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Binding domain-immunoglobulin fusion proteins

A technology of immunoglobulin, human immunoglobulin, application in binding domain-immunoglobulin fusion protein, treatment of malignant conditions and B cell diseases, single chain Fv-immunoglobulin fusion protein, molecularly engineered binding domain-immunoglobulin In the field of globulin fusion proteins, it can solve problems such as short half-life, loss of anti-tumor activity, and growth arrest

Inactive Publication Date: 2004-03-31
阿普泰沃研发有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Shedding or internalization of target antigens following antigen binding by immunoglobulins can allow tumor cells to escape destruction, limiting the effectiveness of serotherapy
Fourth, binding of immunoglobulins to target antigens conveying an activation signal can result in a functional response in tumor cells leading to growth arrest and apoptosis
Rituximab TM The large size of the molecule prevents optimal diffusion of the molecule into lymphoid tissues containing malignant B cells, thereby limiting these antitumor activities
Protease cleavage to Fab or F(ab') as previously discussed 2 Fragments make them smaller and allow easier penetration into lymphoid tissue, but lose effector functions that are important for antitumor activity
Although CD20mAb fragments may be more effective than intact antibodies in delivering radioisotopes, there is still a need to construct effector function retaining Fc parts, but smaller CD20mAb derivatives to facilitate better tumor penetration and result in shorter half-lives

Method used

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  • Binding domain-immunoglobulin fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0165] Cloning of 2H7 variable region and sequencing of 2H7SCFV-IG

[0166] This example illustrates the cloning of cDNA molecules encoding the heavy and light chain variable regions of monoclonal antibody 2H7. This example also demonstrates the construction, sequencing and expression of 2H7 scFv-Ig.

[0167] Hybridoma cells expressing the 2H7 monoclonal antibody specifically binding to CD20 were provided by Ed Clark, University of Washington, Seattle, WA. Prior to harvest, hybridoma cells were maintained in logarithmic phase in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with glutamine, pyruvate, DMEM non-essential amino acids, and penicillin-streptomycin. Resuspend the cells from the medium by centrifugation with 2 x 10 7 cells make RNA. RNA was isolated from 2H7-producing hybridoma cells using the Pharmingen (San Diego, CA) Total RNA Isolation Kit (Cat# 45520K) according to the manufacturer's instructions accompanying the kit. cDNA was prepared b...

Embodiment 2

[0173] Expression of 2H7 SCFV-IG in Stable CHO Cell Lines

[0174] This example demonstrates the expression of 2H7 scFv-Ig in eukaryotic cells and the identification of expressed 2H7 scFv-Ig by SDS-PAGE and functional assays including ADCC and complement fixation.

[0175] 2H7scFv-Ig HindIII-XbaI (about 1.6 kb) with the correct sequence was inserted into mammalian expression vector pD18, and DNA of positive clones was amplified with QIAGEN therapeutic preparation kit (QIAGEN, Valencia, CA). Recombinant plasmid DNA (100 μg) was then linearized in non-essential regions by digestion with AscI, purified by phenol extraction, and resuspended in tissue culture medium Excell302 (Catalog No. 14312-79P, JRH Biosciences, Lenexa, KS). The cells used for transfection, CHO DG44 cells, were in logarithmic growth, and 10 cells were harvested for each transfection reaction. 7 cells. Linearized DNA was added to CHO in a total volume of 0.8 ml for electroporation.

[0176] Stable production ...

Embodiment 3

[0184] Effects of simultaneous ligation of CD20 and CD40 on normal B cell growth, as well as on CD95 expression and induction of apoptosis

[0185] This example illustrates the effect of cross-linking of cell surface expressed CD20 and CD40 on cell proliferation.

[0186] Dense resting B cells were isolated from human tonsils by a Percoll step gradient and T cells were depleted by E-rosetting. Through the last 12 hours of the 4-day experiment 3 [H]-thymidine uptake measured in dense tonsil B cells. Proliferation was measured in quadruplicate cultures, mean and standard deviation are indicated. Mouse anti-human CD20 mAb 1F5 (anti-CD20) alone or cross-linked with anti-mouse kappa mAb 187.1 (anti-CD20XL) was used. CD40 activation was accomplished using soluble human CD154 fused to murine CD8 (CD154) (Hollenbaugh et al., EMBO J 11:4212-21 (1992)), and CD40 crosslinking was accomplished using anti-mouse CD8 mAb 53-6 (CD154XL). This procedure allows simultaneous cross-linking of...

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Abstract

The invention relates to novel binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a hinge region polypeptide having either zero or one cysteine residue, and immunoglobulin CH2 and CH3 domains, and that are capable of ADCC and / or CDC while occurring predominantly as monomeric polypeptides. The fusion proteins can be recombinantly produced at high expression levels. Also provided are related compositions and methods, including immunotherapeutic applications.

Description

Background of the invention [0001] The present invention generally relates to recombinant binding proteins with immunological activity, in particular to molecularly engineered binding domain-immunoglobulin fusion proteins, including single-chain Fv-immunoglobulin fusion proteins. The invention also relates to compositions and methods for treating malignant conditions and B cell diseases, including diseases characterized by autoantibody production. [0002] Immunoglobulin molecules consist of two identical light chains and two identical heavy chains linked by interchain disulfide bonds into a macromolecular complex. Intrachain disulfide bonds link different regions of the same polypeptide chain, which results in the formation of loops which, with adjacent amino acids, constitute immunoglobulin domains. Each light chain and each heavy chain has a single variable region that exhibits wide variation in amino acid composition between antibodies. light chain variable region V L a...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61K38/16A61K39/00A61P3/10A61P5/14A61P21/04C12N15/09A61P25/00A61P29/00A61P35/00A61P37/06C07K16/28C07K16/30C07K16/42C07K19/00C12N5/10C12N15/62C12P21/02
CPCC07K16/2803C07K2316/52C07K2317/622C07K2319/00C07K16/2896C07K2317/52C07K2317/734C07K2317/732A61K2039/505C07K16/30A61P1/04A61P17/00A61P17/06A61P19/02A61P19/06A61P21/04A61P25/00A61P29/00A61P35/00A61P37/00A61P37/02A61P37/06A61P43/00A61P5/14A61P7/00A61P3/10C07K19/00
Inventor J·A·勒贝特尔M·海登-勒贝特尔
Owner 阿普泰沃研发有限责任公司
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