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Proteins binding NKG2D, CD16, and EGFR, CCR4, or PD-L1

A PD-L1, protein technology, applied in the direction of animal/human protein, anti-animal/human immunoglobulin, immunoglobulin, etc.

Pending Publication Date: 2021-06-22
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 EGFR, CCR4, or PD-L1
  • Proteins binding NKG2D, CD16, and EGFR, CCR4, or PD-L1
  • Proteins binding NKG2D, CD16, and EGFR, CCR4, or PD-L1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0434] Example 1 - NKG2D binding domains bind to NKG2D

[0435] NKG2D binding domain bound to purified recombinant NKG2D

[0436] Nucleic acid sequences of human, mouse or cynomolgus NKG2D ectodomains are fused to nucleic acid sequences encoding the human IgGl 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 domains were 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 chains to avoid Fc cross-reactivity. Horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added to the wells to visualize the binding signal, measured at 450 nm and corrected at 540 nm. To each well was added an NKG2D ...

Embodiment 2

[0441] Example 2 - NKG2D binding domains block native ligand binding to NKG2D

[0442] Competition with ULBP-6

[0443] Recombinant human NKG2D-Fc protein was adsorbed to wells of a microplate and the wells were blocked with bovine serum albumin to reduce nonspecific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells followed by the NKG2D binding domain clone. After 2 hours of incubation, wells were washed and ULBP-6-His-biotin still bound to NKG2D-Fc-coated wells was detected by streptavidin-conjugated horseradish peroxidase and TMB substrate . Absorbance values ​​were measured at 450 nM and corrected at 540 nM. Specific binding of the NKG2D binding domain to NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin blocked from binding to NKG2D-Fc protein in the wells after background subtraction. Positive control antibodies (comprising heavy and light chain variable domains selected from the group consisting of SEQ ID NOs: 101...

Embodiment 3

[0450] Example 3 - NKG2D binding domain cloning activates NKG2D

[0451] The nucleic acid sequences of human and mouse NKG2D were fused to nucleic acid sequences 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 virus containing NKG2D-CAR with 8 μg / mL polybrene. Twenty-four hours after infection, the expression levels of NKG2D-CAR in the EL4 cells were analyzed by flow cytometry, and clones that expressed high levels of NKG2D-CAR on the cell surface were selected.

[0452] To determine whether NKG2D binding domains activate NKG2D, they were adsorbed to wells of a microplate and NKG2D-CAR EL4 cells were plated on antibody fragment-coated wells in the presence of Brefeldin-A and monensin Incubate for 4 hours. This indication of NKG2D activation was determined by fl...

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Abstract

Multi-specific binding proteins that bind NKG2D receptor, CD16, and a tumor-associated antigen selected from EGFR, HLA-E, CCR4, and PD-L1 are described, as well pharmaceutical compositions and therapeutic methods useful for the treatment of cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims US Provisional Patent Application No. 62 / 546,300, filed on August 16, 2017, US Provisional Patent Application No. 62 / 546,297, filed on August 16, 2017, and US Provisional Patent Application No., filed on August 30, 2017 62 / 552,152 and U.S. Provisional Patent Application No. 62 / 555,114, filed September 7, 2017, the contents of each of which are hereby incorporated by reference in their entirety for all purposes. [0003] sequence listing [0004] This application contains a Sequence Listing electronically filed in ASCII format and incorporated herein by reference in its entirety. The ASCII copy generated on August 15, 2018 is named DFY-033WO_SL.txt and is 214,413 bytes in size. technical field [0005] The present invention relates to multispecific binding proteins that bind to NKG2D, CD16 and tumor associated antigens selected from the group consisting of EGFR, HLA-E, CCR4 and PD-L1. Background...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K16/28C07K16/30A61K47/68
CPCC07K14/705C07K16/28C07K16/30A61K47/68C07K14/70532C07K14/70539C07K14/7056C07K16/2851C07K16/2863C07K2317/21C07K2317/31C07K2317/33C07K2317/35C07K2317/55C07K2317/622C07K2317/70C07K2317/73C07K2317/732C07K2317/92C07K2317/94C07K2319/03C07K2319/33C07K16/2833C07K16/2866A61P35/00C07K2317/522C07K2317/53C07K16/283C07K16/2827C07K2317/524
Inventor 格雷戈里·P·常安·F·张杜金燕阿斯亚·格林贝格威廉·哈尼达鲁夫·卡姆·赛提尼古拉·瓦格曼布拉德利·M·伦德比昂卡·普林茨
Owner DRAGONFLY THERAPEUTICS INC
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