Application of humanized antibody for resisting 4-1BB

A technology of antibodies and antigens, applied in the direction of antibodies, anti-animal/human immunoglobulins, applications, etc., can solve the problems that cannot meet the needs of immunotherapy methods, and are few in number, and achieve the effect of a wide range of therapeutic uses

Active Publication Date: 2021-05-14
HEFEI HANKEMAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no 4-1BB agonist drug on the market, the fastest is still in the clinical development stage, and there are not many, which cannot meet the needs of various diseases including cancer for immunotherapy

Method used

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  • Application of humanized antibody for resisting 4-1BB
  • Application of humanized antibody for resisting 4-1BB
  • Application of humanized antibody for resisting 4-1BB

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Embodiment 1, the preparation of hybridoma antibody

[0123] 1.1 Immunization of mice

[0124] Three Balb / c mice and three C57b1 / 6 mice aged 4 to 6 weeks were selected, and the mice were immunized with human 4-1BB extracellular segment (positions 24-186 of SEQ ID No.5) as antigens. Immunization once every 2 weeks, a total of 3 times of immunization. After the third immunization, the blood was collected from the tail vein, and the antibody production was detected by indirect ELISA. The appropriate immunized mice were selected, and the eyes were removed and then sacrificed by decapitation. The spleen cells of the mice were aseptically removed, and the cells were fused according to the conventional method to prepare the antibody. Human 4-1BB monoclonal antibody hybridoma cell line.

[0125] 1.2 Hybridoma Screening

[0126] Anti-human 4-1BB monoclonal antibody hybridoma cells were screened by ELISA. ELISA plates were coated with 10 μg / ml human 4-1BB (SEQ ID No. 5), over...

Embodiment 2

[0146] Example 2. Antibody humanization and scFv-Fc antigen-binding fragment property identification

[0147] Secreted antibody 37G10F4 was selected from panning as described in the present invention. Analyze the antibody gene sequence, and then design a reasonable humanized antibody through a series of analyzes such as homology modeling and optimization of antibody Fab, surface scanning to determine the humanized mutation site, virtual mutation and molecular dynamics simulation, and determination of key amino acids. Through humanization transformation, 12 candidate antigen-binding fragments in the form of scFv-Fc (human IgG4) were obtained for further identification as described in this example. Express and purify the candidate scFv-Fc molecule, and then identify it in ELISA (including identifying its ability to recognize human and monkey 4-1BB, see the relevant steps in Example 1 for specific methods), as shown in Table 4, and the ELISA results are as follows Figure 5 and ...

Embodiment 3

[0152] Example 3, IgG humanized antibody

[0153] The exemplary antibody Hanke10F4 identified in this example was humanized from the hybridoma-secreted antibody 37G10F4, that is, converted from scFvB60103 in Table 4. This example will detail the expression and purification of Hanke10F4, followed by identification in ELISA, FACS, luciferase reporter gene assay, T cell activation system.

[0154] 3.1 Antibody expression and purification

[0155] According to the gene sequence obtained by sequencing, the full-length gene sequence was synthesized. The heavy chain coding gene of the Hanke10F4 antibody is shown in SEQ ID No.3, and the light chain coding gene of the Hanke10F4 antibody is shown in SEQ ID No.4.

[0156] The 1-351th positions of SEQ ID No.3 are the coding genes of the heavy chain variable region VH, wherein the coding sequences of CDR1, CDR2 and CDR3 are respectively as the 91-105th and 148-192nd positions of SEQ ID No.3 , as shown in bits 292-318.

[0157] The 1-321...

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Abstract

The invention discloses an application of a humanized antibody for resisting 4-1BB. The antibody can be used for detecting 4-1BB, blocking a 4-1BB/4-1BBL signal channel, stimulating T cell activation, promoting T cell secretion of IFN-gamma, inhibiting colon cancer tumor and cell growth, and treating and/or preventing colon cancer. The HCDR1, the HCDR2 and the HCDR3 of the antibody are sequentially shown as the 31st site to the 35th site, the 50th site to the 64th site and the 98th site to the 106th site of SEQ ID No.1, and the LCDR1, the LCDR2 and the LHCDR3 are sequentially shown as the 24th site to the 34th site, the 50th site to the 56th site and the 89th site to the 97th site of SEQ ID No.2. The antibody provided by the invention has high affinity to human 4-1BB, can effectively enhance T cell response, can be used for regulating T cell and antibody mediated immune response, and can be used as an immunomodulator for treating cancers, autoimmune diseases, inflammatory diseases, infectious diseases and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of an anti-4-1BB humanized antibody. Background technique [0002] 4-1BB, also known as CD137, TNFRSF9, etc., is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), which was first discovered by Kwon and Weissman during the differentiation screening of activated murine T cell clones . Schwarzh et al. isolated human 4-1BB (hu4-1BB) with homology to mouse 4-1BB from human activated T cells, and named it CD137. 4-1BB is a type I membrane glycoprotein, which is different from PD -1 / PD-L, the expression of 4-1BB is activation-dependent, it mediates the costimulatory signal of T cell activation, and widely exists in immune cells including activated NK cells, activated T cells, dendritic cells (DC ), mast cells, monocytes, and neutrophils. Human 4-1BB consists of 255 amino acid residues, is expressed on the cell surface in the form of monomers and d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/68G01N33/577A61K39/395A61P35/00A61P37/04A61P37/02A61P29/00A61P31/00
CPCC07K16/2878G01N33/68G01N33/577A61P35/00A61P37/04A61P37/02A61P29/00A61P31/00G01N2333/70578C07K2317/24C07K2317/76C07K2317/565C07K2317/92A61K2039/505
Inventor 程联胜刘雯婷赵群张大艳
Owner HEFEI HANKEMAB BIOTECH CO LTD
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