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Anti-B7-H3 monoclonal antibody, anti-B7-H3*CD3 bispecific antibody, preparation method and application of anti-B7-H3*CD3 bispecific antibody

A bispecific antibody, B7-H3 technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve problems such as short half-life of antibodies

Pending Publication Date: 2022-05-27
SUZHOU RES INST OF TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies with BITE structure have a short half-life, and antibodies prepared by chemical coupling method have relatively large molecular weight and strong immunogenicity, so antibodies with these two structures have limitations in subsequent applications

Method used

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  • Anti-B7-H3 monoclonal antibody, anti-B7-H3*CD3 bispecific antibody, preparation method and application of anti-B7-H3*CD3 bispecific antibody
  • Anti-B7-H3 monoclonal antibody, anti-B7-H3*CD3 bispecific antibody, preparation method and application of anti-B7-H3*CD3 bispecific antibody
  • Anti-B7-H3 monoclonal antibody, anti-B7-H3*CD3 bispecific antibody, preparation method and application of anti-B7-H3*CD3 bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Production of Anti-B7-H3 Monoclonal Antibodies and Bispecific Antibodies

[0127] Bivalent anti-B7-H3 antibodies were prepared using hybridoma technology. BALB / c mice were immunized with recombinant human B7-H3-ECD protein (11188-H08H, SinoBiological) and screened for parental monoclonal antibodies against B7-H3 using ELISA for protein binding and flow cytometry for cell binding. Chimeric monoclonal antibody (10-2#c) is produced by fusing the light chain variable region (VL) sequence to the human Kappa constant region and the heavy chain variable region (VH) sequence to the human IgG1 constant region .

[0128] The anti-B7-H3×CD3 structure is a heterodimeric structure developed by knob-into-holes (KIH) technology and removes the effector function of IgG1 Fc by removing glycosylation in Fc (N297A). The Hole chain is the heavy chain of 10-2#c with mutations in the Fc region (N297A, T366S, L368A and Y407V). The knob chain is an IgGl Fc fragment with mutations ...

Embodiment 2

[0129] Example 2 Antibody expression and purification

[0130] The DNA fragments encoding the heavy and light chains were synthesized by Genewiz (Azenta Life Sciences) and cloned into the pcDNA3.1+ expression vector. Antibodies were expressed in FreeStyle™ 293T cells (Thermo Fisher Scientific) by PEI transfection. The supernatant was collected 7 days after transfection and the antibody was purified by protein A affinity chromatography (Thermo Fisher Scientific), after which the affinity chromatography product was further purified using anti-flag affinity gel (Biyuntian) (knob Fc fragment C-terminal with flag tag), protein samples were analyzed using SDS electrophoresis.

Embodiment 3

[0131] Example 3 Preparation of primary cells

[0132] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors using Ficoll-Hypaque density gradient centrifugation (Tbdscience, Tianjin, China). PBMCs were cultured in IMDM medium (Hyclone) supplemented with 100 U / mL penicillin, 100 U / mL streptomycin and 10% heat-inactivated FBS. Human CD3+ T cells were isolated from PBMCs by negative selection using the Human CD3 T Cell Isolation Kit (480022; BioLegend) according to the manufacturer's protocol. Pre- and post-selection (positive and negative fractions) purity was tested using flow cytometry.

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Abstract

The invention relates to the field of immunity and antibody drugs, in particular to an anti-B7-H3 monoclonal antibody, an anti-B7-H3 * CD3 bispecific antibody and application of the anti-B7-H3 * CD3 bispecific antibody. The invention provides a mouse anti-human B7-H3 monoclonal antibody, and the binding activity of the mouse anti-human B7-H3 monoclonal antibody with B7-H3 is proved through a protein-based and cell-based immune method. The invention also provides novel bispecific antibodies targeting CD3 and B7-H3, and demonstrates that the antibodies mediate in vitro effective cytotoxic activity against various B7-H3 positive cancer cell lines by promoting activation and proliferation of T cells. The bispecific antibody also shows effective anti-tumor activity in an adoptive transfer xenograft mouse model. The result shows that the novel anti-B7-H3 * anti-CD3 bispecific antibody has the potential of being used for treating B7-H3 positive solid tumors.

Description

technical field [0001] The present invention relates to the field of immunization and antibody medicine, in particular to anti-B7-H3 monoclonal antibody, anti-B7-H3×CD3 bispecific antibody and a preparation method and application thereof. Background technique [0002] The number of reports demonstrating the development and efficacy of therapeutic bispecific antibodies (bsAbs) has increased rapidly over the past few years. Approaches involving the simultaneous modulation of two molecular targets on tumor cells or redirecting immune effector cells to kill tumor cells may show great potential in cancer immunotherapy. Blinatumomab is a marketed bispecific antibody that targets CD3 on T cells and CD19 on B cells in patients with acute lymphoblastic leukemia and has shown potent therapeutic effects in the clinic. These promising clinical data encourage researchers to efficiently synthesize and produce stable bispecific antibodies. [0003] Currently, various bispecific antibodie...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/28C12N15/13G01N33/574G01N33/577G01N33/68A61K39/395A61K47/68A61P35/00
CPCC07K16/2809C07K16/2827G01N33/57484G01N33/577G01N33/6893A61K47/6849A61P35/00C07K2317/31C07K2317/565C07K2317/56C07K2317/74C07K2317/73G01N2333/7051G01N2333/70532G01N2800/24A61K2039/505
Inventor 房健民冯艳尹衍新
Owner SUZHOU RES INST OF TONGJI UNIV
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