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Bispecific antibody targeting NKG2A and PD-L1 and application

A multispecific antibody and antibody technology, applied in the field of antibody drugs and tumor therapeutic antibodies, can solve the problems of insufficient antigen affinity and poor therapeutic effect, and achieve the effect of improving affinity

Pending Publication Date: 2021-11-02
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the therapeutic antibody against NKG2A has insufficient affinity to the antigen, and combined with a single target of NKG2A has poor therapeutic effect on tumors, and there is no marketed drug against NKG2A in the world

Method used

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  • Bispecific antibody targeting NKG2A and PD-L1 and application
  • Bispecific antibody targeting NKG2A and PD-L1 and application
  • Bispecific antibody targeting NKG2A and PD-L1 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Expression Analysis of Anti-NKG2A Specific Antibody Z270 Antibody

[0070] The coding genes of NKG2A (genebank: AF461812.1, 157aa-233aa) and CD94 (genebank: AB009597.1, 4aa-148aa) were connected through the GGSGGS coding gene to synthesize the coding gene of NKG2A-CD94 (SEQ ID NO.1), and Further cloned into eukaryotic transient expression vectors with mFc tag at the N-terminal by enzyme digestion method, and transferred into Escherichia coli to amplify, isolated and obtained mFc-NKG2A-CD94 expression plasmid, and transfected according to the transfection reagent 293fectin (Cat: 12347019 , Gibco), the plasmid was transferred into HEK293 cells for recombinant expression. 5-6 days after the cells were transfected, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column to obtain pure mFc-NKG2A-CD94 recombinant protein.

[0071] Obtain the light and heavy chain amino acid sequences of the antibod...

Embodiment 2

[0074] Example 2. Affinity maturation of anti-NKG2a antibody hum270

[0075] Using yeast display technology, the anti-NKG2a antibody hum270 heavy chain variable region and 6 CDR region amino acids of the light chain variable region were randomly mutated to build a library, and candidate antibody molecules with higher affinity were screened.

[0076] 1. Mutant antibody library design and construction

[0077]The humanized antibody hum270 was selected as a template for affinity maturation, and its heavy chain variable region and light chain variable region were connected by GS linker to construct a single-chain variable fragment (scFv). The CDR region of the single-chain antibody is the target of affinity maturation transformation, while the framework region will remain unchanged in the affinity maturation transformation. A total of 6 amino acids in the CDR region of the light and heavy chains (Table 1) were mutated, and mutation libraries were constructed respectively.

[0078...

Embodiment 3

[0107] Example 3. Expression and affinity analysis of anti-NKG2a and PD-L1 bispecific antibody

[0108] 1. Bispecific antibody construction and affinity analysis

[0109] By PCR, the N-terminal of the nucleotide sequence encoding the anti-PD-L1 blocking humanized Nanobody F2 was connected to the C-terminal of the heavy chain nucleotide sequence of the hum270 antibody via a linker peptide to obtain a heavy chain containing hum270 antibody. The coding sequence hum270-H-F2 of chain-F2, the coding sequence of hum270 antibody heavy chain-F2 (hum270-H-F2) and the coding sequence of hum270 antibody light chain hum270-L were respectively cloned into eukaryotic transient In the expression vector, the obtained expression plasmid was transferred into Escherichia coli for amplification, and the hum270-H-F2 and hum270-L expression plasmids were isolated, and according to the operation instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the plasmid Transformed into H...

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Abstract

According to the invention, based on the reported light and heavy chain amino acid sequences of the anti-NKG2A antibody in the prior art, the method comprises the steps of: constructing a light and heavy chain mutation antibody library for affinity maturation to obtain the mutant antibody with improved affinity and / or improved dissociation constant; and constructing a human Fab heavy chain gene expression vector and a mammalian cell expression vector containing a human kappa subtype light chain constant region gene in a CDRs region of a mutant antibody, carrying out cross pairing on a heavy chain vector and a light chain vector of an antibody with mature affinity, screening to obtain an anti-NKG2A mutant Fab antibody, and connecting an Fc segment of the human antibody; and connecting a second antigen binding molecule (such as an anti-PD-L1 antibody) to the C end of the Fc segment through linker to obtain the bispecific antibody.

Description

[0001] This patent application claims the priority right of the Chinese invention patent application with application number CN202010369911.7 filed on April 30, 2020, the entire contents of which are again incorporated by reference. technical field [0002] The invention relates to the field of antibody medicine, especially the field of tumor therapeutic antibody. Specifically, the present invention relates to a high-affinity humanized antibody targeting human NKG2A, and the preparation and use of a bispecific antibody composed of it and an anti-human PD-L1 nanobody. Background technique [0003] In recent years, tumor immunotherapy has achieved unprecedented success, and still only a small number of patients have shown durable efficacy. Improving clinical response and overcoming drug resistance mechanisms are ongoing challenges in the field of tumor immunotherapy, and blocking other inhibitory immune receptors may be a feasible strategy. [0004] NKG2A (killer cell lectin ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46C12N15/13C12N15/62A61K39/395A61P35/00A61P37/04
CPCC07K16/2827C07K16/2803A61P35/00A61P37/04C07K2317/31C07K2317/92C07K2317/56C07K2317/565A61K2039/505A61K39/395C07K16/00C07K16/28C07K16/46C12N15/62
Inventor 王双桂勋王晋王荣娟蔺利娟焦莎莎徐晓红张畅朱戬李祥烽吴建任红媛毕建军王骊淳
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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