Proteins binding nkg2d, cd16 and flt3

A protein and binding site technology, applied in the direction of hybrid immunoglobulin, anti-animal/human immunoglobulin, immunoglobulin, etc.

Pending Publication Date: 2020-05-08
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 challengi

Method used

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  • Proteins binding nkg2d, cd16 and flt3
  • Proteins binding nkg2d, cd16 and flt3
  • Proteins binding nkg2d, cd16 and flt3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-N

[0256] Example 1 - NKG2D binding domains bind to NKG2D

[0257] NKG2D binding domain binds to purified recombinant NKG2D

[0258] The nucleic acid sequence of the extracellular domain of human, mouse or cynomolgus NKG2D is fused to the nucleic acid sequence encoding the human IgGl Fc domain and introduced into the mammalian cells to be expressed. After purification, the NKG2D-Fc fusion protein was adsorbed to the wells of the microplate. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D binding domain was titrated and added to 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 visually observe the binding signal, its absorbance measured at 450 nM...

Embodiment 2

[0263] Example 2 - NKG2D Binding Domains Block the Binding of Natural Ligands to NKG2D

[0264] Competition with ULBP-6

[0265] 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 a 2-hour incubation, the wells were washed and ULBP-6-His-organisms still bound to NKG2D-Fc-coated wells were detected by streptavidin and TMB substrate conjugated to horseradish peroxidase white. 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 in the wells whose binding to the NKG2D-Fc protein was blocked after background subtraction. A positive control antibody (comprising heavy and light chain variable dom...

Embodiment 3-N

[0271] Example 3 - NKG2D binding domain cloning activates NKG2D

[0272] The nucleic acid sequences of human and mouse NKG2D were fused to the nucleic acid sequence encoding the CD3ξ 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 retrovirus production. EL4 cells were infected with NKG2D-CAR-containing virus and 8 μg / mL polybrene. At 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.

[0273] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed to microplate wells and coated with antibody fragments in the presence of brefeldin-A and monensin. NKG2D-CAR EL4 cells were cultured on the wells for 4 hours. Intracellular TNF-[alpha] production, an indicator of NKG2D activatio...

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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 FLT3 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 / 539,421, filed July 31, 2017; the contents of which are hereby incorporated by reference in their entirety for all purposes. [0003] sequence listing [0004] This application contains a Sequence Listing, which was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on July 30, 2018, is named DFY-027WO_SL.txt and is 103,731 bytes in size. technical field [0005] The present invention relates to a multispecific binding protein binding to NKG2D, CD16 and tumor-associated antigen FLT3. Background technique [0006] Despite numerous research efforts and scientific advances reported in the literature aimed at treating cancer, the disease remains an important health problem. Cancers of the blood and bone marrow are frequently diagnosed types of cancer, includ...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/28C07K16/46
CPCA61K39/395C07K16/28C07K16/46C07K2317/31C07K2317/622C07K16/2863C07K16/283C07K16/2851C07K2317/92A61P35/00C07K16/30C07K2317/75A61P35/02A61K2039/505C07K16/2896C07K16/468C07K2317/21C07K2317/524C07K2317/53C07K2317/569
Inventor 格雷戈里·P·张安·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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