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Anti-B7-H3 antibody as well as preparation method, conjugate and application thereof

A B7-H3, immunoconjugate technology, applied in chemical instruments and methods, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Tumor efficacy needs to be further improved, immunogenicity risk and other issues

Pending Publication Date: 2021-01-19
TAIZHOU FUDAN-ZHANGJIANG PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on the fact that B7-H3 can inhibit T cell activity and thus mediate tumor cell escape from immune surveillance, it is effective to block the binding of B7-H3 to unknown receptors to mediate T cell activation and inhibit tumor cell activity, such as Enoblituzumab The existing clinical results show that it has different degrees of remission for different tumors, and has a good curative effect, but there are still many patients with disease progression, so the simple development of monoclonal antibodies against B7-H3 still cannot meet clinical expectations ; and the above-mentioned antibodies were screened by hybridomas and then humanized. Although the hybridomas were humanized, they still contained murine sequences, which had potential immunogenicity risks, and the existing clinical results showed that they The anti-tumor effect needs to be further improved

Method used

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  • Anti-B7-H3 antibody as well as preparation method, conjugate and application thereof
  • Anti-B7-H3 antibody as well as preparation method, conjugate and application thereof
  • Anti-B7-H3 antibody as well as preparation method, conjugate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Preparation of Example 1 Human B7-H3 Protein

[0083] Select the nucleic acid sequence of amino acids 29-245 of human 2Ig B7-H3, in which a purified 10His tag is added to the N-terminal and a detection Myc tag is added to the C-terminal, named H2M; the nucleic acid sequence of amino acids 27-461 of human 4Ig B7-H3 is selected , where the N-terminus adds a purification tag 10His and the C-terminus adds a detection Myc tag, named H4M; select the nucleic acid sequence of amino acids 27-461 of human 4Ig B7-H3, wherein the N-terminus adds a detection myc tag and the C-terminus adds a purified 10His tag , named M4H. The gene plasmids H2M-pUC57, M4H-pUC57, and H4M-pUC57 of the above three B7-H3 antigens were synthesized, respectively, and pv81 expression vector plasmids were synthesized, respectively digested by EcoRI and SmalI, and ligated to transform Escherichia coli competent cells Trans-T1, The correct clones were screened and amplified by PCR to extract a large number o...

Embodiment 2

[0085] Example 2 Library Preparation

[0086]Take 1ml library Lambda size 2.91×10 9 and the library Kappa size is 3.72×10 9 Add 2.0L fresh culture medium 2YT+100μg / ml Amp+2% glucose, the initial OD600 of the library bacteria solution is less than 0.1, and culture at 37°C and 200rpm. When the OD600 is 0.5-0.6, add 365 μL of M13K07 with a titer of 9.6×10 12 / ml, the amount added is 10 times the amount of bacteria, the amount of bacteria = OD600 × 8.0 × 10 8 / ml×Shaking bacteria volume, assisting phagemid infection. After adding helper phage, culture at 37°C for 30min, then at 200rpm for 30min, and finally at 4000rpm for 10min, resuspend with 2.0L equal volume of 2YT+100μg / mlAmp+50μg / ml Kana, and culture at 30°C 16h, for expression. After the expression culture was completed, the bacterial solution was taken and centrifuged at 8000 rpm for 30 minutes at 4°C, and the supernatant was removed. Centrifuge at high speed, remove the bacteria, add 1 / 5 volume of PEG / NACL to the sup...

Embodiment 3

[0087] Example 3 Anti-B7-H3 Antibody Phage Library Screening

[0088] Liquid-phase panning: 150 μL magnetic beads Dynabeads M-280 were blocked with 1% casein at room temperature for 1 hour, then 100 μL of the prepared phage library Phage was added, shaken gently, and blocked for 1 hour at room temperature. After the completion of blocking, add 30 μg of biotinylated B7-H3 antigen H4M and incubate at room temperature for 1 h. After the incubation is completed, the complex of antigen H4M and phage antibody is incubated with the blocked magnetic beads for 15 min to make the complexes bind to the magnetic beads. Wash with PBST and PBS 15 times respectively. Then add 1ml of trypsin (10μg / ml) to elute the phages bound to the antigen, the elution volume is 1ml, infect TG1 which is growing in the logarithmic phase, measure the titer, and amplify the phages for the next round of panning . A total of 3 rounds of panning were performed, and the last 2 rounds of panning were the same as ...

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Abstract

The invention discloses an anti-B7-H3 antibody as well as a preparation method, conjugate and application thereof. The anti-B7-H3 antibody comprises a complementary determining region which is one ormore of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and / or, one or more of a light chain CDR1, a light chain CDR2 and a light chain CDR3, and a sequence is as described in the invention. The anti-B7-H3 antibody disclosed by the invention is a fully humanized antibody screened from a phage library, and has a unique antigen binding epitope; and the antibody can specifically bind to a B7-H3 antigen on a tumor cell, can be quickly internalized into the cell after binding to the tumor cell, can be used for ADC drug development, and is expected to obtain the better antitumor activity and efficacy so as to achieve the purpose of treating cancers.

Description

technical field [0001] The present invention belongs to the field of antibodies, and in particular relates to an antibody specifically binding to mammalian, especially human, B7-H3, a preparation method thereof, a conjugate thereof and applications thereof; in particular, a fully human antibody and a fully human antibody conjugate for treating cancer United things. Background technique [0002] B7-H3, also known as CD276, was first reported in 2001 (Chapoval AI et al., Nat Immmunol 2001, 2(3):269-274), and its protein is not considered to be a heptad structure and B30.2 domain because it lacks a heptad structure and a B30.2 domain. Milk fat protein and myelin oligodendrocyte glycoprotein, and identified as members of the immunoglobulin superfamily of the B7 family (Chapoval AI et al., Nat Immunol 2001, 2(3):269-274). Different from other family members such as PD-L1, B7-H4, CD80, CD86, etc.: B7-H3 exists in two different variants in the human body: 2IgB7-H3 and 4IgB7-H3, of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K47/68A61P35/00A61K31/4045
CPCC07K16/2827A61K47/6803A61P35/00A61K31/4045C07K2317/565C07K2317/56C07K2317/52C07K2317/24C07K2317/622C07K2317/77C07K2317/92
Inventor 郭青松吴芳杨彤沈毅珺
Owner TAIZHOU FUDAN-ZHANGJIANG PHARM CO LTD
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