Constrained conditionally activated binding proteins

A constrained, fusion protein technology

Pending Publication Date: 2022-04-22
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many antibodies (including scFv and other constructs) exhibit "on target / off tumor" effects, where the molecule is active on non-tumor cells, causing side effects, some of which may is toxic

Method used

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  • Constrained conditionally activated binding proteins
  • Constrained conditionally activated binding proteins
  • Constrained conditionally activated binding proteins

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0225] I. fusion protein of the present invention

[0226] Fusion proteins of the invention have many different components (generally referred to herein as domains) linked together in a variety of ways. Some of the domains are binding domains that each bind to a target antigen (eg, TTA or CD3). Because they bind to more than one antigen, they are referred to herein as "multispecific"; for example, the prodrug constructs of the invention can bind to TTA and CD3, and are therefore "bispecific". Proteins can also have higher specificity; for example, if the first αTTA binds to EGFR, the second αTTA binds to EpCAM, and an anti-CD3 binding domain is present, this would be a "trispecific" molecule. Similarly, adding an anti-HSA binding domain to this construct would be "tetraspecific", as in Figure 3B shown in .

[0227] As will be appreciated by those skilled in the art, proteins of the invention may have different valences and be multispecific. That is, proteins of the invent...

Embodiment 1

[0438] A. Example 1: Construction and purification of Pro constructs

[0439] transfection

[0440] Each protein (eg, single proteins of formats 1, 2 and 4) or paired constructs (format 3) was expressed from individual expression vectors (pcDNA3.4 derivatives). Equal amounts of plasmid DNA encoding a pair of half-Cobra or single-stranded constructs were mixed and transfected into Expi293 cells following the manufacturer's transfection protocol. Five days after transfection, conditioned medium was harvested by centrifugation (6000rpm x 25') and filtration (0.2uM filter). Protein expression was confirmed by SDS-PAGE. The construct was purified and the final buffer composition was: 25 mM citrate, 75 mM arginine, 75 mM NaCl, 4% sucrose, pH 7. The final formulation was stored at -80°C.

[0441] Activation of MMP9

[0442] Recombinant human (rh)MMP9 was activated according to the following protocol. Recombinant human MMP-9 (R&D #911-MP-010) is 0.44mg / ml (4.7uM). Phenylmer...

Embodiment 2

[0450] B. Example 2: T cell-dependent cell cytotoxicity (TDCC) assay

[0451] Firefly luciferase-transduced HT-29 cells were grown to approximately 80% confluency and detached with Versene (0.48 mM EDTA-Ca-Mg in PBS). Cells were centrifuged and resuspended in TDCC medium (5% heat-inactivated FBS in RPMI 1640 with HEPES, GlutaMax, sodium pyruvate, non-essential amino acids and β-mercaptoethanol). Purified human P pan-T cells were thawed, centrifuged and resuspended in TDCC medium.

[0452] A co-culture of HT-29_Luc cells and T cells was added to a 384-well cell culture plate. Serial dilutions of COBRA were then added to the co-culture and incubated at 37 °C for 48 h. Finally, an equal volume of SteadyGlo Luciferase Assay Reagent was added to the plate and incubated for 20 minutes. Plates were read on a Perkin Elmer Envision with an exposure time of 0.1 s / well. Total fluorescence was read and data analyzed on GraphPad Prism 7 or version 8.3.1 (depending on timing).

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Abstract

The present invention relates to a conditionally bispecific redirected activation construct or COBRA administered in the form of an active prodrug. Upon exposure to a tumor protease, the constructs are cleaved and activated such that they are able to bind both tumor target antigens (TTAs) and CD3, thereby recruiting CD3-expressing T cells to the tumor for treatment. In some embodiments, the tumor target antigen is B7H3.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 814,210, filed March 5, 2019; U.S. Provisional Application No. 62 / 814,744, filed March 6, 2019; and U.S. Provisional Application No. 62 / 826,523, the disclosure of which is hereby incorporated by reference in its entirety. [0003] Background of the invention [0004] In various clinical settings, it is often desirable to selectively destroy individual cells or specific cell types. For example, a major goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues as intact as possible. One such approach is by inducing an immune response against the tumor so that immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL), attack and destroy tumor cells. [0005] The use of intact monoclonal antibodies (mAbs), which provide excellent binding specificity and affinity for tumor-associated antige...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P35/00C07K16/28C07K16/30C07K16/18
CPCC07K16/18C07K16/2809C07K16/2863A61P35/00C07K16/30C07K16/2827C07K2317/22C07K2317/31C07K2317/52C07K2317/53C07K2317/56C07K2317/569C07K2317/60C07K2317/626C07K2319/50A61K2039/505C07K16/2806C07K2317/94C07K2319/30
Inventor C·梅R·B·迪布瑞吉M·维诺格拉多沃娃A·潘沙尔
Owner TAKEDA PHARMA CO LTD
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