PD-1 monoclonal antibody and application thereof

An antibody and carrier technology, applied in the field of biomedicine, can solve problems such as unsatisfactory antibody performance

Active Publication Date: 2016-06-01
PERSONGEN BIOMEDICINESUZHOUCO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the performance of current antibodies is still unsatisfactory

Method used

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  • PD-1 monoclonal antibody and application thereof
  • PD-1 monoclonal antibody and application thereof
  • PD-1 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0141] Preparation of monoclonal antibodies

[0142] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol. 6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).

[0143] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (avai...

Embodiment 1

[0172] The preparation of embodiment 1 monoclonal antibody

[0173] 1.1 Animal immunity

[0174] 6-8 weeks old female BALB / C mice were taken, and the immunogen was recombinantly expressed PD-1 protein 293T cells, the amino acid sequence was as follows:

[0175] DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQIDNO.:1),免疫程序见表2。 Before each immunization, blood was collected from the tail of the mice, and 293T cells expressing PD-1 protein by gene recombination were used as the detection antigen coating indirect ELISA method to detect the titer of mouse serum. Splenocytes were taken for fusion when the serum titer titer of the immunized mice reached the maximum and no longer increased.

[0176] Table 2 Mouse immunization program

[0177] immune time

...

Embodiment 2

[0188] Example 2 Affinity analysis of PD1-1375 to soluble human PD-1

[0189] The soluble antigen protein PD1-Fc was dissolved in PBS at a concentration of 5 μg / ml for antigen coating;

[0190] Antibody incubation with PD1-1375 in different concentration gradients;

[0191] Dissolve goat anti-mouse IgG-HRP (ProSci) in diluent (1%BSA+0.05%PBST20) at 1:5000, incubate with secondary antibody, and develop color; the experimental results are as follows figure 1 As shown, the EC50 of the PD1-1375 antibody was obtained = 29.4 ng / mL.

[0192] PD1-Fc is composed of the extracellular domain of human PD1 molecule plus the Fc fragment of human IgG1, and its amino acid sequence is as follows:

[0193] DSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL P KSCDKTHTCPPCPAPELLGGPSVF...

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Abstract

The present invention provides a PD-1 monoclonal antibody and application thereof, and the monoclonal antibody has high specificity and strong affinity, can significantly stimulate the secretion of IFN-gamma, and can effectively activate CD8 + and CD4 + T cells.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a PD-1 monoclonal antibody and its application. Background technique [0002] The specific immune response of T cells is stimulated by the first stimulating signal formed by the combination of T cell receptors and antigen peptide MHC complexes; at the same time, the intensity and magnitude of the immune response mediated by T cells is determined by immune checkpoint proteins (including co-repressors and co-stimulatory molecules). After more than 20 years of research, immune checkpoint molecules have been found to play a key role in regulating T cell immune responses, these molecules include: cytotoxic lymphocyte antigen 4 (Cytotoxiclymphocyte antigen-4, CTLA-4), programmed death protein 1 (Programmeddeath-1, PD-1), T cell immunoglobulin and mucin structure-3 (Tcellimmunoglobulinandmucindomain3, TIM-3), T cell immunoglobulin and ITIM structure (TcellIgandITIMdoma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62A61K49/00A61K51/10A61K47/48A61K39/395A61P35/00
Inventor 杨林褚福亮宗云辉
Owner PERSONGEN BIOMEDICINESUZHOUCO
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