Novel angiopoietin 2, VEGF dual antagonists

An antagonist, antibody technology, applied in
[0010] Contains areas of priority with SEQ ID NO: 4 to 436

Pending Publication Date: 2021-01-22
ASKGENE PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This may require a concentration of 120mg/ml,

Method used

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  • Novel angiopoietin 2, VEGF dual antagonists
  • Novel angiopoietin 2, VEGF dual antagonists
  • Novel angiopoietin 2, VEGF dual antagonists

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 - Production of chimeric molecules comprising VEGF antibody and Ang-2 binding peptide in HEK293 cells.

[0099] HEK-293 cells express chimeric molecules named AMD A, B, C, D and E by transient expression (see Table 2). Briefly, DNAs (SEQ ID NOs: 58, 59, 60 and 63) comprising fusion proteins of VEGF antibody light chains with or without Ang2-binding peptides and fusions comprising VEGF antibody heavy chains with Ang2-binding peptides were synthesized protein DNA (SEQ ID NOs: 57, 61 and 62) and cloned into an expression vector. The complete expression construct containing the gene was verified by DNA sequencing. The DNA constructs were transformed into E. coli DH5alfa competent cells (Invitrogen). Single clones were picked and grown in LB liquid medium containing antibiotics (kanamycin, 25 μg / mL). DNA plasmids were extracted using the Qiagen PlasmidMaxi Kit (Qiagen) according to the manufacturer's instructions. Plasmid concentrations were determined by NanoD...

Embodiment 2

[0107] Example 2 - Preparation of Chimeric Molecules Comprising VEGF Trap and Ang2 Binding Peptide in CHO Cells

[0108] A chimeric molecular DNA comprising a VEGF receptor-Fc fusion protein (VEGF trap) and an Ang-2 binding peptide (SEQ ID NO: 64, designated ASKB-E06) was synthesized and cloned into an expression vector. The complete expression construct containing the DNA gene was verified by DNA sequencing. Expression constructs were amplified by transforming E. coli DH10B and culturing cells overnight. DNA for expression constructs was prepared by endotoxin-free mini-plasmid extraction kit (from ) for purification.

[0109] The expression constructs were transfected into GS- / - Chinese hamster ovary cells (CHO) by electroporation and glutamine-free selective medium ( CD CHO fusion growth medium) screened the transfected CHO cells to obtain a cell line stably expressing ASKB-E06. In this way, 32 or more stable cuvettes were established and preferred cuvettes were select...

Embodiment 3

[0111] Example 3 - Molecular Assays to Assess Dual Antagonist Activity of Chimeric Molecules

[0112] Molecular assays (Octet binding affinity, affinity ELISA and blocking ELISA) were developed to assess the direct binding ability of chimeric molecules to ANG-1, Ang-2 and / or VEGF, as well as the ability of chimeric molecules to interact with Ang1:Tie-2 effect, Ang-2:Tie-2 interaction and / or VEGF:VEGF receptor interaction. These in vitro assays are described below:

[0113] Octet Affinity

[0114]Purified recombinant human VEGF protein was purchased from Life-Technologies (Cat. #PHC9391). Human Ang1 or Ang2 proteins were ordered from R&D System. Analysis was performed using Pall ForteBio's Octet Red96. Using an anti-human IgGFc sensor, samples of the chimeric molecules AMD-B, AMD-D, AMD-E or the control antibody bevacizumab were added to kinetic buffer at 3 μg / mL and loaded for 300 s. Ligand ANG1, ANG2 or VEGF samples were correlated for 300 sec using dilution series start...

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Abstract

The present invention relates to fusion molecules and chimeric molecules comprising two components: Ang2 antagonist peptides linked to VEGF binding moieties. Further disclosed are methods of treatingcancer, proliferative retinopathy, neovascular glaucoma, macular edema, wet age-related macular degeneration (wAMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), ordiabetic retinopathy (DR) in a patient using the chimeric molecules.

Description

[0001] cross-referenced application [0002] This application claims US Provisional Application No. 62 / 336,552, filed May 13, 2016, US Provisional Application No. 62 / 459,046, filed February 14, 2017, and US Provisional Application No. 62 / 448,998, filed January 21, 2017 Interests, each of which is hereby incorporated by reference in its entirety. This application also claims priority to US Patent Application US20170327569A1, filed on May 11, 2017, the entire contents of which are incorporated by reference. This application further claims priority to US Patent Application 62 / 655,436, filed April 10, 2018, the entire contents of which are incorporated by reference. Background technique [0003] The present application relates to novel molecules comprising domains that bind to both VEGF and Ang2. [0004] Angiogenesis has been implicated in the pathogenesis of a variety of diseases, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or ag...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/395A61K47/55A61P35/00A61P9/10A61P27/02A61P3/10
CPCC07K16/22C07K2317/24C07K2317/55C07K2317/622C07K2317/76C07K2319/00C07K2319/32A61K2039/505A61P27/02C07K2317/56
Inventor 吕越峰卢建丰
Owner ASKGENE PHARM INC
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