Antibodies for the detection of integrin complexes in FFPE material

• US 8,420,348 B2
• Filed: 07/15/2010
• Issued: 04/16/2013
• Est. Priority Date: 08/19/2009
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Goodman, Simon, Wilm, Claudia, Mitjans, Francesc
• Primary Examiner(s): Shafer, Shulamith H
• Application Number:
  US13/390,791
• Publication Number:
  US 20120171699A1
• Time in Patent Office: 1,006 Days
• Field of Search: None
• US Class Current: 435/40.5
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2839 against the integrin superfamily
• CPC Class Codes: G01N 2333/70546 Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention relates to antibodies that are capable to bind the extracellular domain of integrin. Another object of the invention concerns the use of said antibodies for detecting integrins in archival formalin fixed paraffin embedded (FFPE) tissue. The invention also relates to methods for preparing monoclonal rabbit antibodies, wherein the immunogen is an insect expression culture-derived recombinant extracellular integrin domain, and another method for screening anti-integrin antibodies that discriminate between closest integrin homologues and that are especially suited for immunohistochemistry in FFPE material.

Claims

1. A monoclonal rabbit antibody, or antigen-binding fragment thereof, against integrin with insect-derived glycosylation pattern and/or integrin with another eukaryotic glycosylation pattern, each comprising at least a light chain variable region (VL) and a heavy chain variable region (VH), wherein the antibody has antigen binding specificity for an exposed epitope of an extracellular integrin domain or extracellular integrin chain domain, and wherein the antibody is able to bind to intact heterodimers of integrin in formalin fixed paraffin embedded (FFPE) material and on viable cells with the substantially same specificity wherein said antibody is selected from the group consisting of:

an antibody that binds to the extracellular domain of integrin α

vβ

3 and V_L comprises the amino acid sequence of SEQ ID NO;

95 (V_L-α

vβ

3) and V_H comprises the amino acid sequence of SEQ ID NO;

96 (V_H-α

vβ

3);

an antibody that binds to the extracellular domain of integrin α

vβ

5 and V_L comprises the amino acid sequence of SEQ ID NO;

15 (V_L-α

vβ

5) and V_H comprises the amino acid sequence of SEQ ID NO;

16 (V_H-α

vβ

5);

an antibody that binds to the extracellular domain of integrin α

vβ

6 and V_L comprises the amino acid sequence of SEQ ID NO;

135 (V_L-α

vβ

6) and V_H comprises the amino acid sequence of SEQ ID NO;

136 (V_H-α

vβ

6);

an antibody that binds to the extracellular domain of integrin α

vβ

8 and V_L comprises the amino acid sequence of SEQ ID NO;

175 (V_L-α

vβ

8) and V_H comprises the amino acid sequence of SEQ ID NO;

176 (V_H-α

vβ

8); and

an antibody that binds to the extracellular domain of integrin chain α

v and V_L comprises the amino acid sequence of SEQ ID NO;

215 (V_L-α

v) and V_H comprises the amino acid sequence of SEQ ID NO;

216 (V_H-α

v).

2. A polynucleotide which encodes the antibody, or antigen-binding fragment thereof, of claim 1.

3. A method for obtaining the monoclonal antibody according to claim 1, comprising

immunizing a rabbit with an immunogen which comprises an extracellular integrin domain with insect-derived glycosylation pattern, optionally coupled as delta-trans membrane form;

obtaining a polyclonal antiserum comprising polyclonal antibodies from said rabbit; and

preparing the monoclonal antibodies.

5. A method for the detection of integrins in formalin fixed paraffin embedded (FFPE) material, comprising contacting said FFPE material with the antibody or an antigen binding fragment thereof of claim 1.

4. A method for manufacturing a recombinant monoclonal antibody comprising a light chain variable region (V_L) and a heavy chain variable region (V_H) with the steps of:

(a) introducing at least one vector comprising antibody-encoding nucleic acid sequences of

(i) SEQ ID NO;

115 (V_L-α

vβ

3) and SEQ ID NO;

116 (V_H-α

vβ

3),

(ii) SEQ ID NO;

35 (V_L-α

vβ

5) and SEQ ID NO;

36 (V_H-α

vβ

5),

(iii) SEQ ID NO;

155 (V_L-α

vβ

6) and SEQ ID NO;

156 (V_H-α

vβ

6),

(iv) SEQ ID NO;

195 (V_L-α

vβ

8) and SEQ ID NO;

196 (V_H-α

vβ

8), or

(v) SEQ ID NO;

235 (V_L-α

v) and SEQ ID NO;

236 (V_H-α

v) into a host cell,

(b) cultivating the host cell in a culture medium, thereby expressing the encoded antibody, and

(c) purifying the expressed antibody.