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Novel multivalent immunoglobulins

Inactive Publication Date: 2010-02-25
F STAR BIOTECHNOLOGISCHE FORSCHUNGS & ENTWICKLUNGS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005]These features have profound functional consequences. The variable regions of both the heavy and light chains (VH) and (VL) lie at the “tips” of the Y, where they are positioned to react with antigen. This tip of the molecule is the side on which the N-terminus of the amino acid sequence is located. The stem of the Y projects in a way to efficiently mediate effector functions such as the activation of complement and interaction with Fc receptors, or ADCC and ADCP. Its CH2 and CH3 domains bulge to facilitate interaction with effector proteins. The C-terminus of the amino acid sequence is located on the opposite side of the tip, which can be termed “bottom” of the Y.
[0018]To enhance the potency of mAbs that exert their effect through the clustering of target molecules, various multivalent Ab formats have been designed. Covalently linked full-length IgGs that form tetravalent Abs and naturally occurring IgM and IgG Abs mimicking polymeric IgM and IgA via the use of their secretory tailpiece have been devised. Another tetravalent format was designed by adding Fab at the C terminus of each H chain of a full-length IgG.
[0021]It is the object of the present invention to provide a modular system which allows designing a cell targeting multivalent immunoglobulin according to the respective need, to solve prior art problems.

Problems solved by technology

With scfv and similar formats it is difficult to control formation of the exact multimerization degree, i.e., diners, trimers, tetramers, and larger complexes may form in varying ratios depending on the basic construct and expression method.
Any of the known formats to produce multivalent immunoglobulins have certain disadvantages, be it immunogenicity, in vivo-half life or production issues.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of the CH3 Library and Phage Surface Display

[0211]The crystal structure of an IgG1 Fc fragment, which is published in the Brookhaven Database as entry 1OQO.pdb was used to aid in the design of the mutated CH3 domain.

[0212]The sequence which was used as the basis for construction of the CH3 library is given in SEQ ID No. 1. In this sequence, the first amino acid corresponds to Proline 343 of chain A of Brookhaven database entry 1oqo.pdb. The last residue contained in 1oqo.pdb is Serine 102 of SEQ ID No. 1. After detailed analysis of the structure of 1oqo.pdb and by visual inspection of the residues forming the loops which connect the beta strands, it was decided to randomize residues 17, 18 and 19, which are part of the loop connecting beta strand A-B as well as 71, 72, 73, 76, and 77, which are part of the loop connecting beta strand E-F of SEQ ID No. 1. The engineered gene was produced by a series of PCR reactions followed by ligation of the resulting PCR products. To ...

example 2

Construction of the CH3+3 Library

[0214]This library was constructed and cloned in the same way as the CH3 library. The amino acid sequence of the construct is given in SEQ ID No. 10, the corresponding nucleotide sequence in SEQ ID No. 11, and the primers used for construction were SEQ ID No. 4-7, SEQ ID No. 9 and SEQ ID No. 12.

example 3

Construction of the CH3+5 Library

[0215]This library was constructed and cloned in the same way as the CH3 library. The amino acid sequence of the construct is given in SEQ ID No. 13, the corresponding nucleotide sequence in SEQ ID No. 14, and the primers used for construction were SEQ ID No. 4-7, SEQ ID No. 9 and SEQ ID No. 15.

Example 4

Construction of a CH1 Library

[0216]In the human IgG1 CH1 library, Ser93, Ser94, Ser95, Gly98, Thr99 and Gln100 were randomized and 3 random residues additionally inserted using site directed random mutagenesis. Leu96 was not mutated. In another human IgG1 CH1 library, Pro92, Ser93, Ser94, Ser95 Leu96 Thr101, Gly98, Thr99 and Gln100 were randomized and 3 random residues additionally inserted using site directed random mutagenesis. The genes coding for the libraries were cloned in frame with the pelB leader at the N-terminus and in frame with protein III from fd phage at the C-terminus using the restriction sites NcoI and NotI of the phagemid vector pHE...

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Abstract

The present invention provides a multivalent immunoglobulin or part thereof binding specifically to at least two cell surface molecules of a single cell with at least one modification in at least one structural loop region of said immunoglobulin determining binding to an epitope of said cell surface molecules wherein the unmodified immunoglobulin does not significantly bind to said epitope, its use and methods for producing it.

Description

[0001]The present invention provides a multivalent immunoglobulin or part thereof binding specifically to at least two cell surface molecules of a single cell, with at least one modification in at least one structural loop region of said immunoglobulin determining binding to an epitope of said cell surface molecules wherein the unmodified immunoglobulin does not significantly bind to said epitope.[0002]Monoclonal antibodies have found use in many therapeutic, diagnostic and analytical applications.[0003]The basic antibody structure will be explained here using as example an intact IgG1 immunoglobulin. Two identical heavy (H) and two identical light (L) chains combine to form the Y-shaped antibody molecule. The heavy chains each have four domains. The amino terminal variable domains (VH) are at the tips of the Y. These are followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3, at the base of the Y's stem. A short stretch, the switch, connects the heavy chain vari...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07H21/04G01N33/53
CPCC07K16/005C07K16/28C07K2317/52C07K16/468C07K16/2887A61P37/04
Inventor RUKER, FLORIANHIMMLER, GOTTFRIEDWOZNIAK-KNOPP, GORDANA
Owner F STAR BIOTECHNOLOGISCHE FORSCHUNGS & ENTWICKLUNGS GMBH