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Anti-PD-1/EGFR bispecific antibody and application thereof

A technology of PD-1 and bifunctional antibody, applied in the field of tumor immunology

Active Publication Date: 2020-02-25
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many technologies can only solve one or two problems, which makes the development of bispecific antibody products always face technical bottlenecks

Method used

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  • Anti-PD-1/EGFR bispecific antibody and application thereof
  • Anti-PD-1/EGFR bispecific antibody and application thereof
  • Anti-PD-1/EGFR bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Example 1: Molecular sequence design of anti-PD-1 / EGFR bispecific antibody

[0210] The structure of the bispecific antibody that can simultaneously bind PD-1 and EGFR extracellular domain provided by the present invention is as follows: figure 1 shown. The PD-1 nanobody sequence is as described in the patent application CN201610826020.3, the PD-1 nanobody VHH sequence is connected in series through a linker (GGGGSGGGS) to form a PD-1 nanobody dimer configuration, and the specific amino acid sequence is as follows (underlined Part is Linker sequence):

[0211] QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS GGGGSGGGS QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS (SEQ ID No.: 31)

[0212] Then the above PD-1 nanobody dimer was fused with the heavy chain and light chain of cetuximab respectively, and the heavy ch...

Embodiment 2

[0225] Example 2: Expression and purification of anti-PD-1 / EGFR bispecific antibody

[0226] Transiently transfer the plasmid containing the synthetic gene into HEK293F cells together. The specific method is as follows: (1) Use the OMEGA plasmid answering kit to prepare a large amount of the plasmid containing the heavy chain and the plasmid containing the light chain respectively, and place them in the ultra-clean workbench. Filter and sterilize for later use; (2) Cultivate HEK293F cells to 2.0×10 6 each / mL; (3) Mix the heavy chain plasmid and the light chain plasmid according to the mass ratio of 2:3, and mix the mixed plasmid with the transfection reagent PEI at a ratio of 1:3 to the transfection medium F17 (Gibco) Stand in the middle for 20min, then add to HEK293F cells, at 37℃, 6%CO 2 Cultivate in a shaker incubator for 6 days; (4) Centrifuge to obtain the supernatant, combine the supernatant with protein A beads at room temperature for 1 hour; (5) Use pH 7.0 phosphate b...

Embodiment 3

[0232] Example 3: FACS detection of the blocking activity of candidate double antibodies against PD-1 / PD-L1

[0233] Blocking activity of candidate anti-PD-1 / EGFR bispecific antibodies against PD-1 / PD-L1: (1) Take 3×10 for each sample 5 293T / PD-1 stably transfected cells were placed in PBS buffer, and diluted anti-PD-1 / EGFR bispecific antibody, negative control antibody (AF70-Fc, a nanobody Fc fusion protein) and positive antibody (PD- 1 nanobody dimer Fc fusion protein), the antibody concentration is 1.67ug / mL and 6.67ug / mL, each sample is 100uL, 2ug hPD-L1(ECD)-Fc-Biotin is added to all samples at the same time, and incubated at 4°C for 20min (2) Wash the cells twice with PBS, add the SA-PE of eBioscience, incubate at 4°C for 20 min, wash the cells twice with PBS, detect them with a flow cytometer (BD FACS Calibur), and use graphpad prism6 software for data processing.

[0234] The result is as image 3 As shown, compared with the positive control antibody, the blocking ac...

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PUM

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Abstract

The invention provides an anti-PD-1 / EGFR bispecific antibody and an application thereof, and particularly provides the bifunctional antibody, which comprises: (a) an anti-EGFR antibody; and (b) an anti-PD-1 nanobody in a divalent form linked to the anti-EGFR antibody. The bifunctional antibody can be combined with EGFR and PD-1 at the same time, and therefore the treatment effect on EGFR positivetumor cells (especially malignant tumor cells) is achieved.

Description

technical field [0001] The invention belongs to the field of tumor immunology, and in particular relates to an anti-PD-1 / EGFR bispecific antibody and its application. Background technique [0002] Programmed cell death protein 1 (PD-1) is a member of the CD28 superfamily. As a T cell inhibitory receptor, it can limit the function of T cell effectors in tumor cells and plays an important role in tumor immune escape. PD-1 is highly expressed in tumor-infiltrating lymphocytes, tumor-specific T cells, and some injury-related T cells, and its ligand PD-L1 is highly expressed on the surface of various tumors. When PD-L1 on tumor cells and T cells on the surface When PD-1 binds, it forms an inhibitory signal, induces T cell apoptosis, and inhibits the activation and proliferation of T cells, thereby preventing tumor cells from attacking T cells and forming tumor immune escape. Therefore, blocking the interaction between PD-1 and PD-L1 can effectively restore the tumor-killing fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/62C12N5/10A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2818C07K16/2863C07K2317/31C07K2317/569C07K2317/76C07K2317/92
Inventor 万亚坤朱敏沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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