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Antibody for resisting human epidermal growth factor receptor 2 (HER2), as well as medical composition and use of antibody

An antibody and antigen technology, applied in the field of tumor therapy and molecular immunology, can solve the problems of drug resistance, abnormal activation of receptor signaling pathways, poor efficacy of breast cancer, etc., and achieve the effect of inhibiting tumor growth

Inactive Publication Date: 2017-12-08
BEIJING MABWORKS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Trastuzumab is currently used as a first-line drug for the treatment of breast cancer, especially in the treatment of metastatic breast cancer with overexpression of Her2, and the objective response rate of single-drug first-line treatment is 30-50%. The curative effect of cancer is not good, and a considerable number of patients who are initially effective in antibody therapy will develop drug resistance within 1 year of use, which may be related to changes in the expression of certain genes in tumor cells that lead to antibody epitope masking or abnormal activation of receptor signaling pathways
In addition, Her2 can also form ligand-dependent or independent heterodimers with other members of the family (Her1, Her3 and Her4), thereby activating downstream pathways and leading to the proliferation of tumor cells, while trastuzumab There is no inhibitory effect on the formation of heterodimers, which may also be one of the reasons for its drug resistance
[0009] Pertuzumab launched in 2012 It has a certain effect on advanced prostate cancer, non-small cell cancer, ovarian cancer and breast cancer, but its curative effect is still related to the expression level of Her2
Therefore, Pertuzumab can effectively inhibit the formation of Her2 / Her3 heterodimers, but the inhibitory effect on EGFR / Her2 heterodimers is not obvious

Method used

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  • Antibody for resisting human epidermal growth factor receptor 2 (HER2), as well as medical composition and use of antibody
  • Antibody for resisting human epidermal growth factor receptor 2 (HER2), as well as medical composition and use of antibody
  • Antibody for resisting human epidermal growth factor receptor 2 (HER2), as well as medical composition and use of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0157] Embodiment 1: Amino acid sequence design and gene sequence optimization of the heavy chain and light chain of antibodies MIL203 and MIL204 change

[0158] (1) Amino acid sequence of MIL203 light chain and heavy chain

[0159] The heavy chain amino acid sequence of MIL203 is as follows, wherein the underlined part is the amino acid sequence of the heavy chain variable region:

[0160] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKN TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:22)

[0161] The light chain amino acid sequence of MIL203 is as follows, wherein the underlined part is the amino acid sequenc...

Embodiment 2

[0179] Embodiment 2: Construction of 203 antibody eukaryotic expression vector and 204 antibody eukaryotic expression vector

[0180] Using the expression vector pTGS-FRT-DHFR (please refer to the Chinese authorized invention patent ZL200510064335.0 for the construction method), remove the hygromycin selection tag, and add GS (glutamine synthetase, glutamine synthetase) expression through the PshA1 and Xho1 restriction sites box, as a selection marker; where GS cDNA can be obtained by RT-PCR from the cell line CHO expressing GS. The transformed vector was named GS vector.

[0181] On the basis of the GS vector, insert the fully synthetic light chain constant region (the constant region sequence is the non-underlined sequence in SEQ ID NO:24 or SEQ ID NO:27) through the restriction sites of BsiwI and NotI; after that, pass The restriction sites of Nhe I and XhoI were respectively inserted into the fully synthesized 203 heavy chain constant region and 204 heavy chain constant...

Embodiment 3

[0187] Example 3: Host cell fucose knockout and suspension acclimatization

[0188] The CHO-K1 cells purchased from ATCC (ATCC: 58995535) were gene knocked out so that the proteins expressed by themselves had almost or no fucosylation modification, and the obtained fucose knockout host cells were named CHOK1-AF, The specific method is to transform the expression system through genetic engineering technology, and knock out the key protein GFT of the fucose modification pathway in the antibody expression host cell CHO-K1, thereby effectively reducing the level of antibody fucose modification. The method can simultaneously block the classical fucosylation pathway and the compensation pathway, thereby achieving the purpose of completely removing fucosylation. Specific technical routes such as figure 1As shown, using the zinc finger enzyme technology, two GFT zinc-finger nuclease zinc finger enzyme sequences were designed for the sequence of the GFT gene SLC35c1 (GenBank: BAE16...

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Abstract

The invention belongs to the field of tumor treatment and the field of molecular immunology, and relates to an antibody for resisting human epidermal growth factor receptor 2 (HER2), as well as a medical composition and use of antibody, in particular to the antibody for resisting Her2 or an antigen binding fragment of the antibody for resisting Her2. The antibody for resisting Her2 comprises a first heavy chain and a second heavy chain, wherein according to the first heavy chain, a variable region of heavy chain (VH) of the first heavy chain comprises a complementarity-determining region (CDR) of which the amino acid sequence is SEQ ID No: 1-3, and the amino acid sequence of the CH of the first heavy chain is as shown in SEQ ID No: 7; according to the second heavy chain, a variable region of heavy chain (VH) of the second heavy chain comprises a complementarity-determining region (CDR) of which the amino acid sequence is SEQ ID No: 4-6, and the amino acid sequence of the CH of the first heavy chain is as shown in SEQ ID No: 8. Compared with the combination of two antibodies of trastuzumab and pertuzumab, the antibody disclosed by the invention has higher cell direct killing activity and higher antibody dependent cellmediated cytotoxicity (ADCC) activity; the complement-dependentcytotoxicity (CDC) activity of the two antibodies of trastuzumab and pertuzumab is equivalent to that of the antibody disclosed by the invention; the antibody disclosed by the invention adopts a structure of a normal antibody; and fucose is also knocked out, so that the ADCC activity is improved.

Description

technical field [0001] The invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-HER2 antibody, its pharmaceutical composition and application. Background technique [0002] Human epidermal growth factor receptor 2 (Human epidermal growth factor receptor 2, abbreviated as Her2, ERBB2, HER2 / neu or c-erbB2) is a protein encoded by the ERBB2 gene. In normal cells, the expression level of Her2 is very low; but in the embryonic development period, the expression level of Her2 is very high, which plays an important role in regulating cell proliferation, differentiation, development, adhesion and migration (Gutierrez, C. and R. Schiff, HER2: biology, detection, and clinical implications. Arch Pathol Lab Med, 2011.135(1): p.55-62.). [0003] Her2 belongs to the human epidermal growth factor receptor family, which consists of four members: Her1 (EGFR), Her2, Her3 and Her4. Among them, Her2 has no specific ligand, and the activation of it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K16/32C12N15/13G01N33/68G01N33/574A61K39/395A61K49/00A61P35/00A61P35/04
CPCA61K49/0004A61K2039/505C07K16/2863C07K16/32C07K2317/24C07K2317/41C07K2317/565C07K2317/73C07K2317/92A61P15/00A61P35/00A61P35/04C07K2317/31C07K2317/526C07K2317/732C07K2317/734A61K39/39558C07K2317/51C07K2317/515C07K2317/52C07K2317/56C07K2317/76
Inventor 赵健黄思佳金春阳刘慧芳张伯彦李锋
Owner BEIJING MABWORKS BIOTECH
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