CN106039306A - Methods of treating immune disorders with single domain antibodies against TNF-alpha - Google Patents

Abstract

The invention relates to the TNFX binding molecule ozoralizumab (ATN-103), methods of using this molecule to treat immune disorders, including rheumatoid arthritis, and specific dosing regimens for the treatment of rheumatoid arthritis.

Description

Use the method that the single domain antibody for TNF α treats immune disorders

The application is international application no PCT/EP2012/055830, international filing date 2012-03-30, China's application number 201280016811.X, China's day 2013-09-30, invention entitled " use treats immunity for the single domain antibody of TNF α The method of disease " divisional application.

Invention field

The present invention relates to include combining (SDAB) molecule for the single domain antigen of tumor necrosis factor α (TNFa) many The method of the immune disorders of peptide and use SDAB molecular therapy such as rheumatoid arthritis.

Background of invention

For the SDABs of TNFa, such as, describe in PCT Publication WO 04/041862 and WO 06/122786.These Anti-TNF a SDABs may be used for prevention and/or treats the disease relevant to TNFa and disease and/or the disease mediated by TNFa And disease, such as inflammation, rheumatoid arthritis (rheumatoid arthritis), Crohn disease (Crohn ' s Disease), ulcerative colitis (ulcerative colitis), inflammatory bowel syndrome (inflammatory bowel Syndrome), multiple sclerosis (multiple sclerosis), Addison disease (Addison's disease), autoimmune Property hepatitis (autoimmune hepatitis), autoimmunity parotitis (autoimmune parotitis), type 1 diabetes (diabetes type 1), epididymitis (epididymitis), glomerulonephritis (glomerulonephritis), Greif This sick (Graves ' disease), Ji Lan mono-barre syndrome (Guillain-Barre syndrome), chronic lymphocytic thyroiditis (Hashimoto ' sdisease), hemolytic anemia (hemolytic anemia), systemic lupus erythematosus (sle) (systemic Lupus erythematosus), male sterility (male infertility), multiple sclerosis, myasthenia gravis (myasthenia gravis), pemphigus (pemphigus), psoriasis (psoriasis), rheumatic fever (rheumatic Fever), sarcoidosis (sarcoidosis), scleroderma (scleroderma), Sjogren syndrome (Sjogren ' s Syndrome), SpA (spondyloarthropathies), thyroiditis (thyroiditis) and vasculitis (vasculitis)。

Therapeutic scheme use anti-tnf treatment agent improved Disease Spectrum and the life of the patient suffering from inflammatory disease Bioplasm amount, but these treatments need mostly by professional medical health worker periodically (such as, weekly) injection medicine.Door frequently Examine and be administered to medical security system, patient and their caregiver and bring significantly burden.Reduce these outpatient services and to Patient and caregiver provide automedication to select to bring significantly individual and economic interests.It it is not every kind of anti-tnf treatment All by becoming minimizing therapeutic frequency or the material standed for of self-injection, therefore, there is demand for novel anti-tnf treatment agent, institute in agent State novel anti-tnf treatment agent to be administered (such as, monthly or bimonthly (bi-monthly)) at larger time intervals And/or be effective during self-injection.

The patient accepting anti-tumor necrosis factor (TNF) medicine is in by antibacterial, mycobacteria, fungus, virus, parasite Danger with the severe infections (SI) that other opportunistic pathogens cause.These infect may be relevant to underlying diseases or adopt with treatment Medicine be correlated with.Severe infections (SI) causes in hospital or dead or need the medicine of such as antibiotic.Accordingly, it would be desirable to protecting While holding efficacy of drugs, severe infections is down to minimum.

Summary of the invention

The present invention solves one or more in demand mentioned above.Use the spy of the polypeptide including anti-TNF a SDAB Determine dosage regimen treatment to suffer from the people experimenter of rheumatoid arthritis and cause the statistically significant of described experimenter's morbid state Improvement.Therefore, in some embodiments, the present invention includes the method treating the immune disorders in the people of these needs, institute The method of stating includes including two kinds of anti-TNF a SDABs and Anti-Human's serum albumin to what this people used multiple 30-200mg dosage (HSA) polypeptide of SDAB, the most each administration is spaced at least about surrounding in time.Modeling shows to include anti-TNF a SDAB's Polypeptide is effective along with dosage increases the time interval extending, the ill effect the most not dramatically increased.Make simultaneously Disclose further with the comparative modeling of anti-TNF inhibitor and to include favourable effect of polypeptides in combination of anti-TNF a SDAB and right The limit risk of SI effect does not affect.Therefore, in some embodiments, during the present invention includes treating the people of these needs The method of immune disorders, described method includes including two kinds of anti-TNF a SDABs to what this people used multiple 30-400mg dosage With the polypeptide of Anti-Human's serum albumin (HSA) SDAB, the most each administration be spaced in time at least the moon surrounding, all such as from about 8 weeks Or 2 months.In some embodiments, the present invention includes two kinds of Anti-tumor necrosin (TNFa) SDABs and Anti-Human The polypeptide of serum albumin (HSA) SDAB, described polypeptide has the immune disorders in this people needed for treatment, and treatment is by giving This people uses the described polypeptide of multiple 30-400mg dosage and carries out, and wherein said spacing of doses is at least about every surrounding.

In some embodiments, described anti-TNF a SDAB includes 3 complementary determining regions (CDR1, CDR2 and CDR3), Wherein:

A () CDR1 includes

(i) aminoacid sequence DYWMY (SEQ ID NO:22)；

(ii) there is the aminoacid sequence of at least 80% sequence iden with DYWMY (SEQ ID NO:22)；Or

(iii) only there is the aminoacid sequence of 1 aminoacid difference with DYWMY (SEQ ID NO:22)；

B () CDR2 includes

(i) aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23)；

(ii) with EINTNGLITKYPDSVKG (SEQ ID NO:23), there is at least 80%, 90% or 95% sequence same The aminoacid sequence of property；Or

(iii) there is the aminoacid of 2 or 1 aminoacid differences with EINTNGLITKYPDSVKG (SEQ ID NO:23) Sequence；With

C () CDR3 includes

(i) aminoacid sequence SPSGFN (SEQ ID NO:24)；

(ii) there is the aminoacid sequence of at least 80% sequence iden with SPSGFN (SEQ ID NO:24)；Or

(iii) there is the aminoacid sequence of 1 aminoacid difference with SPSGFN (SEQ ID NO:24).

In some embodiments, described anti-HSA SDAB includes 3 CDRs (CDR1, CDR2 and CDR3), wherein

A () CDR1 includes

(i) aminoacid sequence SFGMS (SEQ ID NO:25)；

(ii) there is the aminoacid sequence of at least 80% sequence iden with SFGMS (SEQ ID NO:25)；Or

(iii) only there is the aminoacid sequence of 1 aminoacid difference with SFGMS (SEQ ID NO:25)；

B () CDR2 includes

(i) aminoacid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26)；

(ii) with SISGSGSDTLYADSVKG (SEQ ID NO:26), there is at least 80%, 90% or 95% sequence same The aminoacid sequence of property；Or

(iii) there is the aminoacid of 2 or 1 aminoacid differences with SISGSGSDTLYADSVKG (SEQ ID NO:26) Sequence；With

C () CDR3 includes

(i) aminoacid sequence GGSLSR (SEQ ID NO:27)；

(ii) there is the aminoacid sequence of at least 80% sequence iden with 10GGSLSR (SEQ ID NO:27)；Or

(iii) there is the aminoacid sequence of 1 aminoacid difference with GGSLSR (SEQ ID NO:27).

In particular embodiments, at least one in described anti-TNF a SDABs includes and SEQ ID NO:2 (TNF30) there is the aminoacid sequence of the sequence iden of at least 80%, 90%, 95% or 99%.In specific embodiment In, described anti-HSA SDAB include with SEQ ID NO:5 (ALB8) have the sequence of at least 80%, 90%, 95% or 95% with The aminoacid sequence of one property.In particular embodiments, described polypeptide includes the ammonia of SEQ ID NO:1 (ozoralizumab) Base acid sequence.In particular embodiments, at least one SDABs is humanized.

In some embodiments, each of two anti-TNF a SDABs and anti-HSA SDAB are connected by joint. Described joint can be aminoacid sequence, and it can select the group of free SEQ ID NO:6 and SEQ ID NO:7 composition.

The administration of the present invention can be spaced at least about 2 weeks, 3 weeks, 4 weeks, January, 5 weeks, 6 weeks, 7 weeks, 8 weeks or 2 in time Month.

The administration of the present invention can include 10,30,80,100,120,160,180,200,225,250,275,300,320, 350,375 or 400mg SDAB molecules, and can be with intravenous, subcutaneous, oral, peritoneum, nose or sublingual administration.

The SDAB molecule of the present invention can be configured to pharmaceutical formulation.In some embodiments, described preparation includes:

A () concentration is about 1mg/ml to 250mg/ml, the SDAB molecule of all such as from about 10mg/ml to 250mg/ml；

B () concentration is about the cryoprotective agent (lyoprotectant) of 5% to about 10%, its selected from sucrose, sorbitol or Trehalose；

C () concentration is about the surfactant of 0.01% to 0.6%, its selected from Polyoxyethylene Sorbitan Monooleate or poloxamer- 188；With

D () buffer, it is about the histidine buffering liquid of 10-20mM selected from concentration or concentration is about the Tris buffering of 20mM Liquid, so that the pH of described preparation is about 5.0 to 7.5.

In some embodiments, described preparation include 1mg/ml, 10mg/ml, 30mg/ml, 80mg/ml, 100mg/ml, The polypeptide of 160mg/ml, 180mg/ml, 200mg/ml or even 250mg/ml, 20mM histidine, 10% (w/v) sucrose and 0.02% Polyoxyethylene Sorbitan Monooleate, pH is 6.0.

In some embodiments, described preparation include 1mg/ml, 10mg/ml, 30mg/ml, 80mg/ml, 100mg/ml, The polypeptide of 160mg/ml, 180mg/ml, 200mg/ml or even 250mg/ml, 20mM histidine, 7.5% (w/v) sucrose and 0.01% Polyoxyethylene Sorbitan Monooleate, pH is 6.0.

The SDAB molecule of the present invention can be used may be used for treatment to the polypeptide treating immune disorders and/or the present invention and exempt from Epidemic disease disease, the group of wherein said immune disorders choosing freely following composition: inflammation, arthritis, including rheumatoid arthritis, Psoriatic arthritis (psoriatic arthritis), ankylosing spondylitis (ankylosing spondylitis), teenager Idiopathic arthritis (juvenile idiopathic arthritis) and osteoarthritis (osteoarthritis)), COPD, Asthma (asthma), inflammatory bowel, including Crohn disease and ulcerative colitis, multiple sclerosis, Addison disease, autoimmune Property hepatitis, autoimmunity parotitis, type i diabetes, epididymitis, glomerulonephritis, Graves disease, Ji Lan mono-Ba Lei is comprehensive Levy, chronic lymphocytic thyroiditis, hemolytic anemia, systemic lupus erythematosus (sle), male sterility, multiple sclerosis, myasthenia gravis, pemphigus, silver Bits disease, hidradenitis suppurativa (Hidradenitis suppurativa), rheumatic fever, sarcoidosis, scleroderma, Sjogren is comprehensive Levy, SpA, thyroiditis and vasculitis.

Methotrexate therapy can be used with the people of the SDAB molecular therapy of the present invention simultaneously.

In particular embodiments, the present invention includes the side treating the rheumatoid arthritis in the people of these needs Method, described method includes using, every about surrounding, the polypeptide that 80mg includes the aminoacid sequence of SEQ ID NO:1 to this people, Wherein this people uses methotrexate therapy simultaneously.

In another specific embodiment, the present invention considers to treat the rheumatoid arthritis in this people needed Method, described method includes that using one time 160,200,320 or 400mg to this people every about surrounding includes SEQ ID NO:1 The polypeptide of aminoacid sequence, and wherein this people uses methotrexate therapy simultaneously.

In another specific embodiment, the present invention considers to treat the rheumatoid arthritis in this people needed Method, described method includes using once to this people every about eight weeks or being administered once a month 320 or 400mg including SEQ ID The polypeptide of the aminoacid sequence of NO:1, and wherein this people uses methotrexate therapy simultaneously.

In another embodiment, the present invention relates to include the polypeptide of the aminoacid sequence of SEQ ID NO:1, described many Peptide has the rheumatoid arthritis in this people needed for treatment, and treatment is by using 80mg polypeptide to this people every about surrounding Carrying out, wherein this people uses methotrexate therapy simultaneously.

In another embodiment, the present invention relates to include the polypeptide of the aminoacid sequence of SEQ ID NO:1, described many Peptide has a rheumatoid arthritis in this people needed for treatment, treatment by this people every about surrounding use one time 160, 200,320 or 400mg include that the polypeptide of aminoacid sequence of SEQ ID NO:1 is carried out, and wherein this people uses simultaneously Methotrexate therapy.

In another specific embodiment, the present invention includes the polypeptide of the aminoacid sequence of SEQ ID NO:1, Described polypeptide has the rheumatoid arthritis in this people needed for treatment, and treatment is by using one to this people every about eight weeks Secondary or be administered once a month the polypeptide of the 320 or 400mg aminoacid sequences including SEQ ID NO:1 and carry out, and optionally its In this people use methotrexate therapy simultaneously.

In another embodiment, the immune disorders that this present invention relates to treat in people of these needs is many Peptide, wherein said polypeptide with include SEQ ID NO:1 (ozoralizumab) aminoacid sequence polypeptide competition.

Invention describes

Accompanying drawing describes

Fig. 1 shows that reached rheumatoid in being administered the often group of 6 treatment groups of ozoralizumab closed at the 8th and 16 week The ACR-20 of joint inflammation treatment, 50 and 70 patient's percent of standard.In the dosage regimen of every 4 weeks, experimenter the 1st day, 4, within 8 and 12 weeks, it is administered ozoralizumab or placebo.In the scheme of every 8 weeks, experimenter was administered at the 1st day and the 8th week Ozoralizumab, and used placebo at the 4th and 12 week.

Fig. 2 shows treatment group ACR 20 responsiveness (LOCF) in time.

Fig. 3 shows treatment group DAS28 responsiveness (LOCF) in time.

Fig. 4 shows the dosage-ACR20 response of intermediate value (95%PI) placebo-correction of ATN-103.Shown in dotted line shows Comparison therapy intermediate value simulation response.

Fig. 5 shows the DAS response that the intermediate value (95%PI) of ATN-103 is simulated.Dotted line show shown in comparison therapy in Value response.

Fig. 6 shows the medicinal effectiveness represented with %ACR20 and %SI.Each bubble covers every kind of medicine in shown dosage regimen The 95%PI of analog response.

Definition

In order to the present invention is more easily understood, first define some term.Additionally be defined on whole detailed description Middle elaboration.

When used herein, article " " and " a kind of " (" a " and " an ") refer to that one or more than one is (such as, extremely Few one) grammatical object of this article.

Term "or" is with being here and hereinafter meant that term "and/or", and can exchange use with it, unless context is additionally Clearly indicate.

Term " albumen " and " polypeptide " are used interchangeably herein, and include the SDAB molecule of the present invention.

" about " and " about " generally means that acceptable about measured quantity providing the character of measurement or degree of accuracy Error degree.Exemplary error degree is within 20 percent (%) of given numerical value or numerical range, typically Within 10%, more typically within 5%.

When modify term such as " about ", " about ", " at least " or " at most " before a series of terms time, this term is intended to Each in term listed by modification.Such as, phrase " at least about 10% or 20% " should be construed to " at least about 10% or extremely Few about 20% ".

In the situation of nucleotide sequence, term " substantially the same " includes foot with in this article referring to the first nucleotide sequence Enough or the minimal number of nucleotide identical with the nucleotide of comparison in the second nucleotide sequence, so that the first and second nucleotides sequences Row coding has the polypeptide of common functional activity, or encodes common structure polypeptide domain or common functional polypeptide activity, example As, with canonical sequence have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The nucleotide sequence of 99% homogeneity.

The present invention also includes the fragment of polypeptide, derivant, analog or the variant of the present invention, and their any group Close.Term " fragment ", " variant ", " derivant " and " analog " includes retaining corresponding natural when referring to the albumen of the present invention Any polypeptide of at least some functional characteristic of SDAB molecule.The specific Fab discussed except elsewhere herein Outside, the fragment of the polypeptide of the present invention also includes hydrolysis and deletion fragment.The variant of the polypeptide of the present invention includes as described above Fragment, and also include due to aminoacid replacement, the polypeptide that lacks or insert the aminoacid sequence with change.Variant is permissible It is naturally-occurring or non-naturally-occurring.The variant of non-naturally-occurring can use induced-mutation technique known in the art to produce.Become Body polypeptide can include that conservative or nonconserved amino acid replaces, lacks or add.The derivant of the fragment of the present invention is such Polypeptide, described polypeptide be changed thus show natural polypeptides non-existent other feature.Example includes fusion protein. Variant polypeptide can also be referred to as " polypeptide analog " in this article.When used herein, " derivant " of polypeptide refers to have logical Cross the reaction of functional side group's group and the desired polypeptides of chemically derived one or more residues." derivant " also includes containing one The those polypeptides of the naturally occurring amino acid derivativges of individual or multiple 20 kinds of standard amino acids.Such as, 4-hydroxyproline Proline can be replaced；5-oxylysine can replace lysine；3-Methyl histidine can replace histidine；Homoserine Serine can be replaced；Ornithine can replace lysine.

Term " functional variety " refers to have substantially the same aminoacid sequence with naturally occurring sequence or by substantially Upper the most identical nucleotide sequence coded and can have the polypeptide of one or more activity of naturally occurring sequence.

Homology between sequence or the calculating of sequence iden (this term is used interchangeably herein) by following enter OK.

In order to determine the homogeneity percent of two kinds of aminoacid sequences or two kinds of nucleotide sequences, described sequence is for most preferably to compare Purpose is compared (for example, it is possible to introduce breach in the one or both of the first and second aminoacid or nucleotide sequence, to enter The optimal comparison of row, and for comparative purposes, non-homology sequence can be ignored).In a typical implementation, for comparing The length of the canonical sequence of purpose comparison is at least the 30% of the length of described canonical sequence, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

Then compare at corresponding amino acid position or the amino acid residue of nucleotide position or nucleotide.When first When position in sequence is occupied by the identical amino acid residue in the position corresponding with the second sequence or nucleotide, then two kinds points Son in this position be identical (when with time in this article, aminoacid or nucleic acid " homogeneity " and aminoacid or nucleic acid " homology " Of equal value).

In view of the length (needing to introduce described breach with optimal comparison both sequences) of breach number and each breach, Homogeneity percent between two kinds of sequences is the function of the number of the same position that described sequence has.

The determination of the homogeneity percent between the comparison of sequence and two kinds of sequences can use mathematical algorithm to realize.One In individual embodiment, the homogeneity percent between two kinds of aminoacid sequences uses Needleman&Wunsch, J.Mol.Biol. Algorithm described in (J. Mol. BioL) 48:444-453 (1970) determines, this algorithm has been incorporated into GCG software kit GAP program in (on the Internet gcg.com available), it uses Blossum 62 matrix or PAM250 matrix, and 16,14, The Gap Weight of 12,10,8,6 or 4 and the Length Weight of 1,2,3,4,5 or 6.In another embodiment, two kinds of nucleotide Homogeneity percent between sequence uses the GAP program in GCG software kit to determine (available on the Internet gcg.com), its Use NWSgapdna.CMP matrix and the Gap Weight of 40,50,60,70 or 80 and the Length Weight of 1,2,3,4,5 or 6.One group Typical parameter (and the parameter that should use except as otherwise noted) is Blossum 62 rating matrix, and it has Gap Penalty 12, gap extension penalty 4, and frameshift gap point penalty 5.

Homogeneity percent between two kinds of aminoacid or nucleotide sequence can use E.Meyers and W.Miller Algorithm described in CABIO, 4:11-17 (1989) determines, this algorithm is already integrated in ALIGN program (version 2.0) In, it uses PAMl20 weight residue table, Gap Length Penalty 12 and Gap Penalty 4.

Nucleic acid as herein described and protein sequence can serve as " search sequence " and retrieve public database, example As, thus identify other family members or relevant sequence.Such retrieval can use Altschul et al. .J.Mol.Biol. NBLAST and the XBLAST program (version 2.0) of (J. Mol. BioL) 215:403-10 (1990) Carry out.NBLAST program can be used to carry out BLAST nucleotide search, scoring=100, word length=12, thus obtain and this The nucleotide sequence of the nucleic acid molecule homologous described in bright.XBLAST program can be used to carry out BLAST Protein hits, mark= 50, word length=3, thus obtain and the aminoacid sequence of albumen of the present invention (SEQ ID NO:1) molecule homologous.For terrible To comparison jaggy for comparative purposes, it is possible to use BLAST jaggy (Gapped BLAST), as at Altschul Et al., described in Nucleic Acids Res. (nucleic acids research) 25:3389-253402 (1997).When using BLAST and breach During the blast program changed, it is possible to use the default parameters of described various programs (such as, XBLAST and NBLAST).

" conserved amino acid replacement " is that wherein amino acid residue is had the radical amino acid replacement of similar side chain.In ability Territory has been defined for the family with the amino acid residue of similar side chain.These families include the aminoacid with basic side chain (such as, lysine, arginine, histidine), has the aminoacid (such as, aspartic acid, glutamic acid) of acid side-chain, has not The aminoacid of charged polar side chain (such as, glycine, agedoite, glutamine, serine, threonine, tyrosine, Cysteine), have non-polar sidechain aminoacid (such as, alanine, valine, leucine, isoleucine, proline, benzene Alanine, methionine, tryptophan), there is the aminoacid (such as, threonine, valine, isoleucine) of the side chain of β-side chain And there is the aminoacid (such as, tyrosine, phenylalanine, tryptophan, histidine) of beta-branched side.

Term " SDAB " refers to that the single domain antigen as described in W004/041862 and WO 06/122786 combines and divides Son.Term " SDAB molecule " refers to polypeptide or other molecules of one or more SDABs.Term " SDAB " and " SDAB divides Son " use can be exchanged, represent that only domain antigen-binding units (such as, TNF30 or ALB8) or multivalent single domain resist Former binding structural domain (such as, TNF55, TNF56 or orozoralizumab).

" CDR " of variable domains is cumulative, the AbM according to both Kabat, Chothia, Kabat and Chothia, connects Touch (contact) and/or conformation definition or any CDR well known in the art determines that the aminoacid in the hypervariable region that method is identified is residual Base.Antibody CDRs can be accredited as the hypervariable region initially defined by Kabat et al..For example, with reference to Kabat et al., 1992, Sequences of Proteins of Immunological Interest (protein sequence that immunity is interested), the 5th edition, Public Health Service, NIH, Washington D.C..The position of CDRs can also be accredited as initially by Chothia With the structure ring structure other people described.For example, with reference to Chothia et al., 1989, Nature (naturally) 342:877-883.Its He identifies that the method for CDR includes " AbM definition ", and it is the half-way house between Kabat and Chothia, and uses Oxford Molecular ' s AbM antibody modeling software (is now) derivative, or it is included in MacCallum et al., 1996, J.Mol.Biol. (J. Mol. BioL), described in 262:732-745 based on viewed antigen contact (contact) " the contact definition " of CDRs.In another approach, " the conformation definition " of herein referred as CDRs, the position of CDRs Put to be accredited as and antigen is combined the residue making enthalpy contribution.For example, with reference to Makabe et al., 2008, Journal OfBiological Chemistry (journal of biological chemistry), 283:1156-1166.Also other CDR boundary definitions can not In strict accordance with one of said method, but will at least some of overlapping, although according to specific residue with Kabat CDRs Or organize residues or the prediction of the most whole CDRs not appreciable impact antigen combination or experiment discovery more, it can be shortened or add Long.When used herein, CDR may refer to be defined by method as known in the art (including the combination of various method) CDRs.Method used herein can use the CDRs according to any one definition in these methods.For the bag be arbitrarily given Include the embodiment of more than one CDR, CDRs can according to Kabat, Chothia, extension, AbM, contact and/or conformation fixed Any one definition in justice.

Detailed Description Of The Invention

Single domain antigen combines (SDAB) molecule

Single domain antigen combines (SDAB) molecule and includes such molecule, and its complementary determining region is single domain polypeptide A part.Example includes, but not limited to heavy-chain variable domains, the binding molecule of natural shortage light chain, derives from conventional 4-chain The single domain of antibody, the domain of transformation and the single domain framework being different from the framework deriving from antibody.SDAB molecule can To be any single domain molecule or the single domain molecule in any future of this area.SDAB molecule can derive from any Species, include, but not limited to mice, people, camel, yamma, fish, shark, goat, rabbit and cattle.

According to an aspect, the single domain antigen binding molecules that SDAB molecule is naturally-occurring, it is known as lacking gently The heavy chain of chain.Such as, described single domain molecule is disclosed in WO 94/04678 and Hamers-Casterman et al. .Nature In (naturally) 363:446-448 (1993).For clearly reason, this derives from heavy chain molecule variable of natural shortage light chain Domain is properly termed as VHH, to distinguish the conventional VH of itself and four chain immunoglobulins.Described VHH molecule can derive from camel Section's species (Camelidae species), such as, camel (camel), yamma (llama), dromedary camel (dromedary), Alpaca (alpaca) and maroon alpaca (guanaco).It is light that other species in addition to camellid can produce natural shortage The heavy chain molecule of chain；This type of VHHs is within the scope of the present invention.

Described SDAB molecule can be restructuring, CDR-grafting, humanized, camelized, go immunity and/or (such as, selected by phage display) of external generation, as described in greater detail below.

Term " antigen-combination " is intended to include the part of polypeptide, such as, the part of single domain molecule as herein described, It determinant including forming the interface combined with target antigen (or its epi-position).About albumen (or albumen analogies), antigen-knot Close site and typically comprise one or more rings (at least four aminoacid or the amino Radix rumicis acetosae forming the interface being combined with target antigen Intend the ring of thing).Typically, the antigen binding site of polypeptide, such as, the antigen binding site of described single domain antibody molecule, Including at least one or two CDRs, or more typically include at least three, four, five or six CDRs.

Term " immunoglobulin variable domain territory " is generally understood as and VL or VH in human or animal source in the art Domain is identical or substantially the same.It should be appreciated that in some species, such as, in shark and yamma, immune globulin White variable domains can be evolved, thus different from VL or VH of people or mammal on aminoacid sequence.

But, these domains are still primarily involved in antigen and combine.Term 20 " immunoglobulin variable domain territory " typical case Ground includes at least one or two CDRs, or more typically at least three CDRs.

" constant immunoglobulin domains " or " constant region " are intended to include and CL, CH1, the CH2 in human or animal source, The immunoglobulin domains that CH3 or CH4 domain is identical or essentially similar.For example, with reference to, Hasemann and Capra, Immunoglobulins:Structure and Function (immunoglobulin: 26S Proteasome Structure and Function), at William E.Paul compiles, Fundamental Immunology (basic immunology), the second edition, 209,210-218 (, 1989) in.Term " Fc district " refers to the Fc part of constant immunoglobulin domains, it include immunoglobulin domains CH2 and CH3 or and these Essentially similar immunoglobulin domains.

In certain embodiments, described SDAB molecule be unit price or multispecific molecule (such as, bivalence, trivalent or Tetravalent molecule).In other embodiments, SDAB molecule is specific point of monospecific, bispecific, tri-specific or four Son.Molecule is " monospecific " or " polyspecific ", e.g. " bispecific ", refers to that Binding peptide reacts The number of different epi-positions.Multispecific molecule can be the different epitope specificities to target polypeptide as herein described, or Can be to target polypeptid specificity and specific to heterologous epitope (such as heterologous polypeptide or solid support matter).

When used herein, term " valency " refers to the number of binding structural domain potential present in the SDAB molecule, example As, the number of antigen-binding domains.The specific binding epi-position of each binding structural domain.When SDAB molecule includes more than one During individual binding structural domain, each binding structural domain can be with specific binding identical epi-position, for having two basic change domain Antibody, be referred to as " bivalence monospecific ", or can be with specific binding different epi-position, for having two basic change domain SDAB molecule, be referred to as " bivalent, bispecific ".SDAB molecule can also be bispecific and for every species specificity two (referred to as " the bispecific tetravalent molecule ") of valency.Bispecific bivalent molecule, and the method preparing it, such as, describe in the U.S. The patent No. 5,731,168；5,807,706；In 5,821,333；With U.S. Publication No 2003/020734 and 2002/0155537 In；All these disclosures is incorporated herein by reference.Bispecific tetravalent molecule, and the method preparing it, such as, note Stating in WO 02/096948 and WO 00/44788, the disclosure of the two is incorporated herein by reference.Polyspecific and multivalence Other examples of molecule provide at WO 93/17715；WO92/08802；WO 91/00360；WO 92/05793；U.S. Patent number 4,474,893；4,714,681；4,925,648；5,573,920；With 5,601,819；Tutt et al., J.Immunol. (immunity Learn magazine) 147:60-69 (1991)；With Kostelny et al., J.Immunol. (Journal of Immunology) 148:1547-1553 (1992) in.

In certain embodiments, SDAB molecule is the single chain fusion polypeptide combining one or more target antigens, and it includes The complementary variable domains of one or more shortages or the single domain molecule of constant region for immunoglobulin (such as, Fc district).By institute The exemplary target antigen stating antigen-binding polypeptide identification includes tumor necrosis factor α (TNFa).In specific embodiment In, described Antigen-Binding single domain molecule combines serum albumin, such as, in conjunction with selected from serum albumin (such as, human blood Pure albumen (HSA)) or the human albumin of one or more of transferrins.

TNFa

It is known in the art tumor necrosis factor α (TNFa) relevant to inflammatory disease, described inflammatory disease such as class wind Wet arthritis, Crohn disease, ulcerative colitis and multiple sclerosis.Have studied TNFa and receptor the most in detail (CD120a and CD120b).The biologically active form of TNFa is trimer.Have been developed for some and use anti-TNF a Antibodies Against The strategy of TNFa effect, and be currently and can be purchased, such asWithUnijunction in conjunction with TNFa The multiple example of structure territory antigen binding molecules is disclosed in WO 04/041862, WO04/041865, in WO 06/122786, all These content is fully incorporated in this by quoting.

The other example of single domain antigen binding molecules is disclosed in US 2006/286066, US2008/0260757, In WO 06/003388, US 2005/0271663 and US 2006/0106203, all these contents is tied completely by quoting Together in this.In particular embodiments, the SDAB molecule of the present invention includes the SDABs of the aminoacid sequence of table 1, or bag Include the aminoacid sequence that includes having the sequence iden of about 80%, 85%, 90%, 95% or 99% with the sequence of table 1 SDABs.In alternate embodiment, the SDAB molecule of the present invention can include having 1 with the sequence of table 1,2,3,4,5,6, The aminoacid sequence of 7,8,9,10,11 or 12 aminoacid differences.

In other embodiments, it is considered to for TNFa and the monospecific of serum albumin (such as HSA), bispecific, Tri-specific and other polyspecific single domain antibodies.The multiple example of polyspecific TNFa and HSA Binding peptide is disclosed in WO 06/122786 kind, its content is incorporated herein.The polyspecific polypeptide of the present invention can include the ammonia of table 1 Base acid sequence, or include there is the aminoacid sequence of about 80%, 85%, 90%, 95% or 99% sequence iden with the sequence of table 1 Row.In alternate embodiment, the polyspecific SDAB molecule of the present invention can include having 1 with the sequence of table 1,2,3,4, The aminoacid sequence of 5,6,7,8,9,10,11 or 12 aminoacid differences.

In particular embodiments, the SDAB molecule in conjunction with TNFa includes disclosed in one or more WO06/122786 SDABs.Such as, the SDAB molecule of described combination TNFa can be the unit price disclosed in WO 06/122786, bivalence or trivalent TNFa-Binding peptide.

The SDABs molecule of the exemplary combination TNFa disclosed in WO 06/122786 includes, but not limited to TNF1, TNF2, TNF3, its humanization form (such as, TNF29, TNF30, TNF31, TNF32, TNF33).The table of WO 2006/122786 The other example that unit price combines the SDABs of TNFa is disclosed in 8.Exemplary bivalence combines the SDAB molecule of TNFa and includes, But being not limited to, TNF55 and TNF56, it includes two TNF30SDABs connected by peptide linker, thus forms single fused polypeptide (disclosed in WO 06/122786).Bivalence combines the other example table 19 at WO 06/122786 of the SDAB molecule of TNFa Disclosed in be TNF4, TNF5, TNF6, TNF7, TNF8.

In particular embodiments, anti-TNF a SDAB includes 3 complementary determining regions (CDR1, CDR2 and CDR3), its In: CDR1 includes aminoacid sequence DYWMY (SEQ ID NO:22)；With DYWMY (SEQ ID NO:22), there is at least 80% sequence The aminoacid sequence of row homogeneity；Or with DYWMY (SEQ ID25 NO:22), only there is the aminoacid sequence of 1 aminoacid difference Row；CDR2 includes aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23)；With EINTNGLITKYPDSVKG (SEQ ID NO:23) have at least 80%, the aminoacid sequence of 90% or 95% sequence iden；Or and EINTNGLITKYPDSVKG (SEQ ID NO:23) has the aminoacid sequence of 2 or 1 aminoacid differences；And CDR3 includes aminoacid sequence SPSGFN (SEQ ID NO:24)；With the aminoacid sequence that SPSGFN (SEQ ID NO:24) has at least 80% sequence iden；Or with SPSGFN (SEQ ID NO:24) has the aminoacid sequence of 1 aminoacid difference.

In some embodiments, include the aminoacid sequence of table 1 in conjunction with the SDAB of HSA, or have about with the sequence of table 1 The aminoacid sequence of 80%, 85%, 90%, 95% or 99% sequence iden.In alternate embodiment, the present invention's Anti-HSA SDAB can include having the ammonia of 1,2,3,4,5,6,7,8,9,10,11 or 12 aminoacid difference with the sequence of table 1 Base acid sequence.

In other embodiments, the SDAB molecule in conjunction with HSA includes one or more disclosed in WO 06/122786 SDABs.Such as, the SDAB molecule of described combination HSA can be that the unit price disclosed in WO 06/122786, bivalence or trivalent combine The SDAB molecule of HSA.In other embodiments, the SDAB molecule of described combination HSA can be to have at least one to combine HSA The monospecific of binding specificity or multispecific molecule.The SDABs of exemplary combination HSA includes, but not limited to WO ALB1 disclosed in 06/122786 and humanization form (such as, ALB6, ALB7, ALB8, ALB9, ALB10) thereof.

In particular embodiments, described anti-HSA SDAB includes 3 CDRs (CDR1, CDR2 and CDR3), wherein: CDR1 includes aminoacid sequence SFGMS (SEQ ID NO:25；There is at least 80% sequence together with SFGMS (SEQ ID NO:25) The aminoacid sequence of one property；Or with SFGMS (SEQ ID NO:25), only there is the aminoacid sequence of 1 aminoacid difference；CDR2 Including amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26)；With SISGSGSDTLYADSVKG (SEQ ID NO: 26) have at least 80%, the aminoacid sequence of 90% or 95% sequence iden；Or with SISGSGSDTLYADSVKG (SEQ ID NO:26) there is the aminoacid sequence of 2 or 1 aminoacid differences；And CDR3 includes aminoacid sequence GGSLSR (SEQ ID NO:27)；With the aminoacid sequence that GGSLSR (SEQ ID NO:27) has at least 80% sequence iden；Or and GGSLSR (SEQ ID NO:27) has the aminoacid sequence of 1 aminoacid difference.

In other embodiments, two or more single domain molecules of described SDAB molecule are with or without linker Group is fused to heredity or polypeptide fusion.Described linking group can be any linking group that those skilled in the art know. Such as, described linking group can be the biocompatible polymer of a length of 1 to 100 atom.In one embodiment, Described linking group includes following or is made up of following: polyglycine, polyserine, polylysine, polyglutamine, poly-different bright Propylhomoserin or poly arginine residue or combinations thereof.Such as, polyglycine or polyserine linking group can include at least five Individual, seven, eight, nine, ten, 12,15,20,30,35 and 40 glycine and silk Histidine residue.The exemplary joint that can use includes that Gly-Ser repeats, such as, with one, two, three, four, five Individual, six, seven or (Gly) of more repetition₄-Ser (SEQ ID NO:19) repeats.In some embodiments, joint There is following sequence: (Gly)₄-Ser-(Gly)₃-Ser (SEQ ID NO:20) or ((Gly)₄-Ser) n (SEQ ID NO:21), Wherein n is 4,5 or 6.In some embodiments, joint includes SEQ ID NO:6 (GS9) or SEQ ID NO:7 (GS30).

In an exemplary embodiment, the SDAB molecule of the present invention can by two basic change target antigen (such as TNFa) SDAB domain (such as, two camellid variable regions) and a unijunction combining serum albumin (such as HSA) The single-chain polypeptide fusion body of structure domain antibodies molecule (such as, camellid variable region) is constituted.

Being referred to herein as the polypeptide of " ozoralizumab " is melting of humanized, trivalent, bispecific suppression TNFa Hop protein.This fusion protein derives from camellid and has sequence and the knot of height with human normal immunoglobulin's VH domain Structure homology.Its Single polypeptide chain is made up of two binding structural domains for TNFa and a binding structural domain for HSA, Two nine amino acid whose G-S joints are utilized to connect described domain.The detailed description of Ozoralizumab provides at WO 06/ In 122786 (wherein it is referred to as TNF60), its content is incorporated herein.

Table 1

The preparation of SDAB molecule

The SDAB molecule of the present invention can include that one or more are recombinated, CDR-grafting, humanized, camelized , remove immunity and/or external generation (such as, being selected) single domain molecule (such as SDABs) by phage display. The technology producing antibody and the technology of SDAB molecule and these molecules of recombinant modified is well known in the art, and below Describe in detail.

Multiple method well known by persons skilled in the art may be used for obtaining antibody.Such as, monoclonal antibody can be passed through Produce hybridoma according to known methods and prepare.Then, use standard method, such as enzyme-linked immunosorbent assay (ELISA) and Surface plasma body resonant vibration (BIACORE^TM) hybridoma that formed by this way of Analysis and Screening, thus identify generation specificity knot Close one or more hybridomas of the SDAB of the antigen specified.Any type of antigen specified can serve as immunogen, such as, Recombinant antigen, naturally occurring form, its arbitrary variant or fragment, and its antigenic peptides.

A kind of exemplary method preparing antibody and SDAB molecule includes screening protein expression libraries, such as, phage Or ribosomal-display library.Such as, phage display describes at U.S. Patent number 5,223,409；Smith Science 228: 1315-1317(1985)；WO 92/18619；WO 91/17271；WO 92/20791；WO 92/15679；WO 93/01288； WO 92/01047；WO 92/09690；With in WO 90/02809.

Except using in addition to display libraries, it is intended that antigen can be used to immunizing non-human animals, such as, rodent, example As, mice, hamster or rat.In one embodiment, described non-human animal includes at least some of human normal immunoglobulin's base Cause.For example, it is possible to the large fragment of employment Ig locus is transformed lacks the mouse species that mouse antibodies produces.Use hybridoma skill Art, can produce and select have the specific antigenic specificity monoclonal antibody deriving from gene in need.For example, with reference to XENOMOUSE^TM, Green et al. .Nature Genetics (natural genetics) 7:13-21 (1994), US20030070185, WO 96/34096 and PCT Application No. PCT/US96/05928.

In another embodiment, SDAB molecule is obtained by non-human animal, is then modified, and such as, humanization, goes to exempt from Epidemic disease and/or chimeric, it is possible to use recombinant DNA technology known in the art produces these SDAB molecules.Describe multiple For preparing chimeric antibody and the method for SDAB molecule.For example, with reference to Morrison et al., Proc.Natl.Acad.Sci.U.S.A. (NAS's journal) 81:6851 (1985)；Takeda et al., Nature (naturally) 314:452 (1985), U.S. Patent number 4,816,567 and 4,816,397；European Patent Publication EP171496 and 0173494；And British patent GB 2177096B.For example, it is also possible to but use is expressed human immunoglobulin gene can not table Reach the SDAB molecule that the transgenic mice of endogenous mouse immunoglobulin gene is generating humanized.Winter describes a kind of example The CDR-engrafting method of property, its can be used to prepare humanized SDAB molecule as herein described (U.S. Patent number 5,225, 539).All CDRs of specific SDAB molecule by least some of replacement of inhuman CDR, or can only have some CDRs Can be replaced by inhuman CDRs.Only need to replace described SDAB molecule and be combined required number with predetermined antigen CDRs。

Humanized SDAB molecule the most directly can participate in resisting by replacing with the equivalent sequence from people's variable domains The sequence of the variable domains of former combination and produce.The exemplary method of generating humanized antibodies or its fragment is by Morrison Science (science) 229:1202-1207 (1985)；Oi et al., BioTechniques (biotechnology) 4:214 (1986)；With U.S. Patent number 5,585,089；5,693,761；5,693,762；5,859,205；There is provided with 6,407,213.

These methods include that separating, operate and express coding exempts from from least one heavy chain or all or part of of light chain The nucleotide sequence of epidemic disease immunoglobulin variable domain.Described nucleic acid can be from producing the SDAB molecule for predetermined target Hybridoma obtains, and as described above, and obtains from other sources.It is then possible to by the restructuring of encoding humanized SDAB molecule DNA clone is in suitable expression vector.

In certain embodiments, humanized SDAB molecule is taken by the conservative replacement of introducing, consensus sequence replacement, germline Generation and/or back mutation and optimised.The immunoglobulin molecules of described change can be by several technology known in the art In any one prepare (such as, Teng et al., Proc.Natl.Acad.Sci.U.S.A. (NAS's journal), 80:7308-7312 (1983)；Kozbor et al., Immunology Today (Immunol Today), 4:7279 (1983)； Olsson et al., Meth.Enzymol. (Enzymology method), 92:3-16 (1982)), and can be according to WO92/06193 or EP Prepared by the technology of 0239400.Technology for humanization SDAB molecule also provides in WO 06/122786

It is thin that SDAB molecule can also delete people T by the method specificity disclosed in WO 98/52976 and WO 00/34317 Born of the same parents' epi-position or " going immunization " and be modified.In short, about the peptide being combined with II class MHC, the weight of SDAB molecule can be analyzed Chain variable domains；These peptides represent potential t cell epitope (as defined in WO 98/52976 and WO 00/34317).For The t cell epitope that detection is potential, can apply referred to as the microcomputer modelling side of " peptide threading (peptide threading) " Method, and additionally can retrieve people II class MHC binding peptide data base and find motif present in VH and the VL sequence, as at WO Described in 98/52976 and WO 00/34317.These motifs combine any one in 18 kinds of main II class MHC DR allotypes, Therefore potential t cell epitope is formed.Detected potential t cell epitope can be by replacing minority in variable domains Amino acid residue or preferably replaced by single amino acids and be eliminated.Typically, conservative replacement is carried out.

Generally, but not exclusively, it is possible to use the aminoacid that the position in human germline antibody sequences is total.Such as, Human germ line sequences is disclosed in Tomlinson et al. .J.Mol.Biol. (J. Mol. BioL) 227:776-798 (1992)； Cook et al., Immunol.Today (Immunol Today) 16 (5): 237-242 (1995)；Chothia et al., J.Mol.Biol. (J. Mol. BioL) 227:799-817 (1992)；With Tomlinson et al., EMBO J.14:4628-4638 (1995) In.V BASE catalogue provides the panoramic catalogue of human normal immunoglobulin's variable region sequences (by Tomlinson, LA. et al. writing, MRC Centre for Protein Engineering (protein engineering MRC center), Cambridge, Britain).These sequences can For use as the source of human sequence, such as, for framework region and CDRs.Can also use common somebody's framework region, such as, as Described in U.S.6,300,064.SDAB molecule can produce by being produced the host cell alive of this albumen by genetic modification.Lose Pass the raw albuminiferous method of engineered cells to be well known in the present art.(1990) are compiled for example, with reference to Ausabel et al., Current Protocols in Molecular Biology (current molecular biological method) (Wiley, New York).Such Method includes coding and allows the nucleic acid of protein expression to be incorporated in host cell alive.These host cells can be to cultivate life Long bacterial cell, fungal cell or preferred zooblast.Bacterial host cell includes, but not limited to escherichia coli (Escherichia coli) cell.The example of suitable coli strain includes: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and the arbitrary coli strain of foreign DNA can not be cut.The fungal host that can use Cell includes, but not limited to saccharomyces cerevisiae (Sacchammyces cerevisiae), pichia pastoris phaff (Pichia And Eurotium (Aspergillus) cell pastoris).Some examples of the animal cell line that can use are CHO, VERO, BHK, HeLa, Cos, MDCK, 293,3T3 and WI38.The method of well known to a person skilled in the art can be used (such as, logical Cross conversion, virus infects and/or selects) set up new animal cell line.Optionally, albumen can be by host cell secretes to training Support in base.

The SDAB molecule modified

The preparation of the present invention can include at least one such SDAB molecule, described SDAB molecule have framework region it The ammonia that at least one amino acid position in one is different with the aminoacid sequence of naturally occurring domain (such as, VH domain) Base acid sequence.It should be understood that the aminoacid sequence of some SDAB molecules of the present invention (such as humanization SDAB molecule) can be At least one amino acid position at least one framework region and naturally occurring domain (such as, naturally occurring VHI-I knot Structure territory) aminoacid sequence different.

Present invention additionally comprises the preparation of the derivant of SDAB molecule.Described derivant generally can be by modifying and special It not by chemistry and/or 5 biologys (such as, enzyme) modifying SDAB molecule and/or form the one of SDAB molecule disclosed herein Individual or multiple amino acid residue and obtain.Can repair by this way in the example of described modification, and SDAB molecular sequences The example of the amino acid residue (that is, on protein backbone, but preferably on side chain) of decorations, may be used for introducing such modification Method and technology and the potential purposes of such modification and advantage are clearly for technical staff.

Such as, such modification can include in SDAB molecule or on it introduce (such as, by covalently bound or with appoint What his suitable mode) one or more functional groups, residue or part, and particularly give described SDAB molecule a kind of or The characteristic of multiple needs or functional one or more functional group, residue or part.The example of described functional group is technology people Member institute is clearly.

Such as, described modification can include that introducing (such as, by covalent bond or in any other suitable) increases Add the half-life of SDAB molecule, dissolubility and/or absorption, immunogenicity and/or the toxicity of reduction SDAB molecule, eliminate or alleviate Any unwanted side effect of SDAB molecule and/or give other favourable characteristics of SDAB molecule and/or reduce unwanted One or more functional groups of characteristic；Or two or more combination in any in aforementioned.The example of described functional group and introducing The example of the technology of described functional group be technical staff institute clearly, and may be generally comprised in above-cited general background In all functional groups of mentioning and technology and the modification for pharmaceutical protein be known per se functional group and technology, particularly For modified antibodies or the functional group of antibody fragment (including ScFvs and single domain antibody) and technology, such as, for its reference Remington ' s Pharmaceutical Sciences (Lei Mingdun pharmacopeia), the 16th edition, Mack Publishing Co., Easton, PA (1980).Such as, described functional group to can be directly connected to (such as, being covalently attached to) of the present invention On SDAB molecule, or connect optionally by suitable joint or spacer, this be also technical staff institute clearly.About increasing The immunogenic a kind of wide variety of technology adding half-life and/or minimizing pharmaceutical protein includes connecting suitable medicinal polymerization Thing such as PEG (PEG) or derivatives thereof (such as methoxyl group PEG or mPEG).Usually, can apply appoint The PEGization effect of what appropriate format, (includes but not limited to that (singly) domain resists for antibody and antibody fragment in such as this area Body and ScFvs) PEGization effect；Such as, with reference to Chapman, Nat.Biotechnol. (Nature Biotechnol), 54:531- 545(2002)；Veronese and Harris, Adv.Drug Deliv.Rev. (advanced drug delivery summary) 54:453-456 (2003), Harris and Chess, Nat.Rev.Drug.Discov. (natural drug find summary), 2, (2003) and at WO In 04/060965.Various reagent for albumen PEGization can also be purchased, such as from the Nektar Therapeutics of the U.S. Buy.

Preferably, application orientation PEGization, (see for example Yang etc., Protein especially by cysteine-residue Engineering (protein engineering), 16 (10): 761-770 (2003)).Such as, for the purpose of it, PEG may be coupled to In SDAB molecule on naturally occurring cysteine residues, SDAB molecule can be modified, to be appropriately introduced into one or many The individual cysteine residues for connecting PEG, or, can will include that one or more cysteine for PEG connection is residual The aminoacid sequence of base is fused to N-end and/or the C-end of the SDAB of the present invention, and all these uses are originally as art technology Protein engineering known to personnel.

Preferably, about SDAB molecule, use molecular weight more than 5000, all such as larger than 10,000 and less than 200,000, All such as less than 100, the PEG of 000: such as, the PEG 20,000-80, in the range of 000.

About PEGization, it should be noted that generally present invention additionally comprises at one or more amino acid positions by PEGization Any SDAB molecule, the most by this way, i.e. described PEGization (1) increase Half-life in vivo；(2) immunogen is reduced Property；(3) provide about PEGization one or more other more favourable characteristics known per se；(4) described in having no substantial effect on The affinity of SDAB molecule (such as, when by suitable detection assay, those detections described in the most following embodiment, Do not reduce the described affinity more than 90%, be preferably without reducing the described affinity more than 50%, and more preferably Do not reduce the described affinity more than 10%)；And/or (4) do not affect any other spy needed of described SDAB molecule Property.Technical staff is it should also be apparent that suitable PEG-group and the method that specifically or non-specifically connects them.

Such as, the SDABs of PEGization is disclosed in the U.S. Provisional Application No. 61/265 submitted on July 16th, 2011, in 307, It is incorporated herein by reference.

In some embodiments, the SDAB of PEGization includes the SDAB molecule of the modification being connected with PEG polymer, wherein Described PEG polymer molecule is the side chain PEG polymer molecule of the group selecting free style (a)-(h) to form:

In some embodiments, the SDAB of described PEGization includes:

I one or more single antigen-binding domains that () is combined with one or more targets；

(ii) non-peptide linker；With

(iii) one or more polymer molecules,

Wherein said non-peptide linker is the part of formula (I):

Wherein

W1 and W2 is separately selected from key or NR1；

Y is key, with Ra or pyrrolidine-2, the 5-diketone substituted C1-4 alkylidene of 0-2 existence；

X is O, key or do not exist；

Z is O, NR3, S or key；

R1 and R3 is separately hydrogen or C1-6 alkyl；

R2 does not exists or one or more polymer moieties；

Ra is selected from hydroxyl, C1-4 alkyl or C1-4 alkoxyl；

M is 0 or 1；

N is 0,1,2 or 3；

P is 0,1,2,3 or 4.

In further embodiment, the SDAB of described PEGization is connected on PEG by the joint shown in following formula:

In particular embodiments, the SDAB molecule of described modification includes following structure:

It addition, modification the most less preferably includes the glycosylation that N-connects or O-connects, depend on for expressing described The host cell of SDAB molecule, this is usually used as common translation and/or a part for post translational modification.

The method preparing SDABs

SDAB molecule can produce by being produced the host cell alive of this albumen by genetic modification.Genetically modified cell is raw Albuminiferous method is well known in the present art.(1990) are compiled, Current Protocols for example, with reference to Ausabel et al. In Molecular Biology (current molecular biological method) (Wiley, New York).Such method includes coding and permits The nucleic acid being permitted protein expression is incorporated in host cell alive.These host cells can be to cultivate the bacterial cell of growth, fungus Cell or zooblast.Bacterial host cell includes, but not limited to Bacillus coli cells.The reality of suitable coli strain Example includes: HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and can not cut the arbitrary of foreign DNA Coli strain.The fungal host cells that can use includes, but not limited to saccharomyces cerevisiae, pichia pastoris phaff and song Mould Pseudomonas cell.Some examples of the animal cell line that can use are CHO, VERO, BHK, HeLa, Cos, MDCK, 293,3T3 And WI38.The method of well known to a person skilled in the art (such as, infected by conversion, virus and/or select) can be used to set up New animal cell line.Optionally, albumen can be by host cell secretes to culture medium.

In some embodiments, described SDAB molecule can produce in bacterial cell (such as, Bacillus coli cells). Such as, if SDAB is to be included suppressing biting of termination codon by between displaying entity and phage protein (or its fragment) Sequential coding in phage display vector, then can transfer to suppress the bacterial cell of termination codon by this vector nucleic acid In.In this case, SDAB does not merges with gene III protein, and is secreted in pericentral siphon and/or culture medium.

SDAB molecule can also produce in eukaryotic cell.In one embodiment, SDAB molecule is in yeast cells Expressing, described yeast cells such as Pichia sp. (Pichia) (see, e.g., Powers et al., J Immunol Methods. (J. Immunol. Methods) 251:123-35 (2001)), Hanseula or Sacchammyces.

In one embodiment, SDAB molecule produces in mammalian cell.For expression cloning antibody or its resist The typical mammalian host cell of body-binding fragment includes that Chinese hamster ovary cell (Chinese hamster ovary celI) (includes dhfr-CHO Cell, its description is at Urlaub and Chasin, Proc.Natl.Acad.Sci.USA (NAS's journal) 77: In 4216-4220 (1980), use DHFR selected marker, such as, as at Kaufman and Sharp, Mol.Biol. (molecular biosciences Learn) described in 159:601-621 (1982)), lymphocyte series, such as, NS0 myeloma cell and SP2 cell, COS cell with And the cell from transgenic animal (such as, transgene mammal).Such as, described cell is breast epithelial cell.

In addition to the nucleotide sequence of coding SDAB molecule, recombinant expression carrier can also carry other sequence, such as The sequence (such as, origin of replication) of regulation carrier duplication in host cell and selectable marker gene.Selectable marker gene promotees Enter the selection to the host cell wherein having been incorporated into carrier (for example, with reference to U.S. Patent number 4,399,216；4,634,665； With 5,179,017).Such as, typically, selectable marker gene gives the drug resistance of the host cell having been incorporated into carrier, all Such as G418, hygromycin or methotrexate resistance.

In the example system of recombinant expressed SDAB molecule, the recombinant expression carrier of coding single domain antibody chain passes through The transfection of calcium phosphate mediation is incorporated in dhfr-CHO cell.In recombinant expression carrier, antibody gene respectively operably with Enhancers/promoters regulating element (such as, derives from SV40, CMV, adenovirus etc., such as cmv enhancer/AdMLP promoter Regulating element or SV40 enhancer/AdMLP modulator promoter element) connect, thus drive this high-level gene to transcribe.Restructuring table Reaching carrier and also carry DHFR gene, it allows to utilize methotrexate to select/expand to select the CHO having transfected this carrier thin Born of the same parents.The expression with permission antibody chain of the selected transformant host cell can be cultivated, and reclaim complete from culture medium Single domain.The Protocols in Molecular Biology of standard can be used to prepare recombinant expression carrier, and transfection host cell, selection converts Body, cultivates host cell and reclaims antibody molecule from culture medium.Such as, some SDAB molecules can be divided by affinity chromatograph From.

In one embodiment, described SDAB molecule such as purification described in WO 10/056550.Exemplary at one Embodiment in, by following, SDAB is come with one or more pollutant purification: make SDAB with one or more pollute The mixture of thing is allowing SDAB to be attached to described holder with holder based on a-protein and/or ion exchange holder Above or contact under conditions of being adsorbed onto on described holder；By washing under conditions of keeping being combined on described holder at SDAB Wash combined holder and remove one or more pollutant, and divided by the SDAB adsorbed with elution buffer eluting Son and from described holder selective elution SDAB.

SDAB molecule can also be produced by transgenic animal.Such as, U.S. Patent number 5,849,992 describe a kind of turning The method expressing antibody in the mammary gland of gene mammal.Building transgenic, it includes breast specificity promoter and encoding antibody The nucleic acid of molecule and the signal sequence for secretion.Secretion is included by the milk of the female generation in described transgenic animal Purpose single domain in milk.Antibody molecule can from milk purification, or for some apply, can directly make With.

Preparation

The SDABs of the present invention can be configured to any pharmaceutical formulation.Described preparation can be liquid or dry product.Described preparation Can by mixing, be dried, lyophilizing, vacuum drying or the method for arbitrarily known preparation Pharmaceutical composition and produce.

Pharmaceutical formulation can be configured to the orderly of solution, microemulsion, dispersion liquid, liposome or other applicable high protein concentrations Structure.Aseptic parenteral solution can be by following preparation: be combined in suitable solvent by the reagent as herein described of requirement, needs When wanting, there is a kind of composition listed above or the combination of Multiple components, then filtration sterilization.Generally, by by described herein Reagent be combined in sterile excipient and prepare dispersion liquid, described excipient includes basic disperse medium and arranges from below Other the required compositions of those lifted.The appropriate mobility of solution can be maintained, such as, by using coating such as ovum Phospholipid, in the situation of dispersion liquid by maintain required for granularity and by use surfactant maintain.

By including the reagent postponing to absorb, such as, Monostearate and gelatin in the composition, injection group can be caused The absorption of the prolongation of compound.

The preparation of SDAB molecule include SDAB molecule, can be as the compound of cryoprotective agent and buffer agent.Described system The pH of agent is usually pH 5.5-7.0, preferably from about pH 6.In some embodiments, preparation stores as liquid.Real at other Executing in scheme, preparation is prepared as liquid, is dried the most before storing, such as, by lyophilization or spray drying.It is dried Preparation can use as mummification compound, such as, as aerosol or powder, or reconstruct is to its initial concentration or another kind of dense Degree, such as, with water, buffer agent or other suitable liquid reconstruct.

Design SDAB molecular purifications method, the most cryodesiccated suitable to allow SDAB molecule to transfer to as frozen liq In the preparation (such as, using histidine/sucrose preparation) of longer-term storage.Described preparation freezes together with the albumen of certain concentration Dry.Then, when needed, with suitable diluent (such as, water), the preparation of this lyophilizing is reconstructed, thus by original formulation composition Again the concentration needed it is dissolved to, it is common that identical with the concentration before lyophilizing or higher than the concentration before lyophilizing.

The preparation of lyophilizing can reconstruct, and depends on joining in lyophilized products relative to initial cryodesiccated liquid volume Water or the amount of diluent, produce the preparation with the concentration different with initial concentration (that is, concentration before lyophilizing).By measuring antibody One or more parameters of integrity can identify suitable preparation.

The SDABs of the present invention can be as prepared described in WO 10/077422.The SDABs of the present invention can be by following Illustrative methods is prepared: by 10 mixture lyophilizing of SDAB, cryoprotective agent, surfactant and buffer agent；And in dilution Agent reconstructs the mixture of described lyophilizing, thus prepares described preparation.In particular embodiments, the SDABs of the present invention leads to Cross the preparation of following method: in bacterial cultures, express SDAB；By chromatography purification step and/or ultrafiltration/dialysis step purification SDAB；Including that concentration is about the sucrose of 5-10%, concentration is about the polysorbate80 of 0.01-0.02% and concentration is about The histidine of 10-20mM or concentration are about in the preparation of Tris buffer (so that the pH of described preparation is about 5-7.5) of 20mM Adjust the concentration of SDAB to about 10-250mg/ml.

Therapeutic Method

SDAB molecule can individually or be subject to the second reagent (such as, the second treatment or pharmaceutically active agent) combined administration Examination person (such as, people experimenter) treats or prevents (such as, alleviating or improve one or more associated symptoms) TNFa Associated conditions, such as, inflammatory or autoimmune disorder.

The SDAB molecule of the present invention, such as includes two kinds of Anti-tumor necrosin (TNFa) SDABs and anti-human seralbumin The polypeptide of albumen (HSA) SDAB, individually or can have this to need with the second agent combination as herein described for treatment or prevention Immune disorders in the people wanted.

Term " is treated " and is referred to be effectively improved the patient's condition, symptom or the parameter relevant to disease or prevent advancing to of disease The degree of statistically significant or amount, mode and/or pattern to the detectable degree of those skilled in the art implement treatment.Controlling Treating in the situation of application, treatment can improve, cures, the disease that maintains in experimenter or the patient's condition or shorten disease or the patient's condition exists Persistent period in experimenter.In treatment use, experimenter is likely to be of the performance partially or completely of symptom.Typically In situation, treatment improves the disease of experimenter or the patient's condition to the detectable degree of doctor, or prevents disease or the patient's condition from deteriorating.Have Amount, mode or the pattern of effect can be different because of experimenter, and can be experimenter's customization.

When used herein, term " experimenter " and " patient " are used interchangeably.When used herein, " one is subject to term Examination person " and " multiple experimenter " refer to animal, such as, mammal, including non-primate (such as, cattle, pig, horse, donkey, Goat, camel, cat, Canis familiaris L., Cavia porcellus, rat, mice, sheep) and primate (such as, monkey, such as machin, gorilla, Chimpanzee and people).

The limiting examples of treatable immune disorders includes, but not limited to autoimmune disorder, such as, and joint Inflammation (includes rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-relevant Arthritis (lupus-associated arthritis) or ankylosing spondylitis), scleroderma, systemic lupus erythematosus (sle) (systemic lupus erythematosis), Sjogren syndrome, vasculitis, multiple sclerosis, autoimmunity first shape Adenitis (autoimmune thyroiditis), dermatitis (dermatitis) (includes atopic dermatitis (atopic Dermatitis) and eczematoid dermatitis (eczenatous dermatitis)), myasthenia gravis, inflammatory bowel (IBD), Crow Grace is sick, colitis (colitis), diabetes (diabetes mellitus) (I type)；Inflammatory disease, such as, the inflammatory of skin Disease (such as, psoriasis)；Acute inflammatory disorders (such as, endotoxemia (endotoxemia), sepsis (sepsis) and Septicemia (septicemia), toxic shock syndrome (toxic shock syndrome) and infectious disease (infectious disease))；Transplant rejection (transplant rejection) and allergy (allergy).In one embodiment, Described TNFa associated conditions is arthrtic condition, such as, and the disease of one or more in following: rheumatoid joint Inflammation, juvenile rheumatoid arthritis (RA) (such as, moderate is to seriousness rheumatoid arthritis), osteoarthritis, psoriasis Arthritis or ankylosing spondylitis, multi-joint Juvenile idiopathic arthritis (polyarticular juvenile Idiopathic arthritis, JIA)；Or psoriasis, ulcerative colitis, Crohn disease, inflammatory bowel and/or multiple Hardening.

In certain embodiments, described SDAB molecule (or preparation) is used with the second therapeutic combination or is executed for combination With.Such as, for TNF-SDABs, the second reagent can be anti-TNF antibody or its combine the fragment of TNF, wherein the 2nd TNF resists Body has the epitope specificity different from the SDAB molecule of the combination TNF in preparation.Can be with the SDAB co-formulation combining TNF Other nonrestrictive examples of reagent include, such as, cytokine inhibitor, growth factor receptor inhibitors, immunosuppressant, Antiinflammatory, metabolic poison, enzyme inhibitor, cytotoxic agent and cytostatic agent.In one embodiment, additionally Reagent is for arthritic standard care, includes, but not limited to non-steroidal anti-inflammatory agent (NSAIDs)；Corticosteroid, including Prednisolone (prednisolone), prednisone (prednisone), cortisone (cortisone), and triamcinolone (triamcinolone)；With the antirheumatic (DMARDs) palliated a disease, such as methotrexate, oxychloroquine (hydroxychloroquine) (Plaquenil) and sulfur nitrogen sulphur pyridine (sulfasalazine), leflunomide (leflunomide)Tumor necrosis factor inhibitors, including Yi Naxipu (etanercept) Infliximab (infliximab)(with or without methotrexate), and adalimumab (adalimumab)Anti-CD 20 antibodies is (such as,), Soluble IL-1RI gene, such as Antril (Synergen) (asanakinra) (Kineret), gold, minocycline (minocycline)Penicillamine (penicillamine), and cytotoxic reagent, including azathioprine (azathioprine), cyclophosphamide And Cyclosporin A (cyclosporine) (cyclophosphamide).Described combined therapy can be advantageously employed relatively low-dose The therapeutic agent used, therefore avoids the possible toxicity relevant to various single therapies or complication.

When other reagent is methotrexate, the dosage of methotrexate can be in the scope of the most about 7.5 to about 25mg In.

SDAB molecule can be used or for executing with this with the form of liquid solution (such as, injection solution and infusion solution) With.Described compositions can be passed through parenteral pattern (such as, subcutaneous, intraperitoneal or intramuscular injection) or be used by suction.Short Language " parenteral administration " and " passing through parenteral administration " are with being here and hereinafter meant that the mode of administration in addition to intestinal and local application, logical Be often to be used by injection, and include subcutaneously or intramuscularly using, and intravenous, capsule in, ophthalmic, intracardiac, Intradermal, peritoneum In, under trachea, epidermis, under capsule, under arachnoidea, in spinal column, epidural and breastbone inner injection and infusion.An embodiment In, preparation as herein described passes through subcutaneous administration.

The present inventor estimates the ozoralizumab bispecific interaction sites with TNF α for treatment immune disorders It is useful especially.Therefore, the method that the present invention relates to treat immune disorders in this people needed, described method include to This people uses the polypeptide of the polypeptide competition with the aminoacid sequence including SEQ ID NO:1 (ozoralizumab).

Cheat regimen

Ozoralizumab be spaced between twice administration at least surrounding subcutaneous administration time demonstrate treatment rheumatoid close Effect that joint is scorching.By contrast, aboutThe dosage regimen recommended is to use every two weeks, and Must use by intravenous.Accordingly, with respect to prior art situation, the dosage regimen of the present invention provides the advantage that.

The present invention SDAB molecule with the dosage of every 30mg and 80mg used for 4 weeks demonstrated treatment disease merit Effect.Modelings based on these efficacy outcomes show with higher dosage (such as 120-200mg or 200-400mg), the present invention's SDAB molecule is effective to treatment disease at every 6 or 8 weeks or time February uses.This useful pattern causes the injection burden reduced.

It addition, modeling also shows that the severe infections (SI) that ozoralizumab treats is less than Embrel and Infliximab list Anti-severe infections.Especially, compared with anti-TNF a inhibitor of the prior art, it is right that the SDAB molecule of the present invention demonstrates The very favorable benefit (effect) of dangerous (SI effect) ratio.

Therefore, the SDAB molecule of the present invention can be used with the dosage in the range of 30-200mg for every 4,6 or 8 weeks.Have especially The dosage of effect is 30-400mg.In particular embodiments, described dosage includes about 5,10,15,20,25,30,80,100, 120,140,160,180,200,225,250,275,300,320,350,375 or 400mg SDAB molecules.

The single dose of about 30-200mg or even 30-400mg can about every day, every other day, biweekly, every 1, 2,3,4,5,6,7,8,9 or 10 weeks or every 1 or use February.

In particular embodiments, about 30,80,120,160,200,240,280,320,360 or 400mg include SEQ It is tested that the dosage of the polypeptide of the aminoacid sequence of ID NO:1 (ozoralizumab) is administered to people every about 4,6 or 8 weeks or February Person.In some embodiments, described experimenter suffers from rheumatoid arthritis.

In further embodiment, the dosage of described SDAB molecule is about 1, and 5,10,15,20,25,30,35,40,45, 50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150, 155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245, 250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340, 345,350,355,360,365,370,375,380,385,390,395 or 400mg.

In some embodiments, described dosage is about 3.6, and 10.8,36,72,144 or 288mg.

In particular embodiments, described SDAB molecule is PEGization, and dosage is about 3.6,10.8,36,72, 144 or 288mg.

For using to adolescent patient, dosage should be adjusted according to the body weight of described patient.In concrete reality Executing in scheme, described dosage is about 0.1, and 0.38,1,2,3,3.5,4,4.5 or 5mg/kg.

Experimenter can use methotrexate therapy simultaneously.In some embodiments, described experimenter is starting administration Methotrexate has been accepted at least about 6 or 12 weeks before ozoralizumab.Methotrexate can be by the most suitable approach Use, and dosage can be in the range of the most about 7.5-25mg.

The method determining effect

Effect of any concrete SDAB molecule or dosage regimen can by those skilled in the art can method true Fixed.In short, during clinical trial, patient can be watched by medical personnel, and combined by arbitrary standards and assess Disease condition.The improvement of patient disease situation is determined based on these standards at multiple time points, and by these determined combinations Draw to assess the effect treated for PATIENT POPULATION.

In exemplary embodiment, any mark that the progression of disease of rheumatoid arthritis can be listed by table 2 Accurate or all standards are measured.

Table 2

Embodiment

Embodiment 1

In rheumatoid arthritis (RA) patient with ozoralizumab carry out seamless 1/2 phase, random, layering, Double blinding, the research of placebo.A total of 254 experimenters participate in research at random；Wherein an experimenter does not enter Row treatment, remaining 253 experimenter is included in adjustment purpose treatment (modified intent-to-treat (mITT)) crowd In and safety population in.Every experimenter assigns in only one treatment group at random, based on tnf inhibitor (TNFi) first Or TNFi application layering before.A total of six treatment groups, describe in table 3, often organize and have 40-45 name patient at random.

Table 3

Baseline characteristic in these six groups is typically similar；Little still the adding up of only one observed between each group Learning significant difference (p=0.048) is in DAS28, meansigma methods the highest (6.59) DAS28 in 80mg Q4 group.Experimenter's Age is at 18-79 year (average 52.1 years old), and most subjects (80.2%) is women (pre-such as RA patient clinical trial Survey the same).The compound average of disease duration is 8.3 years, and 28.9% patient once accept before TNFi control Treat.

Main foreigner tourists for efficiency analysis is mITT colony, is defined as accepting the ozoralizumab of dosage at least one times All random experimenter.All patients are simultaneously with the methotrexate therapy of 7.5-25mg dosage weekly.

Every experimenter accepts the ozoralizumab of single dose level or the subcutaneous of placebo at the time point specified (SC) injection.

Interested main compare the comparison being each ATN-103 treatment group with placebo group.

Table 4 shows Americanism damp disease institute 20 (ACR20) responsiveness at the 16th week.

Table 4

Based on layering (TNFi or TNFi before application first) Cochran-Mantel-Haenszel inspection obtains P- Value.Minimum risk (MR) weight method using Mehrotra and Railkar to propose calculates the difference about placebo-adjustment (TNFi or TNFi before application first) confidence interval of layering.Before obtaining ACR20 response, last observation is used to advance The data of any omission of ACR composition are entered by method (Last observation carried forward (LOCF) approach) Row attribution.

Fig. 1 shows at the 8th week and within the 16th week, reaches the percent of experimenter of ACR20, ACR50 and ACR70 standard.Under Stating terminal, some dosage regimens relatively placebo has benefit: ACR20 (Tu1 &2), an ACR50 (Fig. 1), DAS28 (Fig. 3), ACR-N, TJC, SJC, pain VAS, HAQ-DI, CRP, doctor's comprehensive assessment, Patient Global assesses, holistic health VAS, and EULAR response (data do not show).

In short, as shown in FIG. 1, at the 8th week, for 10mg Q8, the experimenter of 36.6% reaches ACR20, The experimenter of 9.8% reaches ACR50, and the experimenter of 0% reaches ACR70；For 10mg Q4, the experimenter of 45.2% reaches ACR20, the experimenter of 11.9% reaches ACR50, and the experimenter of 7.1% reaches ACR70；For 30mg Q4,50% tested Person reaches ACR20, and the experimenter of 32.5% reaches ACR50, and the experimenter of 15% reaches ACR70；For 80mg Q8, The experimenter of 54.8% reaches ACR20, and the experimenter of 23.8% reaches ACR50, and the experimenter of 4.8% reaches ACR70；And For 80mg Q4, the experimenter of 65.1% reaches ACR20, and the experimenter of 25.6% reaches ACR50, and the experimenter of 2.3% reaches To ACR70.At the 16th week, for 10mg Q8, the experimenter of 48.8% reaches ACR20, and the experimenter of 24.4% reaches ACR50, With 8.9% experimenter reach ACR70；For 10mg Q4, the experimenter of 52.4% reaches ACR20, and the experimenter of 21.4% reaches To ACR50, and the experimenter of 4.8% reaches ACR70；For 30mg Q4, the experimenter of 60% reaches ACR20,32.5% be subject to Examination person reaches ACR50, and the experimenter of 20% reaches ACR70；For 80mg Q8, the experimenter of 59.5% reaches ACR20, The experimenter of 31% reaches ACR50, and the experimenter of 19% reaches ACR70；And for 80mg Q4, the experimenter of 72.1% Reaching ACR20, the experimenter of 37.2% reaches ACR50, and the experimenter of 11.6% reaches ACR70.

As shown in FIG. 3, at the 4th week, about DAS28 standard, it was observed that the mean change from baseline (placebo) For: it is 1.39% about 10mg Q4, is 1.05% about 10mg Q8, is 1.62% about 30mg Q4, about 80mg Q4 is 1.63%, and be 1.67% about 80mg Q8.At the 8th week, about DAS28 standard, it was observed that the mean change from baseline For: it is 1.59% about 10mg Q4, is 1.01% about 10mg Q8, is 1.78% about 30mg Q4, about 80mg Q4 is 2.00%, and be 1.85% about 80mg Q8.At the 12nd week, about DAS28 standard, it was observed that the mean change from baseline For: it is 1.61% about 10mg Q4, is 1.72% about 10mg Q8, is 2.31% about 30mg Q4, about 80mg Q4 is 2.30%, and be 2.20% about 80mg Q8.At the 16th week, about DAS28 standard, it was observed that the mean change from baseline For: it is 1.60% about 10mg Q4, is 1.70% about 10mg Q8, is 2.09% about 30mg Q4, about 80mg Q4 is 2.46%, and be 1.93% about 80mg Q8.At the 20th week, about DAS28 standard, it was observed that the mean change from baseline For: it is 1.02% about 10mg Q4, is 1.03% about 10mg Q8, is 1.62% about 30mg Q4, about 80mg Q4 is 2.00%, and be 1.36% about 80mg Q8.

In general, the safety profile of ozoralizumab seems the safety with other tnf inhibitors (TNFi) reagent Sexual norm is suitable.The SAEs of report is also consistent with the safety profile of other anti-TNF reagent (data do not show).

Embodiment 2:ATN-103 is relative to five kinds of commercially available anti-TNF s ratio in rheumatoid arthritis Relatively effect

2.1 purposes:

Ozoralizumab (ATN-103) is a kind of novel tumor necrosis factor inhibitors (TNFi), and it is a kind of bag Humanized, trivalent, the bispecific that include two the people's TNF binding structural domains being connected with human serum albumin's binding structural domain are received Meter Kang Ti.Carrying out meta-analysis based on model (model-based meta analysis, MBMA), to assess ATN-103 relative (effect (ACR20/50/ of the various dose/dosage regimen of anti-TNF a) medicine is blocked in five kinds of commercially available tumor necrosis factor αs 70, DAS, HAQ) and safety/toleration (severe infections rate, %SI), and evaluate illustrative co-variation (explanatory Covariates) effect.

2.2 methods:

Use the data announced, study that sum up in branch (study-arm) level and internal IIa issue according to 5 kinds Relatively agent (infliximab, adalimumab, Embrel, training house pearl monoclonal antibody (certolizumab pegol) He Geli Wood monoclonal antibody) carry out comparison effect based on model and safety analysis.

Specifically, ACR20/50/70 data set includes from 7, the data of 474 experimenters, and described experimenter participates in 20 Individual test, has 63 branches.DAS is observed in 21 branches of 8 tests.Severe infections rate data set includes from 6, The data of 209 experimenters, described experimenter participates in 14 tests, has 46 branches.Data set includes following measurement: medicine Expose (dose intensity, dosing interval), baseline disease severity co-variation (tenderness and the Joint Count of swelling, it was observed that DAS, Patient and the comprehensive assessment of doctor, CRP concentration, disease duration), the feature of test colony (lives through the tested of anti-TNF alpha The percent of person, and geographic (Asia is relative to non-Asia experimenter)).

ACR20/50/70 and DAS model is used to describe three ACR terminals and DAS terminal respectively.

2.3 results:

2.3.1Dosage-AC20/50/70 the response of simulation

With lasting DMARD background medication therapeutic alliance 24 weeks after, in typical patient colony (in placebo group, Live through the ACR20 respondent that average baselining Swollen Joint Count is 19 and 21% of TNF) use final ACR20/50/70 mould Type simulated ATN-103 and five kinds compare the dosage-response relation of the prediction of agent.Fig. 4 shows and other anti-TNF alpha medicines in colony Compare, at the 24th week, about the dosage-response relation of the ATN-103 of ACR20 response prediction.

This simulation shows that 80mg Q4W ATN-103 treatment is equivalent to adalimumab and Ge Li wood monoclonal antibody (golimumab) Response 95%, and anticipated 200mg ATN-103 Q4W produces the response similar to the 95% of the response of Embrel.

2.3.2Dosage-DAS the response of simulation

With united 24 weeks of ongoing DMARD background medication treatment after, about DAS%CfB simulation ATN-103 with Dosage-the response relation of five kinds of predictions comparing agent medicine.Assuming that DAS placebo response for about DAS from baseline change- 16%, this reflects the meansigma methods (data do not show) of unstructured mean placebo model parameter estimation value.

Dosage-the response of the prediction that Fig. 5 is shown in the ATN-103 that other anti-TNF alpha medicines compare about DAS response is closed System.At 200mg Q4W, simulation shows in terms of DAS%CfB, and ATN-103 is better than dagger-axe profit wood monoclonal antibody and Pei She pearl monoclonal antibody (certolizumab)。

2.3.3The severe infections rate of simulation

The %SI of the placebo-correction of simulation shows infection rate and adalimumab and the Ge Li wood of the prediction of ATN-103 The infection rate of monoclonal antibody is suitable, and trains house pearl monoclonal antibody, Embrel and infliximab and be likely to be of higher severe infections rate (data do not show).

Fig. 6 provides ATN-103 effect (ACR) relative to the prediction of other anti-TNF alpha medicines and safety (%SI) mould The comprehensive sketch plan of formula.Specifically, Fig. 6 shows the 95% pre-of the anti-TNF alpha efficacy of drugs (ACR20) simulated in relevant dosage regimen Survey 95% predicting interval of the simulation of interval and corresponding severe infections rate.Display optimal efficacy/safety effectiveness trends towards The upper left corner (high effect, low severe infections rate), reduces to the lower right corner.Blister figure shows effectiveness between multiple anti-TNF alpha medicine Considerable overlap.

This Random effects shows with the expression of safety compared with other anti-TNF alpha medicines, 200mg ATN-103 Q4W May preferably position.

2.3.4 The dosage regimen of 400mg Q8W

Model shows that response does not relies on therapeutic scheme, and can obtain for 200mg Q4W scheme or 400mg Q8W Similar effect.

2.4 conclusions:

ATN-103 is evaluated relative to five kinds of anti-TNF a medicines in effect (ACR/DAS) and peace with meta-analysis based on model The effectiveness of the comparison of full property/toleration (checking infection rate) aspect.Compared with other anti-TNF alpha medicines, ATN-103 shows merit Effect and the advantageous combination of SI effect.

Simulation based on model shows that 200mg ATN-103 Q4W or 400mg ATN-103 Q8W are in effect and according to that Western general, adalimumab is similar with infliximab.

Claims (35)

1. The most kinds of pharmaceutical preparatioies comprising 30-400mg dosage polypeptide have the immunological disease in the people of these needs in preparation for treatment Purposes in the test kit of disease, wherein said polypeptide includes two Anti-tumor necrosin & (TNF α) SDABs and Anti-Human's serum Albumin (HSA) SDAB.
2. 2. the purposes of claim 1, the most each anti-TNF alpha SDAB includes 3 complementary determining regions (CDR1, CDR2 and CDR3), Wherein

   A () CDR1 includes

   (i) aminoacid sequence DYWMY (SEQ ID NO:22)；

   (ii) there is the aminoacid sequence of at least 80% sequence iden with DYWMY (SEQ ID NO:22)；Or

   (iii) only there is the aminoacid sequence of 1 aminoacid difference with DYWMY (SEQ ID NO:22)；

   B () CDR2 includes

   (i) aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23)；

   (ii) with EINTNGLITKYPDSVKG (SEQ ID NO:23), there is at least 80%, 90% or 95% sequence iden Aminoacid sequence；Or

   (iii) there is the aminoacid sequence of 2 or 1 aminoacid differences with EINTNGLITKYPDSVKG (SEQ ID NO:23)； With

   C () CDR3 includes

   (i) aminoacid sequence SPSGFN (SEQ ID NO:24)；

   (ii) there is the aminoacid sequence of at least 80% sequence iden with SPSGFN (SEQ ID NO:24)；Or

   (iii) there is the aminoacid sequence of 1 aminoacid difference with SPSGFN (SEQ5 ID NO:24).
3. 3. the purposes of claim 1 or 2, wherein CDR1 includes aminoacid sequence DYWMY (SEQ ID NO:22), and CDR2 includes ammonia Base acid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23), and CDR3 includes aminoacid sequence SPSGFN (SEQ ID NO:24).
4. 4. the purposes any one of claim 1-3, at least one in wherein said anti-TNF alpha SDABs includes and SEQ ID NO:5 (TNF30) has the aminoacid sequence of at least 80%, 90%, 95% or 99% sequence iden.
5. 5. the purposes any one of claim 1-4, the most each anti-TNF alpha SDAB includes and SEQ ID NO:5 (TNF30) There is the aminoacid sequence of at least 80%, 90%, 95% or 99% sequence iden.
6. 6. the purposes any one of claim 1-5, wherein said anti-HSA SDAB include 3 CDRs (CDR1, CDR2 and CDR3), wherein

   A () CDR1 includes

   (i) aminoacid sequence SFGMS (SEQ ID NO:25)；

   (ii) there is the aminoacid sequence of at least 80% sequence iden with SFGMS (SEQ ID NO:25)；Or

   (iii) only there is the aminoacid sequence of 1 aminoacid difference with SFGMS (SEQ ID NO:25)；

   B () CDR2 includes

   (i) aminoacid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26)；

   (ii) with SISGSGSDTLYADSVKG (SEQ ID NO:26), there is at least 80%, 90% or 95% sequence iden Aminoacid sequence；Or

   (iii) there is the aminoacid sequence of 2 or 1 aminoacid differences with SISGSGSDTLYADSVKG (SEQ ID NO:26)； With

   C () CDR3 includes

   (i) aminoacid sequence GGSLSR (SEQ ID NO:27)；

   (ii) there is the aminoacid sequence of at least 80% sequence iden with GGSLSR (SEQ ID NO:27)；Or

   (iii) there is the aminoacid sequence of 1 aminoacid difference with GGSLSR (SEQ ID NO:27).
7. 7. the purposes of claim 6, wherein CDR1 includes aminoacid sequence SFGMS (SEQ ID NO:25), and CDR2 includes amino Acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26), and CDR3 include aminoacid sequence GGSLSR (SEQ ID NO: 27)。
8. 8. the purposes of claim 6 or 7, wherein said anti-HSA SDAB includes having at least with SEQ ID NO:10 (ALB8) 80%, the aminoacid sequence of 90%, 95% or 99% sequence iden.
9. 9. the purposes any one of claim 1-8, wherein said polypeptide includes the aminoacid sequence of SEQ ID NO:1 (ozoralizumab)。
10. 10. the purposes any one of claim 1-9, at least one in wherein said SDABs is humanized.
11. Purposes any one of 11. claim 1-10, each of wherein said two anti-TNF alpha SDABs and anti-HSA SDAB is connected by joint, the aminoacid sequence composition of the most each joint choosing free SEQ ID NO:6 and SEQ ID NO:7 Group.
12. Purposes any one of 12. claim 1-11, the group of the most following composition of wherein said dosage choosing: about 30,80, The SDAB molecule of 100,120,140,160,180,200,250,275,300,320,350,375 and 400mg.
13. Purposes any one of 13. claim 1-12, wherein said polypeptide subcutaneous or intravenous is used.
14. Purposes any one of 14. claim 1-13, wherein said test kit also comprises methotrexate.
15. Purposes any one of 15. claim 1-14, wherein said polypeptide is the form of pharmaceutical formulation.
16. Purposes any one of 16. claim 1-15, the group of the most following composition of wherein said immune disorders choosing: scorching Disease, arthritis, including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Juvenile idiopathic arthritis and bone Arthritis, COPD, asthma, inflammatory bowel, including Crohn disease and ulcerative colitis, multiple sclerosis, Addison disease, self Autoallergic, autoimmunity parotitis, type i diabetes, epididymitis, glomerulonephritis, Graves disease, Ji Lan-Ba Lei Syndrome, chronic lymphocytic thyroiditis, hemolytic anemia, systemic lupus erythematosus (sle), male sterility, multiple sclerosis, myasthenia gravis, sky bleb Skin ulcer, psoriasis, hidradenitis suppurativa, rheumatic fever, sarcoidosis, scleroderma, Sjogren syndrome, SpA, thyroiditis And vasculitis.
17. The purposes of 17. claim 16, wherein said immune disorders is rheumatoid arthritis.
18. The pharmaceutical preparation of 18. polypeptide comprising 80mg has the rheumatoid arthritis in the people of these needs for manufacturing for treatment The purposes of test kit, wherein said test kit also comprises methotrexate, and described polypeptide includes the aminoacid sequence of SEQ ID NO:1 Row.
19. 19. pharmaceutical preparatioies, described pharmaceutical preparation comprises:

    A () concentration is about 1mg/ml to 250mg/ml, the SDAB molecule of such as from about 10mg/ml to 250mg/ml；

    B () concentration is about the cryoprotective agent of 5% to about 10%, it is selected from sucrose, sorbitol or trehalose；

    C () concentration is about the surfactant of 0.01% to 0.6%, it is selected from Polyoxyethylene Sorbitan Monooleate or poloxamer-188；With

    (d) buffer, it is about the histidine buffering liquid of 10-20mM selected from concentration or concentration is about the Tris buffer of 20mM, So that the pH of described preparation is about 5.0 to 7.5,

    Wherein, SDAB is to include two Anti-tumor necrosin & (TNF α) SDABs and Anti-Human's serum albumin (HSA) SDAB Polypeptide.
20. The pharmaceutical preparation of 20. claim 19, the most each anti-TNF alpha SDAB include 3 complementary determining regions (CDR1, CDR2 and CDR3), wherein

    A () CDR1 includes

    (i) aminoacid sequence DYWMY (SEQ ID NO:22)；

    (ii) there is the aminoacid sequence of at least 80% sequence iden with DYWMY (SEQ ID NO:22)；Or

    (iii) only there is the aminoacid sequence of 1 aminoacid difference with DYWMY (SEQ ID NO:22)；

    B () CDR2 includes

    (i) aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23)；

    (ii) with EINTNGLITKYPDSVKG (SEQ ID NO:23), there is at least 80%, 90% or 95% sequence iden Aminoacid sequence；Or

    (iii) there is the aminoacid sequence of 2 or 1 aminoacid differences with EINTNGLITKYPDSVKG (SEQ ID NO:23)； With

    C () CDR3 includes

    (i) aminoacid sequence SPSGFN (SEQ ID NO:24)；

    (ii) there is the aminoacid sequence of at least 80% sequence iden with SPSGFN (SEQ ID NO:24)；Or

    (iii) there is the aminoacid sequence of 1 aminoacid difference with SPSGFN (SEQ ID NO:24).
21. The pharmaceutical preparation of 21. claim 20, wherein CDR1 includes aminoacid sequence DYWMY (SEQ ID NO:22), CDR2 bag Include aminoacid sequence EINTNGLITKYPDSVKG (SEQ ID NO:23), and CDR3 includes aminoacid sequence SPSGFN (SEQ ID NO:24).
22. Pharmaceutical preparation any one of 22. claim 19-21, at least one in wherein said anti-TNF alpha SDABs includes With the aminoacid sequence that SEQ ID NO:5 (TNF30) has at least 80%, 90%, 95% or 99% sequence iden.
23. Pharmaceutical preparation any one of 23. claim 19-22, the most each anti-TNF alpha SDAB includes and SEQ ID NO:5 (TNF30) there is the aminoacid sequence of at least 80%, 90%, 95% or 99% sequence iden.
24. Pharmaceutical preparation any one of 24. claim 19-23, wherein said anti-HSA SDAB include 3 CDRs (CDR1, CDR2 and CDR3), wherein

    A () CDR1 includes

    (i) aminoacid sequence SFGMS (SEQ ID NO:25)；

    (ii) there is the aminoacid sequence of at least 80% sequence iden with SFGMS (SEQ ID NO:25)；Or

    (iii) only there is the aminoacid sequence of 1 aminoacid difference with SFGMS (SEQ ID NO:25)；

    B () CDR2 includes

    (i) aminoacid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26)；

    (ii) with SISGSGSDTLYADSVKG (SEQ ID NO:26), there is at least 80%, 90% or 95% sequence iden Aminoacid sequence；Or

    (iii) there is the aminoacid sequence of 2 or 1 amino acid differences with SISGSGSDTLYADSVKG (SEQ ID NO:26)； With

    C () CDR3 includes

    (i) aminoacid sequence GGSLSR (SEQ ID NO:27)；

    (ii) there is the aminoacid sequence of at least 80% sequence iden with GGSLSR (SEQ ID NO:27)；Or

    (iii) there is the aminoacid sequence of 1 aminoacid difference with GGSLSR (SEQ ID NO:27).
25. The pharmaceutical preparation of 25. claim 24, wherein CDR1 includes aminoacid sequence SFGMS (SEQ ID NO:25), CDR2 bag Include aminoacid sequence SISGSGSDTLYADSVKG (SEQ ID NO:26), and CDR3 includes aminoacid sequence GGSLSR (SEQ ID NO:27).
26. The pharmaceutical preparation of 26. claim 24 or 25, wherein said anti-HSA SDAB includes having with SEQ ID NO:10 (ALB8) There is the aminoacid sequence of at least 80%, 90%, 95% or 99% sequence iden.
27. Pharmaceutical preparation any one of 27. claim 19-26, wherein said polypeptide includes the aminoacid sequence of SEQ ID NO:1 Row (ozoralizumab).
28. Pharmaceutical preparation any one of 28. claim 19-27, at least one in wherein said SDABs is humanized.
29. Pharmaceutical preparation any one of 29. claim 19-28, each of wherein said two anti-TNF alpha SDABs is with anti- HSA SDAB is connected by joint, and the most each joint selects free SEQ ID NO:6 and the aminoacid sequence group of SEQ ID NO:7 The group become.
30. Pharmaceutical preparation any one of 30. claim 19-29, wherein said pharmaceutical preparation comprises about 30,80,100,120, The SDAB molecule of 140,160,180,200,250,275,300,320,350,375 or 400mg.
31. Pharmaceutical preparation any one of 31. claim 19-30, wherein said pharmaceutical preparation applies to subcutaneous or intravenous and executes Form.
32. The pharmaceutical preparation any one of 32. claim 1-31 purposes in the test kit manufacturing treatment immune disorders.
33. The purposes of 33. claim 32, wherein said test kit also comprises methotrexate.
34. Purposes any one of 34. claim 32-33, the group of the most following composition of wherein said immune disorders choosing: scorching Disease, arthritis, including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Juvenile idiopathic arthritis and bone Arthritis, COPD, asthma, inflammatory bowel, including Crohn disease and ulcerative colitis, multiple sclerosis, Addison disease, self Autoallergic, autoimmunity parotitis, type i diabetes, epididymitis, glomerulonephritis, Graves disease, Ji Lan-Ba Lei Syndrome, chronic lymphocytic thyroiditis, hemolytic anemia, systemic lupus erythematosus (sle), male sterility, multiple sclerosis, myasthenia gravis, sky bleb Skin ulcer, psoriasis, hidradenitis suppurativa, rheumatic fever, sarcoidosis, scleroderma, Sjogren syndrome, SpA, thyroiditis And vasculitis.
35. The purposes of 35. claim 35, wherein said immune disorders is rheumatoid arthritis.