CN1735629B - Single domain antibodies against tumor necrosis factor-alpha and uses thereof - Google Patents
Single domain antibodies against tumor necrosis factor-alpha and uses thereof Download PDFInfo
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- CN1735629B CN1735629B CN200380108511.5A CN200380108511A CN1735629B CN 1735629 B CN1735629 B CN 1735629B CN 200380108511 A CN200380108511 A CN 200380108511A CN 1735629 B CN1735629 B CN 1735629B
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Abstract
The present invention relates to polypeptides derived from single domain heavy chain antibodies directed against tumor necrosis factor-alpha. The invention also relates to single domain antibodies which are camelid VHHs. Also relates to methods of administering the polypeptides. Also contemplated are protocols for screening for agents that modulate the TNF-alpha receptor, and agents resulting from such screening.
Description
Invention field
The invention provides polypeptide, it contains the one or more single domain antibodies to tumor necrosis factor alpha (TNF-α).The invention still further relates to them and be used to the purposes diagnosing and treat.These antibody can have and people's framework sequence height homologous framework sequence.Described and only contained the compsn that tumor necrosis factor alpha (TNF-α) antibody or itself and other drug are united.
Background of invention
Think that tumor necrosis factor alpha (TNF-α) at various diseases, for example, plays an important role in inflammatory diseases such as rheumatoid arthritis, segmental enteritis, ulcerative colitis and multiple sclerosis.Studied in sufficient detail TNF-α and acceptor (CD120a, CD120b).The TNF-α of biologically active form is that tripolymer and the ditch that forms through contiguous subunit are important for cytokine-acceptor interaction.Some strategy and these tactful current various disease states that are used to treat of antagonism effect of cytokines have been developed.
To TNF-α have enough specificitys and optionally the TNF-alpha inhibitor can be effectively prevention or therapeutic drug compounds, it is used for prevention or treats some disease, wherein TNF-α is hinted as the cause of disease.Described and suppressed through Antybody therapy toxicogenic shock (EP 486526), tumour regression, cytotoxicity that (US 6448380 to TNF-α; US 6451983; US 6498237), (EP 663836 for autoimmune disease such as RA and segmental enteritis; US 5672347, and US 5656272), the method for transplant rejection host response (US 5672347), bacterial meningitis (EP 585705).
Yet current available medicine do not have a kind of can treatment autoimmune disease in full force and effect, and most restriction that receives serious toxicity.In addition, exploitation has enough potential and optionally new chemical entities (NCE) is exceedingly difficult and very long process to this target sequence.Therapeutical agent based on antibody has the important potentiality as medicine on the other hand, because their target is had unusual specificity and low intrinsic toxicity.In addition, when with the new chemical entities (NCE) of exploitation when comparing, the development time can significantly shorten.Yet, when the acceptor-binding domains of part is embedded in the ditch (TNF-α comes to this), be difficult to produce conventional antibody to multimeric protein.The heavy chain antibody of describing among the present invention is from Camelidae, and known these these antibody have chamber combination tendency, and (WO 97/49805; People EMBO such as Lauwereys J.17,5312,1998)).Therefore, this heavy chain antibody is suitable for combining the receptors bind structural domain such as the part of TNF inherently.In addition, therefore known this antibody stable for extended periods of time, has increased their storage life (people such as Perez, Biochemistry, 40,74,2001).In addition, use with the mammalian cell cultivation and fermentation and compare cheap expression system, produce this heavy chain antibody fragment (EP 0698097) like yeast or other mikrobes " together " in fermentor tank.
Use from the illness that need regulate inflammation such as antibody and its humanization derivatives for treatment in mouse, sheep, goat, rabbit or the like source because the several reasons existing problems.Conventional antibody is at room temperature unstable, and necessary frozen preparation and preservation, needs necessary freezing experiment chamber equipment, storage and transport, and this has consumed time and expense.Freezing is infeasible sometimes in developing country.In addition, the production of said antibody or to produce on a small scale be expensive, because The expressed and need the high level support of time and equipment by the required mammal cell line system of active antibody, and productive rate is very low.In addition, the large size of conventional antibody will limit, for example, and the tissue penetration at Inflamed tissue position.In addition, the combination activity of conventional antibody depends on pH, and therefore be inappropriate for the environment that is used for outside the common physiological pH scope such as, for example, treatment gastrorrhagia, stomach operation.And conventional antibody is the unstable oral administration that also therefore is inappropriate under low or high pH.Yet, illustrated the anti-harsh condition of Camelidae antibody, like extreme pH, sex change reagent and high temperature people such as (, Protein Science11,500,2002) Dumoulin, therefore make them be suitable for sending through oral administration.In addition, conventional antibody has and combines actively, and it depends on temperature, therefore is inappropriate for being used for mensuration or the test kit under the temperature outside the biological activity TR (for example, 37 ± 20 ℃), implemented.
The polypeptide therapeutical agent, especially the therapeutical agent based on antibody has the important potentiality as medicine, because they have unusual specificity and low intrinsic toxicity to their target.Yet the known treatment modification that upward antibody of useful target need be extra that is directed against that has obtained of technician is used it for people's treatment with preparation, is applied to the undesired immunological response that man-hour takes place thereby avoid working as in individual human.This modification is commonly called " humanization ".The technician be known in antibody that the species that are different from the people produce need humanization in case make this antibody can be used for people's Antybody therapy ((1) CDR grafting: Protein Design Labs:US 6180370, US 5693761; Genentech US6054297; Celltech:460167, EP 626390, and US 5859205; (2) veneer (veneering): Xoma:US 5869619, and US 5766886, and US 5821123).Need a kind of method that produces antibody, this method has been avoided the humanized needs of essence, does not perhaps need humanization fully.Need one type of new antibody, these antibody to have clear and definite framework region or amino-acid residue and can be applied to people experimenter and do not need the essence humanization, perhaps do not need humanization.
The important defective of another of conventional antibody is that they are complicated macromole and therefore unstable relatively, and they are responsive to proteasome degradation.This means the conventional antibody medicine can not per os, hypogloeeis, part, nose, vagina, rectum or suck use, because they can not resist the effect and/or because their large size of proteolytic enzyme in low pH, these positions and the blood at these positions.They must be used to overcome some problems of these problems through injection (intravenously, subcutaneous, etc.).Using through injection needs the expert to train so that correctly and safely use syringe or pin.Also need the liquid preparation of sterile equipment, therapeutical peptide, said polypeptide is aseptic and the bottle of stable form is packed and, needs the suitable position of pin entering for the experimenter.In addition, before accepting injection when injecting the experimenter experience physiology and psychological pressure usually.Therefore, need a kind of method of delivery treatments polypeptide, this method has been avoided the needs to injection, and this method is cost saving/time not only, and more convenient and more comfortable to the experimenter.
Antybody therapy based on the single structure territory has the important potentiality as medicine, because they have unusual specificity and low intrinsic toxicity to their target.Yet the inherence and the function avidity that further improve them cause the many benefits to the patient, like littler therapeutic dose, treatment and littler spinoff faster.
Goal of the invention
The funtion part that the purpose of this invention is to provide the homologue of the polypeptide that comprises the one or more single domain antibodies that combine TNF-α, the homologue of said polypeptide, said polypeptide.When combining TNF-α, said polypeptide changes the BA of TNF-α.Said polypeptide can be incorporated into acceptor-combination ditch of TNF-α, perhaps can be not joined in the receptors bind ditch.This peptide species is a single domain antibody.
Another object of the present invention provides single domain antibody, and it can be any single domain antibody or the single domain antibody in arbitrary future in this area.Instance includes, but not limited to heavy chain antibody, naturally do not have the antibody of light chain, from conventional 4-chain antibody deutero-single domain antibody, engineered antibody and single structure territory support, it is different from from those supports of antibody deutero-.According to an aspect of the present invention, the single domain antibody that place like this is used is naturally occurring single domain antibody, and it is known as the heavy chain antibody (WO 9404678) that lacks light chain.In order to illustrate, be called as VHH or nanobody with conventional VH difference with itself and four chain Tegelines from this variable domains of heavy chain antibody deutero-that lacks light chain.This VHH molecule can come comfortable Camelidae (Camelidae) species, the antibody that for example in camel, llama, dromedary camel, alpaca and pack horse, produces.
Another object of the present invention provides intravenously, subcutaneous, per os, hypogloeeis, part, intranasal, vagina, rectum or uses the method for anti-TNF-α polypeptide through suction.
Another object of the present invention is the binding affinity that strengthens the unit price single domain antibody.
Summary of the invention
One embodiment of the invention are the anti-TNF-α polypeptide that contain at least a anti-TNF-α single domain antibody.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and wherein single domain antibody is corresponding to the sequence of SEQ ID NOs:1 to 16 and 79 to 84 any representative.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and it also contains at least a single domain antibody of serum proteins.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and wherein said serum proteins is one of serum albumin, serum immune globulin, TBP, transferrin or fibrinogen.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and wherein single structure territory antiserum(antisera) albumen single domain antibody is corresponding to the sequence of SEQ ID NOs:26 to 29 and 85 to 97 any representative.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and it is corresponding to the sequence of one of SEQID NOs:30 to 43 representative.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and it also contains at least a single domain antibody that is selected from anti--IFN-γ single domain antibody, anti-TNF-α acceptor single domain antibody and anti--IFN-γ acceptor single domain antibody.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and the number that wherein is directed against the single domain antibody of TNF-α is at least 2.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and it is corresponding to the sequence of one of SEQID NOs:73 to 76 representative.
Another embodiment of the invention is aforesaid anti-TNF-α polypeptide, and wherein at least one single domain antibody is humanized Camelidae VHH.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and wherein humanization Camelidae VHH is corresponding to the sequence of one of SEQ ID NOs:17 to 19 and 21 to 24 representative.
Another embodiment of the present invention is a compsn; It contains as above the anti-TNF-α polypeptide described and from least a single domain antibody of anti--IFN-γ single domain antibody, anti-TNF-α acceptor single domain antibody and anti--IFN-γ acceptor single domain antibody, is used for simultaneously, separately or sequentially be applied to the experimenter.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and perhaps like above-mentioned compsn, wherein at least a resisting-IFN-γ single domain antibody is corresponding to the sequence of one of SEQ ID NOs:44 to 72 representative.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and perhaps like above-mentioned compsn, wherein said single domain antibody is the funtion part of homologous sequence, funtion part or the homologous sequence of total length single domain antibody.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and perhaps like above-mentioned compsn, wherein anti-TNF-α polypeptide is the funtion part of homologous sequence, funtion part or the homologous sequence of the anti-TNF-α of total length polypeptide.
Another embodiment of the present invention is the anti-TNF-α polypeptide of as above describing, and perhaps like above-mentioned compsn, wherein at least a single domain antibody is Camelidae VHH.
Another embodiment of the present invention is the nucleic acid of coding as above-mentioned anti-TNF-α polypeptide.
Another embodiment of the present invention is to identify a kind of compositions and methods; This reagent is regulated the anti-TNF-α polypeptide of as above description and combining of tumor necrosis factor-alpha; The method comprising the steps of: the anti-TNF-α polypeptide that (a) will as above describe and target tumor necrosis factor-alpha are when existing and do not have candidate modulator; Contact under said polypeptide and the target bonded condition allowing; (b) polypeptide of measuring process (a) and the combination between the target, the wherein combination when not having said candidate modulator combines to reduce in the presence of said candidate modulator; Said candidate modulator is accredited as a kind of reagent, and this reagent is regulated the anti-TNF-α polypeptide of as above description and combining of tumor necrosis factor-alpha.
Another embodiment of the present invention is to identify a kind of compositions and methods; This reagent is through the disease that combines to regulate the tumor necrosis factor-alpha mediation of anti-TNF-α polypeptide with the tumor necrosis factor-alpha of as above description; This method comprises: the anti-TNF-α polypeptide that (a) will as above describe and target tumor necrosis factor-alpha are when existing and do not have candidate modulator; Contact under said polypeptide and the target bonded condition allowing; (b) polypeptide of measuring process (a) and the combination between the target, the wherein combination when not having said regulator combines to reduce in the presence of said candidate modulator; Said candidate modulator is accredited as a kind of reagent, and this reagent is regulated the disease of tumor necrosis factor-alpha mediation.
Another embodiment of the present invention is to identify a kind of compositions and methods; This reagent combines combining of adjusting tumor necrosis factor-alpha and its acceptor through the anti-TNF-α polypeptide as above described and tumor necrosis factor-alpha; This method comprises: the anti-TNF-α polypeptide that (a) will as above describe and target tumor necrosis factor-alpha are when existing and do not have candidate modulator; Contact under said polypeptide and the target bonded condition allowing; (b) polypeptide of measuring process (a) and the combination between the target wherein when not having said candidate modulator, combine to reduce in the presence of said candidate modulator; Said candidate modulator is accredited as a kind of reagent, and this reagent adjusting tumor necrosis factor-alpha combines with its acceptor.
Another embodiment of the present invention is to screen the test kit of the reagent of the disease of regulating the tumor necrosis factor-alpha mediation, and this test kit contains anti-TNF-α polypeptide and the tumor necrosis factor-alpha of as above describing.
Another embodiment of the present invention is a kind of unknown reagent of identifying through according to aforesaid method, and this reagent is regulated the anti-TNF-α polypeptide of as above description and combining of tumor necrosis factor-alpha.
Another embodiment of the present invention is a kind of unknown reagent of identifying through according to aforesaid method, and this reagent is regulated the disease of tumor necrosis factor-alpha mediation.
Another embodiment of the present invention is a kind of unknown reagent of as above describing, and wherein said disease is one or more of inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.
Another embodiment of the present invention is the anti-TNF-α polypeptide as above described, or the nucleic acid as above described, or the compsn as above described, the reagent of perhaps as above describing, and they are used to treat and/or prevent and/or alleviate and the inflammatory process diseases associated.
Another embodiment of the present invention is the anti-TNF-α polypeptide as above described or the nucleic acid of as above describing, or the compsn as above described; The purposes of the reagent of perhaps as above describing is used to prepare and treats and/or prevents and/or alleviate the medicine with the inflammatory reaction diseases associated.
Another embodiment of the present invention is the anti-TNF-α polypeptide as above described or the compsn as above described, be used to treat and/or prevent and/or alleviate be subject to TNF-α and regulate the disease that material is regulated, this material can pass gastric environment and this material not by inactivation.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used for the preparation treatment; Prevention and/or alleviate is subject to the medicine that TNF-α regulates the disease symptoms that material regulates, this material can pass gastric environment and this material not by inactivation.
Another embodiment of the present invention is anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to treat and/or prevent and/or alleviate the disease that is subject to the adjusting of TNF-α adjusting material, this TNF-α regulates material and is delivered to vagina and/or rectum road.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to prepare treatment, prevent and/or alleviate and be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates, this TNF-α regulates material and is delivered to vagina and/or rectum road.
Another embodiment of the present invention is anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to treat and/or prevent and/or alleviate the disease that is subject to the adjusting of TNF-α adjusting material, this TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to prepare treatment, prevent and/or alleviate be subject to the medicine that TNF-α regulates the disease symptoms of material adjusting, this TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
Another embodiment of the present invention is anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to treat and/or prevent and/or alleviate and be subject to the disease that TNF-α regulates the material adjusting; This TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease has increased the perviousness of intestines mucosa.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to prepare treatment, prevent and/or alleviate the medicine of the disease symptoms that is subject to the adjusting of TNF-α adjusting material; This TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease has increased the perviousness of intestines mucosa.
Another embodiment of the present invention is anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to treat and/or prevent and/or alleviate the disease that is subject to the adjusting of TNF-α adjusting material, this TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to prepare treatment, prevent and/or alleviate be subject to the medicine that TNF-α regulates the disease symptoms of material adjusting, this TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
Another embodiment of the present invention is anti-TNF-α polypeptide of as above describing or the compsn of as above describing, and is used to treat and/or prevent and/or alleviates be subject to the disease that TNF-α regulates the material adjusting, and this TNF-α instrumentality mass-energy is enough effectively passed skin.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing or the compsn of as above describing; Be used to prepare treatment, prevent and/or alleviate be subject to the medicine that TNF-α regulates the disease symptoms of material adjusting, this TNF-α instrumentality mass-energy is enough effectively passed skin.
The purposes of the purposes of the compsn that another embodiment of the present invention is the purposes of the method as above described, the test kit of as above describing, the nucleic acid of as above describing or reagent, the nucleic acid of as above describing or reagent, the compsn of as above describing, as above describe, anti-TNF-α polypeptide of as above describing, anti-TNF-α polypeptide of as above describing; Wherein said disease is one of inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA (spondyloarthropathies), thyroiditis and vasculitis.
Another embodiment of the present invention is a compsn, and it contains as above the nucleic acid described or reagent, anti-TNF-α polypeptide of as above describing, or compsn and the appropriate drug carrier as above described.
Another embodiment of the present invention is the diagnostic method of disease that is characterized as the dysfunction of tumor necrosis factor-alpha; This method comprises: (a) sample is contacted with anti-TNF-α polypeptide of as above describing; (b) detect combining of said polypeptide and said sample; With detected combination and standard in the step (b) relatively, can diagnostic characteristic be the disease of the dysfunction of tumor necrosis factor-alpha wherein (c) with respect to the difference in the combination of said sample.
Another embodiment of the present invention is to use the method for as above describing to screen the test kit of the disease of as above quoting.
Another embodiment of the present invention is to screen the test kit of the disease of as above quoting, and this test kit contains separative anti-TNF-α polypeptide of as above describing.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing, is used for the said tumor necrosis factor-alpha of purifying.
Another embodiment of the present invention is the purposes of anti-TNF-α polypeptide of as above describing, is used to suppress the interaction between tumor necrosis factor-alpha and one or more tumor necrosis factor-alpha acceptors.
Another embodiment of the present invention is the method that produces as above anti-TNF-α polypeptide of describing, and the method comprising the steps of: (a) obtain encoding to the double-stranded DNA of the Camelidae VHH of tumor necrosis factor-alpha, (b) clone and express selected DNA in the step (b).
Another embodiment of the present invention is the method that produces anti-TNF-α polypeptide of as above describing; The method comprising the steps of: (a) reclaim the polypeptide that is produced at the host cell of the nucleic acid that allows to cultivate under the condition of this expression of polypeptides anti-TNF-α polypeptide that containing to encode as above describes with (b) from culture.
Another embodiment of the present invention is the method for as above describing, and wherein said host cell is bacterium or yeast.
Another embodiment of the present invention is the test kit that contains as above anti-TNF-α polypeptide of describing, and this test kit is used to screen any of inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome or multiple sclerosis.
Accompanying drawing and subordinate list summary
The contrast of the anti-people TNF VHH that describes among Fig. 1 such as the embodiment 1.
The dilution series of the anti-humanTNF-VHH that checks among the ELISA of Fig. 2 according to embodiment 1.
Fig. 3 uses the antagonistic action of the VHH that the human cell line KYM according to embodiment 1 measures in CTA.
The extracorporeal receptor of Fig. 4 wild-type VHH# 12B and two mutants A74S+Y76N+K83R+P84A combines to measure.
The extracorporeal receptor of Fig. 5 wild-type VHH# 12B and two mutants 1E+Q5LA74S+Y76N+K83R+P84A combines to measure.
Combination among the ELISA of Fig. 6 wild-type VHH# 3E and two mutants VHH.
The extracorporeal receptor of Fig. 7 wild-type VHH# 3E and two mutants VHH combines to measure.
The contrast of the anti-mouse TNF of antagonism property that describes among Fig. 8 such as the embodiment 3.
Fig. 9 uses the antagonistic action of the anti-mouse TNF VHH that the mouse cell line L929 according to embodiment 3 measures in CTA.
The EcoRI-HindIII that Figure 10 is used to produce the carrier pAX11 (pUC119 main chain) of divalence or two special VHH inserts fragment.
The painted PAGE of coomassie (15%) of the unit price of Figure 11 IMAC purifying-(swimming lane 8), divalence-(swimming lane 1), trivalent-(swimming lane 2,3 and 5) and tetravalence (swimming lane 4,6 and 7) anti-TNF a VHH.
Figure 12 through unit price-, divalence-, the tomographic map analyzed of trivalent and the tetravalence VHH gel-filtration on Superdex 75HR.
The unit price of the anti-people TNF of Figure 13 VHH-, divalence-, the comparison of the antagonism characteristic of the product infliximab (Remicade) of trivalent and tetravalence form and clinical use and rhu TNFR:Fc (Enbrel).
Figure 14 to the unit price of mouse TNF α-with the agonistic behavior of divalence VHH.
Figure 15 is from the painted PAGE of coomassie of the human IgG1's who describes among the embodiment 4 VHH-Fc-fusions.
The antagonism usefulness of comparing VHH# 3E deutero-VHH-Fc fusions with the bivalent form of VHH# 3E that Figure 16 confirms in biological assay.
The ELISA (100%, the signal under 1/100 dilution measured) of the TNF3E of Figure 17 reference and pepsin under pH 2.2, pH 3.2 and pH 4.2.
Figure 18 tests setting
Table 1 is to the aminoacid sequence of the peptide of the each side of the present invention of TNF-α.
Table 2 is used for the tabulation of mutagenesis reaction, mutagenic primer and the template of the mutagenesis of VHH# 12B.
Table 3 is used for the tabulation of mutagenesis reaction, mutagenic primer and the template of the mutagenesis of VHH# 3E.
The overview of table 4 humanization and wild-type VHH.
The anti-mice serum white protein of table 5/anti-TNF-α.
Table 6 is to the aminoacid sequence table of the VHH of people IFN-γ.
Table 7 to the divalence of TNF-α (BIV 3E, BIV#m3F), the sequence of trivalent (TRI3E) or tetravalence (TETRA 3E) VHH.
Portion homologous property between the aminoacid sequence of table 8 anti-mice serum white protein VHH of the present invention.
Portion homologous property between table 9 anti-TNF of the present invention-α VHH.
Table 10 of the present invention anti--IFN-γ VHH between the per-cent homology.
Table 11 treatment procedure table
Detail
The present invention relates to anti-tumor necrosis factor-α (TNF-α) polypeptide, it contains the one or more single domain antibodies to TNF-α.The invention still further relates to can coding said polypeptide nucleic acid.
Single domain antibody is such antibody, and their complementary determining region is the part of single structure domain polypeptide.Instance includes, but not limited to heavy chain antibody, naturally do not have the antibody of light chain, from conventional 4-chain antibody deutero-single domain antibody, engineered antibody and single structure territory support, it is different from from those supports of antibody deutero-.Single domain antibody can be any single domain antibody or the single domain antibody in arbitrary future in this area.Single domain antibody can include, but not limited to mouse, people, camel, llama, goat, rabbit, ox from any species.According to an aspect of the present invention, single domain antibody used herein is the naturally occurring single domain antibody that is called the heavy chain antibody of no light chain.This single domain antibody is for example being described among the WO 94/04678.In order to illustrate, be called as VHH or nanobody from natural this variable domains of heavy chain antibody deutero-that lacks light chain, with conventional VH difference with itself and four chain Tegelines.This VHH molecule can come comfortable Camelidae species, the antibody that for example in camel, dromedary camel, llama, alpaca and pack horse, produces.Other species except Camelidae can produce the natural heavy chain antibody that lacks light chain; This VHHs within the scope of the invention.
According to the present invention and the known VHHs of technician be the Tegeline deutero-weight chain variable structural domain that lacks light chain from natural, as describe among the WO 94/04678 from Camelidae deutero-those (after this being called VHH structural domain or nanobodies).The VHH molecule is less than about 10 times of IgG molecule.They are single polypeptides and highly stable, anti-extreme pH and temperature condition.In addition, the effect of their protease inhibitors, and conventional antibody can not the protease inhibitor effect.In addition, the vivoexpression of VHHs produces folding function VHHs high yield, correct.In addition; The antibody that in Camelids, produces will be discerned some epi-position, and these epi-positions are different from through using antibody library to be different from the epi-position (WO 9749805) that antibody that the Mammals of Camelids produces is discerned at the antibody of external generation or through immunity.Equally, anti-TNF-α VHH can more effectively interact with TNF-α than conventional antibody, thereby more effectively blocks the interaction of itself and TNF-α acceptor.
According to the present invention, TNF-α is from any species.The instance of the species relevant with the present invention comprises rabbit, goat, mouse, rat, milk cow, calf, camel, llama, monkey, donkey, cavy, pig, chicken, sheep, dog, cat, horse and people preferably.
TNF-α also is the fragment of TNF-α, and it can cause immunne response.TNF-α also is the fragment of TNF-α, and it can combine the single domain antibody to total length TNF-α generation.
Single domain antibody to TNF-α refers to single domain antibody, and it can combine TNF-α with the avidity that is better than 10-6M.
One embodiment of the invention are anti-TNF polypeptide, and wherein single domain antibody contains the Camelidae VHH to TNF-α.
One or more single domain antibodies to the anti-TNF polypeptide of TNF-α can be identical sequences.Alternatively, they can not be all to have identical sequence.Within the scope of the invention, the anti-TNF polypeptide contains anti-TNF-α single domain antibody, and these antibody not all have identical sequence, but they are to identical target, its one or more antigens.
Another embodiment of the present invention is anti-TNF-α polypeptide, and wherein single domain antibody is corresponding to the sequence of one of SEQ ID NOs:1 to 16 as shown in table 1 and 79 to 84 representative.Said sequence is from Camelidae heavy chain antibody (VHHs), and these antibody are to TNF-α.
The invention still further relates to anti-TNF-α polypeptide, wherein said single domain antibody is the VHH to TNF-α, and wherein VHH belongs to the classification with similar human sequence.Such other be characterised in that VHHs carries an amino acid that is selected from glycocoll, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine(Phe), tyrosine, tryptophane, methionine(Met), Serine, Threonine, l-asparagine or Stimulina at 45; As; For example; L45, and 103 carry tryptophane (according to Kabat numbering).The new classification of the Camelidae single domain antibody of describing among the present invention (table 1, embodiment 1) is by VHH#2B (SEQ ID NO:3) and VHH# 12B (SEQ ID No.14) representative, and they contain hydrophobic residue and are the hydrophobic residue tryptophane at 103 in FR2.
In WO03035694, described by sequence VHH# 1A (SEQ ID NO.1), VHH#4B (SEQ ID NO.12), VHH#8-29 (SEQ ID NO.81), VHH#8-41 (SEQID NO.82), VHH#8-42 (SEQ ID NO.83) and VHH#8-44 (SEQ ID NO.84) (table 1; Embodiment 1) another similar people's the classification of the Camelidae single domain antibody of representative; And these antibody contain the hydrophobic FR2 residue of in people source or the conventional antibody from other species, finding usually; But the charged l-arginine through on 103 has remedied hydrophilic loss, and this arginine residues has been replaced the conservative tryptophan residue that exists among the VH from double-stranded antibody.Equally, belong to this peptide of two types and demonstrate the height amino acid sequence homology with people VH framework region, and said peptide can directly be applied to the people and can not expect and undesired immunne response occurs, and do not had further humanized burden.The invention still further relates to can coding said polypeptide nucleic acid.
Therefore; One side of the present invention allows its patient of needs is directly used anti-TNF-α polypeptide; Wherein single domain antibody belongs to the humanization classification of VHH, and contains the sequence by SEQ ID NO:1, one of 3,12,14,81,82,83 and 84 representatives.
Any of the VHHs that the present invention is used can be the General category or similar people's Camelidae antibody classification.Said antibody can be to complete TNF-α or its fragment, the perhaps fragment of its homologous sequence.These polypeptide comprise total length Camelidae antibody, i.e. Fc and VHH structural domain, the chimeric form of heavy chain Camelidae antibody and people Fc structural domain or VHH self or deutero-fragment.
Antiserum(antisera) white protein VHH can be to interact than more effective mode of conventional antibody and serum albumin, and known serum albumin is a carrier proteins.As carrier proteins, some epi-positions of serum albumin can not be approaching by bonded albumen, peptide and micromolecular compound.Because known VHH is attached in " uncommon " or unconventional epi-position such as chamber (WO 97/49805), so the albuminised avidity of this VHH and round-robin can be increased.
The invention still further relates to following discovery: also contain one or more single domain antibodies to one or more serum proteinss of experimenter like anti-TNF polypeptide described herein; Compare this anti-TNF polypeptide half life significant prolongation astoundingly that in said experimenter, circulates with the half life of anti-TNF-α single domain antibody that is not the part of said construct.The instance of these polypeptide is represented through SEQ ID NOs:30 to 43 in table 5.In addition, find that said polypeptide demonstrates the identical favourable character of single domain antibody, such as the high stability that in mouse, is kept perfectly, extreme pH resistance, high-temperature stability and high target avidity.
Another embodiment of the present invention is the anti--TFN-α polypeptide that also contains to one or more single domain antibodies of one or more serum proteinss, and said resisting-TFN-α polypeptide contains the sequence by any representative of SEQ IDNOs:30 to 43 (table 5).
Another embodiment of the present invention is anti--TFN-α polypeptide, and wherein antiserum(antisera) albumen single domain antibody is corresponding to the sequence like the representative of one of SEQ ID NOs:26 to 29 in the table 5 and 85 to 97.
Serum proteins can be the arbitrary suitable albumen of finding in experimenter's serum.In one aspect of the invention, serum proteins is serum albumin, serum immune globulin, TBP, transferrin or fibrinogen.According to the purposes of being planned, like the compartmentation of desired half life of effective treatment and/or target antigen, the VHH-mating partner can be to one of top serum proteins.
Another aspect of the present invention be as here disclosed anti--TFN-α polypeptide, it also contains at least one polypeptide, said polypeptide is selected from anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides.
One embodiment of the invention are to be directed against the sequence of the single domain antibody of IFN-γ corresponding to any representative of SEQID NOs:44 to 72 as shown in table 6.
According to an aspect of the present invention, single domain antibody is to TNF-α acceptor.Said single domain antibody can be Camelidae VHH.
According to an aspect of the present invention, single domain antibody is to IFN-γ acceptor.Said single structure territory can be Camelidae VHH.
Another aspect of the present invention is treatment as the autoimmune disease of here quoting or the method for illness; This method comprises anti-TNF-α polypeptide of the patient being used significant quantity; This polypeptide also contains at least a polypeptide that is selected from anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides, and these polypeptide such as following description interconnect.
This polyspecific construct can have the bigger potential as the inflammation treatment compound than monospecific construct.
One side of the present invention is a compsn; Said composition contains like disclosed anti-TNF-α polypeptide here and at least a polypeptide that is selected from anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides, be used for simultaneously, separately or order be applied to the experimenter.
One side of the present invention is the method for treatment autoimmune disease, and this method comprises to individuality simultaneously, separately or sequentially use the anti-TNF-α polypeptide of significant quantity and be selected from least a polypeptide of anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides.
Another aspect of the present invention is a test kit, and it contains at least a polypeptide that is useful on simultaneously, separately or in proper order is applied to anti-TNF-α polypeptide of experimenter and is selected from anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides.One aspect of the present invention be can be used according to the invention test kit.One side of the present invention is that test kit can be used for treating the disease of quoting here.
Use simultaneously and refer to that polypeptide is applied to the experimenter simultaneously.For example, the compsn that perhaps comprises said polypeptide as polypeptide mixture.Instance includes, but are not limited to solution, tablet, liquid, the local creme of intravenous administration, or the like, wherein every kind of preparation all contains target polypeptides.
Separate administration refers to that the perhaps substantially the same at the same time time of polypeptide is applied to the experimenter.These polypeptide are present in the test kit as independent, unmixing preparation.For example, different polypeptides can be used as independent tablet and is present in the test kit.Through simultaneously or tablet connect a ground and swallow tablet is applied to the experimenter.
Order is used and is referred to that polypeptide is applied to the experimenter by order.Polypeptide is present in the test kit as independent, unmixing preparation.Lifetime at interval between the preparation.Used when for example, a peptide species can reach 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 another component applied.
Use in order, a peptide species can and/or be used once before another polypeptide used afterwards, perhaps arbitrary number of times and use with various dosage.The order use can with use associating simultaneously or sequentially.
The medical use of following anti-TNF-α polypeptide also is applied to contain like the compsn of disclosed anti-TNF-α polypeptide and at least a polypeptide that is selected from anti--IFN-γ polypeptide, anti-TNF-α receptor polypeptides and anti--IFN-γ receptor polypeptides here, be used for simultaneously, separately or order be applied to like the disclosed experimenter of preceding text.
According to an aspect of the present invention, the anti-TNF-α of anti-IFN-γ polypeptide single domain antibody is to IFN-γ.Said single domain antibody can be Camelidae VHH.
One embodiment of the invention are sequences of representing corresponding to one of SEQ ID NOs:44 to 72 as shown in table 6 to the single domain antibody of IFN-γ.
According to an aspect of the present invention, anti-TNF-α single domain antibody is to TNF-α acceptor.Said single domain antibody can be Camelidae VHH.
According to an aspect of the present invention, the anti-TNF-α of anti-IFN-γ receptor polypeptides single domain antibody is to IFN-γ acceptor.Said single domain antibody can be Camelidae VHH.
Another embodiment of the present invention is like disclosed anti-TNF-α polypeptide here, and wherein the number to the single domain antibody of TNF-α is two or more.The anti-TNF-α of this multivalence polypeptide is compared the uncommon high function avidity that has target, the advantage that demonstrates the inhibition activity higher than the inhibition activity of expection with their unit price counterpart.
The function avidity of multivalence anti-TNF-α polypeptide is than unit price parent anti-TNF-high several magnitude of α polypeptide.The function avidity that the inventor has been found that these multivalence polypeptide is more much higher than the avidity of reporting about divalence and multivalent antibody in the prior art.Astoundingly, anti-TNF of the present invention-α polypeptide directly interconnects (SEQ ID No.77 and 78) or is connected through the short circuit head sequence, and these polypeptide demonstrate the high function avidity that conventional four chain antibodies of multivalence are predicted in theory.
The inventor has been found that and preferably uses the antigen of being made up of Multidomain and multimeric protein, directly combining to measure perhaps functional examination, for example can detect this remarkable enhanced functionally active in the CTA.
Another embodiment of the present invention is like disclosed anti-TNF-α polypeptide here, and wherein the number to the single domain antibody of TNF α is two or more, and said anti-TNF-α polypeptide contains the sequence corresponding to any representative of SEQ ID NOs:73 to 76.
Utilize the method in method as known in the art or any future can single domain antibody be coupled together and form arbitrary polypeptide disclosed herein, this polypeptide contains more than one single domain antibody.For example, through chemically crosslinked with people such as amino-acid residue and Blattler, Biochemistry24,1517-1524; The described organic derivatizing agent reaction of EP294703 can be merged these single domain antibodies.Alternatively, can be on dna level the gene fusion single domain antibody, that is, form polynucleotide constructs, its coding contains the complete polypeptide construct of one or more anti-target single domain antibodies and one or more antiserum(antisera) albumen single domain antibodies.The method that produces divalence or multivalence VHH polypeptide construct is open in PCT patented claim WO 96/34103.A kind of method that connects a plurality of single domain antibodies is the single domain antibody encoding sequence directly to be connected or connect through peptide linker through the gene approach.For example, the C-end of first single domain antibody can be connected to the N-end of next single domain antibody.Can be with the expansion of this connection mode so that connect extra single domain antibody making up and to produce three formation function bodies, four formation function bodies, or the like.
According to an aspect of the present invention, single domain antibody is directly interconnected, and does not use joint.Opposite with the conventional antibody that is connected big volume (wherein needing joint sequence) to keep two combinations in the subunit active; Polypeptide of the present invention can directly connect (SEQ ID No.77 and 78); Thereby avoided the potential problems of joint sequence, the antigenicity when being applied to the human experimenter, the instability of joint sequence cause subunit to be dissociated.
According to a further aspect in the invention, single domain antibody interconnects through the peptide linker sequence.This joint sequence can be the sequence that naturally occurring sequence or non-natural exist.Expection joint sequence right and wrong in the experimenter that this anti-TNF-the α polypeptide is used are immunogenic.Joint sequence can provide enough flexibilities to multivalence anti-TNF-α polypeptide, can resist proteolytic degradation simultaneously.A limiting examples of joint sequence is can be derived from the joint sequence of the hinge area of the VHHs that describes among the WO96/34103.
According to a further aspect in the invention, containing the multivalence single domain antibody of two above single domain antibodies can be directly or interconnect through joint sequence.Be difficult to produce this construct and because big volume subunit sterically hindered with conventional antibody; Functional will the forfeiture perhaps greatly reduces; Rather than compare the functional remarkable increase of seeing with VHH of the present invention (, seeing Figure 12) with the unit price construct for the gel-filtration analysis of this multivalence VHH construct.
The technician can prepare polypeptide construct disclosed herein according to method as known in the art or any method in the future.For example, use method as known in the art,, perhaps utilize the library of Protocols in Molecular Biology clone single domain antibody as known in the art and can obtain VHH through the phage display selection subsequently as obtaining hybridoma through immune camel with from camel.
According to an aspect of the present invention, anti-TNF-α polypeptide can be the homologous sequence of total length anti-TNF-α polypeptide.According to a further aspect in the invention, anti-TNF-α polypeptide can be total length anti-TNF-α polypeptide funtion part.According to a further aspect in the invention, anti-TNF-α polypeptide can be the homologous sequence of total length anti-TNF-α polypeptide.According to a further aspect in the invention, anti-TNF-α polypeptide can be the funtion part of the homologous sequence of total length anti-TNF-α polypeptide.According to an aspect of the present invention, anti-TNF-α polypeptide can contain the sequence of anti-TNF-α polypeptide.
According to an aspect of the present invention, the single domain antibody that is used to form anti-TNF-α polypeptide can be complete single domain antibody (for example, VHH) or its homologous sequence.According to a further aspect in the invention, the single domain antibody that is used to form polypeptide construct can be the funtion part of complete single domain antibody.According to a further aspect in the invention, the single domain antibody that is used to form polypeptide construct can be the homologous sequence of complete single domain antibody.According to a further aspect in the invention, the single domain antibody that is used to form polypeptide construct can be the funtion part of the homologous sequence of complete single domain antibody.
Used as indicated, homologous sequence of the present invention can contain one or more amino acid whose addings, disappearance or displacement, and these addings, disappearance or displacement do not change the functional character of polypeptide of the present invention not substantively.Aminoacid deletion or metathetical number preferably reach 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69 or 70 amino acid.
According to homologous sequence of the present invention can be that said modification is compared with the polypeptide of unmodified and do not changed functional character in fact through amino acid whose adding, disappearance or displacement modified polypeptides.
According to homologous sequence of the present invention can be that said modification is compared with the polypeptide of unmodified and do not changed functional character in fact through amino acid whose adding, disappearance or displacement modified polypeptides.
According to homologous sequence of the present invention can be present in other Camelidae species as, for example, the sequence in camel, dromedary camel, llama, alpaca, the pack horse etc.
When homologous sequence is pointed out sequence identity; Sequence of its expression and parental array have highly sequence identity (more than 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) and preferred feature for to have similar character with parental array; Be avidity, said identity calculates for utilizing known method.
Alternatively, homologous sequence can also be the arbitrary aminoacid sequence that obtains according to the displacement that following formula allows from the position of any number of parental array:
Ser is by Ser, Thr, Gly and Asn displacement;
Arg is replaced by one of Arg, His, Gln, Lys and Glu;
Leu is replaced by one of Leu, Ile, Phe, Tyr, Met and Val;
Pro is replaced by one of Pro, Gly, Ala and Thr;
Thr is replaced by one of Thr, Pro, Ser, Ala, Gly, His and Gln;
Ala is replaced by one of Ala, Gly, Thr and Pro;
Val is replaced by one of Val, Met, Tyr, Phe, Ile and Leu;
Gly is replaced by one of Gly, Ala, Thr, Pro and Ser;
Ile is replaced by one of Ile, Met, Tyr, Phe, Val and Leu;
Phe is replaced by one of Phe, Trp, Met, Tyr, Ile, Val and Leu;
Tyr is replaced by one of Tyr, Trp, Met, Phe, Ile, Val and Leu;
His is replaced by one of His, Glu, Lys, Gln, Thr and Arg;
Gln is replaced by one of Gln, Glu, Lys, Asn, His, Thr and Arg;
Asn is replaced by one of Asn, Glu, Asp, Gln and Ser;
Lys is replaced by one of Lys, Glu, Gln, His and Arg;
Asp is replaced by one of Asp, Glu and Asn;
Glu is replaced by one of Glu, Asp, Lys, Asn, Gln, His and Arg;
Met is replaced by one of Met, Phe, Ile, Val, Leu and Tyr.
Can refer to 50,100,200,300,400,500,600,800 or 1000 nucleotide sequences more than the Nucleotide according to homologous nucleotide sequence of the present invention; Its at stringent hybridization condition (like people such as Sambrook; Molecular Cloning, Laboratory Manuel, Cold Spring; Harbor Laboratory press, the hybridization conditions of describing among the New York) down can with the reverse complementary sequence hybridization of the nucleotide sequence of the parental array of can encoding.
Used as indicated, funtion part refers to the sequence of single domain antibody, interacts that to remain on avidity be 1 * 10 thereby this sequence has enough size targets
-6M or better.
Alternatively, funtion part contains the excalation of complete amino acid sequence and still keeps combining target and combining site and the protein domain required with its interaction.
Used as indicated; Funtion part refer to complete sequence below 100% (for example; 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, etc.), but 5 or more amino acid or 15 or polynucleotide more contained.
The target of being mentioned among this paper such as TNF-α, TNF-α acceptor, serum proteins (for example serum albumin, serum immune globulin, TBP, transferrin, fibrinogen) and IFN-γ, IFN-γ acceptor can be the fragments of said target.Thereby target also is the fragment of said target, and it can cause immunne response.Target still is the fragment of said target, and it can combine with the single domain antibody that produces to the total length target.
As fragment used among this paper refer to sequence below 100% (for example; 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), but contain 5,6,7,8,9,10,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or amino acids more.Thereby fragment sufficiently long target interacts and remains on avidity is 1 * 10
-6M or better.
Fragment as used herein also refers to one or more amino acid whose optional insertions, disappearance and displacement, and these insert, lack and displacement does not change target and the single domain antibody bonded ability that produces to the wild-type target not substantively.Aminoacid insertion disappearance or metathetical number preferably reach 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69 or 70 amino acid.
Homologous sequence of the present invention can comprise by humanized anti-TNF-α polypeptide.The humanization of the antibody of the new classification of VHH will make that the unwanted immunoreactive possibility in the human individual further reduces when using.
One embodiment of the invention relate to the method for preparation based on the modified polypeptides of llama antibody; Through confirming the amino-acid residue in antibody variable territory (VHH); Can the modified antibodies variable domains and do not reduce this structural domain to antigenic natural avidity, can realize this method and reduce its immunogenicity about the allos species; Also relate to be used to be applied to the allos species have the purposes of the VHHs of modification at the residue place that is identified, also relate to the VHH of modification like this.
More specifically, the preparation of the VHHs that the present invention relates to modify is modified this VHHs in order to be applied to the people, also relates to gained VHH self and this " humanization " VHHs purposes in the treatment Human diseases.Humanization means sudden change, makes that when being applied to human patients immunogenicity is very little or does not exist.According to the present invention; The polypeptide humanization comprised one or more Camelidae amino acid are used in their the people's counterpart displacement of finding in people's consensus sequence; And this polypeptide is not lost its characteristic feature, that is, and and the antigen binding capacity of this not remarkably influenced of humanization gained polypeptide.This method is that the technician is known.
The humanization of Camelidae single domain antibody need import in single polypeptied chain and the limited amount amino acid of mutagenesis.The humanization of this and scFv, Fab, (Fab) 2 and IgG is opposite, and the humanization of scFv, Fab, (Fab) 2 and IgG need be at two chains---and import amino acid in light chain and the heavy chain, also need keep the assembling of two chains.
As limiting examples.Will be in FR2 the polypeptide humanization of the VHH# 12B of type of comprising people residue.Humanization needs the residue on 1 and 5 among the mutagenesis FR1, and it imports through the primer that is used for the repertoire clone and does not exist natively in the llama sequence.Mutagenesis in those residues does not cause combining and/or suppressing active loss.Humanization also need the mutagenesis of residue on 74,76,83,84,93 in FR3.Mutagenesis in those residues does not cause combining and/or suppressing active remarkable loss (see figure 4).The sudden change of therefore uniting FR1 and FR3 does not influence and combines and/or suppress active (Fig. 5).Humanization also need the mutagenesis on 108 in FR4.The mutagenesis of Q108L causes production level reduction in intestinal bacteria.108 are exposed to solvent in camelid VHH, and in people's antibody, this position is buried in VH-VL interface (Spinelli, 1996; Nieba, 1997).In isolating VHs, 108 are exposed to solvent.Import the not charged Gln of nonpolar hydrophobic Leu rather than polarity and can/stability folding have remarkably influenced the inherence of this molecule.
As limiting examples, with the polypeptide humanization of representative among the VHH# 3E, the polypeptide of representative contains the significant residue of camelid among the VHH# 3E on 37,44,45 and 47, and they have hydrophilic characteristic.There is not influence through people's hydrophobic residue displacement hydrophilic residue (E44G and R45L) to combining and/or suppressing at 44 and 45.Yet, when importing F37V and F47W, observe combination and/or suppress active loss.Thereby modeling data proof residue 37 is crucial (see figure 6) (all are numbered all according to Kabat) for the integrity that keeps CDR3 ring conformation for active maintenance.
SEQ ID NO:3 and 14 demonstrate with the amino acid sequence homology more than 90% of people VH framework region and therefore said V.HH can directly be applied to the patient and not expect and therefrom produce immunne response, and do not have extra humanization burden.Therefore, one side of the present invention allows the patient of this treatment of needs is directly used polypeptide, its homologous sequence that contains SEQ ID NO:3 and 14, the perhaps funtion part of its homologous sequence.
One embodiment of the invention are methods of humanization VHH, and this method comprises separately or unite one of following residue of displacement:
44 and 45 bit flag amino acid among the FR2,
FR3 residue 74,75,76,83,84,93 and 94 and
Among the FR4 103,104,108 and 111;
Number according to the Kabat method for numbering serial.
One embodiment of the invention are anti-TNF-α polypeptide, nucleic acid that perhaps can coding said polypeptide, and they are used to treat, prevent and/or alleviate the symptom with the inflammatory process diseases associated.TNF-α is relevant with inflammatory process, and the blocking-up of TNF-α effect has anti-inflammatory effect, this anti-inflammatory effect some morbid state such as, for example, be unusual ideal in the segmental enteritis.Our embodiment has illustrated according to VHHs of the present invention, and they combine TNF-α and block itself and the combining of TNF-α acceptor.
Anti-TNF-α polypeptide of the present invention can be applicable to autoimmune disease, like autoimmune disease (ear), the autoimmune disease (eyes) of eyes, autoimmunization hepatitis (liver), autoimmunization parotitis (parotid gland), segmental enteritis (intestines), type i diabetes (pancreas), epididymitis (epididymis), glomerulonephritis (kidney), Exophthalmus goiter (Tiroidina), acute febrile polyneuritis (neurocyte), struma lymphomatosa (Tiroidina), hemolytic anemia (red corpuscle), systemic lupus erythematous (multiple tissue), male fertility (seminal fluid), multiple sclerosis (neurocyte), myasthenia gravis (myoneural junction), pemphigus (mainly being skin), psoriasis (skin), rheumatic fever (heart and joint), rheumatoid arthritis (joint lining), sarcoidosis (many tissues and organ), scleroderma (skin and reticular tissue), xerodermosteosis (exocrine gland and hetero-organization thereof), SpA (Axial sketelon and hetero-organization thereof), thyroiditis (Tiroidina), the vasculitis (blood vessel) of Addison's disease (suprarenal gland), ear.It in the bracket tissue that receives sickness influence.This autoimmune disease is tabulated representative but not is comprised.
The autoimmune disorder that anti-TNF of the present invention-α polypeptide is suitable for comprises; For example; Transplanting, chronic fatigue syndrome, Alzheimer, parkinson's disease, myocardial infarction, apoplexy, autism, epilepsy, arthus' phenomenon, anaphylaxis and alcohol and the dopy of AIDS, atopic allergy, bronchial allergy, eczema, leprosy, schizophrenia, heredity dysthymia disorders, tissue and organ.In the autoimmune disorder of identifying above, affected tissue is main target, and in other cases, this tissue is less important target.These illnesss part or mainly be autoimmunization syndrome.Therefore, when treating them, can use the disclosed same procedure of this paper, perhaps unite with additive method sometimes the aspect of this same procedure.
Another embodiment of the present invention is the purposes according to the nucleic acid of anti-TNF of the present invention-α polypeptide or coding said polypeptide, is used to prepare the medicine of treatment and inflammatory process diseases associated.The instance of disease comprises rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.
Through conventional route, can use according to polypeptide of the present invention and nucleic acid the experimenter like intravenous route.Yet a special character of anti-TNF of the present invention-α polypeptide is that they penetrate such as the barrier of tissue film and/or tumour and act locally on them, and these polypeptide are enough stable and can resist such as the extreme environment in the stomach.Therefore, another aspect of the present invention relates to sending of anti-TNF-α polypeptide.
According to experimenter of the present invention can be any Mammals that is subject to treat the treatment influence of polypeptide.
The oral delivery of anti-TNF of the present invention-α polypeptide causes in colon, receiving providing in the part of sickness influence this molecule of activity form.Can the height inflammation and contains the cell that produces TNF-α in these positions.Anti-TNF of the present invention-α polypeptide combines TNF-α, can partially-neutralized TNF-α, and avoided distribution, thereby limited disadvantageous spinoff at whole body.The mikrobe of genetic modification such as micrococcus acidilactici (Micrococcus lactis) can secretory antibody or its funtion parts.The mikrobe of this modification can be used as carrier and produces and send antibody or its funtion part with part in intestines.Through using the strain system that produces anti-TNF-α polypeptide, can treat inflammatory bowel syndrome.
Another aspect of the present invention comprises use such as the carrier of describing among the WO00/23471 through the non-infective bacterium, like the Gram-positive host living beings, sends the anti-TNF polypeptide like the surface expression or the secretion of lactococcus genus species (Lactococcus spec.).
One embodiment of the invention are like anti-TNF disclosed herein-α polypeptide; It is used to the symptom of treating, preventing and/or palliating a disease; These diseases are subject to the adjusting that TNF-α regulates material, and this TNF-α instrumentality mass-energy is enough passed gastric environment and not by inactivation.
The instance of disease is the arbitrary disease that causes inflammation, includes, but are not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.Known like those skilled in the art, in case have said polypeptide construct, (in the stomach, in the colon, or the like) discharges a large amount of polypeptide in the tram just can to use compounding process.This delivering method is important for the symptom of treating, preventing and/or palliating a disease, and wherein the target of these diseases is arranged in the internal organ system.
One side of the present invention is through to experimenter's oral administration such as anti-TNF disclosed herein-α polypeptide; Treatment, prevent and/or alleviate to be subject to the symptom that TNF-α regulates the disease that material regulates, this TNF-α instrumentality mass-energy is enough passed gastric environment and not by inactivation.
Another embodiment of the invention is the purposes like anti-TNF disclosed herein-α polypeptide; Be used to prepare treatment, prevent and/or alleviate and be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates, this TNF-α instrumentality mass-energy is enough passed gastric environment and not by inactivation.
One side of the present invention is to regulate material and said material not by the method for inactivation through experimenter's oral administration such as anti-TNF disclosed herein-α polypeptide are sent TNF-α to the internal organ system.
One side of the present invention is to regulate material and said material not by the method for inactivation through experimenter's oral administration such as anti-TNF disclosed herein-α polypeptide are sent TNF-α to experimenter's blood flow.
Another embodiment of the invention is like anti-TNF disclosed herein-α polypeptide, and it is used to treat, prevent and/or alleviates be subject to the symptom that TNF-α regulates the disease of material adjusting, and this TNF-α regulates material and is delivered to vagina and/or rectum road.
The instance of disease is the arbitrary disease that causes inflammation, includes, but not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.In limiting examples; Preparation according to the present invention contains anti-TNF disclosed herein-α polypeptide; Said preparation is the form of gel, creme, suppository, film or is the form of sponge or pesseulum, the slow in time release of active ingredients (these preparations are described in EP 707473, EP 684814, US 5629001) of said preparation.
One side of the present invention be through with anti-TNF disclosed herein-α polypeptide transvaginal and/or rectal administration in the experimenter with treatment, prevent and/or alleviate to be subject to the method that TNF-α regulates the symptom of the disease that material regulates, this TNF-α regulates material and is delivered to vagina and/or rectum road.
Another embodiment of the present invention is the purposes of anti-TNF disclosed herein-α polypeptide; Be used to prepare treatment, prevent and/or alleviate and be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates, this TNF-α regulates material and is delivered to vagina and/or rectum road.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide transvaginal and/or rectum road are applied to the experimenter so that TNF-α adjusting material is delivered to vagina and/or rectum road and said material not by the method for inactivation.
One side of the present invention be through anti-TNF disclosed herein-α polypeptide transvaginal and/or rectum road are applied to the experimenter with the blood flow of TNF-α being regulated the said experimenter of substance delivery and said material not by the method for inactivation.
Another embodiment of the present invention is anti-TNF disclosed herein-α polypeptide, and it is used to treat, prevent and/or alleviates be subject to the symptom that TNF-α regulates the disease of material adjusting, and this TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
The instance of disease is the arbitrary disease that causes inflammation, includes, but not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.In limiting examples, preparation according to the present invention contains anti-TNF disclosed herein-α polypeptide, and said preparation is the form of nasal spray (for example, aerosol) or sucker.Because this polypeptide construct is little, so it can more effectively arrive its target than therapeutic IgG molecule.
One side of the present invention is to go into through mouth or snuffing anti-TNF disclosed herein-α polypeptide is applied to the experimenter to treat, to prevent and/or to alleviate the symptom of the disease that is subject to the adjusting of TNF-α adjusting material, and this TNF-α regulates material and is delivered to the upper respiratory tract and lung.
Another embodiment of the present invention is the purposes of anti-TNF disclosed herein-α polypeptide; Be used to prepare treatment, prevent and/or alleviate and be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates; This TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung, and said polypeptide is not by inactivation.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide being applied to experimenter's nose, the upper respiratory tract and/or lung, TNF-α being regulated material be delivered to nose, the upper respiratory tract and lung and the method for non-inactivation.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide being applied to experimenter's nose, the upper respiratory tract and/or lung, and TNF-α is regulated blood flow and the method for non-inactivation that material is delivered to the experimenter.
One embodiment of the invention are anti-TNF disclosed herein-α polypeptide; It is used to treat, prevent and/or alleviates be subject to the symptom that TNF-α regulates the disease of material adjusting; This TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease increases the passing through property of intestines mucosa.Disclosed anti-TNF-α polypeptide in the literary composition is because their small size, so can pass intestines mucosa and more effectively arrive the experimenter's who suffers from disease blood flow, what said disease caused intestines mucosa passes through the property increase, and this disease is for for example, segmental enteritis.
One side of the present invention is the method through the symptom that experimenter's oral administration anti-TNF disclosed herein-α polypeptide is treated, prevents and/or palliated a disease; Said disease is subject to be delivered to the TNF-α adjusting material adjusting of intestines mucosa, and wherein said disease has increased the property of passing through of intestines mucosa.
This method in addition can be by additional aspect of the present invention---using active transport carrier---further strengthen.Aspect this, VHH and carrier merge of the present invention, and this carrier has strengthened and passes the transmission that the intestines wall gets into blood flow.In limiting examples, should " carrier " be another VHH, it is fused to treatment VHH.Utilize method as known in the art to prepare this fusion constructs.Should " carrier " VHH specific combination acceptor to the intestines wall, this zygotic induction passes the active transfer of intestines wall.
Another embodiment of the present invention is to use the medicine of the symptom that the preparation of anti-TNF disclosed herein-α polypeptide is used to treat, prevent and/or palliate a disease; This disease is subject to be delivered to the TNF-α adjusting material adjusting of intestines mucosa, and wherein said disease has increased the permeability of intestines mucosa.
One side of the present invention is to be delivered to intestines mucosa and the method for non-inactivation through experimenter's oral administration anti-TNF disclosed herein-α polypeptide is regulated material with TNF-α.
One side of the present invention is through experimenter's oral administration anti-TNF of the present invention-α polypeptide is regulated blood flow and the method for non-inactivation that material is delivered to the experimenter with TNF-α.
This method in addition can be by additional aspect of the present invention---using active transport carrier---further strengthen.Aspect this, anti-TNF disclosed herein-α polypeptide and carrier merge of the present invention, and this carrier has strengthened and passes the transmission that the intestines wall gets into blood flow.In limiting examples, should " carrier " be the VHH that merges with said polypeptide.Utilize method as known in the art to prepare this fusion constructs.Should " carrier " VHH specific combination acceptor to the intestines wall, this zygotic induction passes the active transfer of intestines wall.
One embodiment of the invention are anti-TNF disclosed herein-α polypeptide, and it is used to treat, prevent and/or alleviates be subject to the symptom that TNF-α regulates the disease of material adjusting, and this TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
The instance of disease is the arbitrary disease that causes inflammation, includes, but not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.The preparation of said polypeptide construct disclosed herein, for example, tablet, sprays, drops are placed in the hypogloeeis and pass through mucosa absorption in the network of capillary tube in hypogloeeis.
One side of the present invention be through with anti-TNF disclosed herein-α polypeptide sublingual administration in the experimenter; Treat, prevent and/or alleviate the method for the symptom of the disease that is subject to the adjusting of TNF-α adjusting material, this TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
Another embodiment of the invention is the purposes of anti-TNF disclosed herein-α polypeptide; Be used to prepare treatment, prevent and/or alleviate and be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates, this TNF-α instrumentality mass-energy is enough passed the tissue in hypogloeeis.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide sublingual administration is regulated tissue and the method for non-inactivation that material is delivered to the hypogloeeis in the experimenter with TNF-α.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide is administered orally in the experimenter TNF-α to be regulated blood flow and the method for non-inactivation that material is delivered to the experimenter.
Another embodiment of the invention is the application of anti-TNF disclosed herein-α polypeptide in the symptom of treating, prevent and/or alleviate the disease that is subject to the adjusting of TNF-α adjusting material, and this TNF-α instrumentality mass-energy is enough effectively passed skin.
The instance of disease is the arbitrary disease that causes inflammation, includes, but not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.The preparation of said polypeptide construct, for example, creme, film, sprays, drops, patch are placed on the skin and pass skin.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide is locally applied to the experimenter, treats, prevents and/or alleviate to be subject to the method that TNF-α regulates the symptom of the disease that material regulates, and this TNF-α instrumentality mass-energy is enough effectively passed skin.
Another embodiment of the invention is the purposes of anti-TNF disclosed herein-α polypeptide, is used to prepare treatment, prevent and/or alleviate be subject to the medicine that TNF-α regulates the symptom of the disease that material regulates, and this TNF-α instrumentality mass-energy is enough effectively passed skin.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide being locally applied to the experimenter TNF-α adjusting material to be delivered to skin and the method for non-inactivation.
One side of the present invention is through anti-TNF disclosed herein-α polypeptide is locally applied to the experimenter TNF-α to be regulated the method that material is delivered to experimenter's blood flow.
In another aspect of this invention, anti-TNF-α polypeptide also contains the carrier single domain antibody (for example, VHH), it is transported to said anti-TNF-α polypeptide the blood from the lung chamber as the active transport carrier.
Acceptor on also carrier-containing anti-TNF-α polypeptide specific combination mucomembranous surface (bronchial epithelial cell), cause this polypeptide from the lung chamber active transport to blood.This carrier single domain antibody can be fused to polypeptide construct.This fusion constructs can utilize method preparation as known in the art and describe in this article.Acceptor on " carrier " single domain antibody specific combination mucomembranous surface, this zygotic induction passes this surperficial active transfer.
Another aspect of the present invention is to confirm that when nasal administration those single domain antibodies (for example VHHs) are by the method for active transport in the blood flow.Similarly, can be with that be used to first test or immune VHH phage library nasal administration, and after using back different time point, can separating blood or organ to save by the phage of active transport in the blood flow.A limiting examples from the lung chamber to the acceptor of the active transport of blood flow is Fc acceptor N (FcRn).One side of the present invention comprises the VHH molecule of identifying through this method.This then VHH can be used as carrier VHH and when nasal administration, will treat VHH and be delivered in the corresponding target in the blood flow.
In one aspect of the invention, can use anti-TNF disclosed herein-α polypeptide, so that this polypeptide and TNF-α bonded reagent are regulated in screening.When in measuring combination or only said polypeptide metathetical mensuration, identifying, reagent must carry out function test to confirm whether they regulate antigenic effect in vivo.Relate generally to the instance that SEQ ID NO:3 has provided screening assay below, yet any anti-TNF-α polypeptide disclosed herein suits all.
In an instance of displacement experiment; In the existence of the candidate modulator of cumulative concentration or when not existing; To express TNF-α or its segmental phage or cell and for example, the polypeptide that has been labeled of SEQ ID NO:3 representative is hatched in binding buffer liquid.In order to verify and calibrate this mensuration, can implement to contrast competing reaction, this reaction uses said polypeptide and this polypeptide of cumulative concentration not to be labeled.After hatching, the thorough washing cell is used for the suitable method of given mark (for example, scintillation counting, fluorescence, etc.) and is measured the bonded labeling polypeptide.Candidate modulator exists down the amount of the polypeptide of bonded institute mark to descend and at least 10% shows that combination replaced by candidate modulator.If the candidate modulator polypeptide (sub-saturated polypeptide dosage) that displacement 50% is labeled under 1 μ M or littler concentration is thought specific combination in this mensurations that this candidate modulator is described in this article or other mensuration so.
Alternatively, can keep watch on combination or bonded displacement through surface plasmon resonance (SPR).Surface plasmon resonance is measured and can be used as quantivative approach measuring two combinations between the molecule, this measurement be through for example from the film on the polypeptide of the SEQ ID NO:3 representative of water and the transmitter between the fixed TNF-α combine or the bonded forfeiture causes near the change realization of quality this transmitter that is fixed.Resonance units and use Biacore biosensor (Biacore AB) with respect to the time after the change of this quality is measured as injection or removes said polypeptide or candidate's wedding agent are measured.According to people such as Salamon (people such as Salamon, 1996, Biophys is J.71:283-294; People such as Salamon, 2001, Biophys.J.80:1557-1567; People such as Salamon, 1999, Trends Biochem.Sci.24:213-219, each piece of writing all is merged in this paper as a reference.) method described, can with TNF-α for example be fixed on sensor chip in the thin film fluid film (for example, research grade CM5 chip, BiacoreAB) on.People such as Sarrio illustrate the combining of GPCR A (1) Adenosine Receptors in the liquid level of SPR on can be used for detector ligand and being fixed on chip (people such as Sarrio, 2000, Mol.Cell.Biol.20:5164-5174 is merged in this paper as a reference).Those skilled in the art use the condition of people's reports such as Sarrio can finely tune SEQ ID NO:3 and TNF-α bonded condition in the SPR mensuration as starting point.
SPR can measure with at least two kinds of methods and combine regulator.At first, the polypeptide of SEQ ID NO:3 representative for example can be attached to the TNF-α that is fixed in advance, and injection concentration is the candidate modulator of 0.1nM to 1 μ M then.The quantitatively displacement of institute's bonded polypeptide combines thereby can detect regulator.Alternatively, film-bonded TNF-α can and for example use with the candidate modulator preincubate, and the polypeptide of SEQ ID NO:3 representative stimulates.Said polypeptide and will be illustrated in the combination or the displacement of said polypeptide under the existence of regulator with the binding affinity difference that the combination between said polypeptide and the TNF-α is compared when not having regulator between the TNF-α of regulator preincubate.In arbitrary mensuration, the amount of the said polypeptide of bonded when not having candidate modulator, when having candidate modulator the amount of the said polypeptide of bonded reduce by 10% or more than show that this candidate modulator suppresses the interaction between TNF-α and the said polypeptide.
The another kind of method that the polypeptide of test example such as SEQ ID NO:3 representative and the bonded between the TNF-α suppress is used FRET (FRET).FRET is a kind of quantum-mechanical phenomenon; If the excitation spectrum of the emmission spectrum of fluorescence donor (D) and fluorescent receptor (A) is overlapping, at mutual very approaching fluorescence donor (D) and fluorescent receptor (A) (being separated by usually＜100
) between FRET takes place.The molecule of being tested, for example, the polypeptide of SEQ ID NO:3 representative and TNF-α are by the complementary pair mark of donor and acceptor fluorescence group.Although by TNF-α: polypeptide interacts and combines closely; But when said polypeptide and TNF-α debond; The fluorescence that the exciting of donor fluorophore sent will with reply the fluorescence that this excitation wavelength takes place and have different wavelengths, thereby through measuring under each wavelength emissive porwer just quantitatively bonded and unconjugated molecule.The donor fluorophore that is used for mark TNF-α is that this area is known.Interested especially is the variant of A.Victoria GFP, and they are known as Cyan FP (CFP, donor (D)) and Yellow FP (YFP, acceptor (A)).As an example, can YFP variant and TNF-α be processed fusion rotein.The reagent (Molecular Probes) that is used to express carrier that the GFP variant is a fusions (Clontech) and fluorophore tagged is as known in the art.Candidate modulator is added the inhibition that the mixture of polypeptide and the YFP-TNF-α of fluorescence-mark will cause energy to shift, and this can be through for example, proves with respect to the reduction of the sample YFP fluorescence that does not have candidate modulator.Using FRET to detect TNF-α: in the interactional mensuration of polypeptide; With respect to the sample that does not have candidate modulator; Contain that the fluorescent emission intensity under the acceptor wavelength reduces by 10% or bigger in the sample of candidate modulator, show that this candidate modulator suppresses TNF-α: polypeptide interacts.
Sample used herein can be the arbitrary biological sample that contains TNF-α; Such as clinical sample (for example; Cell grade branch, whole blood, blood plasma, serum, tissue, cell, etc.), from clinical sample, agriculture sample, forensic samples, study sample or other possible samples.Clinical sample can be originated from the human or animal.The sample of being analyzed can be solid or liquid in essence.Clearly when using solid material, at first they are dissolved in the suitable solution.
A kind of work-around solution of FRET is to use fluorescent quenching to keep watch on interaction of molecules.A molecule of interaction centering can be by fluorophore tagged, and another molecule is by a kind of molecule marker, the fluorescence of this fluorophore of cancellation when this fluorophore and this tagged molecule intimate engagement.The change of fluorescence shows and uses fluorophore when exciting: the variation of the association of the molecule of quencher pairing institute mark.The increase of the fluorescence of the TNF-α that usually, is labeled shows that the anti-TNF-α polypeptide with quencher is replaced.Measure for cancellation, with respect to the sample that does not have candidate modulator, containing in the sample of this candidate modulator fluorescent emission intensity increases by 10% or show that more greatly this candidate modulator suppresses TNF-α: anti-TNF-α polypeptide interacts.
Except surface plasmon resonance and FRET method, also use plate format quantitatively to combine.The fluorescence polarization value of fluorescently-labeled molecule depends on rotation correlation time (rotational correlation time) or reversal rate (tumbling rate).Mixture, compound, labeling polypeptide do not have higher polarization value such as the mixture ratio that combines with fluorescently-labeled anti-TNF-α polypeptide through TNF-α to form.TNF-α: anti-TNF-interactional candidate inhibitor of α polypeptide comprise that fluorescence polarization reduces for the mixture that causes with respect to do not have candidate inhibitor (if candidate inhibitor destroys or suppress the interaction of TNF-α and said polypeptide).Fluorescence polarization is very suitable for identifying destruction TNF-α: the small molecules that anti-TNF-α polypeptide complex forms.With respect to fluorescence polarization in the sample that does not have candidate modulator, contain in the sample of candidate modulator fluorescence polarization reduce 10% or brighter this candidate modulator of multilist suppress TNF-α: anti-TNF-α polypeptide interacts.
Keep watch on TNF-α: interactional another alternatives of anti-TNF-α polypeptide is used the biosensor assay method.The ICS biosensor of having described in the art (Australian Membrane Biotechnology Research Institute; Comell B, Braach-Maksvytis V, King L, Osman P, Raguse B, Wieczorek L and Pace R. " A biosensor that uses ion-channel switches " Nature 1997,387,580).In this technology, thus the ionic channel that gramacidin-promotes in the film bilayer of the combination of TNF-α and anti-TNF-α polypeptide and suspension close coupling and with measurable variation coupling of the admittance (admittance) (being similar to impedence) of biosensor.This method is linear in 6 one magnitude that admittance changes and extensive, high flux screening that be very suitable for the small molecules combinatorial library.With respect to the admittance of the sample that does not have candidate modulator, 10% or the bigger variation (increase or reduce) that contain admittance in the sample of candidate modulator show that candidate modulator suppresses the interaction of TNF-α and said polypeptide.Be important to note that in the interactional mensuration that detects TNF-α and anti-TNF-α polypeptide, maybe interactional regulator needn't be necessarily direct and on physiology with the domain interaction of said polypeptide interacting proteins.Also the possibility regulator interacts in the position of removing from the interaction position and causes, for example, and the conformational change among the TNF-α.Regulator (suppressor factor or agonist) with this mode effect still can be used as the destination agent of regulating TNF-α and its receptors bind.
Described any binding assay all can be used for confirming the existence of reagent in the sample (for example, tissue sample), and this reagent for example combines TNF-α or influence, and the polypeptide of SEQ ID NO:3 representative combines with TNF-α's.For this reason, in the existence of sample or when lacking,, and use and suitable method is measured in employed combination measured the polypeptide combination TNF-α and the reaction of said polypeptide.The combination of said polypeptide reduce 10% or more than show that this sample contains and regulate said polypeptide and TNF-α bonded reagent.Certainly; Above generalized method can easily be applied to screen candidate modulator, the funtion part that this candidate modulator changes any anti-TNF of the present invention-α polypeptide, its homologous sequence, its funtion part or its homologous sequence and TNF-α be combining between its fragment perhaps.
One embodiment of the invention are the unknown reagent of identifying through disclosed method in the literary composition.
One embodiment of the invention are the unknown reagent of identifying through disclosed method in the literary composition, and this reagent is used to treat, prevent and/or alleviates the symptom with the inflammatory process diseases associated.
Another embodiment of the invention is the purposes of the unknown reagent identified through disclosed method in the literary composition, is used to treat, prevent and/or alleviates the symptom with the inflammatory process diseases associated.
The instance of disease comprises rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.
Cell used according to the invention be preferably selected from bacterial cell as, for example, intestinal bacteria, yeast cell as, for example, yeast saccharomyces cerevisiae (S.cerevisiae), finish red saccharomyces pastorianus (P.pastors), insect cell, or mammalian cell.
Cell used according to the invention can be arbitrary cell; The nucleotide sequence of polypeptide that contains the funtion part of anti-TNF of the present invention-α polypeptide, its homologous sequence, its funtion part or its homologous sequence according to the present invention coding can be imported into this cell, expresses thereby this polypeptide can or be higher than natural horizontal with the natural horizontal of definition in as indicated.Normal or the approaching normal pharmacology of definition during the polypeptide of the present invention of preferably, in cell, expressing demonstrates as indicated.Most preferably; The polypeptide of the present invention of in cell, expressing comprises nucleotide sequence, any of the aminoacid sequence that this nucleotide sequence provides in can coding schedule 1 perhaps can encode with table 1 in the identical aminoacid sequence of the aminoacid sequence that provides at least 70%.
According to the preferred embodiments of the invention, cell is selected from COS7-cell, Chinese hamster ovary celI, LM (TK-) cell, NIH-3T3 cell, HEK-293 cell, K-562 cell or 1321N1 astrocytoma cell (astrocytoma cell) and other can be by cells transfected.
Usually, " treatment significant quantity ", " treatment effective dose " and " significant quantity " refer to realize that (regulate TNF-α combines desirable one or more result; Treatment or prevent inflammation) required amount.Those skilled in the art will recognize that potential and therefore " significant quantity " will regulate the TNF-multiple compound of α bonded and become along with being used for the present invention.Those skilled in the art will assess the potential of this compound easily.
Used as indicated; Term " compound " refers to anti-TNF of the present invention-α polypeptide, compsn, or nucleic acid that can coding said polypeptide, reagent of perhaps identifying according to the screening method of describing in the literary composition or the said polypeptide that contains one or more derivative aminos.
" pharmaceutically acceptable " refers to not to be material biologically or is not undesirable material; That is, this material can with this compound be applied to individuality and do not cause any undesirable biological effect or not with pharmaceutical composition in any of other components of being contained interact with deleterious mode.
The compound or the compsn that are used for treating or prevent experimenter's illness and comprise the drug administration significant quantity like anti-TNF disclosed herein-α polypeptide.
Compound or compsn that anti-TNF polypeptide of the present invention is used for treating or prevent experimenter's illness relevant with rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis and comprises the combination TNF-α of drug administration significant quantity.
Compound that anti-TNF disclosed herein-α polypeptide is used for treating or prevent experimenter's illness and comprise co-administered medicine effective quantity and another kind of compound as, for example, Frosst).
Compound that anti-TNF polypeptide of the present invention can be used for treating or prevent illness relevant with rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis among the experimenter and comprise co-administered medicine effective quantity and another kind of compound as; For example, Frosst).
The invention is not restricted to use the preparation that contains simplification compound of the present invention.Combination therapy is provided within the scope of the present invention, and wherein to patient's administered formulation of this treatment of needs, said preparation contains more than one The compounds of this invention.
The alpha mediated illness of TNF-includes, but are not limited to rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome and multiple sclerosis.
Being used for compound of the present invention can be mixed with pharmaceutical composition and be applied to mammalian hosts with the various forms that is suitable for selected route of administration; Such as human patients or livestock animals, this route of administration is per os or parenteral, passes through in the nose or suction, intravenously, intramuscular, part or subcutaneous route.
The gene therapy method that utilization is sent also can be used compound of the present invention.See, for example, U.S. Patent number 5,399,346, this patent is incorporated into as a reference by complete.The gene therapy method that use is sent can also be decided certain organs, tissue, graft, tumour, perhaps cell by tissue-specific promotor transfection with target in addition with the primary cell of the gene transfection of The compounds of this invention.
Therefore, The compounds of this invention can with pharmaceutically acceptable carrier, such as inert diluent or assimilable edible carrier combination systemic administration, like oral administration.They can be closed in hard or the soft shell gelatin capsules, can be compressed into tablet, perhaps can directly mix in the food of patient's meals.Treatment is used for per os, and active compound can use with one or more excipient composition and with the form of ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension agent, syrup, wafer etc.This compsn and preparation will contain at least 0.1% active compound.Certainly, the percentage ratio of compsn and preparation can change and can be about 2 to about 60% of given unit dosage weight expediently.In this treatment in the useful compsn amount of active compound for obtaining the amount of effective dose level.
Tablet, lozenge, pill, capsule or the like can also contain following: wedding agent, like Tragacanth, Sudan Gum-arabic, W-Gum or gelatin; Vehicle such as Lin Suanergai; Disintegrating agent such as W-Gum, yam starch, alginic acid or the like; Lubricant such as Magnesium Stearate; With sweeting agent such as sucrose, fructose, lactose or aspartame or sweetener such as lavender, wintergreen oil, perhaps cherry flavor.When unit dosage was capsule, it can also contain liquid carrier except the material of top type, like vegetables oil or polyoxyethylene glycol.Can have multiple other materials group is dressing or the physical form that changes solid-state unit dosage in addition.For example, can be with gelatin, wax, shellac or sugar or the like with tablet, pill or capsule dressing.Syrup or elixir can contain active compound, as the sucrose of sweeting agent or fructose, as methyl paraben and propylben, dyestuff and spices such as the cherry or the orange spices of sanitas.Certainly, all should be pharmaceutically acceptable and nontoxic in fact in consumption with the arbitrary material in the arbitrary unit dosage of preparation.In addition, active compound can be impregnated in sustained release preparation and device.
Can also use active compound through perfusion or injection intravenously or intraperitoneal.The solution that can in water, prepare active compound or its salt, it is chosen wantonly and mixes with nontoxic surfactants.Can also in glycerine, liquid polyethylene glycol, triactin and their mixture and oil, prepare dispersion agent.Under the usual conditions of preserving and using, these preparations contain and are useful on the sanitas that prevents microorganism growth.
Be suitable for injecting or dabbling pharmaceutical formulation can comprise aseptic aqueous solution or dispersion agent or the sterile powder that contains activeconstituents, it is suitable for preparing aseptic parenteral solution or perfusion liquid or dispersion agent temporarily, optional being encapsulated in the liposome.In all situations, final formulation must be aseptic, liquid and stable under production and preservation condition.
Liquid carrier or vehicle can be solvent or liquid dispersion medium, and it contains, for example, and water, ethanol, polyvalent alcohol (for example, glycerine, Ucar 35, liquid polyethylene glycol, or the like), vegetables oil, nontoxic glyceryl ester and their suitable mixture.Through for example, form liposome, for the situation of dispersion agent, through keeping needed particle size or can keeping adequate liquidity through the use tensio-active agent.Through multiple antibacterium and anti-mycotic agent, for example, parabens, butylene-chlorohydrin, phenol, Sorbic Acid, Thiomersalate, or the like can prevent action of microorganisms.In many cases, preferably include isotonic agent, for example, sugar, buffer reagent or sodium-chlor.Through in compsn, using the reagent that postpones absorption, for example, aluminum monostearate and gelatin can prolong the absorption of Injectable composition.
Active compound and above-named multiple other compositions (if desired) of needed amount in the appropriate solvent are merged, and filtration sterilization can prepare aseptic parenteral solution then.For the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preferred preparation method is vacuum-drying and frost drying technology, and these technology produce the powder that the activeconstituents that exists in front sterile filtration solution adds extra desirable composition.
For topical application, this compound can be used (that is, when they are liquid) with pure form.Yet as compsn or preparation, acceptable carrier is applied to skin on the associating dermatology with them in hope usually, and these carriers can be solid or liquid.
The available solid-state carrier comprises the solid of segmentation, like talcum, clay, Microcrystalline Cellulose, tripoli, alumina, or the like.The available liquid carrier comprises water, hydroxyalkyl or divalent alcohol or water-ethanol/divalent alcohol foreign body, and wherein this compound can be chosen wantonly through the help of nontoxic surfactants and dissolve or dispersion with level of significance.Can add adjuvant such as perfume compound or biocide to optimize the character of given purposes.The gained fluid composition can be used from absorption pad, is used to flood bandage or other dressings, perhaps uses pump type or aerosol atomizer to be sprayed onto affected zone.
Thickening material such as synthetic polymer, lipid acid, soap and ester, Fatty Alcohol(C12-C14 and C12-C18), modified-cellulose or modified mineral material can use paste, gelifying agent, ointment, the soap agent that can smear to form with liquid carrier; Or the like, to directly apply to user's skin.
The instance that can be used for this compound is delivered to the useful dermatological compositions of skin is as known in the art; For example, see people (U.S. Patent number 4,559,157) and Wortzman (U.S. Patent number 4,820,508) such as people such as Jacquet (U.S. Patent number 4,608,392), Geria (U.S. Patent number 4,992,478), Smith.
The useful dosage that can confirm this compound through the external activity and the activity in vivo in the animal model of comparative compound.The method that effective dose in mouse and other animals is extrapolated to the people is as known in the art; For example, see U.S. Patent number 4,938,949.
Usually, compound concentrations will be about 0.1-25wt-%, preferably about 0.5-10wt-% in fluid composition such as the lotion.Concentration in semi-solid state or solid-state composition such as gelifying agent or the pulvis will be about 0.1-5wt-%, preferably about 0.5-2.5wt-%.
Be used to treat required compound; Perhaps the amount of its active salt or verivate will be not only becomes along with selected concrete salt but also along with the character of route of administration, the illness of being treated and patient's age and situation, and will be finally by physician in charge surgeon in charge attending doctor doctor in charge or clinician's judgement decision.And the dosage of this compound will be according to target cell, tumour, tissue, graft, perhaps organ and becoming.
Desired dose can provide as single agent or provide as the dosage that separates expediently, should use with suitable interval by dosage separately, for example, as every day 2,3,4 or a plurality of sub-doses.Itself for example can also be divided into this sub-doses, many discrete loose using of separating; Such as repeatedly sucking perhaps of insufflator through being applied to the repeatedly drops of eyes.
Secular, treatment every day that application program can comprise." for a long time " referred to at least two weeks, preferred several weeks, several months or time length several years.Those of ordinary skills only use the normal experiment method in the instruction here just can confirm modification necessary in this dosage range.See Remington ' s Pharmaceutical Sciences (Martin, E.W., the 4th edition), Mack Publishing Co., Easton, PA.If any complication takes place, individual physician can also be regulated dosage.
The invention provides reagent, this reagent is TNF-α/interactional regulator of TNF-alpha-receptor.
Candidate agent can be a synthetic agent, and perhaps the mixture of reagent perhaps can be natural product (for example, plant milk extract or a culture supernatant liquid).Candidate agent according to the present invention comprises can the synthetic small molecules, natural extract, peptide, protein, carbohydrate, lipid etc.
Can from the big library of synthetic or natural agent, screen candidate modulator.The current several different methods of utilizing is at random with the synthetic sugar of orientation, peptide with based on the reagent of nucleic acid.Can obtain the synthetic agent library from many companies through commercial sources; These companies comprise Maybridge Chemical Co. (Trevillet; Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack; NH) and Microsource (New Milford, CT).(Milwaukee WI) can obtain rare chemicals library from Aldrich.Can obtain and can prepare combinatorial library.Alternatively, the library of the natural agent of bacterium, fungi, plant and animal form of extract can be from for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC) obtain, perhaps easily prepare through the method for knowing in this area.In addition, modify natural and synthetic library and the reagent that produces easily through conventional chemical, physics and biochemical method.
In number of chemical article classification, can find useful reagent.Useful reagent can be an organic reagent, perhaps small molecules reagent.But the molecular weight of small molecules reagent preferably less than about 750, is more preferably less than about 350 dalton greater than 50 less than about 2,500 dalton.Representative category comprises heterocyclic, peptide, sugar, steroid, or the like.Can modify these reagent with enhancing usefulness, stability, drug compatibility, or the like.The structure of reagent is identified and can be used for identifying, produces or screen additional agents.For example, when having identified peptide reagent, can they be modified to strengthen their stability, such as using alpha-non-natural amino acid with the whole bag of tricks; Like D-amino acid, D-L-Ala especially, amino or C-terminal through functionalization; For example, for amino, acidylate or alkylation; For carboxyl, esterification or amidation, or the like.
For preliminary screening, arrive about 100 μ M or above (that is, 1mM, 10mM, 100mM, 1M etc.) for about 10mM according to the useful concentration of candidate agent of the present invention.Preliminary screening concentration will be as the upper limit, and 9 kinds of extra concentration, wherein through with preliminary screening concentration with semilog at interval (for example, for 9 kinds of extra concentration) reduce to be used for postsearch screening or be used to produce concentration curve to confirm extra concentration.
The high flux screening test kit
High flux screening test kit according to the present invention contains method that detection was necessary and the medium of implementing reagent, and this reagent is regulated TNF-α/TNF-α acceptor interaction through interacting with TNF-α at preferred 1 μ M in the presence of the polypeptide of 1mM concentration.
This test kit contains following: reconstitution cell of the present invention, and it contains and expresses the nucleotide sequence of coding TNF-α, according to known method among those skilled in the art; The method of particularly describing among the WO 00/02045, this reconstitution cell is grown in solid support according to test kit, such as microtiter plate; More preferably on the 96 hole microtiter plates; Alternatively, those skilled in the art provide TNF-α with the form of purifying, and it for example is fixed on the 96 hole microtiter plates.Alternatively, TNF-α is provided in test kit, its by predetermined fixed for example, on the 96 hole microtiter plates.TNF-α can be complete TNF-α or its fragment.
Concentration is that about 1 μ M is added in the hole of qualification to 1m or above regulator according to the present invention; Have the funtion part of anti-TNF-α polypeptide, its homologous sequence, its function sequence or its homologous sequence of proper concn in the hole, the said concentration of said polypeptide is preferably 1 μ M to 1mM.Test kit can contain one or more anti-TNF-α polypeptide (for example, the funtion part of one or more polypeptide of one of SEQ ID NOs:1 to 15 representative or other anti-TNF-α polypeptide, its homologous sequence, its function sequence or its homologous sequence).
Combine to measure and result and baseline values are compared according to disclosed method enforcement in the literary composition; Baseline values is for for example, and TNF-α combines the level of the funtion part of anti-TNF-α polypeptide, its homologous sequence, its funtion part or its homologous sequence when not having the regulator that is added.Activity level is compared when not having regulator, and the TNF-alpha polypeptides combines (for example) to increase or is reduced by at least 2 times, preferred 5 times, and more preferably 10 times, most preferably 100 times or above hole are selected for further analysis.
Other test kits used according to the invention
The invention provides the test kit of the regulator that is used to screen TNF-α/TNF-α receptors bind, and be used for the test kit that diagnostic characteristic is the disease of TNF-α dysfunction.The present invention also provides the test kit of the regulator that is used to screen disease and their diagnostic kit, and the characteristic of said disease is one or more processes relevant with TNF-α.Test kit used according to the invention can comprise isolating TNF-α.Alternatively, perhaps extraly, test kit can contain the cell of being expressed TNF-α by conversion.The polynucleotide that can contain in another embodiment, coding TNF-α according to test kit of the present invention.In another embodiment, can contain the special primer that is useful on amplification TNF-α according to test kit of the present invention.Test kit used according to the invention can contain separative TNF-α polypeptide, its homologue, perhaps its funtion part.Can contain the cell of being expressed said polypeptide by conversion according to test kit of the present invention.Test kit can contain more than one polypeptide.In another embodiment, can contain the multinuclear former times acid of coding TNF-α according to test kit of the present invention.In another embodiment, according to test kit of the present invention can contain be useful on the amplification macromole as, for example, the special primer of TNF-α.All test kits according to the present invention will contain the article of statement to some extent or the combination and the wrapping material of article.Test kit will also comprise working instructions.
Embodiment
Non-limiting example through following is illustrated the present invention.
Embodiment 1: the embodiment that is directed against the Camelidae antibody of huamn tumor necrosis factory alpha
1) immunity and library construction
Personnel selection TNF-alpha immunization llama (Llama glama).For immunity, cytokine is mixed with emulsion with the friendly adjuvant of suitable animal (Specoll, CEDI Diagnostics B.V.).Use this antigen mixture through the two places injection of neck intramuscular.Animal is accepted 6 emulsion injections with interval weekly, and this emulsion contains 100 μ g TNF-α.Different time points between duration of immunity is collected the 10ml blood sample and is prepared serum from animal.Use serum sample induce (data not shown) with the HI of TNF checking antigen-specific in the ELISA experiment.The 150ml blood sample is collected in back 5 days of last immunity.Separate conventional antibody and heavy chain antibody (HcAbs) such as (Lauwereys people, 1998) and be used for ELISA from this sample classification, ELISA announcement HcAbs is responsible for the HI (data not shown) of antigen-specific.Use Ficoll-Paque gradient (Amersham Biosciences) to separate peripheral blood lymphocyte (PBLs), obtain 5 * 10 as the genetic origin of llama heavy chain immunoglobulin (HcAbs) from the 150ml blood sample
8Individual PBLs.The lymphocytic number of B-that the maximum variety of expection antibody equals to sample, this number is about 10% (5x10 of PBLs number
7).The level of heavy chain antibody is divided up to 20% of bone-marrow-derived lymphocyte number in the llama.Therefore, the maximum variety of HcAbs is calculated as 10 in the 150ml blood sample
7Plant different molecules.Use acid guanidine thiocyanate to extract (Chomczynski and Sacchi, 1987) from the total RNA of these cellular segregation (about 400 μ g).
People such as (, 1999) the de Haard that describes before utilizing prepares cDNA with M-MLV reversed transcriptive enzyme (Gibco BRL) and oligomerization-dT-primer or Hexanucleotide random primer (Amersham Biosciences) from the total RNA of 100 μ g.Be used as template subsequently with specific amplified VHH repertoire with phenol/chloroform extraction associating ethanol sedimentation purifying cDNA and with it.
Use cDNA that oligomerization dT causes to import PstI restriction site (runic) with single degeneracy framework 1 (FR1) primer ABL013 (5 '-GAGGTBCARCTGCAGGASTCYGG-3 ') (SEQ ID NO:98), unite the oligomerization dT primer amplification VHH repertoire of describing among the EP01205100.9 as template.This amplification has produced two fragments of 1650bp and 1300bp, and back one fragment is the product that obtains from the HcAb gene that CH1-lacks.Less PCR product is by gel-purified and subsequently with PstI and BstEII digestion.The BstEII site exists in the segmental FR4 of VHH coding DNA in heavy chain source usually.
Alternatively, use two kinds of Oligonucleolide primers amplification VHH-repertoires that IgG is special with the method that relies on hinge.In a PCR reaction, the known weak point special to HcAbs (5 '-AACAGTTAAGCTTCCGCTT
GCGGCCGCGGAGCTGGGGTCTTCGCTGTGGTGCG-3 ') (SEQ ID NO:99) or long (5 '-AACAGTTAAGCTTCCGCTT
GCGGCCGCTGGTTGTGGTTTTGGTGTCTTGGGTT-3 ') (SEQ ID NO:100) hinge primer and FR1-primer ABL013 (above seeing) associating.PstI and NotI (runic underscore) restriction site is imported into respectively in FR1 and the hinge primer to allow the clone.Subsequently, dna fragmentation is connected among the phagemid carrier pAX004 with PstI-BstEII or PstI-NotI digestion, this carrier and pHEN1 people such as (, 1991) Hoogenboom but identical the coding is respectively applied for the C-terminal (His) of purifying and detection
6-and the c-myc-label.Connect mixture the Microcon filter (YM-50, Millipore) desalination and electroporation to the e. coli tg1 cell to obtain containing 1.8 * 10
7Individual clone's library.Coiled last 37 ℃ of following overnight growth by cell transformed at the 20 * 20cm that the LB that contains 100 μ g/ml Ampicillin Trihydrates and 2% glucose is housed.Strike off bacterium colony and-80 ℃ of preservations 20% glycerine with 2 * TY substratum from plate.
As quality contrast, use based on the combination of the primer of carrier through PCR 24 clone's checkings in every kind of library are contained the percentage ratio that inserts segmental clone.The clone of this analysis announcement 95% contains the insertion fragment of the VHH that encodes.The segmental HinfI fingerprinting inspection of the VHH of the amplification through these 24 clones variability, thus show that all clones are actually different (data not shown).
2) the anti-TNF VHH ' s of selection antagonism
Prepare phage from two kinds of libraries.In order to save polyclone phage repertoire, the library is contained under 37 ℃ among 2 * TY of 100 μ g/ml Ampicillin Trihydrates and 2% glucose and grown into logarithmic phase (OD600=0.5), and use the M13K07 helper phage subsequently 37 ℃ of superingection 30 minutes.With infected cells with centrifugal 5 minutes of 4000rpm and be resuspended among the 2 * TY that contains 100 μ g/ml Ampicillin Trihydrates and 25 μ g/ml kantlex.Through breeding phage with the 250rpm overnight growth at 37 ℃.Overnight culture is hatched 30 minutes deposition phages with centrifugal 15 minutes of 4500rpm and with 1/5 volume [20% polyethylene glycol 6000,1.5M NaCl] solution on ice.With 4000 * g 4 ℃ centrifugal 15 minutes the deposition phages.After phage is resuspended among the PBS, through with in Eppendorf tube with top speed (15000 * g) centrifugal 1 minute sedimentation cell fragments.The supernatant that will contain phage particle is transferred to the phage of also as above describing in the new pipe of deposition once more.Phage is dissolved among the PBS and as mentioned abovely separates from remaining cell debris.After infecting logarithm TG1 cell, selecting to be coated with dull and stereotyped the tiring of phage of confirming on the substratum.
Select the library with external biological elementization TNF-α.Like people such as Magni (Anal Biochem2001,298,181-188) described enforcement biotinylation.Analyze and mixing through SDS-PAGE with vitamin H among Extravidin-SEAP conjugate (Sigma) the detection assessment TNF.Combine the ability of the reorganization p75 acceptor of solid-phase coating to estimate the functional of this modified protein through modified protein.
Select VHH through catching biotinylation TNF-α (following 2 hours of room temperature, every hole 10 to 400ng) on the microtiter plate (with 100 μ l, 10 μ g/ml streptavidins, 4 ℃ encapsulated 16 hours) that encapsulates at streptavidin.Obtain the VHH of antagonism through the cell wash-out of perhaps expressing this receptor with excessive acceptor-extracellular ligand binding domains.After phage and captive cytokine are hatched 2 hours, the non-special phage of eccysis, and will show the cell (＞10 of the specific phage of antagonism VHH with acceptor (ectodomain of CD120b or p75,10 μ M) or usefulness displaying acceptor
5Individual KYM cells/well) wash-out.High enrichment promptly shows greater than 20 with the phage number of acceptor wash-out and ratio with the phage number of serum albumin (50 μ g/ hole) wash-out and has successfully selected TNF-α cloned specific.Alternatively, not to use the acceptor wash-out, but the application standard method, wherein low pH causes VHH and/or antigenic sex change (0.1M glycine buffer, pH 2.5).With the Bacillus coli cells of logarithmic phase growth with wash-out and the phage-infect that is neutralized and be layered on and select on the substratum.Select single clone and on the microtiter plate growth in culture supernatant liquid, to produce VHH.Implement the ELISA screening and disclose about 50% positive colony with being captured in TNF-α on the flat board that Extravidin encapsulates.The HinfI-fingerprinting shows has selected 13 different clones, and these clones are grown with the 50ml scale and induce.Said clone's sequence shows in table 1.
Characterized 5 clones with different sequences (Fig. 1) in more detail, their code name is VHH#1A, #2B, #3E, #3G, #7B and #12B.VHH#3E, #3G and #7B are the single domain antibody fragments, and their in FR2 45 carry typical hydrophilic residue (l-arginine) and 47 displacements with phenylalanine(Phe) to tryptophane, thereby aspect solvability, have given favorable characteristics.VHH#1A contains hydrophobic FR2 residue; It is found in the double-stranded antibody of people source or other species usually; But the charged arginine residues through on 103 has remedied hydrophilic loss, and this residue has been replaced the conservative tryptophan residue (PCT/EP02/07804) that exists among the VH from double-stranded antibody.A new classification of the humanization Camelidae single domain antibody of describing among the present invention is by VHH#2B and VHH#12B representative, and it contains hydrophobic residue and 103 hydrophobic residue tryptophane among the FR2.Through using TALON resin (Clontech) purifying with the more substantial antibody fragment of 50ml scale culture expression and through IMAC.PBS dialysis is removed behind the elutriant imidazoles amount of confirming VHH through OD280; Obtain about 300 μ g VHH from each clone.This material is used for confirming the susceptibility of ELISA (biotinylation) TNF detection.For this reason, the microtiter plate that streptavidin (10 μ g/ml) is encapsulated is used to catch biotinylation TNF (1 μ g/ml), and VHH is diluted in 0.2% casein/PBS and incubated at room 2 hours.With anti--MYC mAB9E10 (0.5 μ g/ml) and anti-mouse AP conjugate (1000 times of dilutions, Sigma) detection bonded VHH.The result shows in Fig. 2.
3) in CTA, confirm antagonistic action with KYM clone
Through Vandenabeele and colleague (Vandenabeele, P., Declercq, W.; Vercammen, D., Van de Craen, M.; Grooten, J., Loetscher, H.; Brockhaus, M., Lesslauer, W.; Fiers, W. (1992) Functional characterization of the human tumor necrosis factor receptor p75in a trasfected rat/moust T cell hybridoma.J.Exp.Med.176, the colorimetric MTT assay method of 1015-1024) describing is confirmed TNF-α inductive cytostasis/cytotoxicity.(3-(4 for MTT; 5-dimethylthiazole-2-yl)-2; 5-phenylbenzene tetrazolium bromide) be a kind of lark substrate, it is produced faint blue first
product by the viable cell cutting.This process need active wire plastochondria, even just dead cell can not cut a large amount of MTT.With KYM cell (Sekiguchi M; Shiroko Y, Suzuki T, Imada; Miyahara M; Fujii G (1985), Characterization of a human rhabdomyosarcoma cell strain in tissue culture.Biomed.Pharmacother.39 372-380.) is inoculated in the 96 hole microtiter plates and in the existence of TNF-α (0.216ng/ml or about 5pM tripolymer) or cultivation not.Except TNF, also comprise the antibody (VHH or infliximab) of different amounts between during cultivation.For this mensuration, MTT is added substratum, its final concentration is 500 μ g/ml, and flat board is hatched to realize the cutting of cyclophorase to MTT at 37 ℃.Add acid isopropyl alcohol (40nM HCl is dissolved in the Virahol) or DMSO and dissolve formed first
product, this product appear in the bottom in hole black, like the crystal of fine hair.Measure absorption at the 570nm place.
MTT measures (Fig. 3) and shows that VHH#1A (it is at 103 hydrophobic residues that combine similar people among the FR2 for l-arginine) has medium antagonistic effect (IC
50～100nM).Though the VHH#7B that in FR2, has a tag type hydrophilic residue in ELISA Sensitive Detection cytokine (curve is for showing) can not prevent combining of TNF-α and its part.On the contrary, the VHH#3E and the #3G that have a significant residue of hydrophilic FR2 are very strong antagonism property VHH ' s (IC
50Be 20nM).VHH#3E and #3G have high homology and relevant on the clone (people such as Harmsen, Mol.Immunol.37,579-590), but VHH#3E than VHH#3G stronger (Fig. 2), this possibly be to have higher avidity owing to VHH#3E.(chimeric) monoclonal antibody infliximab is (IC very effectively
50Be 80pM), but its deutero-Fab fragment has been lost the major part (IC of this usefulness
50Be 3nM, for complete mAB 1/30th).This clearly illustrates that interactional avidity effect between antibody and the cytokine: the mAB with two binding sites combines more effectively and tripolymer TNF interaction of molecules through cooperation.The VHH fragment is strict unit price, infers that therefore merging VHH gene increase avidity through heredity can increase their antagonism usefulness (seeing embodiment 4).
These experiments show that the new classification of similar people's VHH has real combination and functional character, thereby make them can be used for therapeutic purpose.
Embodiment 2: through site-directed mutagenesis humanization VHH# 12B and VHH# 3E
1) VHH# 3E/VHH# 12B and ethnic group are the homology between the heavy chain V-district DP-47
● 1,5,28 and 30 last 4 amino acid whose change among the FR1
● 74,76,83,84 and 93 last 5 amino acid whose changes among the FR3
● 108 last 1 amino acid changes among the FR4
As represent in the sequence alignment below:
DP-47 EVQLLESGGGLVQPGGSLRLSCAASGFTFS?
SYAMS?WVRQAPGKGLEWVS?
AISGSGGSTYY
DP-47
ADSVKG?RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(SEQ?ID?N°101)
(SEQID?N°102)
Therefore, be that special suppressor factor that gene DP-47 has a TNF-α cytokine of high homology is further humanization and estimate the ideal candidates person to the influence that suppresses ability of mutagenesis among the ELISA with ethnic group.
The comparison of VHH# 3E and people VH3 kind system (DP-47) disclose with DP-47 in hydrophobic residue compare among the FR2 of VHH# 3E and have hydrophilic amino acid residue.
DP-47 EVQLLESGGGLVQPGGSLRLSCAAS
GFTFS?SYAMS-----?WVRQAPGKGLEWVS?
AISGSGGSTYY
DP-47
ADSVKG?RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK?-------------?-----------
The effect that replaces hydrophilic residue through the hydrophobic residue evaluation that exists among the people VH is important, because the major part of camelid VHH sequence contains hydrophilic residue.
2) mutagenesis of VHH# 12B
Describe and by the business-like site-directed mutagenesis method sudden change VHH#12B of Stratagene (Quickchange site-directed mutagenesis) through Chen and Ruffner based on non--PCR.DNA is used as template, and uses 2 kinds of mutagenic primers to import desirable sudden change.Each is all complementary with the opposite strand of template plasmid DNA for these two kinds of primers.In the polymeric enzyme reaction that uses the PfuDNA polysaccharase, during using a limited number of round-robin cycling program, every chain extends from primer sequence.This causes the mixture of wild-type and sudden change chain.Can select the external synthetic DNA chain that suddenlys change with DpnI digestion, because only template strand is responsive for digestion.With DNA deposition and be transformed in the intestinal bacteria and and analyze desirable sudden change through sequential analysis.Sudden change VHH ' the s and the mutagenic primer that are produced are listed in table 2.Prepare plasmid and it is transformed into WK6 electroreception attitude cell from mutant clon.Single bacterium colony is used to incubated overnight in the LB that contains 2% glucose and 100 μ g/ml Ampicillin Trihydrates.This overnight culture is diluted 100 times in the 300ml TB substratum that contains 100 μ g/ml Ampicillin Trihydrates, and hatches up to OD600nm=2 at 37 ℃, when adding 1mM IPTG and 5mM MgSO
4Hatched again 3 hours at 37 ℃ when (final concentration) and with culture.
With culture 4 ℃ with 4, centrifugal 20 minutes of 500rpm.The deposition spend the night perhaps freezing-20 ℃ 1 hour.Then, at room temperature thaw and precipitate 40 minutes, it is resuspended among the 20ml PBS/1mMEDTA/1M NaCl and on ice shook 1 hour.Through 4 ℃ with 4,500rpm separated all quality and grade branches in centrifugal 20 minutes.Pack into TALON (ClonTech) and be purified to homogeneity of the supernatant that will contain VHH.Confirm the productive rate of VHH with the optical extinction coefficient that calculates.All expressed sudden change VHH ' s are suitable with wild-type., extracorporeal receptor analyzes the inhibition ability of two mutants in combining to measure.
Spend the night at 4 ℃ with 2 μ g/ml rhu TNFR:Fcs (Wyeth) among the PBS and to encapsulate microtiter plate.This plate was sealed 1 hour in room temperature with PBS-tween washing 5 times and with containing 1% caseic PBS.Wash microtiter plate 5 times with the PBS-tween.With biotinylation humanTNF-(80 μ g/ml) and sudden change or the dilution series of wild-type VHH# 12B preincubate 1 hour and mixture at room temperature hatched in the hole of microtiter plate 1 hour at room temperature.With PBS-tween wash plate 5 times.Detect the bonded humanTNF-with Extravidin-AP (1/1,000 dilution) and p-NP phosphoric acid (pNPP).405nm is measurement signal after 30 minutes.The result provides in Figure 4 and 5.(the Q 1E+Q5L+A74S+Y76N+K83R+P84A of sudden change) increased by 3 times to IC50 from 66nM (wild-type) to 200nM.T93A sudden change in position causes suppressing forfeiture (data not shown).Still need humanized position to be: E28, E30 and Q108.Yet, E28 and E30 be the H1 canonical structure part from but according to the part of the CDR1 of Chothia numbering system.
The aminoacid sequence of sudden change VHHs provides among the SEQ ID NOs:17 to 19 in table 4.
3) mutagenesis of VHH# 3E
Use above-described site-directed mutagenesis method sudden change VHH# 3E based on non-PCR.VHH ' the s and the mutagenic primer of gained sudden change are listed in table 3.
Expression and the wild-type of all sudden change VHH are suitable.In ELISA, analyze purifying sudden change VHH combination and in the receptors bind identical with above-described method measured, analyze the inhibition ability of VHH.
The result of ELISA shows that in Fig. 6 the result that receptors bind is measured shows in Fig. 7.
The aminoacid sequence of sudden change VHHs SEQ ID NOs 21 in table 4 provides to 24.
Embodiment 3: be directed against the separation of the antagonism VHH of mouse TNF-α
1) selects anti-mouse TNF-α VHH
In order in IBD or segmental enteritis mouse model, to implement effect research, select the special VHH of mouse TNF.Therefore, as describe among the embodiment 1 with mouse TNF-alpha immunization llama.From last immunity back the PBL of sampling in the 4th and 10 day and after the 4th day from the examination of living tissue extraction RNA of lymphoglandula.Total RNA is changed into cDNA that causes at random or oligomerization-dT causes and the amplification template that is used as VHH encoding sox sections, and the primer in Ig source or the mixture (seeing embodiment 1) of oligomerization-dT primer and single Ig primer are used in this amplification.Use the Ig primer to produce and contain 8.5 * 10 from first PBL ' s
7The library in individual clone's library has produced 7 * 10 from second PBL sample
6Individual clone's library has produced 5.8 * 10 from lymphoglandula
8Individual clone's library.Use the mixture of oligomerization-dT primer and Ig primer to obtain containing 1.2 * 10 from first PBL sample
8Individual clone's library has obtained 5.7 * 10 from second PBL sample
7Individual clone's library, and the library in lymphoglandula source contains 2 * 10
8Individual clone.Cultivate with the propagation phage according to the combination merging library of used primer and with two libraries of gained, describe like the front.Through be trapped in biotinylation mouse TNF-on the streptavidin that encapsulates (α implements to select, the bonded phage through with people's acceptor p75 competition by wash-out, known p75 and mouse TNF-α cross reaction.Select two kinds of special VHH of different mice TNF-α (VHH#m3F and VHH#m9E) from the library that obtains with Ig deutero-primer amplification, and from obtaining two kinds of closely-related VHH (Fig. 8) through PCR with the library that oligomerization-dT primer and Ig-primer make up.
2) in CTA, confirm the antagonism effect with L929 clone
Like the mensuration of the application same type of description among the embodiment 1, but be to use mouse cell line L929.Proof VHH#m3F and VHH#m4B (Fig. 9) are than more effective 10 times of other two kinds of VHH.
Embodiment 4: strengthen the antagonism effect through using multivalence Camelidae antibody to increase avidity
1) to the antagonism effect of divalence, trivalent and the tetravalence VHH of people and mouse TNF-α
Designed intestinal bacteria and produced carrier pAX11 (Figure 10), this carrier allows two step clone's divalence or two special VHH.At first clone C-terminal VHH with PstI and BstEII, and in second step, insert another VHH through SfiI and NotI, SfiI and NotI be not in first gene fragment incised.Thereby this method has been avoided producing the danger that the PCR mistake has been avoided producing in new site through amplification.
Use this carrier to produce the divalent derivative of antagonism property Anti-Human TNF-α VHH#3E.The plasmid vector of this divalence of encoding VHH is used to produce tripolymer and tetramer verivate, and this accomplishes through partly digest this plasmid with BstEII, and this digestion all takes place in two VHH gene fragments.From gel-purified, also as the segmental acceptor of BstEII of the about 350bp of clone, the BstEII fragment obtains through the identical plasmid of complete digestion dephosphorylation subsequently with linearized vector.Only the BstEII fragment is connected back adding carrier and strengthened the segmental insertion of polymer VHH encoding sox.After in e. coli tg1, transforming, through PCR, with M13Rev and M13Fwd primer screening institute DCRP; Because BstEII is not to becoming nickase (5nt overhang), thus only obtain the insertion fragment of correct direction, this through only with PstI digestion (350bp) or with EcoRI and HindiII double digestion (for divalence (BIV 3E; SEQ ID NO:73) obtains 1000bp; (TRI 3E, SEQ ID NO:74) obtains 1350bp for trivalent, for tetravalence (TETRA 3E; SEQ ID NO:75) obtain 1700bp), data not shown) plasmid confirms.Sequence is listed in table 7.
With 50ml scale growth with induce the clone, prepare the IMAC purifying that all quality and grades are divided and are used to use the TALON resin to implement.Painted PAGE analyzes the good production level (every liter of cell culture 2 is to 10mg) (seeing Figure 11) that purified product has disclosed complete multivalence VHH with coomassie.Confirm through gel-filtration on the Superdex 75HR post IMAC purifying VHH the molecule outward appearance and as desired, the molecule with high affinity more is earlier from pillar come out (seeing Figure 12).
Use based on the assay method of cell and use KYM cell analysis antagonism effect.Cell is inoculated in the microtiter plate and when TNF-α (1.29ng/ml or about 25pM tripolymer) exists or do not exist and cultivates.This mensuration (Figure 13) discloses the monovalent molecule that is used for this research and has the most weak antagonism characteristic.Through their IC
50What value reflected: from the IC of the Fab of chimeric antibody infliximab
50Be 2nM, for VHH#3E, IC
50Be 12nM (also seeing Fig. 3).The avidity of used molecule has remarkable influence to the antagonism effect, and this observes the strong 40 times of (IC of the validity of this molecular ratio Fab through using divalence IgG molecule infliximab
5050pM).TNF-α is the tripolymer molecule; Itself and dimer acceptor interaction and therefore can expect the avidity of IgG allow with this cytokine on two epi-positions mutually combine and be formed on before (the people such as Santora that describes; Anal.Biochem.299,119-129) big mixture.Astoundingly, VHH has much bigger unusual effect to the increase of dimeric avidity than with infliximab is viewed from monomer, because dimeric IC50 (30pM) is than monomeric low 400 times.Increase avidity even more cause better agonistic behavior: the IC50 of tripolymer VHH is 20pM, and the tetravalence form is 6pM.All of VHH more high affinity form are all more effective than infliximab, and the tetravalence form is stronger than rhu TNFR:Fc, and wherein rhu TNFR:Fc is made up of the ectodomain of acceptor p75 and the Fc fusion of IgG, so it is the divalence binding pattern.
VHH with producing to mouse TNF observes the same unexpected influence (Figure 14) of avidity to agonistic behavior.Implement the CTA of same type with MTT as substrate and mouse TNF-α (65pg/ml or 1.3pM), but be to use mouse cell line L929, the special acceptor of this expression of cell lines mouse.Identified three kinds of different antagonisms (unit price) VHH, their code is 9E and 3F, and wherein preceding two kinds IC50 is 25nM, and a kind of IC50 in back is 2nM (also seeing embodiment 3).3F changes into bivalent form (BIV#m3F, SEQ ID NO:76) and causes IC50 to increase by 1000 times (2pM), thereby the avidity that shows the increase of this antibody again causes the unexpected improvement of antagonism characteristic.
The comparison of 2) merging with VHH-Fc
Embodiment 5: calculate the homology between of the present invention resisting-target-single domain antibody
Use Bioedit Sequence Alignment Editor to calculate the amino acid sequence homology degree between of the present invention resisting-target single domain antibody.Through ClustalW. (Thompson; J.D.; Higgins, D.G and Gibson, T.J. (1994) CLUSTAL W: select to improve the sensitivity .Nucleic Acids Research of gradual multisequencing comparison through sequence weighting, special breach point penalty and the weight matrix in position; Submit in June, 1994) aligned sequences, the ratio of the identical residue between the calculating all sequences.Table 8 has been pointed out the portion homologous property between of the present invention resisting-serum albumin VHHs.Table 9 has been pointed out the portion homologous property between anti-TNF of the present invention-α VHH.Table 10 has been pointed out the percentage ratio homology between of the present invention resisting-IFN-γ VHH.
The segmental expression of the VHH-CDR3 of embodiment 6:VHH# 3E
Use is arranged in framework 4 districts, and (forward: CCCCTGGCCCCAGTAGTTATACG) (reverse: TGTGCAGCAAGAGACGG) CDR3 of the antisense primer of (SEQ ID NO:104) amplification VHH# 3E is regional with being positioned at framework 3 districts for the sense primer of (SEQIDNO:103).In order to clone the CDR-3 fragment among the pAXl0, implement second with following primer and take turns pcr amplification, import required restriction site:
Reverse primer Sfi1:
GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCAGCAAGAGACGG(SEQ?ID?NO：105)
Forward primer Not1:
GTCCTCGCAACTGCGCGGCCGCCCCCTGGCCCCAGTAGTTATACG(SEQ?ID?NO：106)
In the 50ml reaction volume, use every kind of primer of 50pmol to implement the PCR reaction.The reaction conditions of first PCR be 94 ℃ 11 minutes, following 30/60/120 second of 94/55/72 ℃ of 30 round-robin and 72 ℃ are 5 minutes then.Use 2.5mM MgCl
2, (Roche Diagnostics, Brussels Belgium) implement institute and respond for 200mM dNTP and 1.25U AmpliTaq GodDNA polysaccharase.
With after Sfi1 and the Not1 cutting PCR product cloning being arrived pAX10.
Embodiment 7: to the stability test of the special antibody fragment of human TNF alpha
The sex change and the pepsic decomposition of the protein receptor of oral administration acid pH in the stomach.We have selected research VHH#3E to pepsic resistance study condition, suppose these condition simulation gastric environments.In intestinal bacteria, produced the special a kind of VHH:VHH#3E of human TNF alpha, this albumen has been purified to homogeneity through IMAC and gel permeation chromatography as recombinant protein.Confirm the protein concn behind the purifying with the molar extinction coefficient of the calculating at 280nm place through spectrophotometry.The diluent of preparation 100 micrograms/ml in the McIIvaine damping fluid (J.Biol.Chem.49,1921,183) that is respectively pH 2, pH3 and 4.Then these solution were hatched 15 minutes at 37 ℃, add pig stomach mucous membrane Pepsin with the 1/30w/w ratio afterwards.Add this proteolytic enzyme after 60 minutes, the collection sample also dilutes 100 times immediately in the PBS that contains 0.1% caseic pH 7.4 makes the stomach en-inactivation.3 times of diluents outside these specimen preparation 7 shares are with existing through ELISA evaluation function antibody fragment.Prepare identical diluent from the aliquots containig of collecting, add this proteolytic enzyme afterwards as reference.In ELISA measures, in the hole of the microtiter plate that encapsulates neutravidin, catch biotinylation TNF α.For the sample and the reference sample of pepsin, the similar serial dilution and the Xiang Kongzhong of preparation sample add those diluents of 100 microlitres.Hatch 1 hour after scouring microtiter plate.In order to detect combining of VHH and captive TNF α, use multi-clone rabbit anti--VHH antiserum(antisera) (R42) and anti-rabbit igg SEAP conjugate.After the washing, make the plate colour developing with p-NP phosphoric acid.The data of in Figure 17, drawing demonstrate similar curve to all samples and the reference sample that is exposed to digestion condition.This shows that VHH#3E keeps its functionally active basically under the condition of all selections.
Embodiment 8: the oral administration of the special VHH of Anti-Human TNF α in the mouse
Preparation contains the antibody-solutions of the special VHH# 3E of Anti-Human TNF α (100 micrograms/microlitre, dilution is 100 times in PBS).Three mouse at first mustn't diversion by 12 hours, in latter two hour, allows free near antibody-solutions then.Afterwards, put to death mouse and dissect their stomach.Wash stomach to obtain the content of stomach through the 500 microlitre PBS that contain 1%BSA immediately.The material of this flushing is used to prepare continuous three times of diluents subsequently, with 1/5 dilution beginning of undiluted material.With these sample transfer of 100 microlitres in each hole of the microtiter plate that encapsulates with human TNF alpha.After hatching 1 hour and thorough washing, with multi-clone rabbit anti--VHH antiserum(antisera) (R42) then hatches the existence of assessing immunologic active material with anti-rabbit SEAP conjugate.Make the ELISA colour developing with the p-nitrophenyl guanidine-acetic acid.Gained ELISA signal clearly illustrates that in the stomach flushing thing of these mouse and has functional VHH# 3E after 10 minutes.Through with typical curve relatively, we confirm for three mouse being tested, the segmental concentration of functional antibodies is respectively 1.5,12.6 and 8.6 micrograms/ml in the stomach washing fluid.Usefulness in the embodiment 9:IBD animal model
1) animal model of chronic colitis
Induce the usefulness of assessing the divalence VHH construct of using through various route of administration in the BALB/c mouse in the chronic colitis model at DSS (DEXTRAVEN SODIUM SULFATE).This model is at first by people such as Okayasu [people Gastroenterology 1990 such as Okayasu; 98:694-702] in describe and by people such as Kojouharoff [people Clin.Exp.Immunol.1997 such as G.Kojouharoff; 107:353-8] improve.Animal obtains from the Charles River Laboratories of Germany, 11 ages in week, and the body weight that remains in the animal facility up to them reaches 21 to 22g.In animal, induce chronic colitis through four DSS cycles of handling.Each cycle is made up of DSS processing interval (7 days), and it is the DSS of 5% (w/v) that concentration wherein is provided in tap water, does not have DSS in the tap water in recovering interval (12 days).The last restore cycle extended to 21 days from 12 days inflammatory conditions to be provided, the chronic rather than acute inflammation of this inflammatory conditions representative when treatment.After recovering at interval at last, mouse is randomized to either in the groups of 8 mouse and begins to treat with the VHH-construct.Treatment was spaced apart for 2 weeks.Treatment is put to death animal at interval in one week of back, and histological examination is dissected and implemented to intestines.Experiment is arranged among Figure 18 and schematically shows.
2) VHH regimen
During VHH treatment, through in the stomach or intravenous application 100 μ g divalence VHH3F to mouse (8 every group) with divalence VHH# 3F (VHH#m3F-VHH#m3F; SEQ ID No.76) treatment every day was treated 14 days continuously.Every other day treat extra animal groups with divalence VHH# 3F rectum ground, continued treatment 14 days.In all treatment groups, use the 100 μ g dosage of divalence VHH# 3F with the 1mg/ml concentration in the damping fluid.Negative control group is being accepted 100 μ l PBS under the same terms in addition.Regimen shows in table 11.
3) result
After putting to death mouse, confirm body weight and dissect colon.Definite colon length of dissecting is also passed through the histology that colon is assessed in hematoxylin-eosin (HE) dyeing (standard conditions).Compare with negative control (PBS processing), demonstrate longer (prorogued) colon length and higher histology score [people such as G.Kojouharoff, Clin.Exp.Immunol.1997 with the group of divalence nanobody 3F treatment; 107:353-8], thus the effect of treatment shown.
Table 1: the aminoacid sequence table that is directed against the each side peptide of the present invention of TNF-α
Table 2: be used for the tabulation of mutagenesis reaction, mutagenic primer and the template of mutagenesis VHH# 12B
Table 3: the mutagenesis reaction, mutagenic primer and the template list that are used for mutagenesis VHH# 3E
Table 4: the overview of the anti-TNF-α of humanization and wild-type VHH
Table 5: anti-mice serum white protein and anti-mice serum white protein+anti-TNF-α VHH
Table 6: the aminoacid sequence table that is directed against the VHH of people IFN-γ
Table 7: to the divalence of TNF-α (BIV 3E, BIV#m3F), the sequence of trivalent (TRI3E) or tetravalence (TETRA 3E) VHH
Table 8: the portion homologous property between the aminoacid sequence of anti-mice serum white protein VHH of the present invention
Table 9: the portion homologous property between anti-TNF-α VHH of the present invention
Table 11: treatment procedure table
Claims (65)
1. anti-TNF-α polypeptide, it contains at least one anti-TNF-α single domain antibody, and said single domain antibody is selected from the group of being made up of following:
(a) have the anti-TNF-α single domain antibody of the sequence of one of SEQ ID NOs:1-5 and 14,
(b) have to have more than the sequence of 85% identity and about combining TNF-α to have and be better than 10 with the sequence of SEQ ID NOs:1
-6The anti-TNF of the avidity of M-α single domain antibody.
2. according to the anti-TNF-α polypeptide of claim 1, wherein said polypeptide contains at least one anti-TNF-α single domain antibody, and sequence that it has and SEQ ID NO:1 possess more than 85% identity.
3. according to the anti-TNF-α polypeptide of claim 1, it also comprises at least one single domain antibody to serum albumin.
4. according to the anti-TNF-α polypeptide of claim 1; It also comprises at least one single domain antibody to serum proteins, and it is selected from the group of being made up of the single domain antibody to serum proteins of the sequence with SEQ ID NOs:26 to 29 and 85 to 97 any representative.
5. according to the anti-TNF-α polypeptide of claim 3, its sequence by any representative of SEQ ID NOs:30 to 43 is formed.
6. according to the anti-TNF-α polypeptide of claim 1, the number that wherein is directed against the single domain antibody of TNF-α is at least 2.
7. according to the anti-TNF-α polypeptide of claim 6, its sequence by any representative of SEQ IDNOs:73 to 76 is formed.
8. according to the anti-TNF-α polypeptide of claim 1, wherein at least a single domain antibody is humanized Camelidae VHH.
9. according to Claim 8 anti-TNF-α polypeptide, wherein at least a humanized Camelidae VHH is made up of the sequence of SEQ ID NOs:17 to 19 and 21 to 24 any representative.
10. according to the anti-TNF-α polypeptide of claim 1, wherein at least a single domain antibody is Camelidae VHH.
11. compsn, it comprises each anti-TNF-α polypeptide and pharmaceutical carrier of claim 1-10.
12. nucleic acid, its coding is according to each anti-TNF-α polypeptide of claim 1 to 10.
Combine with tumor necrosis factor-alpha 13. the method for an indentifying substance, said reagent are regulated each anti-TNF-α polypeptide of claim 1 to 10, the method comprising the steps of:
(a) existing when not having candidate modulator, allowing under said polypeptide and the target bonded condition, with each anti-TNF-α polypeptide of claim 1 to 10 contact with the target tumor necrosis factor-alpha and
(b) polypeptide of measuring process (a) and the combination between the target; The wherein combination when not having said candidate modulator; Bonded reduces said candidate modulator is accredited as a kind of reagent in the presence of said candidate modulator, and this reagent is regulated each anti-TNF-α polypeptide of claim 1 to 10 and combined with tumor necrosis factor-alpha.
14. one kind through each the method that combines indentifying substance of anti-TNF-α polypeptide and tumor necrosis factor-alpha of claim 1 to 10, said reagent is regulated the disease that tumor necrosis factor-alpha mediates, and this method comprises:
(a) existing when not having candidate modulator, allowing under said polypeptide and the target bonded condition, with each anti-TNF-α polypeptide of claim 1 to 10 contact with the target tumor necrosis factor-alpha and
(b) polypeptide of measuring process (a) and the combination between the target; The wherein combination when not having said candidate modulator; The bonded reduction is accredited as a kind of reagent with said candidate modulator in the presence of said candidate modulator, and this reagent is regulated the disease of tumor necrosis factor-alpha mediation.
15. method according to claim 14; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
16. one kind through each the method that combines indentifying substance of anti-TNF-α polypeptide and tumor necrosis factor-alpha of claim 1 to 10, said reagent adjusting tumor necrosis factor-alpha combines with its acceptor, and this method comprises:
(a) existing when not having candidate modulator, allowing under said polypeptide and the target bonded condition, with each anti-TNF-α polypeptide of claim 1 to 10 contact with the target tumor necrosis factor-alpha and
(b) polypeptide of measuring process (a) and the combination between the target; The wherein combination when not having said candidate modulator; The bonded reduction is accredited as a kind of reagent with said candidate modulator in the presence of said candidate modulator, and this reagent adjusting tumor necrosis factor-alpha combines with its acceptor.
17. a test kit that screens the reagent of the disease of regulating the tumor necrosis factor-alpha mediation, this test kit contain each anti-TNF-α polypeptide, tumor necrosis factor-alpha and its wrapping material and working instructions of claim 1 to 10.
18. test kit according to claim 17; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
19. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and the inflammatory process diseases associated.
20. anti-TNF-α polypeptide according to claim 19; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
21., be suitable for treating and/or preventing and/or alleviate and the inflammatory process diseases associated according to the compsn of claim 11.
22. compsn according to claim 21; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
23., be suitable for treating and/or preventing and/or alleviate and the inflammatory process diseases associated according to the nucleic acid of claim 12.
24. nucleic acid according to claim 23; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
25. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and is subject to TNF-α and regulates the disease that material is regulated, said TNF-α instrumentality mass-energy enough pass gastric environment and this material not by inactivation.
26. the anti-TNF-α polypeptide of claim 25; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
27. according to the compsn of claim 11, be suitable for treating and/or preventing and/or alleviate and be subject to TNF-α and regulate the disease that material is regulated, said TNF-α instrumentality mass-energy enough pass gastric environment and this material not by inactivation.
28. the compsn of claim 27; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
29. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and is subject to TNF-α and regulates the disease that material is regulated, said TNF-α regulates material and is delivered to vagina and/or rectum road.
30. the anti-TNF-α polypeptide of claim 29; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
31. the compsn of claim 11 is suitable for treating and/or preventing and/or alleviating the disease that is subject to the adjusting of TNF-α adjusting material, said TNF-α regulates material and is delivered to vagina and/or rectum road.
32. the compsn of claim 31; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
33. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and is subject to TNF-α and regulates the disease that material is regulated, said TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
34. the anti-TNF-α polypeptide of claim 33; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
35. the compsn of claim 11 is suitable for treating and/or preventing and/or alleviating the disease that is subject to the adjusting of TNF-α adjusting material, said TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
36. the compsn of claim 35; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
37. each anti-TNF-α polypeptide of claim 1 to 10; Be suitable for treating and/or preventing and/or alleviate and be subject to the disease that TNF-α regulates the material adjusting; Said TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease has increased the permeability of intestines mucosa.
38. the anti-TNF-α polypeptide of claim 37; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
39. the compsn of claim 11 is suitable for treating and/or preventing and/or alleviating the disease that is subject to the adjusting of TNF-α adjusting material, said TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease has increased the permeability of intestines mucosa.
40. the compsn of claim 39; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
41. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and is subject to TNF-α and regulates the disease that material is regulated, said TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
42. the anti-TNF-α polypeptide of claim 41; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
43. the compsn of claim 11 is suitable for treating and/or preventing and/or alleviating the disease that is subject to the adjusting of TNF-α adjusting material, said TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
44. the compsn of claim 43; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
45. each anti-TNF-α polypeptide of claim 1 to 10 is suitable for treating and/or preventing and/or alleviates and is subject to TNF-α and regulates the disease that material is regulated, said TNF-α instrumentality mass-energy is enough effectively passed skin.
46. the anti-TNF-α polypeptide of claim 45; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
47. the compsn of claim 11 is suitable for treating and/or preventing and/or alleviating the disease that is subject to the adjusting of TNF-α adjusting material, said TNF-α instrumentality mass-energy is enough effectively passed skin.
48. the compsn of claim 47; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
49. method according to claim 14; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
50. the test kit of claim 16; Wherein said disease be inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis and vasculitic any.
51. a compsn, it contains each the anti-TNF-α polypeptide of nucleic acid, claim 1 to 10 of with good grounds claim 12, perhaps according to the compsn and the appropriate drug carrier of claim 11.
52. each anti-TNF-α polypeptide of claim 1 to 10 is used for the application of test kit that diagnostic characteristic is the disease of tumor necrosis factor-alpha dysfunction in preparation, each anti-TNF-α polypeptide of wherein said claim 1 to 10 is used for contacting with sample,
Be used for the combining of said polypeptide and said sample and standard relatively, be the disease of the dysfunction of tumor necrosis factor-alpha wherein with respect to the difference diagnostic characteristic in the combination of said sample.
53. test kit that screens disease; Wherein said disease is inflammation, rheumatoid arthritis, segmental enteritis, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmunization hepatitis, autoimmunization parotitis, type i diabetes, epididymitis, glomerulonephritis, Exophthalmus goiter, acute febrile polyneuritis, struma lymphomatosa, hemolytic anemia, systemic lupus erythematous, male fertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, xerodermosteosis, SpA, thyroiditis; With vasculitic any; This test kit contains each isolating anti-TNF-α polypeptide of claim 1 to 10, its wrapping material and working instructions.
54. one kind produces each the method for anti-TNF-α polypeptide of claim 1 to 10, the method comprising the steps of:
(a) obtain encoding to the DNA of the Camelidae VHH of tumor necrosis factor-alpha,
(b) resulting DNA in clone and the expression step (a).
55. one kind produces each the method for anti-TNF-α polypeptide of claim 1 to 10, this method comprises:
(a) allow to express under the condition of said polypeptide, cultivate contain coding claim 1 to 10 each anti-TNF-α polypeptide nucleic acid host cell and
(b) reclaim the polypeptide that is produced from culture.
56. according to the method for claim 55, wherein said host cell is bacterium or yeast.
57. each anti-TNF-α polypeptide of claim 1-10 perhaps according to the nucleic acid of claim 12, perhaps is used for treating and/or preventing and/or alleviates the application with the medicine of inflammatory reaction diseases associated in preparation according to the compsn of claim 11.
58. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11, said TNF-α instrumentality mass-energy enough pass gastric environment and this material not by inactivation.
59. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11, said TNF-α regulates material and is delivered to vagina and/or rectum road.
60. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11, said TNF-α regulates material and is delivered to nose, the upper respiratory tract and/or lung.
61. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11; Said TNF-α regulates material and is delivered to intestines mucosa, and wherein said disease has increased the permeability of intestines mucosa.
62. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11, said TNF-α instrumentality mass-energy is enough effectively passed the tissue in hypogloeeis.
63. each anti-TNF-α polypeptide of claim 1 to 10; Perhaps be used for treating, prevent and/or alleviate being subject to the application of medicine that TNF-α regulates the symptom of the disease that material regulates in preparation according to the compsn of claim 11, said TNF-α instrumentality mass-energy is enough effectively passed skin.
64. each anti-TNF-α polypeptide of claim 1 to 10 is used for the application of the medicament of the said tumor necrosis factor-alpha of purifying in preparation.
65. each anti-TNF-α polypeptide of claim 1 to 10 is used for suppressing the application of the interactional medicament between tumor necrosis factor-alpha and one or more tumor necrosis factor-alpha acceptors in preparation.
Applications Claiming Priority (14)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US42506302P | 2002-11-08 | 2002-11-08 | |
| US42507302P | 2002-11-08 | 2002-11-08 | |
| US60/425,073 | 2002-11-08 | ||
| US60/425,063 | 2002-11-08 | ||
| EP03447005.4 | 2003-01-10 | ||
| EP033447005.4 | 2003-01-10 | ||
| EP03447005 | 2003-01-10 | ||
| EPPCT/EP03/06581 | 2003-06-23 | ||
| EPPCT/EP03/06581 | 2003-06-23 | ||
| EPPCT/EO03/06581 | 2003-06-23 | ||
| EPPCT/EO03/07313 | 2003-07-08 | ||
| EPPCT/EP03/07313 | 2003-07-08 | ||
| EPPCT/EP03/07313 | 2003-07-08 | ||
| PCT/BE2003/000192 WO2004041862A2 (en) | 2002-11-08 | 2003-11-07 | Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100074434A Division CN102584997A (en) | 2002-11-08 | 2003-11-07 | Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1735629A CN1735629A (en) | 2006-02-15 |
| CN1735629B true CN1735629B (en) | 2012-05-09 |
Family
ID=36077529
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200380108511.5A Expired - Lifetime CN1735629B (en) | 2002-11-08 | 2003-11-07 | Single domain antibodies against tumor necrosis factor-alpha and uses thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1735629B (en) |
| ZA (2) | ZA200503618B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2593476A2 (en) * | 2010-07-16 | 2013-05-22 | Ablynx N.V. | 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 |
| AU2013310924B2 (en) * | 2012-08-31 | 2017-08-03 | Argenx Bvba | Method for producing antibody molecules having inter-species, intra-target cross-reactivity |
| JP2015531350A (en) * | 2012-09-13 | 2015-11-02 | ノバルティス アーゲー | Antigen-binding molecules with terminal modifications |
| 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 |
-
2003
- 2003-11-07 CN CN200380108511.5A patent/CN1735629B/en not_active Expired - Lifetime
-
2005
- 2005-05-05 ZA ZA200503618A patent/ZA200503618B/en unknown
-
2008
- 2008-04-30 ZA ZA200803817A patent/ZA200803817B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200803817B (en) | 2009-11-25 |
| CN1735629A (en) | 2006-02-15 |
| ZA200503618B (en) | 2009-02-25 |
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Correction item: First application number Correct: 03447005.4 PCT/EP03/06581 PCT/EP03/07313 False: 033447005.4|PCT/EO03/06581|PCT/EO03/07313 Number: 07 Volume: 22 |
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Correction item: First application number Correct: 03447005.4 PCT/EP03/06581 PCT/EP03/07313 False: 033447005.4|PCT/EO03/06581|PCT/EO03/07313 Number: 07 Page: The title page Volume: 22 |
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