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Preparation method of tri-specific antibody based on structure optimization protein activity

A protein activity and specificity technology, applied in the direction of antibodies, hybrid immunoglobulins, anti-tumor drugs, etc., can solve problems such as limiting the clinical application of bispecific antibodies, reduce the number of administrations and doses, and simplify expression and purification. Long half-life effect

Pending Publication Date: 2022-07-19
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Brennan et al. constructed the first bispecific antibody by chemical coupling of two monoclonal antibodies in 1985 (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science. 1985 Jul. 5;229(4708):81-3.), bispecific antibodies with different targets and different forms have been widely studied, but due to problems such as heavy chain mismatch, antibody inactivation and non-human related immunogenicity, etc. Broader clinical application of bispecific antibodies (Thakur A, et al. Bispecific antibody based therapeutics: Strengths and challenges. Blood Rev. 2018 Jul; 32(4): 339-347.)

Method used

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  • Preparation method of tri-specific antibody based on structure optimization protein activity
  • Preparation method of tri-specific antibody based on structure optimization protein activity
  • Preparation method of tri-specific antibody based on structure optimization protein activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1 Construction and eukaryotic expression of bi / trispecific antibodies

[0146] 1.1 Vector construction

[0147] Construction of CD3-HC-HER2-VHH and CD3-LC expression vectors:

[0148] The heavy chain of the HER2 / CD3 bispecific antibody is linked by CD3-HC (SP34) and HER2-VHH, respectively, in the following manner: HER2-VHH is linked to the CD3-HC variable region (VH) structure by a rigid linker peptide HE Linker Domain N-terminal 1E; or HER2-VHH is linked to the CD3-HC variable region (VH) domain N-terminal 1E by a rigid linker peptide PD Linker; or HER2-VHH is chimeric to CD3 by a coiled coil linker peptide Coiled Coil Linker - Between 184S-187L of the HC constant region (CH1) domain, while deleting 185S and 186G; or connecting HER2-VHH to the C-terminal 228C of the CD3-HC constant region (CH1) domain through a flexible linker (G4S) 3 Linker ; Light chain CD3-LC without any modification. The coding genes of CD3-HC-HER2-VHH and CD3-LC were synthesized accordi...

Embodiment 2 3

[0176] Example 2 Purification of trispecific antibodies

[0177] 2.1 Protein G affinity purification

[0178] Take 400 mL of the cell supernatant from 1.2 in Example 1 above, centrifuge at 15,000 rpm for 30 min at 4°C, collect the supernatant, filter with a 0.45 μm filter, and place it on ice for later use. Take 4 mL of Protein G (20% ethanol / Protein G 1:1) into the column, rinse with Binding buffer three times, and press the resin surface with a gasket. Equilibrate the Protein G column with 20 mL of Binding buffer. Every 10 mL was loaded, and the sample was passed through the Protein G column at a constant speed (about 0.5 mL / min). Wash the Protein G column with 40 mL of Binding buffer at a constant speed (about 1 mL / min). First, add 10% of the eluate volume of neutralization buffer to the collection tube of the elution tube, add Elution buffer to the column, and elute once with 5 mL until the protein concentration cannot be quantified. The collected protein samples were ...

Embodiment 3

[0179] Example 3 Biological function evaluation of HER2 / VEGFR2 / CD3 trispecific antibody

[0180] 3.1 Antigen binding validation of HER2 / CD3 bispecific antibodies with different structures

[0181] The HER2 ECD-Fc antigen protein was diluted with PBS to 10 μg / mL and coated in a 96-well ELISA plate, 100 μL / well, and coated overnight at 4°C. Discard the liquid in the plate, wash the plate twice with PBST, add 200 μL / well of PBST solution of 5% skim milk, block at room temperature for 2 hours, and drain the blocking solution in the well. The bispecific antibodies to be tested HER2 / CD3 HNT (HE Linker), HER2 / CD3 HNT (PDLinker), HER2 / CD3 H184-187, HER2 / CD3 HCT were diluted 10 times with blocking solution for 8 gradients, and the initial antibody concentration was 100 nM, 3 duplicate wells for each concentration gradient, 100 μL / well was added to the well plate, and incubated at room temperature for 2 h. The plate was washed three times with PBST and then drained. The HRP-labeled an...

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Abstract

The invention relates to the technical field of biology, in particular to a preparation method of a tri-specific antibody based on structure optimization protein activity. According to the construction method, Fab of a monoclonal antibody of a first structural domain is taken as a structural basis, and antibody fragments targeting two tumor specificity or related antigens, namely a second structural domain and a third structural domain, are added at the same time; the second structural domain and the third structural domain can be connected to the N end of a Fab heavy chain variable region, between structural domains 184S-187L of a heavy chain constant region (CH1) and to the C end of the heavy chain constant region through a linker; or is connected to the Fab light chain variable region N end, between the light chain constant region (CL) structural domain 171S-173D and the light chain constant region C end. By adopting the method provided by the invention, the prepared three-specific antibody with the optimized structure design can effectively mediate the targeted recognition and inhibition killing activity of T cells on hematological tumors and solid tumors, prevents the problems of tumor antigen immune escape, drug resistance and the like, and has the functions of collaborative targeted therapy and enhancement of immunotherapy effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a trispecific antibody based on structure-optimized protein activity. Background technique [0002] In recent years, advances in tumor immunotherapy have greatly expanded the treatment methods for malignant tumors. Common immunotherapy includes immune checkpoint blockade therapy or the use of genetic engineering to transform T cells so that T cells have a higher and sustained killing ability to tumor cells. Although immunotherapy has a good effect on the treatment of some malignant tumors, most malignant tumors still do not respond to immunotherapy, and there is an urgent need to develop new and more effective immunotherapy methods (O'Donnell JS, et al. Cancer immunoediting and resistance to T cell-based immunotherapy. NatRev Clin Oncol. 2019 Mar; 16(3): 151-167.; Chandran SS, et al. T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61P35/00A61P35/02
CPCC07K16/468C07K16/2809C07K16/2863C07K16/32C07K16/2803A61P35/00A61P35/02C07K2317/92C07K2317/56C07K2317/31C07K2317/622C07K2317/569C07K2317/73C07K2317/522C07K2317/52C07K2317/55A61K2039/505
Inventor 曹宇刘东赵丽君赵丽丽刘忠
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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