Modified binding molecules comprising connecting peptides

a technology of connecting peptides and binding molecules, which is applied in the direction of antibody mimetics/scaffolds, fusion polypeptides, dna/rna fragmentation, etc., can solve the problems of increasing the effort required for downstream processing, affecting the overall yield, and difficult separation of form a and form b, so as to enhance the formation of form a

Inactive Publication Date: 2005-07-28
BIOGEN MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] In one embodiment, the binding molecules of the invention are tetravalent. The connecting peptides of the invention can be included in any dimeric molecule that tends to form both Form A and Form B, e.g., antibody molecules, domain deleted antibody molecules (e.g., lacking all or part of a CH2 domain), minibodies, diabodies, fusion proteins, etc. In a preferred embodiment, the formation of Form A is enhanced.

Problems solved by technology

Form A and Form B are extremely difficult to separate even after MAb purification, since they are composed of identical amino acids and, therefore, have identical molecular weight and similar physical and chemical properties.
Current manufacturing processes discard at least 50% of the total antibody produced, having a negative impact on overall yield.
Moreover, the presence of the two isoforms increases efforts required for downstream processing.

Method used

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  • Modified binding molecules comprising connecting peptides
  • Modified binding molecules comprising connecting peptides
  • Modified binding molecules comprising connecting peptides

Examples

Experimental program
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Effect test

example 1

Identification of A and B Isoforms

[0403] Solutions of antibody molecules comprise two different isoforms. One form, Form A comprises heavy chain molecules that are linked via at least one disulfide linkage. The other form, Form B, comprises heavy chain molecules that are not linked via at least one disulfide linkage. Form B does not appear or appears at a very low frequency in with intact gamma 1 MAbs, such as Rituxan®. However with domain deleted (dd) constructs having a similar hinge, the frequency of Form B is much higher. These forms can be distinguished using denaturing, non-reducing SDS page. In domain deleted antibody preparations, Form A appears as a 120 kDa dimer while form B appears as a 60 kDa monomer (FIG. 1).

example 2

Identification of Hinge Region Heterogeneity in CH2 Domain Deleted MAb Fragments

[0404] Hinge domains can be subdivided into three distinct regions: upper, middle, and lower hinge regions (Roux et al. J. Immunol. 1998 161:4083). Polypeptide sequences encompassing these regions for IgG1 and IgG3 hinges are shown in Table 1. The IgG3 hinge middle region contains, in addition to the two conserved cysteine residues, a 15 amino acid motif that repeats three times. Amino acid sequences from these regions were used to design synthetic IgG1 / IgG3 connecting peptides. These consisted of IgG1 upper hinge residues corresponding to positions 226 through 238, an IgG1 middle hinge corresponding to positions 239 through 241, and a single IgG3 middle hinge repeat motif corresponding to positions 241 EE through 242 combined with either an added proline at position 243 or an added proline, alanine, proline at positions 243, 244, and 245, respectively (Kabat numbering system), followed by a flexible Gl...

example 3

Construction of Connecting Polypeptides and Preferential Synthesis of Isoforms

[0406] Nucleic acid sequences encoding the hinge region connecting peptides shown in Table 2 were introduced into CH2 domain deleted huCC49 gene sequences using the Splicing by Overlap Extension (SOE) method (Horton, R. M. 1993 Methods in Molecular Biology, Vol 15: PCR Protocols: Current Methods and applications. Ed. B. A. White). Correct modifications to the hinge region were confirmed by DNA sequence analysis. Plasmid DNA was used to transform CHO DG44 cells for stable production of antibody protein.

[0407] CH2 domain deleted huCC49 antibodies containing the eight designed synthetic connecting peptides indicated in Table 2 were constructed and antibody produced in CHO DG44 cells. Supernatants were collected from isolated cell lines and concentration of antibody in the culture supernatants determined by immunoassay. Supernatants containing antibody ranging from 0 to 30 ng of total antibody protein from e...

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Abstract

The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. Ser. No. 60 / 483877, titled “Purification and Preferential Synthesis of Polypeptides,” filed on Jun. 27, 2003 and to U.S. Ser. No. 60 / 508,810, titled “Purification and Preferential Synthesis of Antigen Binding Polypeptides,” filed Oct. 3, 2003. This application also claims priority to U.S. Ser. No. 60 / 515,351, titled “Modified Antibody Molecules Comprising Connecting Peptides,” Oct. 28, 2003 and to U.S. Ser. No. 60 / 516,030, titled “Modified Antibody Molecules Comprising Connecting Peptides,” filed Oct. 30, 2003. This application is also related to U.S. Ser. No. XX / XXXXXX, titled “Purification and preferential synthesis of Binding Polypeptides” filed on Jun. 28, 2004. The contents of these applications are incorporated in their entirety by this reference.BACKGROUND OF THE INVENTION [0002] Antibodies are dimeric molecules; each monomer making up the dimer comprises one light and one heavy chain. Solutions of antibody ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00C07K16/00C07K16/18C07K16/28C07K16/30C12N15/62
CPCA61K2039/505C07K16/00C07K16/18C07K16/2851C07K16/2896C07K2317/52C07K2317/24C07K2317/53C07K2317/622C07K2317/624C07K2319/00C07K16/30A61P1/04A61P1/16A61P3/00A61P3/04A61P3/10A61P5/00A61P5/14A61P7/00A61P7/02A61P7/04A61P7/06A61P9/00A61P9/10A61P9/14A61P11/00A61P11/02A61P11/06A61P13/02A61P13/12A61P15/00A61P15/08A61P17/00A61P17/02A61P17/04A61P17/06A61P17/14A61P19/02A61P21/00A61P21/04A61P25/00A61P25/08A61P27/02A61P29/00A61P31/04A61P31/10A61P31/12A61P35/00A61P35/02A61P35/04A61P37/00A61P37/02A61P37/06A61P37/08A61P43/00
Inventor GLASER, SCOTTREFF, MITCHELLYANG, TZUNG-HORNGWU, XIUFENGCHINN, PAUL
Owner BIOGEN MA INC
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