fragment having a hinge region, a CH2 structural domain and a CH3 structural domain, wherein the fusion peptide is selected from a T cell, an NKT cell or a CIK cell against the immune cell; and

the fusion peptide has specific binding capability against a surface antigen CD3 of the immune cell.

2. The bispecific antibody according to claim 1, characterized in that the CH2 structural domain of the single-chain unit is positioned between the hinge region and the CH3 structural domain;

the bispecific antibody does not contain a CH1 structural domain; and

the hinge region of the single-chain unit is positioned between the ScFv and the CH2 structural domain.

3. The bispecific antibody according to claim 1, characterized in that said single-chain variable fragment consists of a light chain variable region and a heavy chain variable region, both of which are targeted to the antigen epitope CD3.

4. The bispecific antibody according to claim 1, characterized in that in the monovalent unit, both a light chain constant region structural domain and a light chain variable region structural domain of said light chain are targeted to the tumor antigen epitope HER2;

both a heavy constant structural domain CH1 and a heavy chain variable structural domain of said heavy chain are targeted to the tumor antigen epitope HER2;

said light chain binds to said heavy chain through a disulfide bond; and

said heavy chain binds to said fusion peptide through one or more disulfide bonds.

5. The bispecific antibody according to claim 1, characterized in that the single-chain unit comprises an anti-CD3 antibody directed to CD3, and the monovalent unit comprises an anti-HER2 antibody directed to HER2;

and

the amino acid sequence of the heavy chain of said anti-HER2 antibody is an amino acid sequence as shown in SEQ ID NO. 1, the amino acid sequence of the light chain of said anti-HER2 antibody is an amino acid sequence as shown in SEQ ID NO. 3, and the amino acid sequence of the ScFv-Fc of said anti-CD3 antibody is an amino acid sequence as shown in SEQ ID NO. 5;

in addition, cysteine of the heavy chain of said anti-HER2 antibody on the site 233 and cysteine of the light chain of said anti-HER2 antibody on the site 214 are connected in a disulfide bond manner, cysteines of the heavy chain of said anti-HER2 antibody on the sites 229 and 232 and cysteines of the ScFv-Fc of said anti-CD3 antibody on the sites 255 and 258 are connected respectively in a disulfide bond manner, the sites 395 and 412 of the heavy chain of said anti-HER2 antibody and the sites 428 and 397 of the ScFv-Fc of said anti-CD3 antibody are connected in a salt bridge manner, and the site 369 of the heavy chain of said anti-HER2 antibody and the site 436 of the ScFv-Fc of said anti-CD3 antibody are connected in a hump-indent-cavity manner.

6. The bispecific antibody according to claim 1, characterized in that the heavy chain in said monovalent unit contains a human or humanized Fc fragment, and the Fc fragment of the heavy chain contains a human IgG1 Fc fragment;

and the Fc fragment of said fusion peptide contains a human or humanized Fc fragment, and the FC fragment of the fusion peptide contains a human IgG1 Fc fragment.

7. The bispecific antibody according to claim 6, characterized in that the human IgG1 Fc fragment of said monovalent unit and the IgG1 Fc fragment of said single-chain unit are connected through a salt bridge and a hump-indent-cavity structure.

8. A method for preparing said bispecific antibody according to claim 1, characterized by comprising the following steps:

(1) respectively establishing a heavy chain and a light chain of the monovalent unit onto a first expression vector and establishing the single-chain unit onto a second expression vector;

(2) co-transfecting the first expression vector and the second expression vector into cells, culturing and taking supernatant; and

(3) separating the expression supernatant to obtain the purified bispecific antibody;

said cells are CHO-S cells;

or said separation step comprises the substeps of capturing all antibodies with Fc structural domains from the expression supernatant by a protein A affinity column, separating the target bispecific antibody from byproducts through SP cation exchange chromatography, then passing through a Q column and finally concentrating and displacing a buffer solution PBS.

9. The method according to claim 8, wherein, said first expression vector is pCHO1.0, and said second expression vector is pCHO01.0-hygromycin.

10. The method according to claim 8, characterized in that in the step (1):

said monovalent unit is an anti-HER2 antibody, primers for amplifying a light chain of said antibody are Kozak(EcoRV)F, MK-leader(EcoRV)F and hIgK(PacI)R, and the Kozak sequence, the MK-leader and restriction enzyme cutting sites EcoRV and Pad are introduced into the light chain through overlap PCR amplification;

primers for amplifying a heavy chain of said antibody are Kozak(AvrII)F, MK-leader(AvrII)F and hIgG1(BstZ17I)R, and the Kozak sequence, the MK-leader and restriction enzyme cutting sites AvrII and BstZ17I are introduced into the heavy chain through overlap PCR amplification;

the amplified LC gene fragment is subject to homologous recombination with a pCHO1.0 expression vector suffering restriction enzyme cutting via EcoRV and Pad to obtain an anti-HER2 light chain-loaded expression vector, and is then subject to homologous recombination with HC after suffering restriction enzyme cutting via AvrII and BstZ17I to obtain an anti-HER2 pCHO1.0 expression vector of which the plasmid is named as pCHO1.0—

Herceptin-HL-KKW; and

said single-chain unit an anti-CD3ScFv-Fc antibody, primers for amplifying said antibody are Kozak(AvrII)F, MK-leader(AvrII)F, L2K-VH(MK)F1 and hIgG1(BstZ17I)R, and an anti-CD3 ScFv-Fc-loaded expression vector of which the plasmid is named as pCHO1.0-hygromycin-L2K-ScFv-Fc-LDY is obtained through implementing overlap PCR amplification of the anti-CD3ScFv-Fc structural domain, introducing the Kozak sequence, MK-leader and restriction enzyme cutting sites AvrII and BstZ17I into ScFv-Fc and carrying out homologous recombination on the amplified gene fragment and the pCHO1.0-hygromycin expression vector suffering restriction enzyme cutting.

11. The bispecific antibody according to claim 1 or the use of the bispecific antibody prepared through the method for preparing the bispecific antibody according to claim 8 in preparation of drugs, wherein, said drugs are used to treat tumor or related diseases caused by HER2 specific antigen expression or kill cells expressing HER2.