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human-mouse chimeric Siglec-15-resistant neutralizing full-molecule IgG and preparation method and application thereof

A molecular, DNA molecular technology used in the field of biopharmaceuticals

Active Publication Date: 2020-07-07
SHENZHEN INNOVATION CENT OF SMALL MOLECULE DRUG DISCOVERY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antibody prepared by the present invention can effectively bind to the specific fragment of the extracellular region of Siglec-15 protein, and block its binding to sTn protein, showing good neutralization properties, and there is no anti-Siglec-15 with related functions Antibody patent report

Method used

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  • human-mouse chimeric Siglec-15-resistant neutralizing full-molecule IgG and preparation method and application thereof
  • human-mouse chimeric Siglec-15-resistant neutralizing full-molecule IgG and preparation method and application thereof
  • human-mouse chimeric Siglec-15-resistant neutralizing full-molecule IgG and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Siglec-15 Extracellular Region Peptide Design

[0038] Siglec-15 has a total of 328 amino acids, and the extracellular region consists of an N-terminal V-set domain (A49-A165) including a sialic acid binding site and a type 2 constant region (IgC2) region (A168-A251), in which IgV The region is the key site for the combination of Siglec-15 and Sialyl-Tn. Using Siglec-15A37-A180 amino acids as a template, Overlap polypeptides were designed and named A1~A12 and B1~B11, respectively. After the peptides were synthesized, the fractions were mixed and coupled to OVA and KLH respectively.

[0039] A1 HSSPAQRWSMQV B1 RWSMQVPPEVSA A2 PPEVSAEAGDAA B2 EAGDAAVLPCTF A3 VLPCTFTHPHRH B3 THPHRHYDGPLT A4 YDGPLTAIWRAG B4 AIWRAGEPYAGP A5 EPYAGPQVFRCA B5 QVFRCAAARGSE A6 AARGSELCQTAL B6 LCQTALSLHGRF A7 SLHGRFRLLGNP B7 RLLGNPRRNDLS A8 RRNDLSLRVERL B8 LRVER LAL ADDR A9 ALADDRRY FCRV B9 RYFCRVEFAG...

Embodiment 2

[0040] Example 2: Preparation of mouse-derived anti-Siglec-15 hybridoma cells

[0041] Customize the recombinant Siglec-15 whole gene protein according to the human Siglec-15 gene. The protein and the mixed polypeptide coupled with OVA were used as immunogens to immunize the abdomen of pure BALB / c mice by subcutaneous injection, 100 μg / ml each time, five times in total, of which recombinant Siglec- 15 whole gene proteins, the 2nd and 4th use the mixed polypeptide coupled with OVA. When positive serum with an OD greater than 2 can be detected in the peripheral serum, the cells are fused, and the mixed peptide coupled with OVA is used to boost immunization 3 days before the fusion. On the day of fusion, the mouse spleen was taken, and a single-cell suspension was prepared with DMEM medium (GIBCO, USA). In the presence of 50% PEG (PH 8.0), the spleen cells and SP2 / 0 mouse myeloma cells were fused, and used HAT selective medium (DMEM medium 98ml, HT stock solution 1ml, A stock s...

Embodiment 3

[0042] Example 3: Screening, strain determination and identification of murine anti-Siglec-15 antibodies

[0043] The enzyme-linked immunoassay (ELISA) detection and screening was carried out according to the growth condition of the hybridoma cells, and the specific method is as follows. The recombinant Siglec-15 protein was used to coat the plate, and the cells in the positive wells were cloned and cultured again. After three times of subcloning, the positive clones were determined. The plates were then coated with Siglec-15 mixed polypeptides coupled with KLH to detect positive clones. After all the monoclonal cells in the wells were positive for Siglec-15-KLH and negative for KLH, several wells were taken for expansion and cultured and partially frozen.

[0044] ELISA method to screen Siglec-15 positive but normal mouse seronegative monoclonal antibody, the specific method is as follows:

[0045] (1) The chemically synthesized Siglec-15-KLH polypeptide was coated with ELI...

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Abstract

The invention discloses human-mouse chimeric Siglec-15-resistant neutralizing full-molecule IgG and a preparation method and application thereof, and belongs to the field of biological pharmacy. Miceimmune to Siglec-15-specific peptide are adopted, a mouse-derived Siglec-15-resistant monoclonal antibody is prepared through a hybridoma technology, and the recombinant human-mouse chimeric Siglec-15-resistant full-molecule IgG is prepared by using a genetic engineering technology and an antibody engineering technology. Specific amino acid segments in the extracellular region of Siglec-15 can berecognized effectively through the chimeric antibody, and combination of Siglec-15 protein with Sialyl-Tn protein is inhibited. The invention discloses the human-mouse chimeric Siglec-15-resistant full-molecule IgG, the human-mouse chimeric Siglec-15-resistant full-molecule IgG includes a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 6 or a conservative variant which is obtained through conservative mutation of the amino acid sequence by using addition, deletion, substitution and modification of one or more amino acids. The invention also discloses a DNA molecule, expression vector, host cells andapplication of the full-molecule IgG antibody.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a human-mouse chimeric anti-Siglec-15 full-molecule IgG antibody, and also relates to the DNA molecule, expression vector, host cell and application of the above-mentioned full-molecule IgG antibody. Background technique [0002] The sialic acid-binding Ig-like lectin family, the Siglecs family, is a classical family of immunoglobulin-like lectin proteins. Under physiological conditions, only 15 Siglec molecules are expressed on the surface of myeloid and immune cells that mediate immunosuppression and have immunosuppressive properties. When Siglecs recognize sialic acid-containing glycans expressed by all mammalian cells, they help immune cells distinguish self from non-self. Sialylated pathogens can also destroy and promote immune responses through Siglec-dependent interactions, or downregulate immune cell responses and evade immune surveillance. Siglecs have important roles i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00G01N33/68G01N33/574
CPCA61K2039/505A61P35/00C07K16/2803C07K16/2851C07K2317/56C07K2317/565G01N33/574G01N33/68
Inventor 唐奇毛圆唐小军冯振卿朱进肖雪筠
Owner SHENZHEN INNOVATION CENT OF SMALL MOLECULE DRUG DISCOVERY CO LTD
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