Proteins binding nkg2d, cd16, and c-type lectin-like molecule-1 (cll-1)

A CLL-1, protein technology, applied in the field of multispecific binding proteins

Pending Publication Date: 2020-07-17
DRAGONFLY THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other types of cancer also remain challenging to treat with existing treatment options

Method used

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  • Proteins binding nkg2d, cd16, and c-type lectin-like molecule-1 (cll-1)
  • Proteins binding nkg2d, cd16, and c-type lectin-like molecule-1 (cll-1)
  • Proteins binding nkg2d, cd16, and c-type lectin-like molecule-1 (cll-1)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0249] Example 1 - NKG2D binding domains bind to NKG2D

[0250] NKG2D binding domain binds to purified recombinant NKG2D

[0251] The nucleic acid sequence of human, mouse or cynomolgus NKG2D extracellular domains (ectodomains) 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 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. The substrate for horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine (TMB), was added to the wells to visualize the binding signal, measured at 450 nM and corrected at 54...

Embodiment 2

[0256] Example 2 - NKG2D Binding Domains Block Natural Ligand Binding to NKG2D

[0257] Competition with ULBP-6

[0258] The recombinant human NKG2D-Fc protein was adsorbed to the wells of a 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 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 streptavidin-coupled horseradish peroxidase and TMB substrate . 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 from the percentage of ULBP-6-His-biotin blocked from binding to the NKG2D-Fc protein in the wells after background subtraction. Positive control antibodies (comprising heavy and light chain variable domains selected from SEQ ID NO: 101-104) and vari...

Embodiment 3

[0265] Example 3 - NKG2D binding domain cloning activates NKG2D

[0266] 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 Gibson assembly and transfected into expi293 cells for retroviral production. EL4 cells were infected with NKG2D-CAR-containing virus together with 8 μg / mL polybrene. 24 hours after infection, the expression level of NKG2D-CAR in the EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0267] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed to wells of microplates and NKG2D-CAR EL4 cells were incubated on antibody fragment-coated wells in the presence of brefeldin-A and monensin Incubate for 4 hours. Intracellular TNF-[alpha] production, an indicator of N...

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Abstract

The invention provides multi-specific binding proteins that bind to a tumor-associated antigen CLEC12A and to the NKG2D receptor and CD16 receptor on natural killer cells. One aspect of the inventionprovides a protein that incorporates a first antigen-binding site that binds NKG2D; a second antigen-binding site that binds CLEC12A; and an antibody Fc domain, a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16. The antigen-binding sites may each incorporate an antibody heavy chain variable domain and an antibody light chain variable domain, or one or moreof the antigen-binding sites may be a single domain antibody, such as a VHH antibody or a VNAR antibody. Another aspect of the invention provides a method of treating cancer in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of the multi-specific binding protein.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 558,510, filed September 14, 2017. [0003] sequence listing [0004] This application contains a Sequence Listing, filed electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy generated on September 11, 2018 is named DFY-041WO_SL.txt and is 89,993 bytes in size. technical field [0005] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16 and tumor-associated antigen C-type lectin-like molecule-1 (CLL-1). Background technique [0006] Despite numerous research attempts and scientific advances in treating cancer that have been reported in the literature, the disease remains a significant health problem. Some of the most frequently diagnosed cancers include prostate, breast, lung and colorectal cancers. Prostate cancer is the most common fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395A61P35/00C07K16/28C07K16/46
CPCA61K39/395A61K2039/505A61P35/00C07K16/283C07K16/2851C07K2317/33C07K2317/75C07K2317/94C07K2317/31C07K2317/524C07K2317/565C07K2317/569C07K2317/73
Inventor 格雷戈里·P·常安·F·张威廉·哈尼布拉德利·M·伦德比昂卡·普林茨
Owner DRAGONFLY THERAPEUTICS INC
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