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Fms-like tyrosine kinase 3 ligand (FLT3L)-based chimeric proteins

A tyrosine kinase, chimeric protein technology, applied in the field of chimeric proteins based on FMS-like tyrosine kinase 3 ligand (FLT3L), which can solve problems such as limiting dose levels

Pending Publication Date: 2021-11-12
ORIONIS BIOSCI INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therapeutic use of cytokines is often associated with systemic toxicity and deleterious side effects, thus limiting the dose levels at which these agents can be used

Method used

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  • Fms-like tyrosine kinase 3 ligand (FLT3L)-based chimeric proteins
  • Fms-like tyrosine kinase 3 ligand (FLT3L)-based chimeric proteins
  • Fms-like tyrosine kinase 3 ligand (FLT3L)-based chimeric proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Example 1: AcTaferon in vivo studies of Flt3 targeting

[0200] To assess the in vivo efficacy of Flt3-targeted AcTaferon, the Flt3L_linker_human IFNa2(R149A)_GGS_his9 fusion protein was expressed in HEKT cells and purified by metal affinity chromatography. The purified protein was subsequently evaluated in tumor models in humanized mice. Briefly, neonatal NSG mice (1-2 days old) were sublethally irradiated with 100 cGy followed by intrahepatic delivery of 10 5 CD34+ human stem cells (from HLA-A2 positive umbilical cord blood). At 13 weeks after stem cell transfer, mice were inoculated subcutaneously with 25.10E5 human RL follicular lymphoma cells (ATCC CRL-2261; insensitive to direct antiproliferative effects of IFN). Mice were treated intraperitoneally with 30 μg of human Flt3L protein daily from day 8 to day 18 after tumor inoculation. On day 10 after tumor inoculation, when palpable tumors were visible, daily perilesional injections of buffer or Flt3L-AFN (30 μ...

Embodiment 2

[0205] Example 2: FLT3L-AFN fusion forms dimers

[0206] The Flt3L_linker_human IFNa2(R149A)_GGS_his9 fusion protein of Example 1 was recloned into the pcDNA3.4 vector for expression in CHO cells, resulting in plasmid P-2373. Production was performed in ExpiCHO cells (ThermoFisher) according to the manufacturer's instructions. Seven days after transfection, supernatants were harvested and cells were removed by centrifugation. Proteins were purified on 1 ml HisTrap Excel columns (GE Healthcare) on AKTA pure instruments (GE Healthcare). The eluted protein was desalted by Sephadex G25 (5 ml column) into PBS-H 8.0 buffer. Finally, use size exclusion chromatography on a Superdex 75Increase 10 / 300 column (GE Healthcare) on AKTA in 10 mM NH containing 123.5 mM NaCl 4 - Samples were further analyzed in acetate pH 5.0 buffer. SEC profile and subsequent analysis of peak fractions by SDS-PAGE showed that the protein was present in SEC ( figure 2 ) appears as a protein of about 15...

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Abstract

FMS-like tyrosine kinase 3L (FLT3L) fused to human cytokines, which find use in, e.g., cancer treatments, is described. Accordingly, in some aspects, the present invention relates to a chimeric protein comprising a targeting moiety which comprises a single copy of FMS-like tyrosine kinase 3 ligand (FLT3L), or a portion thereof. In various embodiments, the targeting moiety functionally modulates the antigen or receptor of interest. In some embodiments, the targeting moiety binds but does not functionally modulate the antigen or receptor of interest.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 825,579, filed March 28, 2019, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] FMS-like tyrosine kinase 3 ligand (FLT3L) fused to signaling agents such as but not limited to human IFNα2, IFNβ and IL-1β is described, which is useful eg in cancer therapy. [0004] sequence listing [0005] This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is hereby incorporated by reference in its entirety. The ASCII copy, created on March 23, 2020, is named ORN-062PC_A_Sequence_Listing_ST25.txt and is 28,672 bytes in size. Background technique [0006] FMS-like tyrosine kinase 3 (FLT3) is expressed on the surface of many hematopoietic progenitor cells. Signaling by FLT3 is important for the normal development of hematopoietic stem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00C07K19/00C12N15/12C12N15/62
CPCA61K38/00C07K2319/00C07K14/52C07K14/56C07K2319/01C07K2319/21C07K14/4747C07K14/545C07K14/565C07K2319/02
Inventor N·克雷J·塔威尼尔A·范帕里斯
Owner ORIONIS BIOSCI INC
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