Methods for humanizing rabbit monoclonal antibodies

A monoclonal antibody and humanized technology, applied in chemical instruments and methods, anti-animal/human immunoglobulin, organic chemistry, etc., can solve difficult model analysis and cannot easily use rabbit monoclonal antibody human source issues such as

Inactive Publication Date: 2006-09-27
EPITOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Precise model analysis is difficult due to previous lack of understanding of rabbit CDR structure
[0012] Therefore, current methods for humanizing rodent antibodies cannot be readily applied to humanize rabbit mAbs due to the unique nature of rabbit mAbs

Method used

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  • Methods for humanizing rabbit monoclonal antibodies
  • Methods for humanizing rabbit monoclonal antibodies
  • Methods for humanizing rabbit monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Rabbit monoclonal antibody

[0145] figure 2 A procedure is described for the sequence alignment of the variable heavy and kappa chains cloned from various rabbit monoclonal antibodies developed by Epitomics. It shows that the structural features of the rabbit chain are distinct from those of human and murine antibodies. Most of the VK chains and half of the VH chains lack one residue at the N-terminus (compared to other rabbit antibody sequences relative to human antibody sequences). Most heavy chains also lack one or two residues in the D-E loop region. All isolated kappa chains have a cysteine ​​residue at position 80. The CDR3 sequences of many kappa chains are longer than the corresponding sequences of previously known human or murine antibodies. A few kappa chains have an extra pair of cysteine ​​residues within their third complementarity determining region. An extra pair of cysteines is also present in some VH regions. Finally, the fact...

Embodiment 2

[0147] Humanization of Rabbit Monoclonal Antibody B1

[0148] The variable region κ chain and heavy chain of rabbit anti-integrin β-6 monoclonal antibody B1 were cloned according to the following polymerase chain reaction (PCR) procedure. Several independent PCR products were sequenced, and polymerase chain reaction (PCR) with one set of primers was generally sufficient.

[0149] Preparation of a suspension of hybridoma cells

[0150] - 1 ml of grown B1 cells, centrifuged at 1100 rpm for 5 min

[0151] - Rinse with 1×PBS

[0152] - Check the number of cells and adjust to 400,000 cells per ml

[0153] Preparation of RNA

[0154] -Add 1ul cells to 9ul buffer A and place on ice

[0155] -Add 5ul of cold buffer B

[0156] -Heat to 65°C for 1 minute

[0157] -Gradual cooling on the thermal cycler

[0158] 55°C 45°C 35°C 23°C Freezing point

[0159] 30 seconds 30 seconds 30 seconds 2 minutes

[0160] - Add cooling buffer C 5ul per tube

[0161] -Incubate ...

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Abstract

The invention provides a method for humanizing a rabbit monoclonal antibody. In general, the method involves comparing an amino acid sequences of a parent rabbit antibody to the amino acid sequences of a similar human antibody, and altering the amino acid sequence of the parent rabbit antibody such its framework regions are more similar in sequence to the equivalent framework regions of the similar human antibody. In many embodiments, amino acids in the parent rabbit antibody that are not CDR contact residues, interchain contact residues, or buried residues, are not modified. The invention further provides nucleic acids encoding the subject antibodies, as well as vectors and host cells comprising the nucleic acids and methods for producing a subject antibody. The subject antibodies, nucleic acid compositions and kits find use in a variety of applications, including diagnostics and therapeutic treatment and research of conditions and diseases.

Description

technical field [0001] The field of the invention is antibodies, particularly methods for humanizing rabbit monoclonal antibodies. Background technique [0002] Because monoclonal antibodies can target virtually any molecule with some specificity, monoclonal antibodies and their conjugates and derivatives will likely become a major therapeutic agent in the future. Although this possibility has long been recognized, the first attempts to realize it have failed, mainly because even the monoclonal antibodies used in the therapy are injected only once in a single low dose (Dillman, Cancer Biother 19949: 17-28), and monoclonal antibodies can also generate a strong immune response in patients (Schroff, 1985 Cancer Res 45: 879-85, Shawler. J Immunol 1985 135: 1530-5). Scientists predict that human antibodies will not produce this adverse immune response, but current hybridoma technology cannot produce human monoclonal antibodies. Since then, some other alternative techniques for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N5/12C12N15/85C12N15/86C12N1/20C12N15/63C07K16/00C07K16/28C07K16/46
CPCC07K16/2839C07K2317/567C07H21/04C07K2317/565C07K16/461
Inventor F·J·R·D·柯特
Owner EPITOMICS INC
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