A human-mouse chimeric anti-Siglec-15 full-molecule IgG with neutralizing effect and its preparation method and application

A molecular and antibody technology, applied in the field of biopharmaceuticals, to achieve high binding activity and affinity, and good neutralizing properties

Active Publication Date: 2021-02-09
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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  • A human-mouse chimeric anti-Siglec-15 full-molecule IgG with neutralizing effect and its preparation method and application
  • A human-mouse chimeric anti-Siglec-15 full-molecule IgG with neutralizing effect and its preparation method and application
  • A human-mouse chimeric anti-Siglec-15 full-molecule IgG with neutralizing effect and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Siglec-15 extracellular domain 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) containing a sialic acid binding site and a type 2 constant region (IgC2) region (A168-A251), in which IgV The region is the key site where Siglec-15 binds to Sialyl-Tn. Using Siglec-15A37-A180 amino acids as templates, the Overlap polypeptides were designed and named as A1-A12 and B1-B11, respectively. After peptide synthesis, a portion was 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 LRVERLALADDR A9 ALADDRRYFCRV B9 RYFCRVEFAGDV A10 EFAGDVHDRYE...

Embodiment 2

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

[0041] Customized recombinant Siglec-15 whole gene protein according to human Siglec-15 gene. Using this protein and OVA-conjugated mixed polypeptides as immunogens, pure-line BALB / c mice were immunized by subcutaneous injection in the abdomen at 100 μg / ml each time, a total of five times, of which the 1st, 3rd, and 5th times used recombinant Siglec- 15 Whole gene protein, mixed polypeptide coupled with OVA was used for the 2nd and 4th times. When the peripheral serum can detect positive serum with an OD greater than 2, cell fusion is performed, and the mixed polypeptide coupled with OVA is used for boosting immunization 3 days before fusion. On the day of fusion, the mouse spleen was taken, and a single-cell suspension was prepared in DMEM medium (GIBCO, USA), and in the presence of 50% PEG (PH 8.0), the spleen cells and SP2 / 0 mouse myeloma cells were fused, using HAT selective medium (98 ml of DMEM me...

Embodiment 3

[0042] Example 3: Screening, Straining and Identification of Murine Anti-Siglec-15 Antibodies

[0043] Enzyme-linked immunosorbent assay (ELISA) detection and screening were carried out according to the growth of hybridoma cells, and the specific method was shown below. The plates were coated with recombinant Siglec-15 protein, and the cells in the positive wells were cloned and cultured again. After three subcloning, the positive clones were determined. Then, the plate was coated with Siglec-15 mixed polypeptide conjugated with KLH to detect the positive clones. After all the monoclonal cells in the wells were detected as positive for Siglec-15-KLH and negative for KLH, several wells were taken to expand the culture and partially cryopreserved.

[0044] The ELISA method was used to screen the monoclonal antibodies that were positive for Siglec-15 but seronegative in normal mice. The specific methods are as follows:

[0045] (1) The chemically synthesized Siglec-15-KLH polyp...

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Abstract

The invention discloses a human-mouse chimeric anti-Siglec-15 full-molecule IgG with neutralizing effect and its preparation method and application, and belongs to the field of biopharmaceuticals; the invention uses Siglec-15 specific polypeptides to immunize mice, adopts hybridization Rat anti-Siglec-15 monoclonal antibody was prepared by tumor technology, and recombinant human-mouse chimeric anti-Siglec-15 full-molecule IgG was prepared by using genetic engineering technology and antibody engineering technology; the chimeric antibody can effectively recognize the extracellular region of Siglec-15 Specific amino acid fragments, and inhibit the combination of Siglec-15 protein and Sialyl-Tn protein; the invention discloses a human-mouse chimeric anti-Siglec-15 whole molecule IgG, comprising a light chain variable region and a heavy chain variable region, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, or the sequence is added or deleted by one or more amino acids Conservative variants obtained by conservative mutations, substitutions, and modifications; the present invention also discloses the DNA molecule, expression vector, host cell, and application of the above-mentioned full-molecule IgG antibody.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, relates to a human-mouse chimeric anti-Siglec-15 full-molecule IgG antibody, and also relates to a DNA molecule, an expression vector, a host cell and an 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 family of classical 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 recognizes sialic acid-containing glycans expressed by all mammalian cells, it helps immune cells distinguish between self and non-self. Sialylated pathogens can also disrupt and promote immune responses through Siglec-dependent interactions, or downregulate immune cell responses and evade immune surveillance. Siglecs have an impor...

Claims

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Application Information

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Patent Type & Authority Patents(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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