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Protein binding nkg2d, cd16 and a fibroblast activation protein

A fibroblast, activating protein technology, applied in the direction of hybrid immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, immunoglobulin, etc., can solve problems such as adverse side effects

Pending Publication Date: 2021-02-26
DRAGONFLY THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current treatment options for these cancers are not effective in all patients and / or may have serious adverse side effects
Treating other types of cancer with existing treatment options also remains challenging

Method used

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  • Protein binding nkg2d, cd16 and a fibroblast activation protein
  • Protein binding nkg2d, cd16 and a fibroblast activation protein
  • Protein binding nkg2d, cd16 and a fibroblast activation protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-N

[0242] Example 1 - NKG2D binding domain binds to NKG2D

[0243] NKG2D binding domain binds to purified recombinant NKG2D

[0244] The nucleic acid sequence of human, mouse or cynomolgus NKG2D ectodomain is fused with the nucleic acid sequence encoding human IgG1 Fc domain, and introduced into mammalian cells for expression. After purification, the NKG2D-Fc fusion protein was adsorbed to the wells of a microwell plate. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D-binding domain was titrated and added to the wells pre-adsorbed with NKG2D-Fc fusion protein. Primary antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and specifically recognizing human kappa light chain to avoid Fc cross-reactivity. 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase was added to the wells to observe the binding signal, its absorbance was measured at 450 nM and corrected at 540 ...

Embodiment 2

[0249] Example 2 - NKG2D Binding Domains Block the Binding of Natural Ligands to NKG2D Compete with ULBP-6

[0250] The recombinant human NKG2D-Fc protein was adsorbed to the wells of the microwell plate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by NKG2D-binding domain clones. After 2 hours of incubation, the wells were washed and ULBP-6-His-biotin still bound to NKG2D-Fc-coated wells was detected by horseradish peroxidase-conjugated streptavidin and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, the specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin blocked from binding to the NKG2D-Fc protein in the wells. Positive control antibodies (comprising heavy and light chain variable domains selected from SEQ ID NO: 101-104) an...

Embodiment 3-N

[0257] Example 3 - NKG2D binding domain cloning activates NKG2D

[0258] The nucleic acid sequences of human and mouse NKG2D were fused to the nucleic acid sequence encoding the CD3ζ signaling domain to obtain chimeric antigen receptor (CAR) constructs. Then, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells to generate retrovirus. EL4 cells were infected with virus containing NKG2D-CAR and 8 μg / mL polybrene. 24 hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0259] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed to wells of microplates, and NKG2D-CAREL4 cells were incubated in antibody fragment-coated wells in the presence of brefeldin A and monensin Incubate for 4 hours. Intracellular TNF-[alpha] production (an indicator of NKG2D activation)...

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Abstract

Multi-specific binding proteins that bind NKG2D receptor, CD16, and fibroblast activation protein (FAP) are described, as well as pharmaceutical compositions and therapeutic methods of the multi- specific binding proteins useful for the treatment of cancer, autoimmune disease, or fibrosis.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 672,299, filed May 16, 2018. [0003] sequence listing [0004] This application contains a Sequence Listing, which is filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 13, 2019, is named DFY_056WO_SL25.txt and is 121,670 bytes in size. technical field [0005] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16 and fibroblast activation protein (FAP). Background technique [0006] Despite numerous research efforts and scientific advances reported in the literature for the treatment of cancer, the disease remains an important health problem. Some of the most commonly diagnosed cancers include prostate, breast, and lung. Prostate cancer is the most common form of cancer in men. Breast cancer remains the lead...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C07K16/28C07K16/46
CPCC07K16/46C07K16/2851C07K16/40C07K2317/31C07K16/283C07K2317/75C07K2317/73C07K2317/94C07K2319/00C07K2319/30C07K2319/33A61P35/00C07K2317/90A61K2239/57A61K39/4613A61K39/464429A61K39/4631A61K39/464499A61K2039/505C07K16/468C07K2317/524C07K2317/53C07K2317/565C07K2317/569
Inventor 格雷戈里·P·常安·F·张杜金燕阿斯亚·格林贝格威廉·哈尼尼古拉·瓦格曼布拉德利·M·伦德比昂卡·普林茨
Owner DRAGONFLY THERAPEUTICS INC
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