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

Inactive Publication Date: 2006-02-16
ABLYNX NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0047] Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above or a composition as described above, for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance which is able pass through the tissues beneath the tongue effectively.
[0048] Another embodiment of the present invention is a use of an anti-TNF-alpha polypeptide as described above or a composition as described above, for the preparation of a medicament for treating, preventing and/or alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance which is able pass through the tissues beneath t

Problems solved by technology

Yet none of the presently available drugs are completely effective for the treatment of autoimmune disease, and most are limited by severe toxicity.
In addition, it is extremely difficult and a lengthy process to develop a new chemical entitiy (NCE) with sufficient potency and selectivity to such target sequence.
However, conventional antibodies are difficult to raise against multimeric proteins where the receptor-binding domain of the ligand is embedded in a groove, as is the case with TNF-alpha.
The use of antibodies derived from sources such as mouse, sheep, goat, rabbit etc., and humanised derivatives thereof as a treatment for conditions which require a modulation of inflammation is problematic for several reasons.
Traditional antibodies are not stable at room temperature, and have to be refrigerated for preparation and storage, requiring necessary refrigerated laboratory equipment, storage and transport, which contribute towards time and expense.
Furthermore, the manufacture or small-scale production of said antibodies is expensive because the mammalian cellular systems necessary for the expression of intact and active antibodies require high levels of support in terms of time and equipment, and yields are very low.
Furthermore the large size of conventional

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
Comparison scheme
Effect test

Example

Example 1

Example of Camelidae Antibodies Against Human Tumor Necrosis Factor Alpha

1) Immunization and Library Constructions

[0283] A llama (Llama glama) was immunized with human TNF-alpha. For immunization, the cytokine was formulated as an emulsion with an appropriate, animal-friendly adjuvant (Specoll, CEDI Diagnostics B.V.). The antigen cocktail was administered by double-spot injections intramuscularly in the neck. The animal received 6 injections of the emulsion, containing 100 μg of TNF-alpha at weekly intervals. At different time points during immunization, 10-ml blood samples were collected from the animal and sera were prepared. The induction of an antigen specific humoral immune response was verified using the serum samples in an ELISA experiment with TNF (data not shown). Five days after the last immunization, a blood sample of 150 ml was collected. From this sample, conventional and heavy-chain antibodies (HcAbs) were fractionated (Lauwereys et al. 1998) and used in a...

Example

Example 2

Humanization of VHH#12B and VHH#3E by Site Directed Mutagenesis

1) Homology Between VHH#3E / VHH#12B and Human Germline Heavy Chain V-Region DP-47

[0297] Alignment of VHH#12B and a human VH3 germline sequence (DP-47) revealed a high degree of homology: [0298] 4 AA changes in FR1 on position 1, 5, 28 and 30 [0299] 5 M changes in FR3 on position 74, 76, 83, 84 and 93 [0300] 1 AA change in FR4 on position 108

[0301] as represented in the following sequence alignment: DP-47EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVS AISGSGGSTYYVHH#12BQVQLQESGGGLVQPGGSLRLSCAASGFEFE NHWMYWVRQAPGKGLEWVS TVNTNGLITRYDP-47ADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK------------- -----------VHH#12BADSVKG RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSRTNAD WGQGTQVTVSS

[0302] A specific inhibitor for the TNF-alpha cytokine, with high homology to the human germline gene DP47 was therefore an ideal candidate to further humanize and evaluate the influence of mutagenesis on inhibition capacity in ELISA.

[0303]...

Example

Example 3

Isolation of Antagonistic VHH Against Mouse TNF-Alpha

1) Selection of Anti-Mouse TNF-Alpha VHH

[0313] In order to perform efficacy studies in mouse models for IBD or Crohn's disease mouse TNF specific VHH were selected. Therefore a llama was immunized with mouse TNF-alpha as described in Example 1. RNA was extracted from PBL's sampled 4 and 10 days after the last immunization, as well as from a biopsy taken from a lymph node after day 4. Total RNA was converted in either random primed or oligo-dT primed cDNA and used as template for the amplification of the VHH encoding gene segments using Ig derived primers or a combination of oligo-dT primer and a single Ig primer (see example 1). With the Ig primers a library containing 8.5×107 clones was generated from the first PBL's, and a library with 7×106 clones for the second PBL sample and 5.8×108 clones for the lymph node. Using the combination of the oligo-dT primer and the Ig primer libraries from the first PBL sample were m...

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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 THE INVENTION [0001] The present invention provides polypeptides comprising one or more single domain antibodies directed towards tumor necrosis factor alpha (TNF-alpha). The present invention further relates to their use in diagnosis and therapy. Such antibodies may have a framework sequence with high homology to the human framework sequences. Compositions comprising antibodies to tumor necrosis factor alpha (TNF-alpha) alone or in combination with other drugs are described. BACKGROUND TO THE INVENTION [0002] Tumor necrosis factor alpha (TNF-alpha) is believed to play an important role in various disorders, for example in inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. Both TNF-alpha and the receptors (CD120a, CD120b) have been studied in great detail. TNF-alpha in its bioactive form is a trimer and the groove formed by neighboring subunits is important for the cytokine-receptor interaction. Several strategie...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/24
CPCA61K2039/505C07K2317/34C07K16/241C07K16/249C07K16/2863C07K16/2875C07K16/36C07K16/40C07K16/4291C07K2316/96C07K2317/22C07K2317/24C07K2317/31C07K2317/565C07K2317/569C07K2317/626C07K2317/92C07K2319/00C07K16/18A61P19/02A61P31/00A61P31/04A61P35/00A61P37/06C07K2317/567C07K2317/76
Inventor SILENCE, KARENLAUWEREYS, MARCDE HAARD, HANS
Owner ABLYNX NV
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