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Anti-TNFR2 antibodies and methods of use thereof

A technology of antibodies and receptors, applied in the direction of antibodies, chemical instruments and methods, anti-tumor drugs, etc., can solve problems such as reducing and hindering signal transmission

Pending Publication Date: 2022-06-17
APEXIGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In humans, known polymorphisms of TNFRSF1B result in reduced TNFR2 expression or reduced binding to TNFα and impede TNFR2-mediated signaling in regulatory T cells

Method used

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  • Anti-TNFR2 antibodies and methods of use thereof
  • Anti-TNFR2 antibodies and methods of use thereof
  • Anti-TNFR2 antibodies and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0391] Immunization and initial screening

[0392] To prepare antibodies for screening, four New Zealand white rabbits were immunized and subsequently boosted with human 293-TNFR2 overexpressing cells or human TNFR2-Fc fusion protein. All rabbits had serum titers specific for human TNFR2 and were used for the use of APXiMAB TM Technology Generation of hybridomas and antibody production by B cell culture method (RevMAb). After initial screening, 460 antibodies were identified that bound to CHO-TNFR2 overexpressing cells. Screening processes to identify potential lead candidates such as figure 2 shown.

[0393] Supernatants from hybridoma and B cell cultures were then screened for antibodies that could block the binding of TNF-α to soluble TNFR2-His (Sino Biological; 10417-H08H) in an ELISA-based receptor ligand binding assay. Of the 460 antibodies identified, 173 showed inhibition of TNF-α binding to TNFR2, which was greater than 75% of maximal binding.

[0394] 110 antib...

Embodiment 2

[0405] Binding and activity characterization of clones h600-25-71 and h600-25-108

[0406] The binding and activity characteristics of humanized clones h600-25-71 and h600-25-108 were tested. To test binding to TNFR family members, target proteins were coated on ELISA plates at 1 μg / mL overnight at 4°C. Plates were washed, blocked, and antibodies were added at the indicated concentrations for 1 hour at room temperature. Positive control antibodies for each protein were used at approximately 1 ug / mL. Antibodies were washed and detected with anti-human IgG HRP for 1 hour. The assay was developed using TMB substrate for 10 minutes. For cell binding, human CD4+ T cells purified from buffy coat were incubated with anti-CD3 / CD28 in flat bottom plates for 24 hours. Cells were harvested and stained for viability, CD4, CD25 and FOXP3. Stain with anti-human IgG APC detection test antibody. Binding titers of test antibodies were assessed on CD4+CD25hiFOXP3+ regulatory T cells and p...

Embodiment 3

[0418] Identification of the epitope site of clone 25-71

[0419] Experiments were performed to determine the epitope of the TNFR2 / 25-71 complex at high resolution.

[0420] First, high-quality MALDI analysis was performed on individual TNFR2 samples and individual clone 25-71 samples to verify integrity and aggregation levels. Measurements were performed using an Autoflex II MALDI ToF mass spectrometer (Bruker) equipped with an HM4 interaction module (CovalX) containing a detection system designed to optimize detection up to 2 Mda with nanomolar sensitivity. The TNFR2 sample powder was dissolved in distilled water to a concentration of 1 mg / ml and 20 μl of each protein sample of TNFR2 and clones 25-71 were pipetted to prepare 8 dilutions with a final volume of 10 μl. Then, 1 μl of each dilution was mixed with 1 μl of matrix consisting of recrystallized sinapic acid matrix ( 10mg / ml) composition. After mixing, 1 μl of each sample was spotted on MALDI plates (SCOUT 384). Af...

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Abstract

Anti-Tumor Necrosis Factor Receptor 2 (TNFR2) antibodies and related compositions are provided that can be used in any of a variety of therapeutic or diagnostic methods, including the treatment or diagnosis of oncological, inflammatory and / or autoimmune diseases, and other diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims US Provisional Application No. 62 / 901,364, filed on September 17, 2019, US Provisional Application No. 62 / 985,509, filed on March 5, 2020, and US Provisional Application No. 63 / 047,824, filed on July 2, 2020 and U.S. Provisional Application No. 63 / 058,016, filed July 29, 2020, under 35 U.S.C. § 119(e), each of which is incorporated by reference in its entirety. [0003] Statement Regarding Sequence Listing [0004] The Sequence Listing pertaining to this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is APEX-025 / 04WO_ST25.txt. The text file is approximately 265KB, created on September 11, 2020, and is being submitted electronically via EFS-Web. [0005] background technical field [0006] The present disclosure relates to anti-tumor necrosis factor recept...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00C07K16/28C07K16/30
CPCC07K16/2878C07K2317/76C07K2317/33C07K2317/24C07K2317/92C07K2317/732C07K2317/34A61P35/00A61K2039/505C07K2317/56C07K2317/565C07K2317/31C07K2317/52C07K2317/522C07K2317/54C07K2317/75
Inventor E·L·菲尔伯特S·克里希南C·谭R·巴贾特X·杨R·阿尔瓦拉多
Owner APEXIGEN INC
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