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Humanized monoclonal antibody against hIL-33 and application of monoclonal antibody

A technology of hil-33 and antibody, applied in the field of humanized monoclonal antibody, can solve the problems of degree of humanization, difference in affinity strength effect, no therapeutic effect, high adverse reaction rate of fully human antibody, etc., and achieve enhanced recruitment , enhance the effect of acute allergic reaction

Active Publication Date: 2020-07-07
NANJING NOVOACINE BIO-TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical trial of REGN3500 combined with Dupixent (anti-IL-4Rα) in the treatment of asthma (NCT03112577) showed that REGN3500 alone performed better than the placebo group, but the combination of REGN3500 and Dupixent did not show better treatment than Dupixent alone effect; in addition, the fully human antibody REGN3500 has a high clinical adverse reaction rate, and the incidence of adverse events (AEs) when REGN3500 is used alone is 61.6%
Although there are other reports on anti-IL33 antibodies in the prior art, there are differences in the effects of humanization degree, affinity strength, and cytokine secretion.

Method used

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  • Humanized monoclonal antibody against hIL-33 and application of monoclonal antibody
  • Humanized monoclonal antibody against hIL-33 and application of monoclonal antibody
  • Humanized monoclonal antibody against hIL-33 and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Determination of the amino acid sequence of the light and heavy chain variable regions of the mouse-derived anti-hIL-33 monoclonal antibody

[0093] 1.1 Animal immunity

[0094] Using human IL33 (NP_254274.1) (Ser112-Thr270) expressed in Escherichia coli and fused with 6 histidine tags at the C-terminus, 6 BALB / c mice were immunized according to the routine Freund's adjuvant immunization procedure, and immunized in two batches. Three animals were immunized in each batch, with two weeks between batches. Each batch of animals was immunized 4 times, and Human IL33-his was used for ELISA detection. If the serum titer of the animal was >1:100,000, the animal was finally immunized, and the spleen was collected 3-4 days later.

[0095] 1.2 B cell panning and culture

[0096] 2 days before the formal experiment, spread the culture medium for feeder cells into four 10cm culture dishes (corning, cat.430167), and 1 day before the formal experiment, use 25µg / mL MMC to ...

Embodiment 2

[0106] Example 2: Recombinant expression and activity detection of murine anti-hIL-33 monoclonal antibody

[0107] 2.1 Recombinant expression of mouse anti-hIL-33 monoclonal antibody

[0108] The light and heavy chains from the same clone were combined to transfect CHO-K1 cells. After 24 hours of transfection, 10 μg / mL MSX was added for pressure selection. After the cell density and viability recovered, inoculated for Feed-batch expression, and the supernatant after the expression was completed was centrifuged. And purified by protein A, the antibody concentration was quantified by BCA method and used for quantitative screening.

[0109] The name of the mouse antibody is indicated by the number of the cell clone from which the light and heavy chain (H+L) is contained, for example, the mouse antibody "8140-82H1+L1" means the mouse antibody from the 8140-82 cell clone, and its heavy chain is 8140-82H1 , the light chain is 8140-82L1.

[0110]2.2 Binding activity of mouse anti-h...

Embodiment 3

[0128] Example 3: Humanization of murine anti-hIL-33 monoclonal antibody 8140-82H1+L1

[0129] The heavy and light chain variable region sequences of the murine antibody 8140-82H1+L1 were compared to the human germline sequence using a blast search of the IMGT database. Redundant genes as well as those with unpaired cysteines were removed from the set of human germline genes. The remaining closest matching human germline genes were selected as recipient human frameworks in both framework and CDR regions. FR-4 was selected based on sequence similarity to the IGHJ / IGJK germline genes. The anti-hIL-33 humanized antibody 8140-82H2L1 was obtained through humanized design and screening, and the amino acid sequences of the variable regions of the heavy and light chains are shown in Table 8.

[0130] Table 8 Humanized anti-hIL-33 antibody 8140-82H2L1

[0131]

[0132] Note: The amino acid sequence underlined in bold is the CDR region in the heavy and light chain variable region,...

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Abstract

In order to provide a antibody against hIL-33 with clinical application prospect, the invention provides a high-affinity mouse anti-human IL-33 antibody by adopting a panning technology of immunizingmouse B cells, based on the action mechanism of IL-33 in inflammation and tumors in the prior art, the function of IL-33 in regulating cytokine secretion, and cytokines involved in inflammation and tumors. After recombinant expression of mouse-derived antibodies, preliminary screening of biological activity, humanization transformation, affinity maturation and biological activity rescreening, a humanized monoclonal antibody against hIL-33 is obtained, and the monoclonal antibody has the characteristics of high stability, high affinity, good biological activity, broad clinical application prospects, etc.

Description

technical field [0001] The invention belongs to the field of antibody engineering, and specifically relates to a therapeutic monoclonal antibody against inflammation and / or tumors, in particular to a humanized monoclonal antibody that specifically binds to and inhibits the function of anti-IL-33 and its application. Background technique [0002] Interleukin 33 (IL-33) is a cytokine related to IL-1 and IL-18, also known as NF-HEV or IL-1F11. IL-33 has been described as an "alarmin" because it is present in full-length form in the nuclei of epithelial and endothelial cells during homeostasis, but can be cleaved and released during necrosis. Examples of IL-33-induced cellular responses include the production of inflammatory cytokines such as IL-5, IL-6, IL-13, TNF, IFN-γ, and GM-CSF, and chemotaxis such as CXCL8, CCL17, and CCL24 factor production. IL-33 has also been shown to enhance acute allergic responses by enhancing mast cell and basophil activation elicited by IgE rece...

Claims

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Application Information

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IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P35/00A61P35/02A61P37/02A61P11/06A61P19/02A61P29/00A61P1/00A61P11/02A61P25/00
CPCA61K2039/505A61P1/00A61P11/02A61P11/06A61P19/02A61P25/00A61P29/00A61P35/00A61P35/02A61P37/02C07K16/244C07K2317/24C07K2317/35C07K2317/56C07K2317/565C07K2317/92
Inventor 熊新辉张弢仲恺吴伟黄倩卉徐辉王青潘红
Owner NANJING NOVOACINE BIO-TECH CO LTD
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