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Methods of modifying antibodies, and modified antibodies with improved functional properties

一种氨基酸、编号系统的技术，应用在修饰抗体和具有改善的功能性质的修饰抗体领域，能够解决蛋白质弱稳定性和溶解性、高聚集、增加免疫原性危险等问题

Active Publication Date: 2010-09-29
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conversion of full-length antibodies to scFv usually results in poor protein stability and solubility, low yield and high aggregation tendency, which increases the risk of immunogenicity

Method used

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  • Methods of modifying antibodies, and modified antibodies with improved functional properties
  • Methods of modifying antibodies, and modified antibodies with improved functional properties
  • Methods of modifying antibodies, and modified antibodies with improved functional properties

Examples

Experimental program
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Embodiment approach

[0567] It is to be understood that the present invention also includes any of the methods, references and / or compositions shown in Appendices (A-C) of U.S. Provisional Patent Application Serial No. 60 / 905,365 and Appendices (A-I) of U.S. Provisional Patent Application Serial No. 60 / 937,112 , including but not limited to, identified databases, bioinformatics, in silico data manipulation and interpretation methods, functional assays, preferred sequences, preferred residue positions / changes, framework identification and selection, framework changes, CDR alignment and integration and preferred changes / mutations.

[0568]Additional information on such methods and compositions can be found in U.S.S.N.s 60 / 819,378; and U.S.S.N.s entitled "scFv Antibodies Which Pass Epithelial And / Or Endothelial Layers," filed July 2006 and February 6, 2007, respectively. 60 / 899,907 and PCT Publication No. WO 2008 / 006235; WO06131013A2, filed June 6, 2006, entitled "Stable And Soluble Antibodies Inhibi...

Embodiment 1

[0572] Example 1: Antibody position numbering system

[0573] In this example, a conversion table for two different numbering systems used to identify amino acid residue positions in antibody heavy and light chain variable regions is provided. The Kabat numbering system is further described in Kabat et al. (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The AHo numbering system is further described by Honegger, A. and Plückthun, A. (2001) J. Mol. Biol. 309 : 657-670).

[0574] Heavy chain variable region number

[0575] Table 1: Conversion table for residue positions in the heavy chain variable domain

[0576] Kabat AHo

Kabat AHo

Kabat AHo

1 1

twenty two

3 3

4 4

5 5

6 6

7 7

· 8

8 9

9 10

10 11

11 12

12 13

13 14

14 15

15 16

16 17

17 18

18 1...

Embodiment 2

[0586] Example 2 : Sequence-based analysis of scFv sequences

[0587] In this example, sequence-based analysis of scFv sequences is described in detail. A flowchart outlining the analysis process is shown in figure 1 middle.

[0588] Collection and Alignment of Human Immunoglobulin Sequences

[0589] Human mature antibody and germline variable domain sequences were collected from various databases and entered into custom databases as single letter coded amino acid sequences. Antibody sequences were aligned using the EXCEL tool of the Needleman-Wunsch sequence alignment algorithm (Needleman et al., J Mol Biol., 48(3):443-53 (1970)). The database is then subdivided into 4 different arrays (according to the original data source) as follows to facilitate subsequent analysis and comparison:

[0590] VBase : human germline sequence

[0591] IMGT : human germline sequence

[0592] KDB Database: Mature Antibodies

[0593] QC Database: ESBATech internal database con...

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Abstract

The invention provides methods of using sequence based analysis and rational strategies to modify and improve the structural and biophysical properties of immunobinders, and in particular of single chain antibodies (scFvs), including such properties as stability, solubility, and / or antigen binding affinity. The invention provides methods of engineering immunobinders, and in particular scFvs, by performing one or more substitutions at amino acid positions identified by analysis of a database of selected, stable scFv sequences, wherein preferred amino acid residues for substitution have been identified. The invention also provides immunobinders prepared according to the engineering methods of the invention. The invention also provides preferred scFv framework scaffolds, into which CDR sequences can be inserted, as well as scFv antibodies made using these preferred framework scaffolds.

Description

[0001] This application claims priority to US Provisional Application Serial No. 60 / 937,112, filed June 25, 2007, and entitled "Sequence Based Engineering and Optimization of Single Chain Antibodies." This application also claims priority to U.S. Provisional Application Serial No. 61 / 069,056, filed March 12, 2008, and entitled "Methods of Modifying Antibodies, and Modified Antibodies with Improved Functional Properties." Background of the invention [0002] Antibodies have proven to be very effective and successful therapeutic agents in the treatment of cancer, autoimmune diseases and other conditions. Although full-length antibodies have generally been used clinically, there are many advantages that the use of antibody fragments can provide, such as increased tissue penetration, the absence of Fc effector functions combined with the ability to increase other effector functions, and Less potential for systemic side effects due to short systemic in vivo half-life. The pharmaco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395C12N15/13C07K16/00
CPCC07K16/241C07K2317/567C07K2317/622A61P35/00A61P37/00A61P37/02C07K16/00A61K39/395C12N15/09C12N15/11
Inventor D·尤里奇L·博拉斯
Owner NOVARTIS AG
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