Siglec-15 monoclonal antibody and applications thereof

A monoclonal antibody, recombinant vector technology, applied in the application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin and other directions, can solve the problem of immune system detection difficulties and other issues

Active Publication Date: 2021-01-01
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, tumor cells are sometimes able to avoid detection and destruction by the immune system by reducin

Method used

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  • Siglec-15 monoclonal antibody and applications thereof
  • Siglec-15 monoclonal antibody and applications thereof
  • Siglec-15 monoclonal antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Obtaining of human Siglec-15 hybridoma cell line and preparation of monoclonal antibody

[0045] 1) Animal immunity

[0046] The antigen uses the recombinant protein Siglec-15-Fc fused to the human Siglec-15 extracellular domain of the human IgG1 Fc segment. Female Balb / c and C57bl / 6 mice were immunized subcutaneously with 50 μg Siglec-15-Fc fusion protein in 200 μl Freund's complete adjuvant (Sigma-Aldrich) in a 1:1 emulsion. Mice were subsequently boosted with alternating ip / sc injections of 25 μg Siglec-15-Fc in a 1:1 emulsion in Freund's incomplete adjuvant (Sigma-Aldrich) up to 3 times every two weeks. Serum titers of 10 mice all reached 10 after three immunizations 5 above. 4 days before myeloma fusion, showing the highest antibody titer ( figure 1 ) of two mice (No. 251 and No. 260) received an intraperitoneal booster immunization with 25 μg Siglec-15-Fc (without adjuvant).

[0047] 2) Hybridoma fusion and screening

[0048] Spleens were extracte...

Embodiment 2

[0052] Example 2: Production and purification of hybridoma sequencing and antibody

[0053] After using the fast ELISA mouse antibody subtype identification kit (Clono typing System-HRP, SouthernBiotech) to identify the subtype of the monoclonal antibody, use TRIzol (Ambion) from 3 × 10 6 ~5×10 6 Total RNA was extracted from hybridoma cells, and antibody subtype-specific primers and universal primers (PrimeScript TM 1stStrand cDNASynthesis Kit, Takara) to reverse transcribe it into cDNA. Murine immunoglobulin heavy and light chain V-region fragments were subsequently amplified by RACEPCR and the resulting PCR fragments were subcloned into the pMD18-T vector system (Takara) and the inserts were sequenced using vector-specific primers. Finally, the heavy chain and light chain protein sequences of clones C9, C18, C30, C31, C40, C46, ​​and C52 were obtained.

[0054] Table 1 is the sequence information of the heavy chain complementary region:

[0055]

[0056] Table 2 is t...

Embodiment 3

[0061] Example 3: Binding of monoclonal antibody to human Siglec-15 recombinant protein

[0062] Indirect ELISA was used to assess the binding ability of purified antibodies to Siglec-15-Fc. ELISA plates (Nunc) were coated with 100 μl / well of 0.5 μg / ml recombinant Siglec-15-Fc or human IgG1 in PBS overnight at 4°C. Plates were washed with PBS-T (0.05% Tween), and blocked with 200 μl / well of 1% BSA in PBST at 37° C. for 0.5 hours. Then discard the blocking solution, add 100 μl of 10 μg / ml purified antibody to the first well, and dilute according to 2-fold gradient, a total of 8 test concentration gradients. Then incubate for 1 hour at room temperature. Plates were washed three times with PBST and incubated with 100 μl / well horseradish peroxidase-conjugated goat anti-mouse IgG (Fab-specific) for 0.5 hours at 37°C. Plates were washed five times with PBST, then TMB chromogenic solution was added and incubated for 15 minutes at room temperature in the dark. The reaction was sto...

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Abstract

The invention relates to a Siglec-15 monoclonal antibody and applications thereof. Through a cell fusion and hybridoma technology, the obtained human Siglec-15 monoclonal antibody can highly and specifically bind to the cells expressing human Siglec-15 antigen; the monoclonal antibody targeting human Siglec-15 is simple in preparation method; and the obtained anti-human Siglec-15 monoclonal antibody can well detect Siglec-15 protein expressed on tumor cells. The monoclonal antibody targeting the Siglec-15 is high in antibody specificity and has good application prospects on treating and/or preventing or diagnosing diseases, such as tumor, related to the Siglec-15.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy and biomedicine, and relates to a monoclonal antibody targeting human Siglec-15, its preparation method and application, especially in the treatment and / or prevention or diagnosis of diseases related to Siglec-15 such as tumors Applications. Background technique [0002] As we all know, cancer is considered as one of the leading causes of death. Cancer, also known as malignant tumor, refers to a new organism formed by abnormal proliferation and differentiation of cells in local tissues at the gene level due to the action of various carcinogenic factors. Immunotherapy has become the fourth major cancer treatment after conventional treatments such as surgery, radiotherapy, and chemotherapy. Immunotherapy is considered to be the most promising treatment modality because of its small side effects, high antitumor activity, and wide indications. The immune system has great potential to destroy ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P35/02G01N33/68G01N33/577G01N33/574
CPCA61P35/00A61P35/02C07K16/2803C07K2317/56C07K2317/73C07K2317/92G01N33/574G01N33/57484G01N33/577G01N33/68G01N2333/70503
Inventor 代红久徐慧王梦瑶朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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