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Single domain antibodies directed against tumor necrosis factor alpha and uses therefor

A single-domain antibody, tumor necrosis factor technology, applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, etc., can solve the problem of protease degradation sensitivity, resistance, instability and so on

Active Publication Date: 2006-02-15
ABLYNX NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Another important drawback of conventional antibodies is that they are complex macromolecules and thus relatively unstable, and they are sensitive to protease degradation
This means that conventional antibody drugs cannot be administered orally, sublingually, topically, nasally, vaginally, rectally or by inhalation because they are not resistant to the low pH at these sites, the action of proteases at these sites and in the blood and / or because of their large size

Method used

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  • Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
  • Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
  • Single domain antibodies directed against tumor necrosis factor alpha and uses therefor

Examples

Experimental program
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Embodiment 1

[0290] Example 1: Examples of camelid antibodies against human tumor necrosis factor alpha

[0291] 1) Immunization and library construction

[0292] Llama (Llama glama) was immunized with human TNF-[alpha]. For immunization, cytokines are formulated as emulsions with appropriate animal-friendly adjuvants (Specoll, CEDI Diagnostics B.V.). The antigen mixture was administered by intramuscular dual site injection in the neck. Animals received 6 injections of emulsion containing 100 μg TNF-[alpha] at weekly intervals. At various time points during the immunization, 10 ml blood samples were collected from the animals and serum was prepared. The induction of an antigen-specific humoral immune response was verified with TNF in an ELISA experiment using serum samples (data not shown). 5 days after the last immunization, a 150 ml blood sample was collected. Conventional and heavy chain antibodies (HcAbs) were fractionated (Lauwereys et al., 1998) from this sample and used in ELIS...

Embodiment 2

[0306] Example 2: Humanization of VHH#12B and VHH#3E by site-directed mutagenesis

[0307] 1) Homology between VHH#3E / VHH#12B and human germline heavy chain V-region DP-47 Alignment of VHH#12B and human VH3 germline sequence (DP-47) revealed a high degree of homology :

[0308] 4 amino acid changes at positions 1, 5, 28 and 30 in FR1

[0309] 5 amino acid changes at positions 74, 76, 83, 84 and 93 of FR3

[0310] 1 amino acid change at position 108 in FR4

[0311] As represented in the sequence alignment below:

[0312] DP-47 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYY

[0313] VHH#12B QVQLQESGGGLVQPGGSLRLSCAASGFEFE NHWMY WVRQAPGKGLEWVS TVNTNGLITRY

[0314] DP-47 ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK -------------- -----------

[0315] VHH#12B ADSVKG RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTK VLPPYSDDSRTNAD WGQGTQVTVSS

[0316] Therefore, specific inhibitors of TNF-α cytokines with high homology to the human germline gene DP-47 are ideal c...

Embodiment 3

[0333] Example 3: Isolation of antagonistic VHH against mouse TNF-α

[0334] 1) Select anti-mouse TNF-α VHH

[0335] For efficacy studies in IBD or Crohn's disease mouse models, mouse TNF-specific VHHs were selected. Therefore, llamas were immunized with mouse TNF-[alpha] as described in Example 1. RNA was extracted from PBLs sampled 4 and 10 days after the last immunization and from lymph node biopsies after the fourth day. Total RNA was converted to random-primed or oligo-dT-primed cDNA and used as template for amplification of VHH-encoding gene segments using Ig-derived primers or a mixture of oligo-dT primers and a single Ig primer (see Example 1). From the first PBL's generated using Ig primers containing 8.5 x 10 7 A library of libraries of clones was generated from a second PBL sample of 7 x 10 6 A library of clones yielded 5.8 × 10 8 library of clones. A mixture of oligo-dT and Ig primers was obtained from the first PBL sample containing 1.2 × 10 8 A library of...

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Abstract

The present invention relates to polypeptides derived from single domain heavy chain antibodies directed to Tumor Necrosis Factor-alpha. It further relates to single domain antibodies that are Camelidae VHHs. It further relates to methods of administering said polypeptides. It further relates to protocols for screening for agents that modulate the TNF-alpha receptor, and the agents resulting from said screening.

Description

field of invention [0001] The invention provides polypeptides comprising one or more single domain antibodies directed against tumor necrosis factor alpha (TNF-alpha). The invention also relates to their use for diagnosis and therapy. These antibodies may have framework sequences that are highly homologous to human framework sequences. Compositions containing tumor necrosis factor alpha (TNF-alpha) antibodies alone or in combination with other drugs are described. Background of the invention [0002] Tumor necrosis factor alpha (TNF-α) is believed to play an important role in various diseases, for example, in inflammatory diseases such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. TNF-[alpha] and receptors (CD120a, CD120b) have been studied in great detail. The biologically active form of TNF-α is a trimer and the groove formed by adjacent subunits is important for cytokine-receptor interactions. Several strategies to antagonize the...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46A61K39/395C12N15/13G01N33/577A61P37/06A61P19/02C07K16/18
Inventor 卡伦·西莱恩克马克·劳韦里斯汉斯·德哈德
Owner ABLYNX NV
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