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Cd20-binding immunotoxins for inducing cellular internalization and methods using same

A cell and toxin technology, applied in the field of CD20 binding protein, can solve the problem of insufficient CD20 internalization efficiency

Active Publication Date: 2015-11-11
MOLECULAR TEMPLATES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, treatments based on the delivery of immunotoxins targeting the CD20 antigen were predicted to be ineffective based on insufficient CD20 internalization efficiency

Method used

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  • Cd20-binding immunotoxins for inducing cellular internalization and methods using same
  • Cd20-binding immunotoxins for inducing cellular internalization and methods using same
  • Cd20-binding immunotoxins for inducing cellular internalization and methods using same

Examples

Experimental program
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Embodiment

[0214] The following examples illustrate certain embodiments of the invention. However, it should be understood that these examples are for illustrative purposes only, and are not intended and should not be construed as fully limiting the state and scope of the present invention. The described embodiments are performed using standard techniques, which are well known and conventional to those skilled in the art, except where otherwise described in detail.

[0215] The following examples show the ability of exemplary CD20 binding proteins to selectively kill cells expressing CD20 on their cell surface. An exemplary CD20 binding protein binds to the extracellular antigen on CD20 expressed by the targeted cell type and enters the targeted cell. The internalized CD20 binding protein provides its Shigella toxin effector region with a route to the cytosol to inactivate ribosomes and subsequently cause apoptotic death of targeted cells. Therefore, after the CD20 binding protein forms a...

Embodiment 2

[0223] Example 2-Determination of the dissociation constant of an exemplary CD20 binding protein (K D )

[0224] The cell binding characteristics of both αCD20scFv::SLT-1ACD20 binding protein version 1 and 2 were determined by fluorescence-based flow cytometry. Each sample contains 0.5x10 6 CD20-expressing cells (Raji(CD20+)) or non-CD20-expressing cells (BC1(CD20-)) and 100 μL of CD20 binding protein in various dilutions in Hyclone 1XPBS (Fisher Scientific, Waltham, MA) + 1% BSA ( Incubate in Calbiochem, SanDiego, CA, USA) (hereinafter referred to as "1XPBS+1%BSA") at 4°C for 1 hr. The highest concentration of CD20 binding protein is selected to cause saturation of the reaction. The cells were washed twice with 1XPBS + 1% BSA. Combine cells with 100μl containing 0.3ug anti-Strep mAb-FITC (#A01736-100, Genscript, Piscataway, NJ, USA) in 1XPBS+1%BSA was incubated at 4°C for 1hr. The cells were washed twice with 1XPBS + 1% BSA, suspended in 200 μl of 1XPBS, and subjected to flo...

Embodiment 3-

[0228] Example 3-Determining the half-maximal inhibitory concentration (IC 50 )

[0229] Use The cell-free in vitro protein translation assay of the QuickCoupled Transcription / Translation kit (L1170 Promega Madison, WI, USA) was used to determine the ribosome inactivation capacity of both αCD20scFv::SLT-1ACD20 binding protein versions 1 and 2. The kit includes Luciferase T7 Control DNA (LuciferaseT7ControlDNA) (L4821PromegaMadison, WI, USA) and QuickMasterMix. Prepare the ribosome activity reaction according to the manufacturer's instructions.

[0230] Prepare a series of 10-fold dilutions of the αCD20scFv::SLT-1A version to be tested in a suitable buffer and generate a series of identical TNT reaction mixture components for each dilution. Combine each sample in the dilution series of αCD20scFv::SLT-1A protein with each of the TNT reaction mixture and the luciferase T7 control DNA. The test sample was incubated at 30°C for 1.5 hours. After incubation, luciferase assay reagent ...

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Abstract

The present invention provides cytotoxic proteins comprising immunoglobulin-type binding regions for mediating cell-type specific targeting and Shiga toxin effector regions derived from A Subunits of members of the Shiga toxin family for effectuating cytotoxicity. The cytotoxic proteins have uses for selective killing of specific cell types and as therapeutics for the treatment of a variety of diseases, including cancers, immune disorders, and microbial infections.

Description

Invention field [0001] The present invention relates to a CD20 binding protein that has the ability to bind CD20 antigen and drive the CD20 antigen to be rapidly internalized from a cell surface position to the inside of the cell. These CD20 binding proteins have applications as therapeutic molecules for the treatment of various diseases including cancer and immune disorders. Background of the invention [0002] Immunotoxins are chimeric molecules that combine cell surface binding regions, such as immunoglobulin domains, with toxin regions usually derived from naturally occurring protein toxins, such as those found in bacteria or plants. The effectiveness of immunotoxins mainly depends on the efficiency of their transport from the cell surface to the cytosol, that is, the process that starts with the internalization of the cell (see Pirie et al. (2011), JBiolChem286:4165-72). [0003] CD20 is a member of a polypeptide family called the transmembrane 4A (MS4A) family including at l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K14/25C07K14/005A61K39/00
CPCC07K16/32A61K2039/505C07K16/085C07K16/20C07K16/2863C07K16/2887C07K16/2896C07K16/30C07K2317/569C07K2317/622C07K2317/73C07K2317/92C07K2319/00C07K2319/33A61K38/00C07K2319/55A61P1/04A61P11/06A61P13/02A61P17/00A61P17/06A61P19/02A61P19/08A61P25/00A61P25/28A61P29/00A61P31/04A61P31/18A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00A61P5/14A61P7/00A61P9/00A61P3/10C07K2317/77C07K14/245C07K14/25C07K16/2866C07K16/40C07K2317/56A61K39/395
Inventor 埃里克·波马埃林·威勒特贾森·金杰克·希金森桑吉萨·拉贾戈帕兰
Owner MOLECULAR TEMPLATES
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