ZA200803817B - Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor - Google Patents
Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor Download PDFInfo
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- ZA200803817B ZA200803817B ZA200803817A ZA200803817A ZA200803817B ZA 200803817 B ZA200803817 B ZA 200803817B ZA 200803817 A ZA200803817 A ZA 200803817A ZA 200803817 A ZA200803817 A ZA 200803817A ZA 200803817 B ZA200803817 B ZA 200803817B
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Description
: EGA AFB J : : Ta co -
SINGLE DOMAIN ANTIBODIES DIRECTED AGAINST TUMOUR : ll lil NECROSIS FACTOR-ALPHA AND USES THEREFOR
This is a fresh application (i.e. a so-called divisional application) filed in terms of Section 37 of the South African Patents Act 57/1978 in respect of part of the matter disclosed in South African patent application 2005/03618, filed as a national phase
PCT patent application on 5 May 2005 (i.e. the so-called parent patent application) based on PCT international application PCT/BE2003/000192 filed on 7 November 2003. :
The term "invention" is used herein to describe the subject matter of both the parent and the fresh (divisional) patent applications. :
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 oo 10 in combination with other drugs are described. :
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 timer and the groove formed by neighboring subunits is important for the cytokine- receptor interaction. Several strategies to antagonize the action of the cytokine have been developed and are currently used fo treat various disease states.
A TNF-alpha inhibitor which has sufficient specificity and selectivity to TNF-alpha may be an efficient prophylactic or therapeutic pharmaceutical compound for preventing or treating disorders where TNF-alpha has been implicated as causative agent. Methods of treating toxic shock (EP 486526), tumor regression, inhibition of cytotoxicity (US 6448380,
US 6451983, US 6498237), autoimmune disease such as RA and Crohn's disease (EP : 663836, US 5672347, US 5656272), graft versus host reaction (US 5672347), bacterial meningitis (EP 585705) by means of an antibody to TNF-alpha have been described. _ 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 entity (NCE) with sufficient potency and selectivity to such target sequence. Antibody-based therapeutics on the other hand have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. In addition, the development time can be reduced " considerably when compared to the development of new chemical entities (NCE's).
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
® B.2008/034 1;
TNF-alpha. Heavy chain antibodies described in the invention which are derived from
Camelidae, are known to have cavity-binding propensity (WO87/49805; Lauwereys et al,
EMBO J. 17, 5312, 1998)). Therefore, such heavy chain antibodies are inherently suited to bind to receptor binding domains of such ligands as TNF. In addition, such antibodies are known to be stable over long periods of time, therefore increasing their shelf-life (Perez et al, Biochemistry, 40, 74, 2001). Furthermore, such heavy chain antibody fragments can be produced ‘en-masse’ in fermentors using cheap expression systems compared to mammalian cell culture fermentation, such as yeast or other microorganisms (EP 0698 097).
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. Refrigeration is sometimes not feasible in developing countries. Furthermore, the manufacture or small-scale production of sald 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 antibodies, would restrict tissue penetration, for example, at the site of inflamed tissue. Furthermore, traditional antibodies have a binding activity which depends upon pH, and hence are unstitable for use in environments outside the usual physiological pH range such as, for example, in treating gastric bleeding, gastric surgery. Furthermore, traditional antibodies are unstable at low or high pH and hence are not suitable for oral administration. However, it has been demonstrated that camelidae antibodies resist harsh conditions, such as extreme pH, denaturing reagents and high temperatures (Dumoulin ef al, Protein Science 11, 500, 2002), so making them suitable for delivery by oral administration. Furthermore, traditional antibodies have a binding activity, which depends upon temperature, and hence are : unsuitable for use in assays or kits performed at temperatures outside biologically active- temperature ranges (e.g. 37 + 20°C).
Polypeptide therapeutics and in particular antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxicity. However, it is known by the skilled addressee that an antibody which has been obtained for a therapeutically useful target requires additional modification in order
. B - . to prepare it for human therapy, so as to avoid an unwanted immunological reaction in a human individual upon administration thereto. The modification process is commonly termed "humanisation". It is known by the skilled artisan that antibodies raised in species, other than in humans, require humanisation to render the antibody therapeutically useful in humans ( (1) CDR grafting : Protein Design Labs: US 6180370, US 5693761;
Genentech US 6054297; Celltech: 460167, EP 626390, US 5859205; (2) Veneering:
Xoma: US 5869619, US 5766886, US 5821123). There is a need for a method for producing antibodies which avoids the requirement for substantial humanisation, or which completely obviates the need for humanisation. There is a need for a new class of antibodies which have defined framework regions or amino acid residues and which can be administered to a human subject without the requirement for substantial humanisation, or the need for humanisation at all.
Another important drawback of conventional antibodies is that they are complex, large molecules and therefore relatively unstable, and they are sensitive to breakdown by proteases. 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. They have to be administered by injection (intravenously, subcutaneously, etc.) to overcome some of these problems. Administration by injection requires specialist training in order to use a hypodermic syringe or needle correctly and safely. It further requires sterile equipment, a liquid formulation of the therapeutic polypeptide, vial packing of said polypeptide in a sterile and stable form and, of the subject, a suitable site for entry of the needle. Furthermore, subjects commonly experience physical and psychological stress prior to and upon receiving an injection.
Therefore, there is need for a method for the delivery of therapeutic polypeptides which avoids the need for injection which is not only cost/time saving, but which would also be more convenient and more comfortable for the subject.
Single domain antibody-based therapeutics have significant potential as drugs because they have exquisite specificity to their target and a low inherent toxcity. However, improving further their intrinsic and functional affinity can lead to many benefits for a patient such as reduced dose of therapeutic, faster therapy, and reduced side effects.
"THE AIMS OF THE PRESENT INVENTION it is an aim of the present invention is to provide polypeptides comprising one or more single domain antibodies which bind to TNF-alpha, homologues of said polypeptides, functional portions of homologues of said polypeptides. Said polypeptides modify the biological activity of TNF-alpha upon binding. Such polypeptides might bind into the receptor-binding groove of TNF-alpha, or might not bind In the receptor binding groove.
Such polypeptides are single domain antibodies.
It is a further aim of the present invention to provide single domain antibodies which may be any of the art, or any future single domain antibodies. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. According to one aspect of the invention, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains (WO 9404678). For clarity reasons, this variable domain derived from a heavy chain antibody devoid of light chain will be called VHH or nanobody to distinguish it from the conventional :
VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. : It is a further aim of the invention to provide a method of administering anti-TNF-alpha polypeptides intravenously, subcutaneously, orally, subiingually, topically, nasally, vaginally, rectally or by inhalation.
It is a further aim of the invention to enhance the binding affinity of monovalent single domain antibodies.
One embodiment of the present invention is an anti-TNF-alpha polypeptide comprising at least one anti-TNF-alpha single domain antibody.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above wherein a single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 1 to 16 and 79 to 84.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above further comprising at least one single domain antibody directed against a serum protein. 5 Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above wherein said serum protein Is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, transferring, or fibrinogen. : Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above wherein a single domain anti-serum protein single domain antibody correspond to a sequence represented by any of SEQ ID NOs: 26 to 29 and 85 to 97.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above corresponding to a sequence represented by any of SEQ ID NOs: 30 to 43.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above further comprising at least one single domain antibody selected fromthe group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-lFN-gamma receptor single domain antibody.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, wherein the number of single domain antibodies directed against TNF- alpha is at least two.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above corresponding to a sequence represented by any of SEQ ID NOs: 73 to 76.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, wherein at least one single domain antibody is a humanized Camelidae
VHHs.
Another embodiment of the present invention Is an anti-TNF-alpha polypeptide as described above wherein a humanized Camelidae VHH corresponds to a sequence represented by any of SEQ ID NOs: 17 to 19 and 21 to 24.
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Another embodiment of the present invention is a composition comprising an anti-TNF- alpha polypeptide as described above and at least one single domain antibody from the group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-lFN-gamma receptor single domain antibody, for simultaneous, separate or sequential administration to a subject.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, or a composition as described above wherein at least one anti-IFN- : gamma single domain antibody correspond to a sequence represented by any of SEQ ID
NOs:44to72.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, or a composition as described above, wherein said single domain antibody is an homologous sequence, a functional portion, or a functional portion of an homologous sequence of the full length single domain antibody. : Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, or a composition as described above, wherein the anti-TNF-alpha polypeptide is an homologous sequence, a functional portion, or a functional portion of an homologous sequence of the full length anti-TNF-alpha polypeptide.
Another embodiment of the present invention Is an anti-TNF-alpha polypeptide as described above, or a composition as described above wherein at least one single domain antibody is a Camelidae VHH. oo "Another embodiment of the present invention is a nucleic acid encoding an anti-TNF- alpha polypeptide as described above.
Another embodiment of the present invention is a method of identifying an agent that modulates the binding of an anti-TNF-alpha polypeptide as described above, to Tumor
Necrosis Factor-alpha comprising the steps of: (a) contacting an anti-TNF-alpha polypeptide as described above with a target that is
Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in
® the absence of said candidate modulator identified said candidate modulator as an agent that modulates the binding of an anti-TNF-alpha polypeptide as described above and :
Tumor Necrosis Factor-alpha.
Another embodiment of the present invention is a method of identifying an agent that modulates Tumor Necrosis Factor-alpha-mediated disorders through the binding of an anti-TNF-alpha polypeptide as described above to Tumer Necrosis Factor-alpha comprising: (a) contacting an anti-TNF-alpha polypeptide as described above with a target that is
Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease In binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identified, said candidate modulator as an agent that modulates Tumor Necrosis Factor alpha-mediated disorders.
Another embodiment of the present invention is a method of identifying an agent that modulates the binding of Tumor Necrosis Factor alpha to its receptor through the binding of an anti-TNF-alpha polypeptide as described above to Tumor Necrosis Factor-alpha comprising. : (a) contacting an anti-TNF-alpha polypeptide as described above with a target that is
Tumor Necrosis Factor-alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and (b) measuring the binding between the polypeptide and target of step (a), wherein a ~ decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of sald candidate modulator identified said candidate modulator as an agent that modulates the binding of Tumor Necrosis Factor-alpha to its receptor.
Another embodiment of the present invention is a kit for screening for agents that modulate Tumor Necrosis Factor-alpha-mediated disorders comprising an anti-TNF-alpha polypeptide as described above and Tumor Necrosis Factor-alpha.
Another embodiment of the present invention is an unknown agent that modulates the binding of an anti-TNF-alpha polypeptide as described above to Tumor Necrosis Factor- alpha, identified according to the method as described above.
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Another embodiment of the present invention is an unknown agent that modulates Tumor
Necrosis Factor-alpha-mediated disorders, identified according to the methods as described above. :
Another embodiment of the present invention is an unknown agent as described above wherein said disorders are one or more of inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis. : Another embodiment of the present invention is an anti-TNF-alpha polypeptide as described above, or a nucleic acid as described above, or a composition -as described above, or an agent as described above for treating and/or preventing and/or alleviating disorders relating to inflammatory processes.
Another embodiment of the present invention is a use of an anti-TNF-alpha polypeptide as : 15 described above or a nucleic acid as described above, or a composition as described above, or an agent as described above for the preparation of a medicament for treating and/or preventing and/or alleviating disorders relating to inflammatory reactions.
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 gastric environment without the substance being inactivated.
Another embodiment of the present invention is an 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 gastric environment without the substance being inactivated.
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 delivered to the vaginal and/or rectal tract.
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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 andjor alleviating the symptoms of disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the vaginal and/or rectal tract. :
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 delivered to the nose, upper respiratory tract and/or lung.
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 delivered to the nose, upper respiratory tract and/or lung.
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 delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa. :
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 delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.
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.
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 the tongue effectively.
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 skin effectively.
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 skin effectively. ~ Another embodiment of the present invention is a method as described above, a kit as described above, a nucleic acid or agent as described above, use of a nucleic acid or agent as described above, a composition as described above, use of a composition as described above, an anti-TNF-alpha polypeptide as described above, use of an anti-TNF- . alpha polypeptide as described above wherein said disorders are any of inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, Inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis,
Diabetes Type |, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever,
Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome,
Spondyloarthropathies, Thyroiditis, and Vasculitis.
Another embodiment of the present invention is a composition comprising a nucleic acid or agent as described above, an anti-TNF-alpha polypeptide as described above, or a composition as described above, and a suitable pharmaceutical vehicle.
Another embodiment of the present invention is a method of diagnosing a disorder characterised by the dysfunction of Tumor Necrosis Factor-alpha comprising: (a) contacting a sample with an anti-TNF-alpha polypeptide as described above, (b) detecting binding of said polypeptide to said sample, and
® (c) comparing the binding detected in step (b) with a standard, wherein a difference in binding relative to said sample is diagnostic of a disorder characterised by dysfunction of
Tumor Necrosis Factor-alpha.
Another embodiment of the present invention is a kit for screening for a disorder as cited above, using a method as described above.
Another embodiment of the present invention is a kit for screening for a disorder as cited above comprising an isolated anti-TNF-alpha polypeptide as described above.
Another embodiment of the present invention is a use of an anti-TNF-alpha polypeptide as described above for the purification of said Tumor Necrosls Factor-alpha.
Another embodiment of the present invention is a use of an anti-TNF-alpha polypeptide as described above for inhibiting the interaction between Tumor Necrosis Factor-alpha and one or more Tumor Necrosis Factor-alpha receptors.
Another embodiment of the present invention is a method for producing an anti-TNF-alpha polypeptide as described above comprising the steps of: (a) obtaining double stranded DNA encoding a Camelidae VHH directed to Tumor ‘Necrosis Factor alpha, (b) cloning and expressing the DNA selected in step (b). * Another embodiment of the present invention is a method of producing an anti-TNF-alpha polypeptide as described above comprising: (a) culturing host cells comprising nucleic acid capable of encoding an anti-TNF-alpha : polypeptide as described above, under conditions allowing the expression of the polypeptide, and, (b) recovering the produced polypeptide from the culture.
Another embodiment of the present invention Is a method as described above, wherein said host cells are bacterial or yeast.
Another embodiment of the present Invention is a kit for screening for any of inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome or multiple sclerosis comprising an anti-TNF-alpha polypeptide as described above.
Figure 1 Alignment of anti-human TNF VHH’s as described in Example 1
Figure 2 Dilution series of anti-human TNF-alpha VHHSs as tested in ELISA according to
Example 1.
Figure 3 Antagonistic effect of VHH as determined in cytotoxicity assay using human cell line KYM according to Example 1.
Figure 4 In vitro receptor binding assay of wid type VHH#12B and mutant
A74S+YT6N+K83R+P84A
Figure 5 In vitro receptor binding assay of wild type VHH#12B and mutant 1E + Q5LA74S + Y76N + K83R + P84A :
Figure 6 Binding in ELISA of wild type VHH#3E and mutant VHH's
Figure 7 In vitro receptor binding assay of wild type VHH#3E and mutant VHH's
Figure 8 Alignment of antagonistic anti-mouse TNF's as described in Example 3.
Figure 9 Antagonistic effect of anti-mouse TNF VHH as determined in cytotoxicity assay using murine cell line L929 according to Example 3.
Figure 10 EcoRI — Hindlll insert of vector pAX11 (pUC119 backbone) for production of bi- valent or bispecific VHH.
Figure 11 Coomassie-stained PAGE (15%) of IMAC-purified mono-(lane 8), bi- (lane 1), tri- (lanes 2, 3 and 5) and tetravalent (lanes 4, 6 and 7) anti- TNFa VHH.
Figure 12 Chromatogram of the analysis by ge! filtration on Superdex 75HR of the mono-, bi-, tri and tetravalent VHH.
Figure 13 Comparison of the antagonistic characteristics of the mono-, bi, tri- and tetravalent form of the anti-human TNF VHH with the clinically used products Remicade and Enbrel.
Figure 14 Antagonistic behaviour of the mono- and bivalent VHH's directed against mouse TNFalpha . .
Figure 15 Coomassie stained PAGE of VHH-Fc-fusion derived from human lgG1 described in Example 4.
Figure 16 Antagonistic efficacy of VHH-Fc fusion derived from VHH#3E compared with bivalent format of VHH#3E as determined in bioassay.
Figure 17 ELISA of reference and pepsin-treated TNF3E at pH2.2, pH3.2 and pH4.2 (100% Is the signal measured at a 1/100 dilution)
Figure 18 Experimental setting
Table 1 Amino acid sequence listing of the peptides of aspects of present invention directed against TNF-alpha. :
Table 2 List of mutagenesis reactions, mutagenic primers and templates used for mutagenesis of VHH#12B.
Table 3 List of mutagenesis reactions, mutagenic primers and templates used for mutagenesis of VHH#3E.
Table 4 Overview of humanised and wild type VHH
Table 5 Anti-mouse serum albumin/anti TNF-alpha
Table 6 Amino acid sequence listing of VHH's directed against human IFN-gamma.
Table 7 Sequences of bivalent (BIV 3E, BIV#m3F), trivalent (TRI3E) or tetravalent (TETRA 3E) VHH directed against TNF-alpha. "Table 8 Fractional homologies between the amino acid sequences of anti-mouse serum albumin VHHs of the invention.
Table 9 Fractional homologies between anti-TNF-alpha VHHSs of the invention.
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Table 10 Percentage homologies between anti-IFN-gamma VHHs of the invention. : Table 11 Treatment schedule
The present invention relates to an anti-tumour necrosis factor-alpha (TNF-alpha) polypeptide, comprising one or more single domain antibodies which are directed against
TNF-alpha. The invention also relates to nucleic acids capable of encoding said polypeptides.
Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. According to one aspect of the invention, a single domain antibodies as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 94/04678 for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised In Camelidae species, for example in camel, dromedary, llama, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.
VHHSs, according to the present invention, and as known to the skilled addressee are heavy chain variable domains derived from immunoglobulins naturally devoid of light chains such as those derived from Camelidae as described in WO 94/04678 (and referred to hereinafter as VHH domains or nanobodies). VHH molecules are about 10x smaller than lgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHSs. In addition, antibodies generated in
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Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than
Camelids (WO 9749805). As such, anti-TNF-alpha VHH's may interact more efficiently with TNF-alpha than conventional antibodies, thereby blocking its interaction with the
TNF-alpha receptor more efficiently.
According to the invention, TNF-alpha is derived from any species. Examples of species relevant to the invention include as rabbits, goats, mice, rats, COWS, calves, camels, llamas, monkeys, donkeys, guinea pigs, chickens, sheep, dogs, cats, horses, and preferably humans.
TNF-alpha is also a fragment of TNF-alpha, capable of eliciting an immune response.
TNF-alpha is also a fragment of TNF-alpha, capable of binding to a single domain antibody raised against the full length TNF-alpha.
A single domain antibody directed against TNF-alpha means single domain antibody that it is capable of binding to TNF-alpha with an affinity of better than 10% M.
One embodiment of the present invention is an anti-TNF polypeptide, wherein the single domain antibodies comprise Camelidae VHH directed against TNF-alpha.
The one or more single domain antibodies of the anti-TNF polypeptide which are directed : against a TNF-alpha may be of the same sequence. Alternatively they may not all have the same sequence. lt is within the scope of the invention that an anti-TNF polypeptide comprises anti-TNF-alpha single domain antibodies which do not all share the same sequence, but which are directed against the same target, one or more antigens thereof.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide, wherein a single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 1 to 16 and 79 to 84 as shown in Table 1. Said sequences are derived from Camelidae heavy chain antibodies (VHHs) which are directed against TNF-alpha.
The present invention further relates to an anti-TNF-alpha polypeptide, wherein said single domain antibody is a VHH directed against TNF-alpha, wherein the VHH belongs to a class having human-like sequences. The class is characterised in that the VHHs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine,
C proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 and a tryptophan at position 103, according to the Kabat numbering. The new class of Camelidae single-domain antibodies described in this invention (Table 1, Example 1) is represented by VHH#2B (SEQ ID NO: 3) and VHH#12B (SEQ ID No. 14) containing the hydrophobic residues in FR2 in combination with the hydrophobic residue tryptophan at position 103.
Another human-like class of Camelidae single domain antibodies represented by sequences VHH#1A (SEQ ID NO. 1), VHH#4B (SEQ ID NO. 12), VHH#8-29 (SEQ ID NO. 81), VHH#8-41 (SEQ ID NO. 82), VHH#8-42 (SEQ ID NO. 83) and VHH#8-44 (SEQ ID
NO. 84) (Table 1, Example 1) have been described In WO03035694 and contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by the charged arginine residue on position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. The invention also relates to nucleic acids capable of encoding said polypeptides. : 20
Therefore, one aspect of the present invention allows for the direct administration of an anti-TNF-alpha polypeptide, wherein the single domain antbodies belong to the humanized class of VHH, and comprise a sequence represented by any of SEQ ID NO:1, 3, 12, 14, 81, 82, 83, and 84 to a patientin need of the same.
Any of the VHHs as used by the invention may be of the traditional class or of the classes of human-like Camelidae antibodies. Said antibodies may be directed against whole TNF- alpha or a fragment thereof, or a fragment of a homologous sequence thereof. These polypeptides Include the full length Camelidae antibodies, namely Fc and VHH domains, chimeric versions of heavy chain Camelidae antibodies with a human Fc domain or VHH's by themselves or derived fragments.
Anti-serum albumin VHH's may interact in a more efficient way with serum albumin than conventional antibodies which is known to be a carrier protein. As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into ‘unusual’ or non-
C . R conventional epitopes such as cavities (WO 97/49805), the affinity of such VHH's to circulating albumin may be increased.
The present invention also relates to the finding that an anti-TNF polypeptide as described herein further comprising one or more single domain antibodies directed against one or more serum proteins of a subject, surprisingly has significantly prolonged half-life in the circulation of said subject compared with the half-life of the anti-TNF-alpha single domain antibody when not part of said construct. Examples of such polypeptides are represented in Table 5 by SEQ ID NOs: 30 to 43. Furthermore, the said polypeptides were found to exhibit the same favourable properties of single domain antibodies such as high stability remaining intact in mice, extreme pH resistance, high temperature stability and high target affinity.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide further comprising one or more single domain antibodies directed against one or more serum proteins, said anti-TNF alpha polypeptide comprising a sequence corresponding to any : represented by SEQ ID NOs: 30 to 43 (Table 5). :
Another embodiment of the present invention is an anti-TNF-alpha polypeptide, wherein an anti-serum protein single domain antibody corresponds to a sequence represented by any of SEQ ID NOs: 26 to 29 and 85 to 97 as shown in Table 5.
The serum protein may be any suitable protein found in the serum of subject. In one aspect of the invention, the serum protein is serum albumin, serum immunoglobulins, thyroxine-binding protein, transferrin, or fibrinogen. Depending on the intended use such as the required half-life for effective treatment and/or compartimentalisation of the target antigen, the VHH-partner can be directed to one of the above serum proteins.
Another aspect of the invention is an anti-TNF-alpha polypeptide as disclosed herein further comprising at least one polypeptide selected from the group consisting of an anti-
IFN-gamma polypeptide, an anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide.
It is an embodiment of the invention that a single domain antibody directed against IFN- gamma corresponds to a sequence represented by any of SEQ ID NOs: 44 to 72 as shown in Table 6.
According to one aspect of the invention, a single domain antibody is directed against
TNF-alpha receptor. Said single domain antibody may be a Camelidae VHH.
According to one aspect of the invention, a single domain antibody is directed against
IFN-gamma receptor. Said single domain antibody may be a Camelidae VHH.
Another aspect of the invention is a method of treating an autoimmune disease or condition as cited herein, comprising administering to a patient an effective amount of an anti-TNF-alpha polypeptide further comprising a least one polypeptide selected from the group consisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide, such polypeptides Joined to each other as described below.
Such multi-specific constructs may have improved potency as inflammatory therapeutic compound over mono-specific constructs.
One aspect of the Invention Is a composition comprising an anti-TNF-alpha polypeptide as disclosed herein and at least one polypeptide selected from the group consisting of anti-
IFN-gamma polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide, for simultaneous, separate or sequential administration to a subject.
One aspect of the invention is a method for treating autoimmune disease comprising administering to an Individual an effective amount of an anti-TNF-alpha polypeptide and a least one polypeptide selected from the group consisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide, simultaneously, separately or sequentially.
Another aspect of the invention is a kit containing an anti-TNF-alpha polypeptide and a least one polypeptide selected from the group consisting of anti-IFN-gamma polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide for simultaneous, separate or sequential administration to a subject. It is an aspect of the invention that the kit may be used according to the invention. It is an aspect of the invention that the kit may be used fo treat the diseases as cited herein.
By simultaneous administration means the polypeptides are administered to a subject at the same time. For example, as a mixture of the polypeptides or a composition comprising
® said polypeptides. Examples include, but are not limited to a solution administered intraveneously, a tablet, liquid, topical cream, etc., wherein each preparation comprises the polypeptides of interest.
By separate administration means the polypeptides are administered to a subject at the same time or substantially the same time. The polypeptides are present in the kit as separate, unmixed preparations. For example, the different polypeptides may be present in the kit as individual tablets. The tablets may be administered to the subject by swallowing both tablets at the same time, or one tablet directly following the other.
By sequential administration means the polypeptides are administered to a subject sequentially. The polypeptides are present in the kit as separate, unmixed preparations.
There is a time interval between doses. For example, one polypeptide might be administered up to 336, 312, 288, 264, 240, 216, 192, 168, 144, 120, 96, 72, 48, 24, 20, 16, 12, 8,4, 2, 1, or 0.5 hours after the other component.
In sequential administration, one polypeptide may be administered once, or any number of times and in various doses before and/or after administration of another polypeptide.
Sequential administration may be combined with simultaneous or sequential administration.
The medical uses of the anti-TNF-alpha polypeptide described below, also apply to the composition comprising an anti-TNF-alpha polypeptide as disclosed herein and at least one polypeptide selected from the group consisting of anti-IFN-gamma polypeptide, anti-
TNF-alpha receptor polypeptide and anti-IFN-gamma receptor polypeptide, for simultaneous, separate or sequential administration to a subject as disclosed here above.
According to one aspect of the invention, an anti-IFN-gamma polypeptide anti-TNF-alpha a single domain antibody directed against IFN-gamma. Said single domain antibody may be a Camelidae VHH.
It is an embodiment of the invention that a single domain antibody directed against IFN- gamma corresponds to a sequence represented by any of SEQ ID NOs: 44 to 72 as shown in Table 6. .
®
According to one aspect of the invention, anti-TNF-alpha a single domain antibody directed against TNF-alpha receptor. Said single domain antibody may be a Camelidae
VHH.
According to one aspect of the invention, an anti-IFN-gamma receptor polypeptide anti-
TNF-alpha a single domain antibody directed against IFN-gamma receptor. Said single domain antibody may be a Camelidae VHH.
Another embodiment of the present invention is an anti-TNF-alpha polypeptide as disclosed herein, wherein the number of single domain antibodies directed against TNF- alpha is two or more. Such multivalent anti-TNF-alpha polypeptides have the advantage of unusually high functional affinity for the target, displaying much higher than expected inhibitory properties compared to their monovalent counterparts.
The multivalent anti-TNF-alpha polypeptides have functional affinities that are several orders of magnitude higher than the monovalent parent anti-TNF-alpha polypeptides. The inventors have found that the functional affinities of these multivalent polypeptides are much higher than those reported in the prior art for bivalent and multivalent antibodies.
Surprisingly, anti-TNF-alpha polypeptides of the present invention linked to each other directly (SEQ ID No. 77 and 78) or via a short linker sequence show the high functional affinities expected theoretically with multivalent conventional four-chain antibodies.
The inventors have found that such large increased functional activities can be detected preferably with antigens composed of multidomain and multimeric proteins, either in straight binding assays or in functional assays, e.g. cytotoxicity assays.
Another embodiment of the present invention Is an ant-TNF-alpha polypeptide as disclosed herein, wherein the number of single domain antibodies directed against TNF- alpha is two or more, said anti-TNF-alpha polypeptide comprising a sequence corresponding to any represented by SEQ ID NOs: 73 to 76.
The single domain antibodies may be joined to form any of the polypeptides disclosed herein comprising more than one single domain antibody using methods known in the art or any future method. For example, they may be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by
Blattler et al, Biochemistry 24,1517-1524; EP294703. Alternatively, the single domain
® antibody may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more anti-target single domain antibodies and one or more anti-serum protein single domain antibodies. A method for producing bivalent or multivalent VHH polypeptide constructs is disclosed in
PCT patent application WO 96/34103. One way of joining multiple single domain antibodies is via the genetic route by linking single domain antibody coding sequences either directly or via a peptide linker. For example, the C-terminal end of the first single domain antibody may be linked to the N-terminal end of the next single domain antibody.
This linking mode can be extended in order to link additional single domain antibodies for the construction and production of tri-, tetra-, etc. functional constructs.
According to one aspect of the present invention, the single domain antibodies are linked to each other directly, without use of a linker. Contrary to joining bulky conventional antibodies where a linker sequence is needed to retain binding activity in the two subunits, polypeptides of the invention can be linked directly (SEQ ID No. 77 and 78) thereby avoiding potential problems of the linker sequence, such as antigenicity when administered to a human subject, instability of the linker sequence leading to dissociation of the subunits. }
According to another aspect of the present invention, the single domain antibodies are linked to each other via a peptide linker sequence. Such linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. The linker sequence is expected to be non-immunogenic in the subject to which the anti-TNF-alpha polypeptide
Is administered. The linker sequence may provide sufficient flexibility to the multivalent anti-TNF-alpha polypeptide, at the same time being resistant to proteolytic degradation. A non-limiting example of a linker sequences is one that can be derived from the hinge region of VHHs described in WO 96/34103.
According to another aspect of the invention, multivalent single domain antibodies comprising more than two single domain antibodies can be linked to each other either directly or via a linker sequence. Such constructs are difficult to produce with conventional antibodies and due to steric hindrance of the bulky subunits, functionality will be lost or greatly diminished rather than increased considerably as seen with VHH's of the invention compared to the monovalent construct (see Figure 12 for gel filtration analyses of such multivalent VHH constructs). :
Claims (38)
1. An anti-TNF-alpha polypeptide comprising at least one anti-TNF-alpha single domain antibody comprising : (a) a sequence represented by any of SEQ ID NOs: 1 to 16 and. 79 to 84, (b) a homologous sequence which presents a sequence identity of more than 70% with the parent sequence represented by any of SEQ ID NOs: 1 to 16° and 79 to 84, (c) a functional portion of (a) or (b) capable of binding to TNF-alpha with an affinity of 1 x 10-6 or better. :
2. An anti-TNF-alpha polypeptide according to claim 1 further comprising at least one single domain antibody directed against a serum protein.
- 3. An-anti-TNF-alpha polypeptide according to claim 2 wherein said serum protein is any of serum albumin, serum immunoglobulins, thyroxine-binding protein, . : transferrin, or fibrinogen.
4. An anti-TNF-alpha polypeptide according to claims 2 or 3 wherein at least one single domain antibody directed against a serum protein comprises a sequence represented by any of SEQ ID NOs: 26 to 29 and 85 to 97. :
5. “An anti-TNF-alpha polypeptide according to any of claims 2 to 4 comprising a - sequence represented by any of SEQ ID NOs: 30 to 43.
6. An anti-TNF-alpha polypeptide according to any of claims 1 to 5 further : comprising at least one single domain antibody selected from the group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-IFN- gamma receptor single domain antibody. IA An anti-TNF-alpha polypeptide according to any of claims 1 and 6, wherein the number of single domain antibodies directed against TNF-alpha is at least two."
* ”
8. An anti-TNF-alpha polypeptide according to claim 7 comprising a: sequence represented by any of SEQ ID NOs: 73 to 76.
9. An anti-TNF-alpha polypeptide according any of claims 1 to 8, wherein at least one single domain antibody is a humanized Camelidae VHH.
10. An anti-TNF-alpha polypeptide according to claim 9 wherein at least one ~ humanized Camelidae VHH comprises a sequence represented by any of SEQ ID NOs: 17 to 19 and 21 to 24.
11. A composition comprising an anti-TNF-alpha polypeptide according to any of claims 1 to 10 and at least one single domain antibody selected from the group consisting of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor single domain antibody and anti-IFN-gamma ’ receptor single domain antibody, and a pharmaceutically acceptable carrier, suitable for simultaneous, separate or sequential administration to a subject.
12. An anti-TNF-alpha polypeptide according to any of claims 6 to 10, or a composition according to claim 11 wherein at least one anti-IFN-gamma single domain antibody comprises a sequence represented by any of SEQ ID NOs: 44 to 72.
13. An anti-TNF-alpha polypeptide according to any of claims 1 to 10 and 12, or a composition according to claim 11 wherein at least one single domain antibody is a Camelidae VHH.
14. A nucleic acid encoding an anti-TNF-alpha polypeptide according to any of claims 1 to 10, 12 or 13.
15. A method of identifying an agent that modulates the binding of an anti- TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, to Tumor Necrosis Factor-alpha comprising the steps of: (a) contacting an anti-TNF-alpha polypeptide of any of claims 1 to 10, : 12 or 13 with a target that is Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and o 76 ~(b) measuring the binding between the polypeptide and target of step (a), : wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the binding of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 and Tumor Necrosis Factor-alpha.
16. A method of identifying an agent that modulates Tumor Necrosis Factor-alpha- mediated disorders through the binding of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 to Tumor Necrosis Factor-alpha comprising: a) contacting an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 with a target that is Tumor Necrosis Factor alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and a b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate : modulator, relative to the binding in the absence of said candidate - modulator identifies said candidate modulator as an agent that modulates Tumor Necrosis Factor alpha-mediated disorders.
17. A method of identifying an agent that modulates the binding of Tumor Necrosis Factor alpha to its receptor through the binding of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 to Tumor Necrosis Factor-alpha comprising: (a) contacting an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 with a target that is Tumor Necrosis Factor-alpha, in the presence and absence of a candidate modulator under conditions permitting binding between said polypeptide and target, and (b) measuring the binding between the polypeptide and target of step (a), wherein a decrease in binding in the presence of said candidate modulator, relative to the binding in the absence of said candidate modulator identifies said candidate modulator as an agent that modulates the binding of Tumor Necrosis Factor-alpha to its receptor. : 18. A kit for screening for agents that modulate Tumor Necrosis Factor-alpha- mediated disorders comprising an anti-TNF-alpha polypeptide of any of claims
®o mo 1 to 10, 12 or 13, Tumor Necrosis Factor-alpha and packaging materials therefore and instructions for use.
19. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a nucleic acid according to claim 14, or a composition according to claim 11 or 12 suitable for treating and/or preventing and/or alleviating disorders relating to inflammatory processes.
20. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance which is able to pass through the gastric environment without the substance being inactivated.
21. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the vaginal and/or rectal tract. :
22. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the nose, upper respiratory tract and/or lung.
23. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal mucosa.
24, An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance which is able to pass through the tissues beneath the tongue effectively.
25. An anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, suitable for treating and/or preventing and/or alleviating disorders susceptible to modulation by a TNF-alpha modulating substance which is able to pass through the skin effectively.
26. A method according to claim 16, a kit according to claim 18, or an anti-TNF- alpha polypeptide, nucleic acid, or composition according to any of claims 19 to 25, wherein said disorders are any of inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple : sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type 1, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid ‘arthritis, Sarcoidosis, Scleroderrna, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, and Vasculitis.
27. A composition comprising a nucleic acid according to claim 14, an anti-TNF- alpha polypeptide of any of claims 1 to 10, 12 or 13, or a composition according to claim 11, and a suitable pharmaceutical vehicle.
28. An in vitro method of diagnosing a disorder characterised by the dysfunction of Tumor Necrosis Factor-alpha comprising: a) contacting a sample with an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, b) detecting binding of said polypeptide to said sample, and c) comparing the binding detected in step (b) with a standard, wherein a difference in binding relative to said sample is diagnostic of a disorder characterised by dysfunction of Tumor Necrosis Factor-alpha.
29. Ait for screening for a disorder, wherein said disorder is any of inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, : Autoimmune parotitis, Diabetes Type 1, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, ‘Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies,
® 79 Thyroiditis, and Vasculitis, comprising an isolated anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13, packaging materials therefore and instructions for use.
30. A method for producing an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 comprising the steps of: a) obtaining DNA encoding a Camelidae VHH directed against Tumor Necrosis Factor alpha, b) cloning and expressing the DNA obtained in step (a).
31. A method of producing an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 comprising: a) culturing host cells comprising nucleic acid encoding an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 -or 13, under conditions allowing the expression of the polypeptide, and, b) recovering the produced polypeptide from the culture.
32. A method according to claim 31, wherein said host cells are bacterial or yeast. :
33. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a nucleic acid according to claim 14, or a composition according to claim 11for the preparation of a medicament for treating and/or preventing and/or alleviating disorders relating to inflammatory reactions.
34. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a composition according to claim 11, 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 gastric environment without the substance being inactivated.
35. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a : composition according to claim 11, 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 delivered to the vaginal and/or rectal tract.
® | 80
36. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a + composition according to claim 11, 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 delivered to the nose, upper respiratory tract and/or lung.
37. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a composition according to claim 11, 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 delivered to the intestinal mucosa, wherein said disorder increases the permeability of the intestinal . mucosa. :
38. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a composition according to claim 11, 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 to pass through the tissues beneath the tongue effectively.
39. Use of an anti-TNF-alpha polypeptide of any of claims 1 to 10, 12 or 13 or a composition according to claim 11, 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 to pass through the skin effectively. oo
40. An anti-TNF-alpha polypeptide according to claim I, substantially as herein . described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures.
“41. A composition according to claim 11 or claim 27, substantially as herein : described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures.
® o
42. A nucleic acid according to claim 14, substantially as herein described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures.
43. A method according to any one of claims 15 to 17, substantially as herein described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures.
44. A kit according to claim 18 or claim 29, substantially as herein described with reference to and/or as illustrated by any of the examples and/or the sequence : listings and/or the accompanying Figures.
45. A method according to claim 28, substantially as herein described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures.
46. A method according to claim 30 or claim 31, substantially as herein described with reference to and/or as illustrated by any of the examples and/or the sequence listings and/or the accompanying Figures. Dated this 30" day of April 2008. - z= Adams & Adams : : Applicants Patent Attorneys
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US42507302P | 2002-11-08 | 2002-11-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200803817B true ZA200803817B (en) | 2009-11-25 |
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ID=36077529
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200503618A ZA200503618B (en) | 2002-11-08 | 2005-05-05 | Single domain antibodies directed against Tumour Necrosis Factor-alpha and uses therefor |
| ZA200803817A ZA200803817B (en) | 2002-11-08 | 2008-04-30 | Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor |
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| Application Number | Title | Priority Date | Filing Date |
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| ZA200503618A ZA200503618B (en) | 2002-11-08 | 2005-05-05 | Single domain antibodies directed against Tumour Necrosis Factor-alpha and uses therefor |
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| Country | Link |
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| ZA (2) | ZA200503618B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011277983C1 (en) * | 2010-07-16 | 2016-09-29 | Ablynx Nv | Modified single domain antigen binding molecules and uses thereof |
| CN102372779A (en) * | 2011-10-25 | 2012-03-14 | 李江伟 | Camel single domain antibody with allinase activity as well as preparation method and application thereof |
| CN102660551B (en) * | 2012-02-09 | 2014-04-16 | 陈志南 | VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application thereof |
| JP6391574B2 (en) * | 2012-08-31 | 2018-09-26 | アルゲン−エックス ビーブイビーエー | Method for producing an antibody molecule having interspecific intra-target cross-reactivity |
| EP2895510A2 (en) * | 2012-09-13 | 2015-07-22 | Novartis AG | Single domain antibody with c-terminal modification |
| CN106265480A (en) * | 2016-09-03 | 2017-01-04 | 山西纳安生物科技有限公司 | Nano antibody vaginal drug delivery systems and preparation method and application |
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| CN1735629B (en) | 2012-05-09 |
| CN1735629A (en) | 2006-02-15 |
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