Humanized antihuman CTLA4 monoclonal antibody and preparation method and application thereof

A monoclonal antibody, humanized technology, applied in the field of tumor immunotherapy and molecular immunology, can solve problems such as side effects, achieve high affinity, achieve tumor immunotherapy, and high specificity

Inactive Publication Date: 2020-02-07
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is only one CTLA4 monoclonal antibody currently on the market, and CTLA4 monoclonal antibodies also have varying degrees of side effects, including the induction of immunogenicity in some patients, excessive inhibition of CTLA4 signaling may cause autoimmune diseases, and different CTLA4 monoclonal antibodies. Monoclonal antibodies have varying degrees of developability

Method used

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  • Humanized antihuman CTLA4 monoclonal antibody and preparation method and application thereof
  • Humanized antihuman CTLA4 monoclonal antibody and preparation method and application thereof
  • Humanized antihuman CTLA4 monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Humanization of mouse-derived anti-human CTLA4 antibody

[0086] 1) The sequence of the mouse-derived anti-human CTLA4 antibody 42B11G12D3 (the CDR region is underlined) (see, for example, SEQ ID NO: 1-2)

[0087]

[0088]

[0089] 2) Construction of anti-human CTLA4 antibody CDR grafted plasmid

[0090] Select the IMGT human V gene (F+ORF+in-frameP) database, select the human Germline antibody sequence with the highest homology as the humanized receiving vector according to the comparison, and transfer the three heavy chain complementarity determining regions HCDR1 and HCDR2 in the mouse antibody and HCDR3, the three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 were transferred to the corresponding positions, and the post-translational modification sites (PTM) were analyzed, as shown in Table 1. Sequence analysis revealed that two sites, W33 and M63, are hotspots for post-translational oxidative modification (see eg, SEQ ID NO...

Embodiment 2

[0124] Example 2: Recombinant Production of Humanized Antibody

[0125] The selected antibody VH and VL sequences were codon-optimized, connected to the 5' end of the secretory signal peptide, connected to the human antibody IgG1 heavy chain, kappa light chain constant region sequences, respectively cloned into the pTT5 expression vector to prepare the Human antibody DNA sequence expressed and secreted in mammalian cells. The plasmid was co-transfected into HEK293-6E suspension culture cells with PEI for transient expression. During transfection, the cell density was maintained at 1 × 10 6 cells / mL with a PEI:DNA ratio of 3:1. Cells at 37 °C 5% CO 2 Shake culture at 105 rpm in the incubator. 24 hours after transfection, 0.5% Trypton N-1 was added. After 5 days, the cell culture supernatant was collected, and the antibody was purified using protein-A agarose gel, quantified and identified for purity (Table 3).

[0126] Table 3: Recombinant production of humanized antibodie...

Embodiment 3

[0129] Example 3: Affinity Determination of Humanized Monoclonal Antibodies

[0130] Equilibrate the surface of the chip with HBS-EP buffer at a flow rate of 10 μl / min for 5 minutes, then inject a 1:1 mixture of "NHS+EDC" at a flow rate of 10 μl / min for 7 minutes to activate the chip, dilute in 10 mM sodium acetate buffer The capture antibody (Goat anti-mouse IgG) was injected at a flow rate of 10 μl / min for about 7 minutes for coupling, and finally ethanolamine was injected at a flow rate of 10 μl / min for 7 minutes for surface blocking.

[0131] Use HBS-EP buffer as the sample for three pre-circulations to balance the chip to stabilize the baseline, and inject the antibody diluted in HBS-EP buffer at a flow rate of 10 μl / min for 0-5 minutes (control the binding of antibody and antigen by adjusting the capture time Signal at ~100RU), buffer equilibrated for 1 min. Inject low-concentration antigen 0.33nM CTLA4-Fc at a flow rate of 30 μl / min for 5 minutes to bind the antigen to...

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Abstract

The invention belongs to the fields of tumor immunotherapy and molecule immunology, and particularly relates to a humanized antihuman CTLA4 monoclonal antibody. The invention further relates to a preparation method and application of the humanized antihuman CTLA4 monoclonal antibody. Particularly, the humanized antihuman CTLA4 monoclonal antibody provided by the invention has high substantivity and high specificity for CTLA4, can stimulate T cells to secrete cytokine, for example, immunization negative regulation of the CTLA4 can be specially removed, and T cell cytokine can be activated. Thefunctional humanized antihuman CTLA4 monoclonal antibody provided by the invention can block a CTLA4 signal route to activate the T cells, and further the purpose of tumor immunotherapy is realized.

Description

technical field [0001] The invention belongs to the fields of tumor immunotherapy and molecular immunology, and in particular relates to a humanized anti-human CTLA4 monoclonal antibody. The present invention also relates to the preparation method and application of the humanized anti-human CTLA4 monoclonal antibody. Background technique [0002] The immune system of vertebrates is a functional system composed of various organs, tissues, cells and molecules, and it is the most effective mechanism for the body to defend against the invasion of foreign substances (Janeway et al., Immunology: The Immune System in Health and Disease. New York: Garland Science, 2005). Immune organs, tissues, and cells cooperate and check and balance each other. Under the coordination of many immune checkpoint proteins and cytokines, they can protect the body from external infection and maintain homeostasis. The adaptive immune system against foreign pathogens consists of humoral immunity (media...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCC07K16/2818A61P35/00C07K2317/24C07K2317/565C07K2317/92C07K2317/76C07K2317/56C07K2317/567
Inventor 殷刘松周铁林方卓米艳玲吴瑃辰
Owner NANJING GENSCRIPT BIOTECH CO LTD
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