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Anti-PD1 and anti-CD19 bispecific antibody and applications thereof

A bispecific antibody and specific technology, applied in the field of antibody drug development and production, can solve the problem of weakening the immune effect of tumors

Active Publication Date: 2017-07-11
SHUNHAO CELL BIOTECHNOLOGY (TIANJIN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The monoclonal antibody binds to the effector cell surface active receptor PD-L1 through the Fc segment, thereby mediating the killing effect, but the T cells with immune killing effect cannot be effectively mediated because of the lack of the above-mentioned receptors on the surface, thus weakening the body's anti-tumor effect. immune effect

Method used

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  • Anti-PD1 and anti-CD19 bispecific antibody and applications thereof
  • Anti-PD1 and anti-CD19 bispecific antibody and applications thereof
  • Anti-PD1 and anti-CD19 bispecific antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Structural Design of PD-1 / CD19 Bispecific Antibody

[0073] The present invention provides some structural models of bispecific antibodies that can specifically bind to the extracellular domains of PD-1 and CD19 at the same time. The structures of the four main examples of such antibodies are as follows figure 1 as shown, figure 1 The formation of the three heterodimers in (A-C) is based on the CH3 domain mutation technique ("knob and hole mutation") well known to those skilled in the art (VonKreudenstein et al., MAbs.2013 5(5):646- 54). (Among them, (A) light chain (anti-PD-1VL-CL)-heavy chain (anti-PD-1VH-CH1-hinge region-CH2-CH3) of anti-PD-1 specific antibody is paired with anti-CD19 specific antibody Bispecific binding protein composed of light chain (anti-PD-1VL-CL)-heavy chain (anti-PD-1VH-CH1-hinge region-CH2-CH3) pair; (B) light chain of anti-PD-1 specific antibody - a bispecific binding protein consisting of a heavy chain pair fusion protein wit...

Embodiment 2

[0075] Example 2 Preparation of PD-1 / CD19 bispecific antibody antigen (human PD-1 and CD19 extracellular soluble domain)

[0076] The coding gene sequences of human PD-1 and CD19 extracellular domains were obtained from the UniProt database, and the coding genes PD-1ED-CDS and CD19ED-CDS were obtained by whole gene synthesis. -1ED-CDS) and BamH I, Xba I (CD19ED-CDS) double digestion, cloned into pcDNA3.1 vector, using CHO-S cells as host cells for protein expression, using affinity chromatography, ion exchange chromatography and other purification techniques to purify the target protein.

[0077] image 3 It is the SDS-PAGE test results after purification and deglycosylation of human PD-1 and CD19 extracellular domain proteins.

Embodiment 3

[0078] Example 3 Obtaining of anti-human PD-1 and CD19 antibodies

[0079] 1. Immunization of animals

[0080] Mix 100 μl of 1 mg / ml purified human PD-1 or CD19 extracellular domain protein with an equal volume of Freund’s complete adjuvant at room temperature, and then inject into 6-8 week old BALB / C mice subcutaneously at multiple points in the abdomen in vivo. After 7 days, 100 μl of 1 mg / ml protein was uniformly mixed with an equal volume of Freund's incomplete adjuvant at room temperature, and injected subcutaneously into the mouse at multiple points in the abdomen. After seven days, repeat the above steps. After seven days, repeat the above steps. Three days after the fourth injection, 15 μl of blood was collected from the tip of the tail of the broken mouse, left at room temperature for 2 hours, 25°C, 6000-8000 rpm, centrifuged for 10-15 minutes, and the supernatant was taken to detect the titer by ELISA experiment. If the titer test is available, take whole blood f...

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Abstract

The present invention relates to anti-PD1 and anti-CD19 bispecific antibody and applications thereof, wherein the anti-PD1 and anti-CD19 bispecific antibody, or a variant, or a functional fragment thereof comprises: a domain capable of specifically recognizing and binding an immune cell surface antigen PD-1, wherein the domain comprises the heavy chain variable region (anti-PD-1 VH) of an anti-PD-1 specific antibody; and a domain capable of specifically recognizing and binding CD19, wherein the domain comprises the heavy chain variable region (anti-CD19 VH) of an anti-CD19 specific antibody. According to the present invention, the bispecific antibody has the whole human sequence, such that the occurrence of HAMA in clinical treatment application is effectively avoided, the occurrence of drug resistance can be effectively reduced, and the utilization efficiency and the treatment effect of the medicine can be improved; and the anti-PD-1 and anti-CD19 bispecific antibody can well mediate T cells to efficiently and specifically kill B-lymphocyte derived tumor cells compared to the existing anti-PD-1 specific antibody and the existing anti-CD19 specific antibody.

Description

technical field [0001] The invention belongs to the field of antibody drug development and production, and relates to anti-PD-1 and CD19 bispecific antibodies and applications thereof. The present invention provides a bispecific antibody or a functional fragment thereof that specifically recognizes and binds to PD-1 and CD19, and also provides a nucleic acid molecule encoding the bispecific antibody or a functional fragment thereof for expressing the Expression vectors and host cells of bispecific antibodies or functional fragments thereof, their application in the preparation of drugs for treating cancer, and a preparation method of the bispecific antibodies or functional fragments thereof. Background technique [0002] The PD-1 gene is a 55kDa transmembrane protein, and its molecule is composed of 4 subunits: δ, ε, γ, ζ, and their respective molecular masses are 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, which are A member of the Ig gene superfamily. PD-1 is an inhibitory membe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCA61K2039/505C07K16/2803C07K16/2818C07K2317/24C07K2317/31
Inventor 王继明张云赵青朱泽李镜李向臣李相国钟殿胜陈镭王立祥王华庆李忠廉刘卫平
Owner SHUNHAO CELL BIOTECHNOLOGY (TIANJIN) CO LTD
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