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Proteins binding nkg2d, cd16 and a tumor-associated antigen

A tumor-associated antigen and protein technology, which is applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc., and can solve problems such as adverse side effects

Pending Publication Date: 2021-07-23
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 for all patients and / or may have significant adverse side effects
Treating other types of cancer with existing treatment options also remains challenging

Method used

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  • Proteins binding nkg2d, cd16 and a tumor-associated antigen
  • Proteins binding nkg2d, cd16 and a tumor-associated antigen
  • Proteins binding nkg2d, cd16 and a tumor-associated antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-N

[0460] Example 1 - NKG2D binding domain binds to NKG2D

[0461] NKG2D binding domain binds to purified recombinant NKG2D

[0462] 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 microplate. 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 by the 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) (substrate for horseradish peroxide) was added to the wells to observe the binding signal, whose absorbance was measured at 450 nM and corrected at 540 nM . ...

Embodiment 2

[0467] Example 2 - NKG2D Binding Domains Block the Binding of Natural Ligands to NKG2D Compete with ULBP-6

[0468] The recombinant human NKG2D-Fc protein was adsorbed to the wells of the microplate, 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 the addition of NKG2D-binding domain clones. After 2 hours of incubation, the wells were washed, and ULBP-6- that remained bound to the NKG2D-Fc-coated wells was detected by streptavidin and TMB substrate conjugated to horseradish peroxidase. His-Biotin. Absorbance was measured at 450 nM and corrected at 540 nM. The specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated based on the percentage of ULBP-6-His-biotin that was blocked from binding to the NKG2D-Fc protein in the well after background subtraction. Positive control antibodies (comprising heavy and light chain variable dom...

Embodiment 3-N

[0475] Example 3 - NKG2D binding domain cloning activates NKG2D

[0476] 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. The NKG2D-CAR construct was then cloned into a retroviral vector using the Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with virus containing NKG2D-CAR together with 8 μg / mL polybrene. 24 hours after infection, NKG2D-CAR expression levels in EL4 cells were analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0477] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed to wells of microplates, and NKG2D-CAR EL4 cells were incubated with antibody fragment-coated cells in the presence of brefeldin A and monensin. Wells were incubated for 4 hours. Intracellular TNF-α production, an indicator...

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PUM

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Abstract

Multi-specific binding proteins that bind NKG2D receptor, CD 16, and a tumor- associated antigen selected from c-MET, KIT, F3, IGF1R, Lewis Y, MUC13, MUC4, MCAM, LRRC32, sialyl-Tn, gpA33, GD3, GM2, EPHA3, TNFRSF10A, TNFSF11, CD74, and PMEL are described, as well as pharmaceutical compositions and therapeutic methods useful for the treatment of cancer.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 716,110, filed August 8, 2018, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. [0003] sequence listing [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on August 6, 2019, is named DFY-061WO_ST25.txt and is 342,008 bytes in size. technical field [0005] The present invention relates to multispecific binding proteins that bind NKG2D, CD16 and at least one tumor-associated antigen. Background technique [0006] Despite numerous research efforts and scientific advances reported in the literature for the treatment of cancer, the disease remains a major health problem. Cancers of the blood and bone marrow are frequently diagnosed cancer type...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C07K16/28
CPCA61K2039/505C07K2317/31C07K16/2851C07K2317/33C07K2317/76C07K2317/70C07K2317/94C07K16/283C07K16/2863A61P35/00C07K2317/52C07K2317/622C07K2317/565
Inventor G·P·常张莺娥杜瑾焱A·格林贝格W·汉尼B·M·伦德B·普林茨
Owner DRAGONFLY THERAPEUTICS INC
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