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Anti-tenascin-c a2 antibodies and methods of use

A tenascin and antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulins, immunoglobulins, etc.

Inactive Publication Date: 2013-06-19
ROCHE GLYCART AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FcγR binding also requires the presence of oligosaccharides in the CH2 region

Method used

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  • Anti-tenascin-c a2 antibodies and methods of use
  • Anti-tenascin-c a2 antibodies and methods of use
  • Anti-tenascin-c a2 antibodies and methods of use

Examples

Experimental program
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Embodiment 1

[0290] recombinant DNA technology

[0291] DNA was manipulated using standard methods, as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. DNA sequences were determined by double-strand sequencing. In some cases, desired gene segments were prepared from synthetic oligonucleotides and PCR products by automated gene synthesis by Geneart AG (Regensburg, Germany). The gene segment flanked by unique restriction endonuclease cleavage sites was cloned into the pGA18(ampR) plasmid. Plasmid DNA was purified from transformed bacteria and concentrations were determined by UV spectroscopy. The DNA sequence of the subcloned gene fragment was confirmed by DNA sequencing. Gene segments were designed with appropriate restriction sites to allow subcloning into corresponding expression vectors.

[0292]For gener...

Embodiment 2

[0306] Construction of Universal Fab Libraries

[0307] Construction of a universal antibody library in Fab format based on human germline genes using the following V domain pairings: Vk3_20 kappa light chain and VH3_23 heavy chain for the DP47-3 library, and Vk1_17 kappa light chain and VH1_69 heavy chain for DP88 -3 library. See SEQ ID NOs 1 and 2.

[0308] Both libraries were randomized in the light chain CDR3 (L3) and heavy chain CDR3 (H3), and each library was assembled from 3 fragments by splicing by overlapping extension (SOE) PCR. Fragment 1 contains the 5' end of the antibody gene, including the randomized L3, Fragment 2 is the central constant fragment spanning L3 to H3, and Fragment 3 contains the randomized H3 and the 3' portion of the antibody gene.

[0309] The following primer combinations were used to generate library fragments of the DP47-3 library: Fragment 1 (LMB3-LibL1b_new), Fragment 2 (MS63-MS64), Fragment 3 (Lib2H-fdseqlong). See Table 3. The followi...

Embodiment 3

[0318] Selection of anti-TNC A2 clone 2B10 (primary selection)

[0319] Subclones were selected against human TNC-A2 expressed in E. coli at 5' of the avi tag and 6xhis tag. See SEQ ID NO:97.

[0320] Antigens are biotinylated in vivo upon expression. Select in solution according to the following scheme: (i) about 10 12 A library of DP88-3 phagemid particles and 100 nM biotinylated human TNC A2 were combined for 0.5 hours in a total volume of 1 ml; (ii) by adding 5.4x10 7 Streptavidin-coated magnetic beads for 10 minutes to capture biotinylated human TNC-A2 and attached phage; (iii) wash the beads with 5x1ml PBS / Tween20 and 5x1ml PBS; amine) for 10 min to elute the phage particles and neutralize by adding 500 μL 1M Tris / HCl pH 7.4; and (v) reinfect log-phase E. coli TG1 cells with helper phage VCSM13, followed by PEG / NaCl precipitation of the phage Bacteroid pellets for subsequent rounds of selection.

[0321] Selection was performed over 3 rounds using a constant antigen...

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Abstract

The invention provides antibodies against the A2 domain of tenascin-C and methods of using the same.

Description

field of invention [0001] The invention relates to an antibody specific to the A2 domain of tenascin-C (A2 domain of tenascin-C, TNC A2). In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for generating such antibodies and methods of using them in the treatment of diseases. Background of the invention [0002] Tenascin C and anti-tenascin C antibodies [0003] Tenascins are a highly conserved family of larger multimeric extracellular matrix (ECM) glycoproteins found in vertebrates. Four tenascin paralogues have been identified in mammals, termed tenascin-C, tenascin-R, tenascin-X and tenascin-W. The common primary structure of tenascin family proteins, which includes N-terminal heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats and C-terminal fibronectin-like globular domains. Via the N-seg...

Claims

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Application Information

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IPC IPC(8): C07K16/30A61K39/395G01N33/53
CPCC07K2317/41C07K2317/55C07K2317/72C07K2317/92A61K47/6843A61K47/6851C07K16/30A61P35/00A61K39/00A61K39/395C07K16/28C12N15/1138C07K16/18A61K39/39558C07K2317/56A61K49/0058A61K51/1018C07K2317/565
Inventor A.弗里莫瑟-格伦德舍伯R.霍塞C.克莱恩E.莫斯纳V.G.尼科里尼P.尤马纳
Owner ROCHE GLYCART AG
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