Fused drug constructs and conjugates

FIELD: medicine.

SUBSTANCE: what is described are drug conjugates which contain an incretin therapeutic or diagnostic agent who is fused or conjugated with an antigen-binding antibody fragment which binds serum albumin.

EFFECT: conjugates have prolonged in vivo half-life in comparison with an unconjugated or unfused therapeutic or diagnostic agent.

27 cl, 19 dwg, 8 tbl, 18 ex

 

RELATED APPLICATIONS

This application takes priority under provisional patent application U.S. No. 60/632361, filed December 2, 2004, and priority under the patent of great Britain No. 0511019.2. The contents of the aforementioned applications are fully incorporated herein by reference.

PRIOR art

Many drugs having biological activity, which could be useful for therapeutic and/or diagnostic purposes, have limited value, since they are rapidly excreted in the introduction. For example, many polypeptides that are therapeutically useful activity, are rapidly eliminated from the circulation by the kidneys. Accordingly, it is necessary to introduce large doses to achieve the desired therapeutic effect. There is a need for improved therapeutic and diagnostic agents that have improved pharmacokinetic properties. Polypeptides that bind serum albumin, known in the prior art. (See, for example, EP 0486525 B1 (Network Bioteknik AB); US 6267964 B1 (Nygren et al.); WO 04/001064 A2 (Dyax, Corp.); WO 02/076489 A1 (Dyax, Corp.); WO 01/45746 (Genentech, Inc.).)

One such class of drugs that have a short half-life in the body or the big circle of blood circulation, is incredilbe hormone is, such as glucagon-like peptide 1, and Peptide YY.

Glucagon-like peptide (GLP)-1 is incrediby hormone with pronounced glucosazone insulinotropic and glucagonostatic types of action, trophic effect on pancreatic cells and inhibitory effects on gastrointestinal secretion and motility, in addition to the ability to lower the level of glucose in the plasma and to reduce the amplitude of the variations in the concentration of glucose. In addition, through its ability to increase satiety, GLP-1 reduces food intake, thereby limiting the increase of body weight, and may even cause weight loss. All together, these actions give GLP-1 unique profile, which are considered highly desirable for antidiabetic agent, especially due to the fact that the dependence of glucose from its antihyperglycemic effects should minimize any risk of severe hypoglycemia. However, its pharmacokinetic/ pharmacodynamic profile is such that the natural GLP-1 is not therapeutically useful. Thus, while GLP-1 is most effective with the constant introduction, a single subcutaneous injection have short-lived effects. GLP-1 is highly susceptible to enzymatic destruction in vivo, and splitting dipeptidyl what tigati IV (DPP-IV) is probably the most plausible, because it is fast and leads to the formation of reincarination metabolite. Therefore, strategies for mobilizing therapeutic potential of GLP-1-based understanding of the factors that affect its metabolic stability and pharmacokinetic/ pharmacodynamic profile, located in the centre of intensive research.

Huge work has been done to attempt to inhibit peptidase or modify GLP-1 thus, to slow its destruction while maintaining, however, the biological activity. In WO 05/027978 disclosed derivatives of GLP-1 with prolonged profile of action (and included by reference in this document as examples of derivatives and analogues of GLP-1 that can be used in the present invention). In WO 02/46227 described heterologous fused proteins containing polypeptide (e.g., albumin), merged with GLP-1 or analogues (data description analogues incorporated by reference in this document as examples of analogues of GLP-1 that can be used in the present invention). In WO 05/003296, WO 03/060071, WO 03/059934 disclosed uminosity protein, where GLP-1 fused to albumin to increase the half-life of this hormone.

However, despite these efforts, was not received GLP-1, remain active for a long prom is dreadful time.

Essentially, especially in the area of diabetes and obesity, there is a tremendous need for improved GLP-1 peptides or other agents that similarly have insulinotropic effect, suitable for the treatment, in particular, diabetes and obesity. Therefore, there is a need for modification of GLP-1 and other insulinotropic peptides to provide a longer duration of action in vivo while maintaining their low toxicity and therapeutic benefits.

SUMMARY of the INVENTION

The invention relates to fused structures of drugs and drug conjugates, which have improved the elimination half-life in serum. In one aspect, the slit structure of the medicinal product is a continuous polypeptide chain having the formula:

a-(X)n1-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n1-d

where X is a polypeptide drug that has binding specificity with respect to the first target;

Y represents the variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or variable domain of the light chain of immunoglobulin (VL)that has binding specificity in Rel is against serum albumin;

Z is a polypeptide drug that has binding specificity for a second target;

each of a, b, C and d independently is absent or represents from one to about 100 amino acid residues;

n1 is from one to about 10;

n2 is from one to about 10; and

n3 equals from zero to about 10,

provided that, if both n1 and n2 are equal to one, and n3 is zero, X does not contain a chain of the antibody or fragment chain antibodies.

In some embodiments of the incarnation, Y contains an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In specific embodiments, embodiments, X is a GLP-1 or an analogue of GLP-1.

In another aspect, the slit structure of the medicinal product contains a continuous polypeptide chain, and the chain contains groups X' and Y', where

X' is a polypeptide drug, provided that X' does not contain a chain of the antibody or fragment chain antibodies; and

Y' represents the variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in which the compared serum albumin, or variable domain of the light chain of immunoglobulin (VL)that has binding specificity in relation to serum albumin. In some embodiments, embodiments, Y' contains amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In specific embodiments, embodiments, X' is a GLP-1 or an analogue of GLP-1.

In another aspect, the invention is a conjugate of a medicinal product containing the variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or variable domain of the light chain of immunoglobulin (VL)that has binding specificity in relation to serum albumin, and the drug that is covalently associated with the specified VHor VL. In some embodiments embodiment, the variable domain of the heavy chain immunoglobulin contains an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from the group consisting whom her from SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In specific embodiments, embodiments, the drug is a GLP-1 or an analogue of GLP-1.

The invention also suggested that recombinant nucleic acid constructs encoding the merged design of the medicinal product described herein and cells of the host containing the recombinant nucleic acid and/or design. The invention is additionally a method for obtaining a merged design of a medicinal product, comprising maintaining a host cell that contains a recombinant nucleic acid and/or design, which encodes the merged design drugs described herein, under conditions suitable for expression of the indicated recombinant nucleic acid, whereby get merged design drugs.

The invention also suggested that the composition (e.g., pharmaceutical compositions)containing the merged design drugs or drug conjugate according to the invention. The invention is also a method of treatment of an individual having the disease or disorder, such as any described herein, including the introduction of a specified individual a therapeutically effective amount is CTB conjugate of a drug or merged design of a medicinal product according to the invention. In some embodiments embodiment, the disease or disorder is an inflammatory disease such as arthritis (e.g. rheumatoid arthritis). In an additional variant embodiment, the disease or disorder is a metabolic disease such as diabetes or obesity. The invention also suggested that the conjugate of a drug or merged design of a medicinal product according to the invention for the manufacture of drugs for the treatment of diseases or disorders, such as inflammatory disease (such as arthritis (e.g. rheumatoid arthritis)or diabetes or obesity. The invention also relates to the use of fused design drugs or drug conjugate as described herein for use in therapy, diagnosis or prevention.

In another aspect, the invention is a non-covalent conjugate of a medicinal product containing the variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or variable domain of the light chain of immunoglobulin (VL)that has binding specificity in relation to serum albumin, and medicine, which ecovalence associated with asanam V Hor VL. In some embodiments embodiment, the variable domain of the heavy chain immunoglobulin contains an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

In an additional variant embodiment, the invention proposed an inactivated variant of Dom7h-8, iDom7h-8, which does not bind serum albumin, which is used as a tool for research and which is predictive option for active binding serum albumin Dom7h-8.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

On Figa presents the alignment of amino acid sequences of the three VKselected by binding to serum albumin mice (MSA). Aligned amino acid sequences belong to VKmarked MSA 16, which also indicate DOM7m-16 (SEQ ID NO:1), MSA 12, which also indicate DOM7m-12 (SEQ ID NO:2), and MSA 26, which also indicate DOM7m-26 (SEQ ID NO:3).

Figure 1 In the present alignment of amino acid sequences of six VKselected by binding with serum albumin of rats (RSA). Aligned amino acid members is Telenesti belong to V Kmarked DOM7M (SEQ ID NO:4), DOM7r-3 (SEQ ID NO:5), DOM7r-4 (SEQ ID NO:6), DOM7r-5 (SEQ ID NO:7), DOM7r-7 (SEQ ID NO:8), and DOM7r-8 (SEQ ID NO:9).

On Figs presents the alignment of amino acid sequences of six VKselected by binding to serum albumin human (HSA). Aligned amino acid sequences belong to VKdesignated DOM7h-2 (SEQ ID NO:10), DOM7h-3 (SEQ ID NO:11), DOM7h-4 (SEQ ID NO:12), DOM7h-6 (SEQ ID NO:13), DOM7h-1 (SEQ ID NO:14), DOM7h-7 (SEQ ID NO:15).

On Fig.1D presents the alignment of amino acid sequences of the seven VHselected by binding to serum albumin human and consensus sequence (SEQ ID NO:23). Aligned amino acid sequences belong to VKdesignated DOM7h-22 (SEQ ID NO:16), DOM7h-23 (SEQ ID NO:17), DOM7h-24 (SEQ ID NO:18), DOM7h-25 (SEQ ID NO:19), DOM7h-26 (SEQ ID NO:20), DOM7h-21 (SEQ ID NO:21), and DOM7h-27 (SEQ ID NO:22).

On Five presents the alignment of amino acid sequences of the three VKselected by binding to serum albumin human and serum albumin in rats. Aligned amino acid sequences belong to VKdesignated DOM7h-8 (SEQ ID NO:24), DOM7r-13 (SEQ ID NO:25), and DOM7r-14 (SEQ ID NO:26).

On Figa and 2 presents a schematic map of the vector used for the expression of the slit structures MSA16IL-1ra (also called DOM7m-16/IL-1ra) and IL-1raMSA16 (also called IL-1ra/DOM7m-16), sootvetstvenno is.

On Figs-2D presents the nucleotide sequence (SEQ ID NO:27)encoding a fused structure of IL-1raMSA16 (also called IL-1ra/DOM7m-16)and amino acid sequence (SEQ ID NO:28) merged structure.

On Five-2F presents the nucleotide sequence (SEQ ID NO:29)encoding a fused design MSA16IL-1ra (also called DOM7m-16/IL-1ra), and amino acid sequence (SEQ ID NO:30) merged structure.

On Fig.2G-2N presents the nucleotide sequence (SEQ ID NO:31)encoding a fused design DummyIL-1ra, which does not bind serum albumin, and amino acid sequence (SEQ ID NO:32) merged structure.

Figa is an illustration showing that IL-1 induces the production of IL-8 in HeLa cells, and showing the detection of IL-8 by ELISA method.

Figure 3 represents a graph showing that each of IL-1 hectare (♦), MSA16IL-1ra (▀) and IL-1raMSA16 (▲ ) inhibited IL-1-induced secretion of IL-8 in cultured MRC-5 cells. The observed inhibition was wearing a dose-dependent for IL-1ra, MSA16IL-1ra and IL-1raMSA16.

Figa-4C are graphs showing that IL-1ra (♦), and MSA16IL-1ra (▀) inhibited IL-1-induced secretion of IL-8 in cultured MRC-5 cells in analyses that did not include serum albumin mice (4A)included 5% serum albumin mice (4B) ili% serum albumin mice (4C). The observed inhibition was wearing a dose-dependent for IL-1ra and MSA16IL-1ra in all testing conditions.

Figure 5 schematically presents the results of ELISA (enzyme-linked immunosorbent assays), demonstrating that the merged design MSA16IL-1ra, and merged the design of the IL-1raMSA16 tied serum albumin, and merged design DummyIL-1ra did not.

On Figa-6C presents sensogram and tables showing data BIACORE affinity for clone DOM7h-1 that binds serum albumin human (HSA) (6A), DOM7h-7, HSA binding (6V), DOM7M that binds serum albumin in rats (RSA).

Figure 7 presents a table showing the affinity DOM7h-1, DOM7r-1, DOM7h-2, DOM7r-3, DOM7h-7, DOM7h-8, DOM7r-8, DOM7r-13, DOM7r-14, DOM7m-16, DOM7h-22, DOM7h-23, DOM7h-26, DOM7r-16, DOM7m-26, DOM7r-27 and DOM7R-31 to serum albumin, which they bind.

On Figa presents the nucleotide sequence (SEQ ID NO:33) a nucleic acid that encodes a receptor antagonist to human interleukin-1 (IL-1ra), placed in GenBank under registration number NM_173842. Nucleic acid has an open reading frame starting at position 65.

On FIGU presents the amino acid sequence of human IL-1ra (SEQ ID NO:34)encoded by the nucleic acid shown in Figa (SEQ ID NO:33). The Mature protein consists of 152 amino acid residues (amino acid mod and 26-177 of SEQ ID NO:34).

Figure 9 shows the graphical dependence of concentration (µg/ml) MSA-binding fused protein with dAb/HA-epitope tag in the serum of mice after a single intravenous (IV) injections (dose was approximately 1.5 mg/kg) animals male strain CD1 from time (day). Serum concentration was determined by ELISA method using reagents detection of capture of goat anti-(Abeam, UK) and protein L-HRP (Invitrogen, USA). Standard curves of known concentrations of MSA-binding merged design dAb/HA were established in the presence of 1x mouse serum to ensure comparability with the test samples. Modeling using onecompartment model (WinNonlin Software, Pharsight Corp., USA) showed that MSA-binding protein with dAb/HA-epitope tag was the end-of-phase t1/2 equal to 29.1 per hour, and the area under the curve equal to 559 h-ág/ml

Figure 10 presents the amino acid sequence of the VKselected by binding with serum albumin of rats (RSA). The illustrated sequence belong to VKdenoted DOM7r-15 (SEQ ID NO:37), DOM7r-16 (SEQ ID NO:38), DOM7M7 (SEQ ID NO:39), DOM7r-18 (SEQ ID NO:40), DOM7r-19 (SEQ ID NO:41).

On Figa-11B presents the amino acid sequence of the VKthat bind serum albumin in rats (RSA). The illustrated sequence belong to VKdenoted DOM7r-20 (SEQ ID NO:42), DOM7r-21 (SEQ I NO:43), DOM7r-22 (SEQ ID NO:44), DOM7r-23 (SEQ ID NO:45), DOM7r-24 (SEQ ID NO:46), DOM7r-25 (SEQ ID NO:47), DOM7r-26 (SEQ ID NO:48), DOM7r-27 (SEQ ID NO:49), DOM7r-28 (SEQ ID NO:50), DOM7r-29 (SEQ ID NO:51), DOM7r-30 (SEQ ID NO:52), DOM7r-31 (SEQ ID NO:53), DOM7r-32 (SEQ ID NO:54), DOM7r-33 (SEQ ID NO:55).

On Fig presents a graphical dependence of concentration (% of initial dose) DOM7m-16, DOM7m-26 or dAb control, which does not bind MSA, each of which is contained ON-epitope tag in the serum of mice after a single intravenous (IV) injections (dose was approximately 1.5 mg/kg) animals male strain CD1 from time to time. Serum concentration was determined by ELISA method using reagents detection of capture of goat anti-(Abeam, UK) and protein L-HRP (Invitrogen, USA). Standard curves of known concentrations of MSA-binding merged design dAb/HA were established in the presence of 1x mouse serum to ensure comparability with the test samples. Modeling using onecompartment model (WinNonlin Software, Pharsight Corp., USA) showed that the control dAb had the end-of-phase t1/2a, is equal to 20 minutes at a time as DOM7m-16, DOM7m-26 remained in the serum significantly longer.

On Fig graphically represented, DOM7m-16/IL-1ra was more effective than IL-1ra or ENBREL® (interecept; Immunex Corporation) for the treatment of arthritis in a model of collagen-induced arthritis (CIA) in mice. Induced arthritis and, beginning with the 21st day, was administered to mice with dexamethasone in the amount of 0.4 mg/kg (Stero the d), DOM7m-16/IL-1ra in the amount of 1 mg/kg (IL-1ra/anti-SA 1 mg/kg) or 10 mg/kg (IL-lra/aHTH-SA 10 mg/kg), IL-1ra in the amount of 1 mg/kg or 10 mg/kg ENBREL® (interecept; Immunex Corporation) in an amount of 5 mg/kg or saline. The results show that DOM7m-16/IL-1ra was more effective than IL-1ra or ENBREL® (interecept; Immunex Corporation) in this study. The response to IL-1ra was dose-dependent, as expected, and the response to DOM7m-16/IL-1ra was also dose-dependent. The average in the case of treatment with DOM7m-16/IL-1ra in the amount of 1 mg/kg were consistent with lower than average scores obtained in the case of treatment of IL-1ra in the amount of 10 mg/kg Results show that treatment with DOM7m-16/IL-1ra was 10 times more effective than the use of IL-1ra in this study.

On Figa-14G presents the amino acid sequence Suprinovich of polypetides. On Figa presents the amino acid sequence of the precursor saporin-2, registered as Swissprot Accession Number P27559 (SEQ ID NO:60). The signal peptide is amino acids 1 to 24 of SEQ ID NO:60. On Fig In the submitted amino acid sequence saporin-3, registered as Swissprot Accession Number P27560 (SEQ ID NO:61). On Figs presents the amino acid sequence of the precursor saporin-4, registered as Swissprot Accession Number P27561 (SEQ ID NO:62). The signal peptide is amino acids 1 to 24 of SQ ID NO:62. On Fig.14D presents the amino acid sequence saporin-5, registered as Swissprot Accession Number Q41389 (SEQ ID NO:63). On Five presents the amino acid sequence of the precursor saporin-6, registered as Swissprot Accession Number P20656 (SEQ ID NO:64). The signal peptide is amino acids 1 to 24 of SEQ ID NO:64, and the potential propeptide represents amino acids 278 at 299 of SEQ ID NO:64. The Mature polypeptide is amino acids 25 to 277 of SEQ ID NO:64 (SEQ ID NO:65). On Fig.14F presents the amino acid sequence saporin-7, registered as Swissprot Accession Number Q41391 (SEQ ID NO:66). On Fig.14G presents a consensus amino acid sequence spanning several variants and isoforms saporin-6 (SEQ ID NO:67).

On Fig presents amino acid sequences of several Camelid VHHthat bind serum albumin mice, which are described in WO 2004/041862. The sequence A (SEQ ID NO:72), In Sequence (SEQ ID NO:73), the Sequence (SEQ ID NO:74), the Sequence D (SEQ ID NO:75), the Sequence E (SEQ ID NO:76), the Sequence F (SEQ ID NO:77), the Sequence G (SEQ ID NO:78), the Sequence H (SEQ ID NO:79), the Sequence I (SEQ ID NO:80), the Sequence J (SEQ ID NO:81), The sequence (SEQ ID NO:82), the Sequence L (SEQ ID NO:83), the Sequence M (SEQ ID NO:84), serial is inost N (SEQ ID NO:85), The sequence Of (SEQ ID NO:86), Sequence P (SEQ ID NO:87), the Sequence Q (SEQ ID NO:88).

On Figa presents the nucleotide sequence encoding [Pro9]GLP-1-Dom7h8 merged structure (SEQ ID NO:177), and amino acid sequence fused constructs (SEQ ID NO:179).

On FIGU presents the nucleotide sequence encoding [Pro9]GLP-1-PSS-Dom7h8 merged structure (SEQ ID NO:180), and amino acid sequence fused constructs (SEQ ID NO:182).

On Figs presents the nucleotide sequence encoding [Pro9]GLP-l-PSSGAP - Dom7h8 merged structure (SEQ ID NO:183), and amino acid sequence fused constructs (SEQ ID NO:185).

Fig is a graph showing that [Pro9]GLP-1-PSSGAP-Dom7h8 merged design (□) was equivalent to the dose-dependent activity of cell proliferation to GLP-1 control (▲), on the Basis of 4 (▼). Basal zero control is shown (♦).

Fig is a graph showing that [Pro9]GLP-1-PSSGAP-Dom7h8 merged design (□) was equivalent to a dose-dependent release of insulin / GLP-1 control (▲), on the Basis of 4 (▼). Basal zero control is shown (♦).

On Figa-19S presents the amino acid sequence of Dom7h-8 PYY (3-36) (SEQ ID NO:186), PYY (3-36). Dom7h-8 (SEQ ID NO:187) and [Pro9]GLP-1(3-27)-Dom7h-8 PYY (3-36) (SEQ ID NO:188) merged structures, respectively.

FOR THE DETAILED description of the INVENTION

In this description and options embodiments have been described in such a way as to create a clear and lucid description, it is understood and it should be understood that variations of the embodiments are several ways to unite or separate without deviating from the scope of invention.

As used in this description, "drug" refers to any compound (e.g., small organic molecule, nucleic acid, polypeptide, which you can enter the individual to obtain the best therapeutic or diagnostic effect by binding with and/or changes in the biological function of a target molecule in the individual. Molecule-target may represent an endogenous molecule-target encoded by the genome of an individual (e.g., enzyme, receptor, growth factor, cytokine encoded by the genome of an individual), or exogenous molecule-target encoded by the genome of a pathogen (for example, the enzyme encoded by the genome of a virus, bacteria, fungus, nematodes or other pathogen).

As used in the present description, the term "basis of the medicinal product" refers to the activity of the compositions of drugs and medicines, which are normalized based on the number of drugs (or drug combinations), used to assess and what to measure or establish a activity. Typically, the composition of the medicinal product according to the invention (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) have a greater molecular weight than the drug they contain. Thus, an equivalent amount of the composition of medicines and drugs, by weight, will contain different amounts of drugs at the molecular or molar basis. For example, if the composition of the medicinal product according to the invention has a molecular mass of nearly twice the molecular mass of a medicinal product which it contains, the activities can be set on the basis of "principles of medicinal product, using 2 μg of the composition of the medicinal product and 1 µg medicines, as these amounts would contain the same amount of drug (in the form of free medicines or as part of the composition of the medicinal product). Activities may be normalized and expressed on the basis of "principles of medicinal product" by using appropriate calculations, for example, expressing the activity-based website, which links the target, or, for enzymatic medicines, on the basis of the active site.

p> As used in the present description, "the composition of the medicinal product" refers to a composition containing the drug that is covalently or ecovalence associated with polipeptidnaya group, where polipeptidnaya grouping contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. The composition of the medicinal product may be a conjugate, where the drug is covalently or ecovalence associated with polipeptidnaya group. The drug can be covalently or ecovalence associated with polipeptidnaya group directly or indirectly (for example via a suitable linker and/or non-covalent binding of complementary binding partners (e.g., Biotin and avidin)). If used complementary binding partners, one of the binding partners can be covalently linked to drug directly or through a suitable linker group, and the complementary binding partner can be covalently linked to polipeptidnaya group directly or through a suitable linker group. If the medicinal product represents polypeptide or peptide, the composition of the medicinal product may be a protein, where the polypeptide or peptide drug and polipeptidnaya grouping are provided as separate parts (groups) continuous polypeptide chain.

As used in the present description, a "conjugate" refers to a composition containing antigennegative fragment of the antibody that binds serum albumin, and which is associated with the drug. Such conjugates include conjugates medicines, which contain antigennegative fragment of the antibody that binds serum albumin, which are covalently linked drug, and noncovalent drug conjugates, which contain antigennegative fragment of the antibody that binds serum albumin, which ecovalence related drug.

As used in this description, "drug conjugate" refers to a composition containing antigennegative fragment of the antibody that binds serum albumin, which are covalently linked drug. The drug can be covalently linked to antigennegative fragment directly or indirectly through a suitable linker group. Medication is the main means may be associated with antigennegative fragment in any suitable position, such as aminocore, carboxylic or through suitable amino acid side chains (for example, the e amino group of lysine or Tolna group of cysteine).

As used in this description, "non-covalent conjugate of a medicinal product" refers to compositions containing antigennegative fragment of the antibody that binds serum albumin, which ecovalence related drug. The drug can be ecovalence associated with antigennegative fragment directly (for example, electrostatic interaction, hydrophobic interaction) or indirectly (for example, through non-covalent binding of complementary binding partners (e.g., Biotin and avidin), where one partner is covalently associated with the drug, and the complementary binding partner is covalently bonded to the antigen-binding fragment). If used complementary binding partners, one of the binding partners can be covalently linked to drug directly or through a suitable linker group and the complementary binding partner can be covalently linked to antigennegative fragment of an antibody that binds serum albumin, either directly or through a suitable linker group.

As used in n the standing description the slit design drugs" refers to fused protein, which contains antigennegative fragment of the antibody that binds serum albumin, and a polypeptide drug. Antigennegative fragment of the antibody that binds serum albumin, and the polypeptide drug is present in the form of separate parts (groups) one continuous polypeptide chain.

The term "albuminaemia residue", as used in the present description, means a residue that binds ecovalence with human serum albumin. Albuminaemia residue attached to therapeutic polypeptide typically has a binding affinity of less than 10 μm human serum albumin and, preferably, less than 1 PM. In one variant embodiment, the known number of albuminaemia residues among linear and branched lipophilic groups containing 4-40 carbon atoms, compounds with cyclopentanophenanthrene skeleton, peptides having 10-30 amino acid residues, etc.

As used in this description, "receptor antagonist interleukin-1" (IL-1ra) refers to natural or endogenous IL-1ra mammalian proteins and to proteins having amino acid sequence which is the same as aminokislot the I sequence in natural or corresponding endogenous protein IL-1ra mammals (for example, recombinant proteins, synthetic proteins (i.e., obtained by the methods of synthetic organic chemistry)). Accordingly, as indicated in the present description, the term includes Mature protein, polymorphic or allelic variants and other isoforms of IL-1ra (e.g., through alternative splicing or other cellular processes), and modified or unmodified forms of the above (for example, with attached lipids, glycosylated, paglierani). Natural or endogenous IL-1ra include proteins of the wild type, such as Mature IL-1ra, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates). Such proteins can be extracted or isolated from a source that, for example, produces IL-1ra in nature. The data of proteins and IL-1ra proteins having the same amino acid sequence as natural or corresponding endogenous IL-1ra, indicate the name of the corresponding mammal. For example, if the mammal is a human protein known as the human IL-1ra.

"Functional variants" of IL-1ra include functional fragments, functional mutant proteins, and/or functional fused proteins, which can be obtained using appropriate methods (in the example, mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis), methods of recombinant DNA). "Functional variant" prevents receptor type 1 interleukin-1. Generally, fragments or sections of IL-1ra include those having a deletion and/or addition (i.e. one or more deletions and/or additions of amino acids) amino acids (i.e. one or more amino acids) relative to the Mature IL-1ra (such as N-terminal, C-terminal or internal deletions). Also provides fragments or sections, which were removed only contiguous amino acids, or which have been removed noncontiguous amino acids relatively Mature IL-1ra. A functional variant of human IL-1ra may have at measures about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with the Mature form of human IL-1ra, consisting of 152 amino acids, and prevents receptor type 1 human interleukin-1. (See Eisenberg et al., Nature 343:341-346 1990). A variant can contain one or more additional amino acids (e.g., to contain 153, or 154, or more amino acids). For example, a variant of IL-1ra may have amino acid p is the sequence, which consists of aminoanisole methioninamide residue, followed by residues from 26 to 177 of SEQ ID NO:33. (KINERET® (anakinra), Amgen).

As used in the present description, the term "about" is possible, but, preferably, is interpreted as meaning plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, even more preferably, plus or minus 2%, most preferably plus or minus 1%.

The term "analog"as used in the present description, related polypeptide, means a modified polypeptide in which one or more amino acid residues of the peptide have been substituted by other amino acid residues, and/or where one or more amino acid residues of the peptide have been deleted from the peptide and/or where one or more amino acid residues of the peptide have been deleted from the peptide and/or where one or more amino acid residues of the peptide were added to the peptide. Such addition or deletion of amino acid residues may occur at the N-end of the peptide and/or at the C-end of the peptide, or they may occur within the peptide. Use a simple system to describe analogues of GLP-1: for Example, [Arg34] GLP-1 (7-37) Lys denotes the analogue of GLP-1, where natural lysine at position 34 has been substituted with arginine, and lysine residue added to the C-end (position 38). The formula obtained is s peptide and its derivatives paint using standard single letter abbreviations for amino acids, used according to the nomenclature of the IUPAC-IUB.

The term "GLP-1 peptide", as used in the present description, means GLP-1 (7-37) (SEQ ID NO:158), or GLP-1 (7-36) (SEQ ID NO:159), analogue of GLP-1, a derivative of GLP-1 or a derivative of an analog of GLP-1. Such peptides, analogs and derivatives are insulinotropic agents.

The term "insulinotropic agent", as used in the present description, means a compound that can stimulate, or cause the stimulation of the synthesis, or expression, or activity of the hormone insulin. Well-known examples insulinotropic agents include, but are not limited to, glucose, GIP (glucosidation insulinotropic peptide), GLP, the Basis and OHM (oxyntomodulin).

The term "incretin", as used in the present description, means a type of gastrointestinal hormone that causes an increase in the amount of insulin released when glucose levels are normal, or, especially, when they are raised. As an example, they include GLP-1, GIP and OHM.

The term "peptide on the Basis of 4", as used in the present description, means on the Basis of 4 (1-39), a similar Basis-4, derived on the Basis of 4 or derived similar Basis 4. In one variant embodiment, the Basis-4 peptide is insulinotropic agent. Such peptides, analogs and derivatives are insulinotropic agents.

Those who min "DPP-IV protected", as used in this description, related polypeptide, means polypeptide that has been modified (e.g., chemically modified)to make the specified connection resistant to peptidase plasma dipeptidylpeptidase-4 (DPP-IV). DPP-IV enzyme in plasma, known to be involved in the destruction of several peptide hormones such as GLP-1, GLP-2, etc. So put serious efforts to develop analogues and derivatives of the polypeptides susceptible to DPP-IV-mediated hydrolysis to reduce the speed and/or the degree of destruction under the action of DPP-IV.

As used in this description, "saporin" belongs to the family of single-chain polypeptides, inactivating the ribosome produced by the plant Saponaria officinalis. (Stirpe, F., et al., Biochem. J. 216:617-625 (1983), Bagga, S. et al., J. Biol. Chem. 278:4813-4820 (2003).) Sapronova polypeptides exist in several forms that differ in length and/or amino acid sequence. (See, for example, Id. and Barthelemy, L et al., J. Biol. Chem. 268:6541-6548 (1993).) Saporin-6 is the most active form saporin. (Bagga, S. et al., J. Biol. Chem. 278:4813-4820 (2003).) Describe at least four naturally occurring isoforms saporin-6, in which the amino acid at position 48 of the Mature polypeptide (SEQ ID NO:65) represents Asp or Glu, and amino acid at position 91 of the Mature polypeptide (SEQ ID NO:65) represents the t of an Arg or Lys (Barthelemy, L et al., J. Biol. Chem. 268:6541-6548 (1993).) Additional forms saporin-6 include polypeptides in which the amino acid at position 99 of the Mature polypeptide (SEQ ID NO:65) is a Ser or Leu; the amino acid at position 134 of the Mature polypeptide (SEQ ID NO:65) represents Gln or Lys; the amino acid at position 147 of the Mature polypeptide (SEQ ID NO:65) is a Ser or Leu; the amino acid at position 149 of the Mature polypeptide (SEQ ID NO:65) is a Ser or Phe; the amino acid at position 162 of the Mature polypeptide (SEQ ID NO:65) is an Asp or Asn; the amino acid at position 177 of the Mature polypeptide (SEQ ID NO:65) is Ala or Val; the amino acid at position 188 of the Mature polypeptide (SEQ ID NO:65) is a Il or Thr; the amino acid at position 196 of the Mature polypeptide (SEQ ID NO:65) represents Asn or Asp; the amino acid at position 198 of the Mature polypeptide (SEQ ID NO:65) represents Glu or Asp; the amino acid at position 231 of the Mature polypeptide (SEQ ID NO:65) represents Asn or Ser, and polypeptides in which the amino acid at position 233 of the Mature polypeptide (SEQ ID NO:65) represents Lys or AGD. A consensus sequence, covering these isoforms and variants presented on Fig.14G (SEQ ID NO:67).

Accordingly, the term "saponin" includes the precursor protein, the Mature polypeptide, native protein, polymorphic or allelic variants and other and formy (for example, produced by alternative splicing or other cellular processes), and modified or unmodified forms of the above (for example, with attached lipids, glycosylated, paglierani). Natural or endogenous saporin include proteins of the wild type, such as Mature saporin (for example, Mature saporin-6), polymorphic or allelic variants and other isoforms which occur naturally in Saponaria officinalis. Such proteins can be retrieved or select from Saponaria officinalis, using any suitable methods. "Functional variants" saporin include functional fragments, functional mutant proteins, and/or functional fused proteins, which can be obtained by using suitable methods (e.g., mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis), methods of recombinant DNA). Generally, fragments or sections saporin (for example, saporin-6) include those having a deletion and/or addition (i.e. one or more deletions and/or additions of amino acids) amino acids (i.e. one or more amino acids) relative to the Mature saporin (such as N-terminal, C-terminal or internal deletions). Also provides fragments or sections, which were removed only contiguous amino acids, or which have been removed noncontiguous amino acids relative to the rela saporin. You can get many functional options saporin. For example, were obtained fused proteins saporin-6, which contain aminobenzene elongation and, as shown, which remain fully ribosome-inhibitory activity in the method of analysis of rabbit reticulocyte lysate. (Barthelemy, L. et al., J. Biol. Chem. 268:6541-6548 (1993).) Options saporin-6, in which the residue of the active site, Tug, Tug, Glu176, Arg179 or Thr (amino acids 72, 120, 176, 179 and 208 of SEQ ID NO:65) was replaced by alanine, was reduced cytotoxic activity in in vitro assays. (Bagga, S. et al., J. Biol. Chem. 278:4813-4820 (2003).) Accordingly, if it is desirable to obtain additional functional variants saporin, avoid mutation, substitution, replacement, removal or modification of active site residues. Preferably, a functional variant saporin, which contains fewer amino acids than the natural Mature polypeptide includes at least an active website. For example, option saporin-6, which contains fewer amino acids than the natural Mature saporin-6, may include residues of the active site Mature saporin-6 (Tyr72, Tyr120, Glu176, Arg179 and Thr (amino acids 72, 120, 176, 179 and 208 of SEQ ID NO:65)) and be at least about 137 amino acids in length, at least about 150 amino acids in length, at least about 175 amino acids in length, at least about you in length, at least about 225 amino acids in length, or at least about 250 amino acids in length.

"Functional variant" saporin has the ribosome-inactivating activity (for example, grnc N-glycosidase activity) and/or cytotoxic activity. Such activity can be easily estimated using any suitable method, such as inhibition of protein synthesis, using a well-known method of analysis of rabbit reticulocyte lysate or any of the well known methods of analysis of cytotoxicity, which used cancer cell lines. (See, for example, Bagga, S. et al., J. Biol. Chem. 278:4813-4820 (2003) and Barthelemy, L. et al., J. Biol. Chem. 268:6541-6548 (1993).)

In some embodiments embodiments, a functional variant saporin has at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or, at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with Mature caporino-6 (SEQ ID NO:65).

The invention relates to compositions which contain a drug and polipeptidnaya gr is piroko, which contains antigennegative site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. As described in detail in the present application with respect to compositions that contain antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, drug and binding polypeptide can be conjugated covalently or ecovalence. In some embodiments, embodiment, the composition is a protein that contains a polypeptide drug and polipeptidnaya grouping that contains antigennegative site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. In other embodiments, embodiment, the composition comprises a drug that is covalently or ecovalence associated with polipeptidnaya group that contains antigennegative site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo.

The invention relates to compositions of medicines containing the drug and polipeptidnaya group that contains vyzyvayushe site (for example, antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. As described in detail in the present application with respect to compositions of medicines that contain antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, drug and polipeptidnaya grouping can be linked together covalently or ecovalence. In some embodiments, embodiment, the composition of the medicinal product is a protein, a polypeptide which contains the drug and polipeptidnaya grouping that contains antigennegative site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. In other embodiments, embodiment, the composition of the medicinal product contains a drug that is covalently or ecovalence associated with polipeptidnaya group that contains antigennegative site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo.

Typically, a polypeptide that enhances half-life in serum in vivo, before the hat is a polypeptide, which occurs naturally in vivo and which is resistant to degradation or destruction of the endogenous mechanisms that remove unwanted material from the body (e.g., human). For example, a polypeptide that enhances half-life in serum in vivo, can be selected from extracellular matrix proteins, proteins found in the blood, proteins found in the blood-brain barrier or in the nerve tissue proteins, localized in the kidneys, liver, lungs, heart, skin, or bone, stress proteins, disease-specific proteins or proteins involved in Fc transport.

Suitable polypeptides that increase the half-life in serum in vivo include, for example, fused proteins ligand-neuropharmaceutical agent, specific to transferium receptors (see U.S. Patent. No. 5977307, the contents of which are incorporated herein by reference), the receptor capillary endothelial cells of the brain, transferrin, atransferrinemia receptor (e.g., soluble atransferrinemia receptor, insulin receptor insulin-like growth factor 1 (IGF 1)receptor, insulin-like growth factor 2 (IGF 2), insulin receptor, coagulation factor X, α1-antitripsin and HNF 1α. Suitable polypeptides that increase the half-life in serum, also include alpha-1 glycopro the ID (orosomucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1 microglobulin (protein NS; AIM), antithrombin III (AT III), apolipoprotein a-1 (APO a-1), apolipoprotein B (APO B), ceruloplasmin (CP), a component of complement C3 (C3), a component of complement C4 (C4), C1 esterase inhibitor (C1 INH), C-reactive protein (CRP), ferritin (FER), hemopexin (HPX), lipoprotein(a) (Lp(a)), manservisi protein (ICBMs), myoglobin (Muo), prealbumin (transthyretin; PAL), reynoldsville protein (RBP) and rheumatoid factor (RF).

Suitable proteins of the extracellular matrix include, for example, collagens, laminin, integrins and fibronectin. The collagens are the major proteins of the extracellular matrix. Currently, there are about 15 types of collagen molecules found in various parts of the body, for example, collagen type I (constituting 90% of the collagen of the body), found in bone, skin, tendons, ligaments, cornea, internal organs, or collagen type II, found in cartilage, vertebral discs, the chord and the ocular vitreous body.

Suitable proteins from the blood include, for example, plasma proteins (e.g., fibrin, α-2 macroglobulin, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B), serum amyloid protein a, haptoglobulin, profilin, ubiquitin, uteroglobin and α-2-microglobulin), enzymes and enzyme inhibitors (e.g. the measures the plasminogen, lysozyme, cystatin C, alpha-1-antitripsin and pancreatic trypsin inhibitor), proteins of the immune system, such as immunoglobulin proteins (e.g., IgA, IgD, IgE, IgG, IgM light chain of immunoglobulin (Kappa/lambda)), transport proteins (e.g., reynoldsville protein, α-1 microglobulin), defensin (for example, beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2 and neutrophil defensin 3), and the like.

Suitable proteins found in the blood-brain barrier or in the nervous tissue include, for example, melanocortin receptor, myelin, ascorbate conveyor and the like.

Suitable polypeptides that increase the half-life in serum in vivo, also include proteins that are localized in the kidney (for example, polycystin, type IV collagen, K1 conveyor organic ion, the antigen of Heymann), proteins localized in the liver (e.g., alcohol deshydrogenase, G250)protein, localized in the lungs (e.g., secretory component, which binds IgA), proteins localized in the heart (e.g., HSP 27, which is associated with dilated cardiomyopathy), proteins localized in the skin (e.g., keratin), proteins that are specific for bone tissue, such as morphogenetic proteins (BMPs), which are a subclass of the superfamily of proteins, transforming growth factor P,which demonstrate osteogenic activity (for example, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8), specific proteins neoplasms (for example, trophoblastic antigen, perceptively receptor, estrogen receptor, cathepsins (e.g., cathepsin In, which can be detected in the liver and spleen)).

Suitable disease-specific proteins include, for example, antigens expressed only on activated T-cells, including LAG-3 (gene lymphocyte activation), the ligand of osteoprotegerin (OPGL; see Nature 402, 304-309 (1999)), H (member of the TNF family of receptors expressed on activated T cells and is specifically activated in the cells of the host T-lymphotropic human virus of the first type (HTLV-I); see Immunol. 165 (1):263-70 (2000)). Suitable disease-specific proteins also include, for example, metalloproteases (associated with arthritis/cancer), including CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH, and angiogenic growth factors, including acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor-α (TGF-a), factor-alpha tumor necrosis (TNF-α), angiogenin, interleukin 3 (IL-3), interleukin 8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF), placental growth factor (P1GF), dimeric isoform BB platelet factor Rho is the (PDGF), fractalkine.

Suitable polypeptides that increase the half-life in serum in vivo, also include stress proteins such as heat shock proteins (HSPs). HSPs have generally found inside the cells. When they are extracellular, it is an indication that the cell has died and gave out its contents. This non-programmable cell death (necrosis) occurs when, as a result of injury, disease, or damage to, extracellular HSPs trigger a response from the immune system. Binding of extracellular HSP can lead to localization of the composition of the invention at the site of the disease.

Suitable proteins involved in Fc transport, include, for example, the Brambell receptor (also known as FcRB). This Fc receptor has two functions, both of which are potentially useful for the delivery. Functions are (1) the transfer of IgG from mother to child through the placenta, and (2) the protection of IgG from degradation, thereby extending its half-life in serum. It is believed that receptor recycling of IgG from endosomes. (See, Holliger etal., Nat Biotechnol 15(7):632-6 (1997).)

The composition of the medicinal product according to the invention can contain any polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide, which increases per the od half-life in serum in vivo. Preferably, polipeptidnaya grouping contains at least 31, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids or at least about 110 amino acids in the form of individual molecular structures. Preferably, polipeptidnaya grouping binds a polypeptide that enhances half-life in serum in vivo, with a KD of at least about 5 mm KD (KD=Koff(kd)/Kon(ka)). In some embodiments of the incarnation, polipeptidnaya grouping binds a polypeptide that enhances half-life in serum in vivo, with a KD of from about 10 to about 100 nm, or from about 100 nm to about 500 nm, or from about 500 nm to about 5 mm, as determined by the method of surface plasma resonance (for example using the BIACORE instrument). In specific embodiments of the incarnation, polipeptidnaya grouping binds a polypeptide that enhances half-life in serum in vivo, with a KD of about 50 nm, or about 70 nm, or about 100 nm, or about 150 nm, or about 200 nm.

Preferably, polipeptidnaya grouping that contains a binding site (e.g., the Antiga the binding site), which has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, is not prokaryotic or bacterial polypeptide or peptide. Preferably, polipeptidnaya grouping is a polypeptide or peptide of eukaryotic cells, of a mammal or human.

In specific embodiments of the incarnation, polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, represents a domain of the folded protein. In other variants of the embodiment polipeptidnaya group has a molecular weight of at least about 4 kDa, at least about a 4.5 kDa, at least about 5 kDa, at least about a 5.5 kDa, at least about 6 kDa, at least about 6,5 kDa, at least about 7 kDa, at least about a 7.5 kDa, or at least about 8 kDa, as separate molecular structure.

Suitable polipeptidnaya groups that contain a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide, which increases Perry is on the half-life in serum in vivo, can be identified by any suitable means, such as screening natural or not naturally occurring polypeptides in a suitable method of analysis of adhesion. As described herein, the preferred polipeptidnaya groups that have antigennegative site for a polypeptide that enhances half-life in serum in vivo, are antigennegative fragments of antibodies that have binding specificity in relation to serum albumin. However, in the invention can be applied antigennegative fragments of antibodies that have binding specificity in relation to other polypeptides that increase the half-life in serum in vivo.

If desired, one or more hypervariable sites (CDR) of the antibody or its antigennegative fragment that binds a polypeptide that enhances half-life in serum in vivo, can be formatted in nimmanahaeminda structure that stores antigennegative specificity of the antibodies or antigennegative fragment. The composition of the medicinal product according to the invention may contain such nimmanahaeminda linking group. Such nimmanahaeminda linking groups can be obtained using any suitable JV is soba, for example, natural bacterial receptors, such as the SpA, were used as structural frames for CDR grafting to produce polipeptidnaya groups that specifically bind the epitope. Details of this technique are described in Application for U.S. Patent No. 5831012, the contents of which are incorporated herein by reference. Other suitable structural frames include those based on fibronectin and affitech. Details of suitable methods are described in WO 98/58965. Other suitable structural frames include lipocalin and CTLA4, as described in van den Beuken et al., J. Mol. Biol. 310:591-601 (2001), and structural frameworks, such as those described in WO 00/69907 (Medical Research Council), which is based, for example, on the ring structure of the bacterial Chaperonin or other chaperone polypeptides.

In some embodiments, embodiment, the composition of the medicinal product according to the invention contains nimmanahaeminda linking group that has binding specificity in relation to serum albumin, where nimmanahaeminda linking group contains one, two or three of the CDR of the VHVKor VHHas described herein, and a suitable structural frame. In specific embodiments of the incarnation, nimmanahaeminda linking group contains CDR3, but not CDR1 or CDR2 of the VH VKor VHHas described herein, and a suitable structural frame. In other variants of the embodiment nimmanahaeminda linking group contains CDR1 and CDR2, but not CDR3 of VHVKor VHHas described herein, and a suitable structural frame. In other variants of the embodiment nimmanahaeminda linking group contains CDR1, CDR2 and CDR3 from VHVKor VHHas described herein, and a suitable structural frame. In other embodiments, embodiment, the composition of the medicinal product contains only the CDR3 of the VHVKor VHHdescribed in this document, and medicine.

The composition of the medicinal product according to the invention can be obtained by using appropriate methods such as the methods described herein to obtain a merged structures medicines, drug conjugates and non-covalent conjugates of the drug. Additionally, the composition of the medicinal product according to the invention have advantages and properties, which have been described herein in relation to the slit structures medicines, drug conjugates and non-covalent conjugates of the drug.

In the proposed invention is oppozitsii medicines (for example, the drug conjugates, noncovalent drug conjugates, merged design drugs)that have improved pharmacokinetic properties (e.g., increased half-life in serum and other advantages compared to single drug (unconjugated drug, naslite drug). The drug conjugates, noncovalent drug conjugates and merged designs medicines contain antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, and one or more desired medicines.

As described herein, the composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs) according to the invention can be considerably increased in vivo half-life in serum and/or increased area under the curve (AUC) compared to individual drug. Furthermore, the activity of medicines usually do not have substantial changes in the composition of the medicinal product (e.g., drug conjugate, noncovalent conjugate the drug is tion means, fused design drugs). However, some changes in the activity of the composition of the medicinal product compared to individual drug is acceptable and is usually compensated by the improved pharmacokinetic properties of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds, mergers medicines). For example, the composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged structures medicines) can bind the target drugs with a lower affinity than the drug itself, but can be almost equivalent or superior efficiency compared with single drug with improved pharmacokinetic properties (e.g., increased in vivo half-life in serum, a large AUC) of the composition of the medicinal product. In addition, you can enter a smaller number of compositions, medicinal product (e.g., drug conjugates, noncovalent drug conjugates and the slit structures medicines) to achieve the desired therapeutic or diagnostic E. the reaction. Preferably, the activity of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the medicinal product) is different from that of the individual drugs is not more than about 100 times, or no more than about 50 times, or no more than about 10 times, or no more than about 5 times, or no more than about 4 times, or no more than about 3 times, or no more than about 2 times. For example, the drug may have KD, Ki, or the neutralization dose (ND50)equal to 1 nm, and the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) may have KD, Ki or ND50, equal to about 2 nm, or about 3 nm, or about 4 nm, or about 5 nm, or about 10 nm.

Preferably, the activity of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug) is not reduced significantly compared with the activity of the drug. In specific embodiments embodiment, the activity of the composition of the medicinal product is reduced compared to the activity of lekarstvennogo not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, not more than about 1%, or is substantially unchanged. Alternative stated, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) retains at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least approximately 99% of the activity of the medicinal product, or substantially the same activity as a drug. Preferably, the activity of the compositions of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs and medicines install and/or compared on the basis of "principles of medicinal product".

As described and shown herein, the composition of the medicinal product (e.g., drug conjugate cf is DSTV, non-covalent conjugate drugs, merged design drugs) according to the invention can have a lot of activity (e.g. activity in vivo)than a single drug. For example, as shown in Example 6, DOM7m-16/IL-1ra was more effective in the treatment of arthritis model in mice than IL-1ra, with the introduction of data agents in the same dose in terms of weight (10 mg/kg or 1 mg/kg). DOM7m-16/IL-1ra was more effective even though its molecular weight is approximately two times greater than the molecular weight of IL-1ra. Thus, mice that received DOM7m-16/IL-1ra, received only about half of the IL-1ra (as groups in the composition of DOM7m-16/IL-1ra), compared with mice that received IL-1ra.

In specific embodiments, embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) has greater activity (e.g. activity in vivo)than a drug, for example, the composition of the medicinal product may be at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least approximately 500% of the activity of the drug. Preferably, the activity of the compositions of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs and medicines install and/or compared on the basis of "principles of medicinal product". The activity of the compositions of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structures medicines and drugs can be installed using suitable in vitro or in vivo systems. In specific embodiments, embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) is more active than the drug, which it contains, as determined in vivo. In other embodiments, embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) is more active than the drug, which it contains, as determined in vitro.

The composition of the medicinal product (e.g. the R, the drug conjugates, noncovalent drug conjugates, merged design drugs)that contain a domain antibody (dAb)that has binding specificity in relation to serum albumin, provide additional benefits. Domain antibodies are very stable, have a small size compared to antibodies and other antihistamine fragments of antibodies, they can be obtained in high yields by expression in E. coli or yeast (e.g., Pichia pastoris), and as described herein, antigennegative fragments of antibodies that bind serum albumin, you can easily select from libraries of human origin or of any desired form. Accordingly, the composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), which contain a dAb that binds serum albumin, can't get any easier than therapeutic agents that are normally produced in mammalian cells (e.g., human, humanized or chimeric antibodies), and can be used not immunogenic dAbs (for example, human dAb can be used to slit the design of a drug or conjugate of a drug the frame means for treatment or diagnosis of diseases in humans).

Immunogenicity of the drug can be reduced if the drug is a part of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the medicinal product, which contains polipeptidnaya group that binds serum albumin (e.g., antigennegative fragment of the antibody that binds serum albumin). Accordingly, the drug may be less immunogenic than the individual drug) or to be not significantly immunogenic in the context of the composition of the medicinal product, which contains polipeptidnaya group that binds serum albumin (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs). Thus, such compositions drugs (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs) you can enter the subject again during the period of time with minimal loss of efficiency due to the generation antiscarring of antibodies by the immune system of the subject.

Additional is about, the composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described herein, may have an improved safety profile and fewer side effects than a single drug. For example, in the activity of binding to serum albumin inherent antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, merged design drugs and conjugates (drug conjugate, noncovalent drug conjugate funds) have increased the residence time in the vascular blood flow. Additionally, the conjugates and the slit structures medicines largely unable to overcome the blood-brain barrier and accumulate in the Central nervous system after systemic administration (e.g., intravascular injection). Accordingly, the conjugates (drug conjugate, noncovalent drug conjugate funds) and merged design drugs that contain medicinal product, which has a neurological toxicity or undesirable psychotropic effects, you can enter more be the danger and reduced side effects compared to individual drug. Alternatively, the conjugates (drug conjugate, noncovalent drug conjugate funds) and merged design drugs may have reduced toxicity to specific organs (e.g. kidney or liver)than a single drug. Conjugates and merged design drugs described herein can also be applied to sekvestirovanija drug or target that binds the drug (e.g., toxin), vascular blood flow, thus reducing the effects provided by the medicinal product or the target tissue (e.g., inhibiting the effects of the toxin).

Appropriate methods for pharmacokinetic analysis and a determination of half-life in vivo are well known in this field. Such methods are described, for example, Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and Peters er al, Pharmacokinetic analysis: A Practical Approach (1996). Also refer to "Pharmacokinetics", M Gibaldi &D Perron, published by Marcel Dekker, 2ndRev. edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half life t 1/2 alpha, t 1/2 beta) and area under the curve (AUC).

Half life t 1/2 alpha and t 1/2 beta) and AUC can be determined from a curve of serum concentration of conjugate or fused design on the relative time. You can use, for example, analytical package WinNonlin (available from Pharsight Corp., Mountain View, CA 94040, USA) to simulate the curve. In the first phase (alpha phase) composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) undergoes mainly distribution in the body of the patient, with some destruction. The second phase (beta phase) represents the final phase, when the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design of the medicinal product has already been distributed, and serum concentration decreases as the composition of the medicinal product withdrawn from the body of the patient, t alpha half-life is the half-life period of the first phase, and t beta half-life is the half-life period of the second phase. Thus, in the present invention proposed composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds, the merger of the medicinal product or composition containing the composition of the medicinal product (e.g., drug conjugate, noncovalent conjuga the medicinal product, fused design drugs) in accordance with the invention, having a tα half-life in the range of 15 minutes or more. In one variant embodiment, the lower limit of the interval is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. Additionally, or alternatively, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design of the medicinal product or composition according to the invention will have to half-life in the range up to and including 12 hours. In one variant embodiment, the upper boundary of the interval is 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable interval represents from 1 to 6 hours 2 to 5 hours, or from 3 to 4 hours.

Preferably, in the present invention proposed composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), having a tβ half-life in the range of 2.5 hours or more. In one variant embodiment, the lower limit of the interval is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In some embodiments, embodiment, composition and medicines (for example, the drug conjugates, noncovalent drug conjugates, merged design drugs), have a tβ half-life in the range up to and including 21 days. In one variant embodiment, the upper boundary of the interval is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. In specific embodiments, embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) in accordance with the invention will have a tβ half-life in the range from 12 to 60 hours. In an additional variant embodiment, it will be in the range from 12 to 48 hours. In yet another additional variant embodiment, it will be in the range of from 12 to 26 hours.

Additionally, or alternatively the above criteria, the present invention proposed composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), having a value of AUC (area under the curve) in the range of 0.01 mg·min/ml or more, or 1 mg·min/ml or more. In one variant embodiment, the lower limit of the interval is 0,01, 0,1, 1, 5, 10, 15, 20, 30, 100, 200 or 300 mg·min/ml In specific embodiments, voploscheni is, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) has AUC in the range of up to 600 mg·min/ml In one variant embodiment, the upper boundary of the interval is 500, 400, 300, 200, 150, 100, 75 or 50 mg·min/ml In other embodiments, embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) has AUC in the range selected from the group consisting of the following: 15 up to 150 mg·min/ml, from 15 to 100 mg·min/ml, 15 to 75 mg·min/ml, from 15 to 50 mg·min/ml, from 0.01 to 50 mg·min/ml, from 0.1 to 50 mg·min/ml, from 1 to 50 mg·min/ml, from 5 to 50 mg·min/ml and from 10 to 50 mg·min/ml

The invention relates to compositions of drugs (e.g., drug conjugates, noncovalent the conjugates of drugs, merged structures medicines), which contain the drug and polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. In preferred embodiments, embodiments of compositions of Lech the only means polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, has binding specificity in relation to serum albumin.

In some embodiments, embodiment, the composition of the medicinal product contains a drug that is associated covalently with polipeptidnaya group that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo. In these cases embodiment, the drug may be associated covalently with polipeptidnaya domain in any suitable position, such as aminocore, carboxylic or through suitable amino acid side chains (for example, ¼ of the amino group of lysine or Tolna group of cysteine).

In other embodiments, embodiment, the composition of the medicinal product contains a drug that is associated ecovalence with polipeptidnaya group that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide, which increased the AET half-life in serum in vivo. In such embodiments, embodiments, the drug can be ecovalence associated with antigennegative fragment directly (for example, by electrostatic interaction, hydrophobic interaction) or indirectly (for example through non-covalent binding of complementary binding partners (e.g., Biotin and avidin), where one partner is covalently associated with the drug, and the complementary binding partner is covalently bonded with antigennegative fragment). If use of complementary binding partners, one of the binding partners can be covalently linked to drug directly or through a suitable linker group, and the complementary binding partner can be covalently linked to polipeptidnaya domain directly or through a suitable linker group.

In other embodiments, embodiment, the composition of the medicinal product is a protein that contains polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, and a polypeptide drug. Slit proteins contain a continuous polypeptide CE the e l e C when the chain contains polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, as the first group, and a polypeptide drug as a second group, which are present in the form of individual parts (groups) polypeptide chains. The first and second groups can be directly connected to each other by peptide bond or bonded by means of a suitable amino acid, or a peptide or polypeptide linker. If necessary, there may be additional groups (e.g., third, fourth and/or linker sequences. The first group may be N-terminal position, the C-terminal position or be positioned inside relative to the second group (i.e. polypeptide drugs). In specific embodiments of the incarnation, the protein contains one or more polipeptidnaya groups that contain a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, and one or more groups polypeptide, in addition to the state funds. In these variants of the embodiments, a protein may contain from one to about ten (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) groups of polypeptide drugs, which may be the same or different, and from one to about twenty (such as, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) polipeptidnaya groups that contain a binding site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, which may be the same or different.

Polipeptidnaya groups that contain a binding site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, and grouping polypeptide drugs can be located in any desired position. For example, moving from aminobenzo to carboxylic, grouping can be in the following order: one or more polipeptidnaya groups, one or more groups of polypeptide drugs, one or more polipeptidnaya groups. In another example, moving from aminobenzo to carboxylic, grouping can be in the following order: one or more polipeptidnaya groups, one or more groups is polypeptides medicines one or more polipeptidnaya groups, one or more groups of polypeptide drugs, one or more polipeptidnaya groups. As described herein, polipeptidnaya groups and groups of polypeptide drugs can be directly connected to each other by peptide bond or bonded by means of a suitable amino acid, or a peptide or polypeptide linker.

In specific embodiments, embodiments, a protein is a continuous polypeptide chain, which has the formula (aminobenzo to carboxylic):

a-(P)n2-b-(X)n1-c(Q)n3-d or a-(Q)n3-b-(X)n1-c(P)n2-d,

where X is a polypeptide drug;

each of P and Q independently represents polipeptidnaya grouping that contains a binding site that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo;

each of a, b, C and d independently is absent or consists of from one to about 100 amino acid residues;

n1, n2 and n3 represent the number attending X, P or Q groups, respectively;

n1 is from one to about 10;

n2 equals from zero to about 10; and

n3 equals from zero to about 10,

provided that both n2 and n3 is not equal to zero;and

provided that, when both n1 and n2 is equal to one and n3 is zero, X does not contain a chain of the antibody or fragment chain antibodies.

In some embodiments, embodiment, n2 is equal to one, two, three, four, five or six, and n3 is equal to zero. In other embodiments, embodiments, n3 is equal to one, two, three, four, five or six, and n2 is zero. In other variants of the embodiment, each of n1, n2 and n3 is equal to one.

In specific embodiments, embodiments, X does not contain a chain of the antibody or fragment chain antibodies.

In preferred embodiments the embodiment, each of P and Q independently represents polipeptidnaya group, which has binding specificity in relation to serum albumin.

In particularly preferred embodiments of embodiment, the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs), contains polipeptidnaya grouping that contains a binding site (e.g., antigennegative site)that has binding specificity in respect of the polypeptide that enhances half-life in serum in vivo, where polypeptidesee domain is antigennegative fragment of the antibody that has binding specificity from which Oseni serum albumin.

Antigennegative fragment of the antibody that binds serum albumin

The drug conjugates, noncovalent drug conjugates and merged design of a medicinal product according to the invention contain (i.e. one or more) antigennegative fragment of the antibody that binds serum albumin. Antigennegative fragment can have binding specificity in relation to serum albumin animal, which will introduce a conjugate of a drug or merged design drugs. Preferably, antigennegative fragment has binding specificity in relation to serum albumin human. However, provided veterinary use, and antigennegative fragment can have binding specificity in relation to serum albumin from the desired animal, such as serum albumin from dogs, cats, horses, cows, chicken, sheep, pig, goat, deer, mink and the like. In some embodiments of the incarnation, antigennegative fragment has binding specificity in relation to serum albumin of more than one species. For example, as described in this document were obtained human dAbs that have the binding specificity against serum albumin is the Rys and serum albumin mice, and dAb that has binding specificity in relation to serum albumin in rats, mice and humans (table 1 and 7). Such dAb prefer preclinical and clinical trials, using the same conjugate of a drug or merged design drugs, and eliminate the need for pre-clinical trials with appropriate surrogate merged design drug or conjugate of the drug.

Antigennegative fragments, suitable for use in the invention include, for example, Fab fragments, Fab' fragments, F(ab')2fragments, Fv fragments (including single-chain Fv (scFv) and Fv, linked by a disulfide bond), a single variable domain and dAbs (VHVL). Such antigennegative fragments can be obtained in any suitable way, such as proteolysis of antibodies using pepsin, papain or other proteases, having the desired specificity of cleavage, or using recombinant technology. For example, you can get Fv fragments by cleavage of antibodies with a suitable protease, or using recombinant DNA technology. For example, you can get a nucleic acid which encodes a variable area light chain and variable plot heavy chain that soy is inany suitable peptide linker, such as a chain of from two to about twenty gliceridih residues. Nucleic acid can be introduced in a suitable host (e.g., E.li) using any suitable technique (e.g., transfection, transformation, infection), and the owner can be kept under conditions suitable for expression of single-chain Fv fragment. You can get a lot antigenspecific fragments of antibodies using antibody genes in which one or more termination codons were introduced above natural termination site. For example, you can design expressing a construct encoding a F(ab')2the plot of the heavy chain of the immunoglobulin, by introducing a translational termination codon at the 3' end of the sequence encoding the hinge section of the heavy chain. The drug conjugates, noncovalent drug conjugates and merged design of a medicinal product according to the invention may contain separate heavy and light chains of antibodies that bind serum albumin, or parts of some of the chains that bind serum albumin (e.g., single VHVKor Vλ).

Antibodies and their antigennegative fragments that bind the desired serum albumin (such as human serum albumin), you can select the ü from a suitable collection of natural or artificial antibodies or raised against an appropriate immunogen in a suitable host. For example, antibodies can be obtained by immunizing a suitable host (e.g., a mouse, a transgenic mouse, capable of producing human antibodies, rat, rabbit, chicken, goat, not APE (e.g., macaque)) serum albumin (e.g., isolated or purified human serum albumin) or peptide serum albumin (for example, a peptide containing at least about 8, 9, 10, 11, 12, 15, 20, 25, 30, 33, 35, 37 or 40 amino acid residues). Antibodies and antigennegative fragments that bind serum albumin, can also be selected from libraries of recombinant antibodies or antigenspecific fragments, such as phage display library. Such libraries may contain antibodies or antigenspecific antibody fragments that contain natural or artificial amino acid sequence. For example, a library may contain Fab fragments that contain artificial CDRs (e.g., a random amino acid sequence) and human framework region (immunoglobulin). (See, for example, U.S. Patent No. 6300064 (Knappik, et al.).) In other examples, the library contains scFv fragments or dAbs (single VHsingle VKor single Vλwith the diversity of sequences in one or more CDRs. (See, for example, WO 99/20749 (Tomlinson Winter), WO 03/002609 A2 (Winter et al.), WO 2004/003019 A2 (Winter et al.).)

Suitable antibodies and their antigennegative fragments that bind serum albumin, include, for example, human antibodies and their antigennegative fragments, gumanitarnye antibodies and their antigennegative fragments, chimeric antibodies and their antigennegative fragments, antibodies and their antigennegative fragments rodents (e.g. mice, rats) and Camelid antibodies and their antigennegative fragments. In specific embodiments of the incarnation, drug conjugates, noncovalent drug conjugates and merged designs medicines contain Camelid VHHthat binds serum albumin. Camelid VHHs are polypeptides single variable domain of the immunoglobulin derived from antibody heavy chains, which are naturally devoid of light chains. Such antibodies exist in the Camelid species including camel, llama, Alpaca, DROMEDARY, llama. Molecules VHHabout ten times smaller than IgG molecules, and as single polypeptides, are very stable and resistant to the harsh conditions of pH and temperature. Suitable Camelid VHHthat bind serum albumin, include those described in WO 2004/041862 (Ablynx N.V.) and in this document (Fig and SEQ ID NOs:77-88).THE specific variants of the embodiment, Camelid VHHbinds human serum albumin and contains an amino acid sequence that has at least about 80%, or at least about 85%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 or SEQ ID NO:88. The identity of the amino acid sequence preferably set using a suitable algorithm for sequence alignment and parameter values by default, such as BLAST P (Karlin and Altschul, Pmc. Natl. Acad. Sci. USA 87(6):2264-2268 (1990)).

Preparation of immunizing antigen and obtain polyclonal and monoclonal antibodies can be performed using any suitable techniques. It has been described many ways. (See, for example, Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al" Nature 266: 550-552 (1977); Koprovski et al., U.S. patent No. 4172124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol.2 (Supplement 27, Summer 94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991).) Usually, if you want to get a monoclonal antibody, get hybridoma merge the appropriate cells of immortalizing cell lines (e.g. the R, cell myeloma line such as SP2/0, Rad or heteromalla) cells by host antibodies. Cell host antibodies can be obtained from the peripheral blood or, preferably, spleen or lymph nodes human transgenic animals capable of producing human antibodies, or other suitable animals immunized with interest antigen. Cells that produce antibodies of human origin (e.g., human antibody), can be obtained using suitable methods, for example, the fusion of host cell of human antibodies and heteromyinae or creamy, or immortality activated human cells by infection with Epstein van-virus. (See, for example, U.S. Patent No. 6197582 (Trakht); Niedbala et al., Hybridoma, 17:299-304 (1998); Zanella et al., J. Immunol. Methods 156:205-215 (1992); Gustafsson et al., Hum Antibodies Hybridomas, 2:26-32 (1991).) Flushed or immortalized cells of host antibodies (hybridoma) can be identified with the use of selective culture conditions and clone by the method of serial dilution. Cells which produce antibodies with the desired specificity can be identified using an appropriate method of analysis (e.g., ELISA).

Antibodies can also be obtained directly (for example, to synthesize or clone) of an isolated antigen-specific host cell an is the body (for example, the cell from peripheral blood or, preferably, spleen or lymph nodes, deterministic for producing antibodies with the desired specificity) human transgenic animals capable of producing human antibodies, or other suitable animals immunized with interest antigen (see, for example, U.S. Patent No. 5627052 (Schrader)).

If the drug conjugate, noncovalent drug conjugate funds or merged design of a medicinal product intended for administration to humans, the antibody or antigennegative fragment that binds serum albumin (e.g., human serum albumin), can be a human, humanitariannet or chimeric antibody or antigennegative fragment of such antibody. These types of antibodies and antigenspecific fragments are less immunogenic or not immunogenic in humans and provide the well-known advantages. For example, drug conjugates, noncovalent drug conjugates, or merged design drugs that contain antigennegative fragment of a human, gumanitarnogo or chimeric antibodies, you can enter the person repeatedly with lower efficiency or no loss of efficiency the activity (in comparison with other fully immunogenic antibodies) due to the formation of human antibodies which bind the conjugate of a drug or merged design drugs. If the drug conjugate, noncovalent drug conjugate funds or merged design drugs intended for veterinary injection, it can be applied to similar antibodies or antihistamie fragments. For example, CDRs from murine or human antibodies can be transferred to the frame region of the immunoglobulin from the desired animal such as a horse or cow.

Human antibodies and nucleic acids, encoding, can be obtained, for example, from a person or from transgenic animals capable of producing human antibodies. Transgenic animals capable of producing human antibodies (e.g., a mouse), are animals capable of producing a set of human antibodies, such as XENOMOUSE (Abgenix, Fremont, CA), HUMAB-MOUSE, KIRIN TC MOUSE or KM-MOUSE (MEDAREX, Princeton, NJ). Generally, the genome of transgenic animals capable of producing human antibodies, has been modified to include a transgene containing DNA from a locus of human immunoglobulin, which can undergo functional rearrangement. Endogenous immunoglobulin locus in transgenic animal capable of producing human antibodies, may be destroyed or UDA is Yong, in order to eliminate the ability of the animal to produce the antibody encoded by the endogenous gene. Suitable methods of obtaining transgenic animals capable of producing human antibodies are well known in this field. (See, for example, U.S. Patent No. 5939598 and 6075181 (Kucherlapati et al.), U.S. patents№5569825, 5545806, 5625126, 5633425, 5661016 and 5789650 (Lonberg et al.), Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2552-2555 (1993), Jakobovits et al., Nature, 362: 255-258 (1993), Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO 98/24884, Lonberg er al. WO 97/13852, Lonberg et al. WO 94/25585, Lonberg et al. EP 0814259 A2, Lonberg et al. GB 2272440 A, Lonberg er al., Nature 368:856-859 (1994), Lonberg et al., Int Rev Immunol 13(1):65-93 (1995), Kucherlapati et al/. WO 96/34096, Kucherlapati et al. EP 0463151 B1, Kucherlapati et al. EP 0710719 A1, Surani et al. U.S. patent No. 5545807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438474 B1, Taylorera/., Int. Immunol. 6(4)579-591 (1994), Taylorera/., Nucleic Acids Research 20(23):6287-6295 (1992), Green era/.. Nature Genetics 7:13-21 (1994), Mendez er al., Nature Genetics 15:146-156 (1997), Tuaillon e/ al., Proc Natl Acad Sci USA 90(8):3720-3724 (1993) and Fishwild er al., Nat Biotechnol 14(7):845-851 (1996), the content of each of the above publications are fully incorporated herein by reference.)

Transgenic animals capable of producing human antibodies, it is possible to immunize a suitable antigen (e.g., human serum albumin), and cells of host antibodies can be selected and drain for the formation of a hybrid, using conventional methods. Hybridoma that produce human antibodies having the desired is harakteristiki (for example, specificity, affinity), can be identified using any suitable method of analysis (e.g., ELISA), and, if desired, to select and subclinical using suitable methods of cultivation.

Humanized antibodies and other antibodies with transferred CDR can be obtained using any suitable method. CDRs from antibody migrated CDR can be derived from a suitable antibody that binds serum albumin (referred to as donor antibody). Other sources of suitable CDRs include natural and artificial antibodies specific for serum albumin derived from human and non-human source, such as a rodent (e.g. mouse, rat, rabbit, chicken, pig, goat, Primate besides humans (e.g., monkey) or a library.

Frame region gumanitarnogo antibodies, preferably, are of human origin and may be derived from any of the variable segment of the human antibody having affinity sequences with similar or equivalent to plot (for example, variable plot heavy chain or variable area light chain) antigennegative plot of the donor antibody. Other sources of frame areas of human origin include consensus sequences of ilovecosmo variable segment. (See, for example, Kettleborough, C.A. et al., Protein Engineering 4:773-783 (1991); Carter et al., WO 94/04679; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)). Other types of antibodies transferred CDR can contain a frame region of a suitable origin, such as a framework region encoded by gene segments antibodies embryonic type from horses, cows, dogs, cats and the like.

Frame of human origin may include the substitution or replacement of amino acids, such as "reverse mutation", which replaces a residue amino acids in the framework region of a human or animal residue of the corresponding position of the donor antibody. You can perform one or more mutations in the frame region, including deletions, insertions or substitutions of one or more amino acids. Options can be obtained using a variety of suitable ways, including mutagenesis of the human chain from a non-human donor or acceptor. (See, for example, U.S. Patent No. 5693762 (Queen et al.) and 5859205 (Adair et al.), the contents of which are fully incorporated herein by reference.)

Permanent plots antibodies, antibody chains (e.g., heavy chain, light chain) or their fragments or parts, if present, can occur from any suitable source. For example, the permanent parts of the human, humanized and specific chimeric antibodies, antibody chains (e.g., heavy chain, light chain) or their fragments or parts, if any are present, can be of human origin and can occur from any suitable human antibody or chain antibodies. For example, the permanent site of human origin or its part can occur from a human κ or λ light chain and/or human γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2), δ or ε heavy chain, including allelic variants. In specific embodiments, embodiments, the antibody or antigennegative fragment (for example, an antibody of human origin, human antibody) may include amino acid substitutions or substitutions that modify or adapt the function (e.g., effector function). For example, you can develop a permanent site of human origin (e.g., γ1 constant area, constant γ2 plot), to reduce activation of complement and/or Fc receptor binding. (See, for example, U.S. Patent No. 5648260 (Winter et al.), 5624821 (Winter et al.) and 5834597 (Tso et al.), the contents of which are fully incorporated herein by reference.) Preferably, the amino acid sequence of the permanent site of human origin, which contains such amino acid Sames the treatment or replacement, is at least about 95% identical to the full length amino acid sequence of an unmodified permanent part of the human origin, more preferably is at least about 99% identical to the full length amino acid sequence of an unmodified permanent part of human origin.

Humanized antibodies, antibodies with transferred CDR or antihistamie fragments gumanitarnogo or antibodies to the migrated CDR can be obtained using any suitable method. Several such methods are well known in this field. (See, for example, U.S. Patent No. 5225539 (Winter), U.S. Patent No. 5530101 (Queen et al.).) Lots gumanitarnogo antibodies or antibodies with transferred CDR (e.g., CDRs, frame area, constant area) can be derived or to be derived directly from the appropriate antibodies (e.g., by de novo synthesis section), or you can receive and Express the nucleic acid encoding the antibody or circuit having the desired property (e.g., binds serum albumin). To get a piece of chain, you can enter one or more termination codons in the desired position. For example, nucleic acid sequences (e.g., DNA)encoding humanized or CDR-shifted variabel the major areas you can construct using PCR (polymerase chain reaction) mutagenetic methods to modify existing DNA sequences. (See, for example, Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989).) PCR primers encoding new CDRs, you can hybreed with DNA matrix pre gumanitarnogo variable segment, which is based on the same, or very similar, the human variable site (Sato, K., et al., Cancer Research 53:851-856 (1993)). If similar DNA sequence is not available for use as a matrix, it is possible to construct a nucleic acid containing a sequence encoding the sequence of the variable segment of synthetic oligonucleotides (see, for example, Kolbinger, F., Protein Engineering 8:971-980 (1993)). In nucleic acid can also include a sequence encoding a signal peptide (for example, by synthetic means, when inserted into the vector). You can use the natural sequence of the signal peptide from the acceptor antibody, the sequence of the signal peptide from the other antibodies or other suitable sequence (see, for example, Kettleborough, C.A., Protein Engineering 4:773-783 (1991)). Using these methods or other suitable methods, it is easy to get options. In one variant embodiment, cloned variable plots can be subjected to mutation is possible to select the sequence, encoding variants with the desired specificity (e.g., from ragovoy libraries; see, for example, U.S. Patent No. 5514548 (Krebber et al.) and WO 93/06213 (Hoogenboom et al.)).

The antibody or antigennegative fragment that binds serum albumin, may be a chimeric antibody or antigennegative the fragment of the chimeric antibody. Chimeric antibody or antigennegative fragment contains a variable section from one species (e.g., mouse) and at least a part of the permanent plot from another species (e.g., human). Chimeric antibodies and antigennegative fragments, chimeric antibodies can be obtained in any suitable way. A number of suitable methods well known in the field. (See, for example, U.S. Patent No. 4816567 (Cabilly et al.), U.S. patent No. 5116946 (Capon et al.).)

The preferred method of obtaining antigenspecific fragments of antibodies that bind serum albumin, includes choice antigennegative fragment (e.g., scFvs, dAb)that has binding specificity against the desired serum albumin, from a set of antigenspecific fragments. For example, as described herein, dAbs that bind serum albumin, can be selected from a suitable ragovoy display library. Has been described a number of suitable bacteriophobic display libraries and the method is in a selection (for example, monovalent and multivalent display display systems). (See, for example, Griffiths et al., U.S. patent No. 6555313 B1 (incorporated herein by reference); Johnson et al., U.S. patent No. 5733743 (incorporated herein by reference); McCafferty et al., U.S. patent No. 5969108 (incorporated herein by reference); Mulligan-Kehoe, U.S. Patent No. 5702892 (incorporated herein by reference); Winter, G. et al., Annu. Rev. Immunol. 12:433-455 (1994); Soumillion, P. et al., Appl. Biochem. Biotechnol. 47(2-3): 175-189 (1994); Castagnoli, L. el al., Comb. Chem. High Throughput Screen, 4(2): 121-133 (2001); WO 99/20749 (Tomlinson and Winter); WO 03/002609 A2 (Winter et al.); WO 2004/003019A2 (Winter et al.).) The polypeptides shown in the bacteriophage library can be displayed on any suitable bacteriophage, such as a filamentous phage (e.g., fd, M13, F1), virulent phage (e.g., T4, T7, lambda) or RNA-phages (MS2), for example, and selected for binding to serum albumin (such as human serum albumin).

As a rule, use of phage library, which displays a set of polypeptides as a fusion protein with a suitable rahovym envelope protein. This library can be obtained by any appropriate means, such as the introduction of a library of phage vectors or pagemenu (phagemid) vectors encoding the displayed antibodies or their antigennegative fragments into suitable bacteria-owners and cultivation of the floor is built in the result of bacteria for the production of phage (e.g., using a suitable helper phage or complementarily plasmid, if desired). Phage library can be isolated from the culture by any suitable means, such as precipitation and centrifugation.

The library can contain a set of antibodies or their antigenspecific fragments, which contains any number of a variety of amino acid sequences. For example, the kit may contain antibodies or their antigennegative fragments, which have amino acid sequences that correspond to the natural antibodies of the desired organism, and/or can contain one or more sections of random or randomized amino acid sequences (e.g., CDR sequence). Antibodies or their antigennegative fragments in this collection or library may contain certain parts of random or randomized amino acid sequences and plots of the normal amino acid sequence. In specific embodiments, embodiments, all or substantially all of the polypeptides in the set represent the desired type antigennegative fragment antibodies (e.g., human VHor human VL. For example, each polypeptide in the set may contain VHVLor Fv (for example, single-chain Fv)./p>

The diversity of amino acid sequences can be implemented in any desired section of the antibodies or their antigenspecific fragments in whatever way is appropriate. For example, a variety of amino acid sequences can be embedded in the plot of a target, such as the hypervariable area variable domain of the antibody by producing a library of nucleic acids that encode diverse antibodies or their antigennegative fragments, using any suitable methods of mutagenesis (e.g., PCR with low precision (low fidelity PCR), oligonucleotide-mediated or site-directed mutagenesis, diversification using NNK codons) or any other suitable method. If desired, you can randomize the plot of antibodies or their antigenspecific fragments, which should diversify.

Suitable phage display library can be used for the selected antibodies or antigenspecific fragments of antibodies that bind serum albumin and have other advantageous properties. For example, to select antibodies or antihistamie fragments that are resistant to aggregation when they are spatially deployed. The aggregation is affected by the concentration of the polypeptide, and, believe, it occurs in many cases due to the partly collapsed is, or expanded intermediate products. Factors and conditions that favor the partially folded intermediate products, such as high temperature and high protein concentration, contribute to irreversible aggregation. (Fink, A.L, Folding &Design 3:R1-R23 (1998).) For example, storage of purified polypeptides in a concentrated form, such as lyophilized drug, often leads to irreversible aggregation, at least part of the polypeptide. Also, the production of polypeptide expression in biological systems, such as E. Li, often leads to the formation of intracellular cells, which contain aggregates of polypeptides. The release of active polypeptides from the intracellular Taurus can be very difficult and may require the use of additional stages to the biological production system, such as stage of refolding.

Antibodies and antigennegative fragments that are resistant to aggregation and reversible unfolding when heated, it is possible to select a suitable ragovoy display library. Typically, phage display library containing a set of displayed antibodies or their antigenspecific fragments, heated to a temperature (Ts)at which at least part of the displayed antibodies or their antigenspecific fragments become expanded, then cooled to a temperature (Te), where Ts>Tc, pore is what STV, at least part of the antibodies or their antigenspecific fragments were subjected to refolding, and part of the polypeptide was subjected to aggregation. Then, antibodies or their antigennegative fragments that unfold reversibly and bind serum albumin, restore at a temperature of (Tg). The recovered antibody or antigennegative fragment that is unfolding is reversible, has a melting temperature (TM), and preferably, the set was heated to Ts, cooled to TC, and the antibody or antigennegative fragment that unfolds reversibly, allocated at Tg, so Ts>Tm>Tc and Ts>Tm>Tr. Typically, phage display library is heated to about 80°C and cooled to about room temperature or about 4°C before sampling. Antibodies or their antigennegative fragment that unfold reversibly and resistant to aggregation, you can also develop or construct replacement of a particular amino acid residue remains, which give the ability to unfold reversibly. (See, WO 2004/101790 (Jespers et al.) and a provisional application for U.S. Patent nos: 60/470340 (announced on may 14, 2003) and 60/554021 (announced on may 17, 2004) for a detailed discussion of the methods of selection and development or design of antibodies or their antigenspecific fragments that unfold reversibly. Containing the s WO 2004/101790 and both of the aforementioned provisional applications U.S. Patent are incorporated herein by reference.).

Antibodies or their antigennegative fragments that unfold reversibly and resistant to aggregation, provide a number of advantages. For example, due to its resistance to aggregation, antibodies or their antigennegative fragments that unfold reversibly can be obtained with high yield in the form of soluble protein expression in suitable biological producing systems, such as E. coli. In addition, antibodies or their antigennegative fragments that unfold reversibly can be formulated and/or stored at higher concentrations than conventional polypeptides, as well as less aggregation and loss of activity. DOM7h-26 (SEQ ID NO:20) is a human VHthat unfolds reversibly.

Preferably, the antibody or antigennegative fragment that binds serum albumin, contains a variable domain (VHVKVλ), in which one or more frame regions (FR) contain (a) the amino acid sequence of a human framework region, (b) at least 8 contiguous amino acids of the amino acid sequence of a human framework region, or (C) the amino acid sequence encoded by gene segment of a human antibody embryonic type, where the specified frame area to depict ablaut a such as set Kabat. In a specific embodiment variants, the amino acid sequence of one or more frame regions is the same as the amino acid sequence of a corresponding framework region encoded by gene segment of a human antibody embryonic type, or the amino acid sequence of one or more specified frame regions together contain up to 5 amino acid differences compared to the amino acid sequence specified corresponding frame region encoded by gene segment of a human antibody embryonic type.

In other variants of the embodiment, the amino acid sequences of FR1, FR2, FR3 and FR4 are the same as the amino acid sequence of the corresponding frame regions encoded by gene segment of a human antibody embryonic type, or amino acid sequences of FR1, FR2, FR3 and FR4 collectively contain up to 10 amino acid differences compared to the amino acid sequences of the respective frame regions encoded by these gene segments of the human antibodies of embryonic type. In other variants of the embodiment, the amino acid sequence of the above FR1, FR2 and FR3 are the same as the last amino acid is successive respective frame areas encode the specified segment of the gene of the human antibodies of embryonic type.

In specific embodiments of the incarnation, antigennegative fragment of the antibody that binds serum albumin, contains a variable domain immunoglobulin (e.g., VHVLbased on human gametes sequence, and optionally can have one or more diverse areas, such as the hypervariable sites. Suitable human gameta sequence for VHincludes, for example, the sequence encoded by the VHsegments of the gene DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP45, DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67, DP68 and DP69, and JH segments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6. Suitable human gameta sequence for VLincludes, for example, the sequence encoded by the VKsegments of the gene DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 and DPK28 and JKsegments JK1, JK2, JK3, JK4 and JK5.

In specific embodiments, embodiments, the drug conjugate, noncovalent drug conjugate funds or merged design of the medicinal product does not contain antibody mouse, rat and/or rabbit, which binds serum albumin, or antigennegative fragment such as is tetela.

Antigennegative fragment can bind to serum albumin with any desired affinity, on-rate and off rate on rate - the rate of Association of two molecules, off rate - the rate of dissociation of two molecules). The affinity (KD), on rate (Konor ka) and off rate (Koffor kd), you can choose to get the desired half-life in serum for specific drugs. For example, it may be desirable to obtain the maximum half-life in serum for the medicinal product, which will neutralize the inflammatory mediator of chronic inflammatory diseases (e.g., dAb that binds and neutralizes inflammatory cytokine), while a shorter half-life in serum may be desirable for a medicinal product which has some toxicity (e.g., chemotherapeutic agent). Usually, it is preferable to fast on rate and rapid or moderate off rate for binding to serum albumin. The conjugates of drugs and merged design drugs that contain antigennegative fragment with these characteristics, will quickly bind serum albumin after the introduction, and will be quickly dissociate and re-bind serum albumin. These characteristics will be reduced if the three excretion of drugs (for example, the kidneys), but, nevertheless, will ensure effective delivery and access to drug target.

Antigennegative fragment that binds serum albumin (e.g., dAb), is usually associated with a KD equal to from about 1 nm to about 500 μm. In some embodiments of the incarnation, antigennegative fragment binds to serum albumin with a KD (KD=Koff(kd)/Kon(ka))equal to from about 10 to about 100 nm, or from about 100 nm to about 500 nm, or from about 500 nm to about 5 mm, as determined surface plasma resonance (for example using the BIACORE instrument). In specific embodiments, embodiments, the drug conjugate, noncovalent drug conjugate funds or merged design of the medicinal product contains antigennegative fragment antibodies (e.g., dAb)that binds to serum albumin (such as human serum albumin) with a KD equal to about 50 nm, or about 70 nm, or about 100 nm, or about 150 nm, or about 200 nm. Improved pharmacokinetic properties (e.g., prolonged 11/2 In increased AUC) of drug conjugates, noncovalent drug conjugates and the slit structures of the medicinal product described in this document may correlate with the affinity antigennegative the its fragment which binds serum albumin. Accordingly, drug conjugates, noncovalent drug conjugates and merged design drugs which have improved pharmacokinetic properties, can usually be obtained using antigenspecific fragment that binds serum albumin (e.g., human serum albumin) with high affinity (e.g., KD of about 500 nm or less, about 250 nm or less, about 100 nm or less, about 50 nm or less, about 10 nm or less, or about 1 nm or less, or about 100 PM or less).

Preferably, the drug that is conjugated or fused with antigennegative fragment that binds serum albumin, binds its target (drug target) with an affinity (KD), which is stronger than the affinity antigennegative fragment to serum albumin, and/or Koff(kd), which is faster thanoffantigennegative fragment with serum albumin that the measured surface plasma resonance (for example using the BIACORE instrument). For example, the drug may bind its target with an affinity that is about 1 to about 100,000, or from about 100 to about 100,000, or from about 1000 is about 100000, or from about 10000 to about 100000 times stronger than the affinity antigennegative fragment that binds SA, SA. For example, antigennegative fragment of the antibody that binds to SA, can be contacted with an affinity equal to about 10 μm, while the drug binds its target with an affinity equal to about 100 PM.

In specific embodiments of the incarnation, antigennegative fragment of the antibody that binds serum albumin, is a dAb that binds human serum albumin. For example, VKdAb having the amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or VHdAb having the amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In other variants of the embodiment antigennegative fragment of the antibody that binds serum albumin, is a dAb that binds human serum albumin and contains CDRs of any of the aforementioned amino acid sequences. In other variants of the embodiment antigennegative fragment of the antibody that binds serum albumin, is a dAb that binds human serum is albumin and contains the amino acid sequence, which has at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23. The identity of the amino acid sequence preferably set using a suitable algorithm for sequence alignment and parameter values by default, such as BLAST P (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87(6):2264-2268 (1990)).

Drugs

The specific composition of the medicinal product according to the invention (e.g., drug conjugates, noncovalent drug conjugates) can contain any drug (e.g., small organic molecule, nucleic acid, polypeptide)that can be entered to the individual to make a favorable therapeutic or diagnostic effect, for example, by binding with and/or changes in the biological function of a target molecule in the individual. Other compositions of the medicinal product according to the invention (for example, the slit structures medicinal medium spans the VA) can contain a polypeptide or peptide drug. In preferred embodiments the embodiment of the slit structures medicinal product medicinal product contains no chain antibody or fragment chain antibodies (e.g., VHVKVλ). In private variants embodiment, the drug is selected from insulinotropic agent and incretin, glucagon-like peptide-1, GLP-1 peptide, a GLP-1 analog, a GLP-1 derivative, PYY, PYY peptide, PYY analog, PYY derivative, on the Basis 3 Basis-3 peptide, on the Basis of 3 similar, on the Basis of 3 is derived, on the Basis 4 Basis-4 peptide, on the Basis of 4 similar, the Basis-4 derivative, or a combination of two or more of these agents (e.g., GLP-1 and PYY peptide peptide).

Suitable drugs for use in the invention include, for example, immunosuppressants (eg, cyclosporine a, rapamycin, FK506, prednisone), antiviral agents (acyclovir, ganciclovir, indinavir), antibiotics (penicillin, minocycline, tetracycline), anti-inflammatory agents (aspirin, ibuprofen, prednisone), cytotoxins or cytotoxic agents (such as paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, terakai is, lidocaine, propranolol, puromycin and analogs or homologs of any of the above agents. Suitable drugs include antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-fluorouracil of dacarbazine), alkylating agents (e.g., mechlorethamine, tiefflieger, CC-1065, melphalan, carmustine (BSNU), lomustin (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin mithramycin and astromicin (AMC)), radionuclides (e.g., iodine-125, -126), yttrium (for example, yttrium-90, -91) and praseodymium (for example, praseodymium-144, -145), and protease inhibitors (e.g., inhibitors of matrix metalloproteinases). Other suitable drugs are nucleic acids, such as antisense-nucleic acids and the RNA. Calicheamicin is also suitable for use according to the invention.

Suitable drugs include analgesic agents, including drugs (e.g. codeine, nalmefene, naloxone, fentanyl, meperidine, morphine, tramadol, propoksifen, oxycodone, methadone, nalbuphine), nonsteroidal anti-inflammatory agents (e.g., indomethacin, Ketorolac, arthrotec, ibuprofen, naproxen, salicylate, celecoxib, rofecoksib), acetaminophen, capsaicin, ziconotide and the like.

In specific embodiments, embodiments, the drug is a polypeptide toxin, such as a toxin, such as abrin, ricin A, pseudomonades exotoxin or diphtheria toxin. Other suitable polypeptide drugs include antibodies or antihistamie fragments (e.g., dAb) of an antibody, polypeptide agonists, activators, promoters secretion, antagonists or inhibitors. For example, polypeptide or peptide drug can bind and agonizing or anlagenservice protein on the cell surface, such as a CD antigen, cytokine receptor (e.g., interleukines receptor, chemokinesis receptor), adhesion molecule, or co-stimulatory molecule. For example, the polypeptide drug can bind cytokines, growth factors, cytokine receptor, growth factor receptor and other ligands to the target, which include, but are not limited to: APOE, Apo-SAA, BDNF, cardiotrophin-1 (Cardiotrophin-1), CEA, CD40, CD40 ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78, Eotaxin (Eotaxin), Eotaxin-2, Exodus-2 (Exodus-2), FAPα, FGF-acidic, FGF-basic, fibroblast factor growth-10, FLT3 ligand, Fractalkine (Fractalkine) (CX3C), GDNF, G-CSF, GM-CSF, GF-β1, human serum al the IOR techniques, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, the receptor for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 amino acids), IL-8 (77 amino acids), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin α (Inhibin α), Inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin (Lymphotactin), Mullerian (Mullerian(inhibitory substance, monotany kolonialiserade factor monotany protein attractant, M-CSF, MDC (67 amino acids), MDC (69 amino acids), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67 amino acids), MDC (69 amino acids), MIG, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, inhibitory factor-1 myeloid progenitor (MPIF-1), NAP-2, Neurturin, nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M (Oncostatin M), PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, factor stem cells (SCF), TARC, TGF-α, TGF-β, TGF-R2, TGF-β3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1, TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4. It is clear that this list is by no means exhaustive.

Suitable drugs include hormones, including pituitary hormone (PTH), adrenocorticotropic hormone (ACTH), renin, luteinizing hormone-releasing factor (LHRH)analogues, gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), aldosterone, and the like. Suitable drugs include keratinocyte growth factor,interferons (e.g., INF-α, INF-β, INF-γ), erythropoietin (EPO), protease, elastase, analogues, agonists and LHRH antagonists, agonists, opioid receptor, such as agonists of the Kappa opioid receptor (for example, dynorphin A), calcitonin and analogs of calcitonin, antidiuretic hormone (vasopressin), antagonists of oxytocin, vasoactive intestinal peptide inhibitors of thrombin, factor von Willebrand's, surfactants and poison snails (e.g., ziconotide).

Suitable drugs include peptides and polypeptides that have antitumor activity (e.g., inhibition of proliferation, inhibition of growth, induction of apoptosis, inhibition of metastasis, inhibition of adhesion, inhibition of revascularization). Several such peptides and polypeptides are known in this field. (See, for example, Janin Y.L., Amino Acids, 25:1-40 (2003). The content of this link, especially peptides and polypeptides described in this document is included in its entirety in the present description by reference.) Amino acid sequences of several of these peptides are presented in Table 8.

Other suitable drugs include peptides and polypeptides that have antiviral activity. Several such peptides and polypeptides are known in this field, for example, peptides and polypeptides disclosed in Gianecchini, et al., J Viro., 77(6):3724-33 (2003); Wang, J., et al., Clin Chem (2003); Hilleman, M.R, Vaccine, 21(32):4626-49 (2003); Tziveleka, LA, et al., Curr Top Med Chem, 3(13):1512-35 (2003); Poritz, M.A., et al., Virology, 313(1):170-83 (2003); Oevermann, A., et al., Antiviral Res, 59(7):23-33 (2003); Cole, A.M. etal.. Curr Pharm Des 9(18):1463-73 (2003); Pinon, J.D., et al., Virol, 77(5):3281-90 (2003); Sia, S.K., et al., Proc Natl Acad Sci USA, 99(23):14664-9 (2002); Bahbouhi, C., etal.. Biochem J, 66(Pt 3):863-72 (2002); de Soultrait, V.R., et al., J Mol Biol, 18(1):45-58 (2002); Witherell, G., Curr Opin Investig Drugs, 2(3):340-7 (2001); Ruff, M.R., et al., Antiviral Res, 52(1):63-75 (2001); Bultmann, H., et al., J. Virol, 75(6):2634-45 (2001); Egal, M., et al., Int J Antimicrob Agents, 13(1):57-60 (1999); and Robinson, W.E., Jr., J Leukoc Biol, 63(1):94-100 (1998). The contents of these links, especially peptides and polypeptides described in these documents is included in its entirety in the present description by reference. These peptides and polypeptides are examples of medicines that can be used in the compositions, the slit structures medicines, drug conjugates, noncovalent the drug conjugates of the present invention.

Polypeptide drug may also be a cytokine, or growth factor or soluble portion of the receptor (e.g., cytokine receptor, growth factor receptor, a hormone receptor), or another polypeptide, such as the polypeptides listed above. For example, a suitable polypeptide drugs also include agonists and antagonists of the receptor (for example, is eceptor growth factor, receptor of the cytokine, hormone receptor), such as receptor antagonist interleukin 1 (Eisenberg et el., Nature 343:341-346 (1990)), agonists of the receptor thrombopoetin (for example, GW395058 (de Serres et al., Stem Cells 17:316-326 (1999)), receptor antagonists melanocortin (e.g., MCR-4 antagonists (Cepoi et al., Brain Res. 1000:64-71 (2004)), engines, 6DBF7 (Mayo et al., J. Biol. Chem. 278:45746-45752 (2003)), the mimetics of the chemokine (e.g., RANTES mimetics (Nardese et al., Nat. Struct. Biol. 8:611-615 (2001)), growth hormone (e.g. human growth hormone), analogs of growth hormone and stimulators of GH secretion (for example, CP-424391 (MacAndrew et al., Eur. J. Pharmacol. 432:195-202 (2001)), the mimetics of growth hormone releasing hormone (for example, MK-677 (Chapman et al., J. Clin. Endocrinol. Metab. 82:3455-3463 (1997)), inhibitors of the interaction of cellular adhesion molecules (e.g., LFA-1/ICAM-1, VLA-1/VCAM-1 (Yusuf-Makagiansar et al., Med. Res. Rev. 22:146-167 (2002)), the mimetics of interferon (e.g., SYR6 (Sato et al., Biochem. J. 371(Pt.2):603-608 (2003), the mimetics of Herceptin (Nature Biotechnol. 18:137 (2000)), inhibitors of antigen presentation (Bolin et al., J. Med. Chem. 43:2135:2148 (2000)), GPIIB/IIIA antagonists (e.g., FK633 (Aoki et al., Thromb. Res. 81:439-450 (1996)), alphavbeta3 antagonists (for example, S (Engleman et al., J. Clin. Invest. 99:2284-2292 (1997)), the mimetics of erythropoietin (for example, AMR (Johnson etal., Biochemistry 37:3699-3710 (1998)), antagonists of the opioid receptor (for example, [(2S, 3R)-TMT1]DPDPE (Liao et al., J. Med. Chem. 41:4767-4776 (1998)), blood-forming (hematopoietic) factors (e.g. erythropoietin (EPO), granularity coloniali uliuli factor (GM-CSF)).

Additional suitable peptide and polypeptide drugs include peptide antagonists that bind the human receptor IL-1 of the first type (for example, AF 11377 (FEWTPGYWQPYALPL, SEQ ID NO:56), AF11869 (FEWTPGYWQJYALPL, SEQ ID NO:57 (J=1 azetidin-2-carboxylic acid), FEWTPGYWQJY (SEQ ID NO:58), FEWTPGWYQJY (SEQ ID NO:59), FEWTPGWYQJYALPL (SEQ ID NO:60) or any of the aforesaid sequences possibly containing acylated aminocore and/or aminirovanie carboxyl end (Akeson et al., J. Biol. Chem. 271:30517-305123 (1996)), peptide antagonists of TNF-alpha-mediated cytotoxicity (for example, those described in Chirinos-Rojas et al, J. Immunol. 161:5621-5626 (1998)), peptide agonists of the erythropoietin receptor (for example, those described in McConnel et al., Biol. Chem. 379:1279-1286 (1998) or Wrighton et al., Science 273:458-464 (1996)), glucagon-like peptide-1 (GLP-1, for example, GLP-1(7-37), GLP-1(7-36)amide and analogues (see, for example, Ritzel U. et al., J. Endocrinology 159:93-102 (1998)) and interferons (e.g., INF-α, INF-β, INF-λ). Additional suitable polypeptide and peptide drugs include inhibitors of integrin (e.g., RGD peptides, such as H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2(Janssen, M.L, et al., Cancer Research 62:6146-6151 (2002)), cyclo(Arg-Gly-Asp-D-Phe-Lys) (Kantlehner, M., et al., Agnew. Chem. Int. Ed. 38:560 (1999)), cyclo(Arg-Gly-Asp-D-Tyr-Lys) (Haubner, R., et al., J. Nucl. Med. 42:326-336 (2001)), proteins, inactivating the ribosome (RIPs)such as Saporin (for example, SEQ ID NO:67), inhibitors of matrix metalloproteinases (n is an example, U.S. patent No. 5616605), and antiviral peptides and polypeptides, such as a fusion inhibitors of HIV (for example, T-1249 and T-20 (FUZEON® (enfuvirtide); Trimeris Inc.), and soluble receptor antagonists, such as immunoadhesin (for example, LFA3-Ig, CTLA4-Ig).

Antimicrobial polypeptide and peptide drugs are also suitable for use according to the invention. Examples of suitable antimicrobial polypeptide and peptide drugs include adenomegaly, dermcidin-1 L, cathelicidin (for example, cathelicidin-like peptide, human LL-37/hCAP-18), defensin, including α-defensin (for example, human neutrophil peptide 1 (HNP-1), HNP-2, HNP-3, HNP-4, human defensin 5, human defensin 6), β-defensin (for example, human β-defensin-1, human θ-defensin-2) and θ-defensin (for example, the θ-defensin-1), histamine (for example, histatin 1, histatin 3, histatin 5), peptide - derived lactoferricin and related peptides (see, Tomita M., et al., Acta Paediatr. Jpn. 36:585-591 (1994) and Strom, M.S., et al. Biochem Cell il. 80:65-74 (2002)).

In a preferred variant embodiment of the invention medicines are insulinotropic medicines. Examples of suitable insulinotropic medicines include GLP-1, a derivative of GLP-1 analogues, GLP-1 or a derivative of an analog of GLP-1. Additionally they include the Basis with 4 equivalents on the Basis of 4 and proizvodnye the Basis 4, and the Basis-3, derived on the Basis of 3 and analogues on the Basis 3.

Other suitable drugs include Peptide YY (3-36) or equivalents. Peptide YY (PYY) is a peptide amide of the 36 residues, highlighted for the first time from porcine intestine and localized in endocrinolo the gastrointestinal tract and pancreas (Tatemoto et al. Proc. Natl. Acad. Sci. 79:2514, 1982). Peptide YY is the N-terminal and C-terminal the tyrosine amide; accordingly, these two tyrosine give PYY his name (Y represents the amino acid tyrosine in the peptide nomenclature). In addition, PYY divides the number of Central and peripheral regulatory functions with its homologous to the peptide neuropeptide Y (NPY), which was first isolated from pig brain (Tatemoto, Proc. Natl. Acad. Sci. 79:5485, 1982). In contrast to the cellular location of PYY, NPY is located in submucosal neurons and the neurons that are related to the muscular coat of the intestine, which innerviews mucous membranes and smooth muscle layers, respectively (Ekblad et al. Neuroscience 20:169, 1987). I believe that PYY and NPY inhibit intestinal motility and blood flow (Laburthe, Trends Endocrinol. Metab. 1:168, 1990), and believe that they also weaken the basal (SOH et al. Br. J. Pharmacol. 101:247, 1990) and induced by stimulators of secretion of intestinal secretion in rats (Lundberg et al. Proc. Natl. Acad. Sci. USA 79:4471, 1982), as well as stimulate the absorption in the intestine (MacFadyen et al. Neuropeptide 7:219, 1986). Together, these observations suggest that PYY and NPY are released into the bloodstream after eating a meal (Adrian et al. Gastroenterology 89:1070, 1985; Balasubramaniam et al. Neuropeptides 14:209, 1989) and, thus, play a physiological role in the regulation of intestinal secretion and absorption.

In the intestinal epithelium of rats was characterized PYY receptor high affinity, which shows a slightly higher affinity to PYY, rather than to NPY (Laburthe et al. Endocrinology 118:1910, 1986) and, as shown, is negatively associated with adenylate-cyclase (Servin et al. Endocrinology 124:692, 1989). Study of the relationship structure-activity using multiple subsequences (partial sequences) resulted in the identification of PYY(22-36) as the active site to interact with intestinal PYY receptors (Balsubramaniam et al. Pept. Res. 1:32, 1988).

Additionally, PYY has been involved in a number of physiological activities, including the uptake of nutrients (Bilcheik et al. Digestive Disease Week 506:623, 1993), cell proliferation (Laburthe, Trends Endocrinol. Metab. 1:168, 1990; Voisin et al. J. Biol. Chem, 1993), lipolysis (breakdown of fats) (Valet et al., J. Clin. Invest. 291, 1990) and narrowing of blood vessels (Lundberg et al., Proc. Natl. Acad. Sci., USA 79: 4471, 1982).

In WO 03/057235 and WO 03/026591 described a method of reducing calorie intake, food intake and decrease appetite by introducing a PYY or agonist and GLP-1. These publications included in this document in all the fullness by reference, especially to bring examples of medicines on the basis of PYY and GLP-1 and methods that can be applied according to the present invention.

More other medicines that are suitable for use in the invention include insulin, Resistin (Resistin), Leptin (Leptin), antagonist MC3R/MC4R, antagonist AgRP, Apolipoprotein A-IV, Enterostatin, Gastrin-Releasing Peptide (GRP), IGF1, BMP-9, IL-22, RegIV, interferon alpha, INGAP peptide, somatostatin, Amylin, nerolin, interferon beta, the hybrid interferon, adiponectin, endocannabinoids, peptide, WNT10b, Orexin (Orexin-A), adrenocorticotropin, Enterostatin (Enterostatin), Cholecystokinin (Cholecystokinin), oxyntomodulin, melanocytestimulating hormone, melanocortin, melaninconcentrating hormone, BB-2, NPY Y2 agonists, NPY Y5/Y1 antagonists, OHM, Gal-1 R antagonists, MCH-1R antagonists, MS-3/4 agonists, BRS-3 agonists, pancreatic polypeptide, fragment antibodies against ghrelin, derived neurotrophic factor on the basis of the brain, human growth hormone, parathyroid hormone, follicle stimulating hormone, gastric inhibitory peptide or its equivalent.

Fused design drugs

Fused design of a medicinal product according to the invention are fused proteins, which contain a continuous polypeptide chain, and the chain contains antigennegative fragment and is tetela, which binds serum albumin as the first grouping associated with the second group, which is a polypeptide drug. The first and second groups can be directly connected to each other by peptide bond or bonded by means of a suitable amino acid, or a peptide or polypeptide linker. Additional groups (e.g., third, fourth and/or linker sequences may be present if necessary. The first group may be N-terminal position, the C-terminal position or in the second group (i.e. polypeptide drug). In specific embodiments, embodiments, each group may be present in more than one copy. For example, the slit structure of the medicinal product may contain two or more first groups, each of which contains antigennegative fragment of the antibody that binds serum albumin (e.g., VHthat binds human serum albumin, and VLthat binds human serum albumin, or two or more VHs or VLs that bind human serum albumin).

In some embodiments embodiment, the slit design medicines submit yet a continuous polypeptide chain, which has the formula:

a-(X)m-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n1-d

where X is a polypeptide drug that has binding specificity with respect to the first target;

Y represents a single-stranded antigennegative fragment of the antibody that has binding specificity in relation to serum albumin;

Z is a polypeptide drug that has binding specificity for a second target;

Each of a, b, C and d independently is absent or has from one to about 100 amino acid residues;

n1 is from one to about 10;

n2 is from one to about 10; and

n3 equals from zero to about 10,

provided that, if both n1 and n2 are equal to one, and n3 is zero, X does not contain a chain of the antibody or fragment chain antibodies.

In one variant embodiment, neither X nor Z does not contain a chain of the antibody or fragment chain antibodies. In one variant embodiment, n1 is equal to one, n3 and n2 is equal to one equal to two, three, four, five, six, seven, eight or nine. Preferably, Y is a variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or variable domain of the light chain of immunoglobulin (V )that has binding specificity in relation to serum albumin. More preferably, Y represents a dAb (for example, VHVKor Vλ), which binds human serum albumin. In a particular variant embodiment, X or Z is a human GLP-1 or a derivative of GLP-1 or the equivalent.

In specific embodiments of the incarnation, Y contains an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In other embodiments, embodiments, Y contains an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

In other variants of the embodiment, the slit structure of the medicinal product contains groups X' and Y', where X' is a polypeptide drug, provided that X' does not contain a chain of the antibody or fragment chain antibodies, and Y' represents a single-stranded antigennegative fragment of the antibody that has binding specificity in relation to serum albumin. Preferably, Y' is a variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or a variable domain of leglize immunoglobulin (V L)that has binding specificity in relation to serum albumin. More preferably, Y' is a dAb (for example, VHVKor Vλ), which binds human serum albumin. X' may be located on aminocore with respect to Y', or Y' can be located on aminocore with respect to X'. In some embodiments, embodiment, X' and Y' are separated by amino acid or a peptide or polypeptide linker, which contains from two to about 100 amino acids. In a particular variant embodiment, X' is a human GLP-1 or a derivative of GLP-1 or their equivalents.

In specific embodiments, embodiments, Y' contains amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In other embodiments, embodiments, Y' contains amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

In specific embodiments embodiment, the slit structure of the medicinal product contains a dAb that binds serum albumin and human IL-1ra (e.g., SEQ ID NO:28). Preferably, the dAb binds human serum albumin and contains human framework region.

In other embodiment variants, fused to the construction of a drug or conjugate of the medicinal product contains a functional variant of human IL-1ra, which has at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with the Mature form of human IL-1ra, consisting of 152 amino acids, and is an antagonist of the receptor type 1 human interleukin-1. (See, Eisenberg et al, Nature 343:341-346 (1990).) A variant can contain one or more additional amino acids (e.g., to contain 153 or 154 or more amino acids). Fused design of a medicinal product according to the invention can be obtained by any appropriate means. For example, some variants of the embodiment can be obtained by inserting a nucleic acid that encodes a fused design medicines in suitable expressing vector. The resulting design is then introduced into a suitable cell host for expression. After the expression of protein you can select or clear from the cell lysate or, preferably, from culture medium or periplasm using any suitable methods. (See, for example, Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Vol.2, Suppl. 26, pp.16.4.1-16.7.8 (1991)).

In an additional variant embodiment, the slit constructioncrossing funds or conjugate of the medicinal product contains insulinotropic agents preferred variant of embodiment, fused design drug or conjugate of the medicinal product contains a GLP-1 or an analogue or peptide GLP-1. In a more preferred variant of embodiment, the slit design drug or conjugate of the medicinal product contains Ser8GLP-1 (7-36) amide.

In an additional variant embodiment, the slit design drugs or drug conjugate contains an analogue of GLP-1 having one or more of the following substitutions: Val8or Pro9.

Preferably, the analogue of GLP-1 is a Pro9GLP-1(7-36) or Pro9GLP-1(7-37). Additionally, the analog or peptide GLP-1 can include any of the following C-terminal extensions: PSS, PSSGAP or PSSGAPPPS.

In another variant embodiment, the slit design drugs or drug conjugate contains an analogue of GLP-1 containing a sequence of the Formula I:

His7-Xaa8-Xaa9-Glyl0-Xaall-Phel2-Thrl3-Xaal4-Aspl5-Xaal6-Xaal7-Xaal8-Xaal9-Xaa20-Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Phe28-Ile29-Xaa30-Xaa31-Xaa32-Xaa33-XAA34-XAA35-XAA36-XAA37-XAA38-XAA39-XAA40-XAA41-XAA42-XAA43-XAA44-XAA45

Formula Is I - SEQ ID NO:172,

where

XAA in position 8 is an Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;

XAA in position 9 represents Glu or Asp;

XAA in position 11 is a Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;

XAA in position 14 is a Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;

XAA at position 16 is a Val, Ala, Gly, Ser, Thr, Leu, Ile, Tight, Glu, Asp, Trp or Lys;

XAA in position 17 represents Ser, Ala, Gly, Thr, Leu, Ile, Val,

Glu, Asp, or Lys; XAA at position 18 is a Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr or Lys;

XAA at position 19 is a Tyr, Phe, Trp, Glu, Asp, Gin or Lys;

XAA in position 20 is a Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp, Tyr or Lys;

XAA at position 21 is a Glu, Asp, or Lys;

XAA in position 22 is a Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;

XAA at position 23 is Gln, Asn, Arg, Glu, Asp, or Lys;

XAA at position 24 is an Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, or Lys;

XAA at position 25 is an Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;

XAA at position 26 represents Lys, Arg, Gin, Glu, Asp, or His;

XAA at position 27 is Leu, Glu, Asp, or Lys;

XAA at position 30 is an Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys;

XAA at position 31 is a Trp, Phe, Tyr, Glu, Asp, Lys;

XAA at position 32 is a Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;

Haa the position 33 is a Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;

XAA at position 34 represents Asn, Lys, Arg, Glu, Asp, or His;

XAA at position 35 is a Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;

XAA at position 36 is a Gly, Arg, Lys, Glu, Asp, or His;

XAA at position 37 is a Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys, or is deleted;

XAA at position 38 is a Ser, Arg, Lys, Glu, Asp, or His, or is deleted;

XAA at position 39 is a Ser, Arg, Lys, Glu, Asp, or His, or is deleted;

XAA at position 40 is a Gly, Asp, Glu or Lys, or is deleted;

XAA at position 41 is an Ala, Phe, Trp, Tight, Glu, Asp, or Lys, or is deleted;

XAA at position 42 is a Ser, Pro, Lys, Glu or Asp or is deleted;

XAA at position 43 is a Ser, Pro, Glu, Asp, or Lys, or is deleted;

XAA at position 44 is a Gly, Pro, Glu, Asp, or Lys, or is deleted;

and XAA at position 45 is an Ala, Ser, Val, Glu, Asp, or Lys, or is deleted, provided that when the amino acid in position 37, 38, 39, 40, 41, 42, 43 or 44 is deleted, then each amino acid after the amino acid is also deleted.

In another variant embodiment, the slit design drugs or drug conjugate contains an analogue of GLP-1, which contains the amino acid sequence of the Formula (II):

Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaal6-Ser-Xaal8-Xaal9-Xaa20 -Glu-Xaa22-Xaa23-Ala-Xaa25-Xaa26-Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-XAA34-XAA35-XAA36-XAA37-XAA38-XAA39-XAA40-XAA41-XAA42-XAA43-XAA44-XAA45-XAA46

Formula (II) SEQ ID NO:173,

where

Haa is an L-histidine, D-histidine, deteministic, 2-amino - histidine, β-hydroxy-histidine, homocysteine, Nα-acetyl-histidine, α-ferometer-histidine, α-methyl-histidine, 3-pyridylamine, 2-pyridylamine or 4-pyridylamine;

Haa represents Ala, Gly, Val, Leu, lie, Lys, Aib, (1-aminocyclopropane)carboxylic acid, (1-aminocinnamate)carboxylic acid, (1-aminocyclopent)carboxylic acid, (1-aminocyclohexane)carboxylic acid, (1-aminocyclopent)carboxylic acid, or (1-aminocyclopent)carboxylic acid;

XAA16represents Val or Leu;

XAA18represents Ser, Lys or Arg;

XAA19is a Tight or Gln;

XAA20represents Leu or Met;

XAA22represents Gly, Glu or Aib;

XAA23represents Gln, Glu, Lys or Arg;

XAA25represents Ala or Val;

XAA26represents Lys, Glu or Arg;

XAA27represents Glu or Leu;

XAA30represents Ala, Glu or Arg;

XAA33represents Val or Lys;

XAA 34represents Lys, Glu, Asn or Arg;

XAA35represents Gly or Aib;

XAA36represents Arg, Gly or Lys;

XAA37represents Gly, Ala, Glu, Pro, Lys, amide or is absent;

XAA38represents Lys, Ser, amide or is absent;

XAA39represents Ser, Lys, amide or is absent;

XAA40represents Gly, amide or is absent;

XAA41represents Ala, amide or is absent;

XAA42is a Pro, amide or is absent;

XAA43is a Pro, amide or is absent;

XAA44is a Pro, amide or is absent;

XAA45represents Ser, amide or is absent;

XAA46is an amide or is absent, provided that if XAA38, XAA39, XAA40, XAA41, XAA42, XAA43, XAA44, XAA45or XAA46is missing, then each subsequent amino acid residue is also missing.

In another variant embodiment of the invention, the slit design drug or conjugate of the medicinal product contains a peptide GLP-1 containing the amino acid sequence of the Formula (III):

Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Glu-Phe-lle-Xaa30-Tip-Leu-Val-Xaa34Ho 35-Xaa36-Xaa37-Xaa38

Formula (III) SEQ ID NO:174,

where

XAA7is an L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homocysteine, N'1-acetyl-histidine, α-ferometer-histidine, α-methyl-histidine, 3-pyridylamine, 2-pyridylamine or 4-pyridylamine;

XAA8represents Ala, Gly, Val, Leu, Ile, Lys, α-aminoadamantane acid (Aib), (1-aminocyclopropane)carboxylic acid, (1-aminocinnamate)carboxylic acid, (1-aminocyclopent)carboxylic acid, (1-aminocyclohexane)carboxylic acid, (1-aminocyclopent)carboxylic acid, or (1-aminocyclopent)carboxylic acid;

XAA18represents Ser, Lys or Arg;

XAA22represents Gly, Glu or Aib;

XAA23represents Gln, Glu, Lys or Arg;

XAA26represents Lys, Glu or Arg;

XAA30represents Ala, Glu or Arg;

XAA34represents Lys, Glu or Arg;

XAA35represents Gly or Aib;

XAA36represents Arg or Lys;

XAA37represents Gly, Ala, Glu or Lys;

XAA38represents Lys, amide or is absent.

In another embodiment, additional embodiments of the invention, the peptide GLP-1 are selected from the group consisting of: GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-36)-amide, GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40), GLP-1 (7-41)or its analogue is whether peptide.

In another variant embodiment of the invention, the peptide GLP-1 is GLP-1 (a-b), where a is an integer from 1 to 7, and is an integer from 37 to 45, or equivalent, containing one albuminaemia residue attached via a hydrophilic spacer to the C-terminal amino acid residue, and possibly the second albuminaemia residue attached to one of the other amino acid residues.

In another variant embodiment, the peptide GLP-1 contains no more than fifteen amino acid residues which have been substituted, added or deleted as compared to GLP-1 (7-37), or no more than ten amino acid residues which have been substituted, added or deleted as compared to GLP-1 (7-37).

In another variant embodiment, the peptide GLP-1 contains no more than six, preferably not more than 5, 4, 3, 2, or 1) amino acid residues which have been substituted, added or deleted as compared to GLP-1 (7-37).

In another variant embodiment, the peptide GLP-1 contains not more than 4 (preferably, not more than 3, 2 or 1) amino acid residues which are not encoded by the genetic code.

In another variant embodiment, the peptide GLP-1 is a DPPIV protected peptide GLP-1.

In another variant embodiment, insulinotropic agent stabilized with the help of DPPIV.

In another variant embodiment, the PE the San GLP-1 contains the remainder of the a-aminoadamantane acid (Aib) in position 8.

In another variant embodiment, the amino acid residue in position 7 of the peptide GLP-1 is selected from the group consisting of D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homocysteine, Nα-acetyl-histidine, α-vermeil-histidine, α-methyl-histidine, 3-pyridylamino, 2-pyridylamine and 4-pyridylamine.

In another variant embodiment, the peptide GLP-1 is selected from the group consisting of:

Arg26,34Lys36GLP-1(7-37), Gly8Arg26,34Lys36GLP-1(7-37), Aib8,35Lys37GLP-1(7-37)-OH, Ala8Arg26,34Lys38GLP-1(7-38), Aib8,22,35Lys38GLP-1(7-38), Aib8Arg26,34Lys36GLP-1(7-36), Gly8Arg26,34Lys36GLP-1(7-37)-OH,

Aib8,22,35Lys37GLP-1(7-37)-NH2, Aib8Arg34GLP-1(7-37), Gly8Arg26,34Lys38GLP-1(7-38), Arg34GLP-1(7-37)-OH, Gly8Glu22,23,30Arg18,26,34Lys38GLP-1(7-38),

imidazolidinedione acid7Asp18Aibto 22.35Lys38GLP-1(7-38), imidazolidinone acid7Aibto 22.35Lys38GLP-1(7-38}, [3-(5-imidazolyl)propionyl]7Aib8Arg26,34Lys38GLP-1(7-38) and Aibby 8.22Lys37GLP-1(7-38).

In another variant embodiment, the peptide GLP-1 is attached to the hydrophilic spacer through amino acid residue at position 23, 26, 34, 36 or 38 of native GLP-1 analogue GLP-1.

In another variant embodiment, insole atrophy agent represents Lys 20The basis-4(1-39)-NH2.

In another variant embodiment, the peptide GLP-1 is a

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-

amide-SEQ ID NO:175.

In another variant embodiment, the peptide GLP-1 is a

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX|

-SEQ ID NO:176,

where X=P or Y or a fragment or analogue.

In another variant embodiment of the invention, the peptide GLP-1 represents Arg18, Leu20, Gln34, Lys33(Nε-(γ-aminobutyryl(Nα-hexadecanoyl))) on the Basis of 4-(7-45)-amide or Arg33, Leu20, Gin34, Lys18(Nα-(γ-aminobutyric(Nα-hexadecanoyl))) on the Basis of 4-(7-45)-amide.

Examples insulinotropic of agentov, which can be useful as analogues of GLP-1 or derivatives of GLP-1 or GLP-1-like medicines according to the present invention, is described in international patent application no WO 87/06941 (The General Hospital Corporation), which refers to a peptide fragment that contains GLP-1 (7-37) and its functional derivatives, and also to its use as insulinotropic agent (incorporated herein by reference, especially in the examples of drugs for use in the present invention).

Additional analogs of GLP-1 is described in international patent application No. 90/11296 (The General Hospital Corporation), which relates to peptide fragments that contain GLP-1 (7-36) and its functional PR is spodnie and have insulinotropic activity which exceeds insulinotropic activity of GLP-1 (7-36) or GLP-1 (7-37), and to their use as insulinotropic agents (incorporated herein by reference, especially in the examples of drugs for use in the present invention).

In the International Patent Application no WO 91/11457 (Buckley et al.) disclosed analogs of active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37, which can also be useful as GLP-1 drug according to the present invention (incorporated herein by reference, especially in the examples of drugs for use in the present invention).

Additional Basis analogues that are useful for the present invention are described in the PCT patent applications WO 99/25728 (Beeley et al.), WO 99/25727 (Beeley et al.), WO 98/05351 (Young et al.), WO 99/40788 (Young et al.), WO 99/07404 (Beeley et al.) and WO 99/43708 (Knudsen et al.) (all incorporated herein by reference, especially in the examples of drugs for use in the present invention).

Suitable expressing vectors can contain a number of components, such as a port Replicator gene breeding marker, one or more elements regulating expression, such as an element of the regulation of transcription (e.g., promoter, enhancer, terminator) and/or one or more broadcast signal, the signal sequence is alnost or leader sequence, and the like. The elements that regulate the expression of, and a signal sequence, if present, can be provided by the vector, or some other source. For example, transcriptional and/or translational control sequence of the cloned nucleic acid that encodes the chain antibodies, can be used to direct the expression.

You can provide a promoter for expression in a desired cell host. Promoters can be constitutive or inducible. For example, the promoter may be functionally linked to the nucleic acid encoding the antibody chain antibody or a part of it, so he directs transcription of the nucleic acid. There are many suitable promoters for prokaryotic (e.g., lac, tac, T3, T7 promoters for E.li) and eukaryotic (e.g., early or late promoter of simian virus 40 promoter of the long terminal repeat of Rous sarcoma virus, cytomegalovirus promoter, adenovirus late promoter) of the hosts.

In addition, expressing vectors usually contain a breeding marker for selection of host cells carrying the vector, and, in the case of the playback expressing vector Replicator. Genes encoding products which give resistance to antibiotics or drug, are conventional breeding markers and which may be used in prokaryotic (e.g., gene lactamase (resistance to ampicillin), Tet gene for resistance to tetracycline) and eukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycofenolate acid), genes for resistance to ampicillin or hygromycin). Digidrofolatreduktaza marker genes allow the selection with the use of methotrexate in different hosts. The genes encoding the genetic product of auxotrophic markers of the host (e.g., LEU2, URA3, HIS3), often used as breeding markers in yeast. Also provided by the use of virus (e.g. baculovirus) or phage vectors and vectors that integrate into the genome of the host cell, such as retroviral vectors. Suitable expressing vectors for expression in mammalian cells and prokaryotic cells (E. Li), insect cells (Drosophila Schnieder S2 cells, Sf9) and yeast (P. methanolica, P. pastoris, S. cerevisiae) are well known in this field.

The proposed recombinant cell host, which Express a fused design of the medicinal product, and method for producing the slit structure of the medicinal product, as described herein. Recombinant a host cell contains a recombinant nucleic acid encoding a fused design drugs. Fused design of medicinal substances which can be obtained by expression of a recombinant nucleic acid, encodes a protein in a suitable cell host, or using other suitable methods. For example, expressing the constructs described herein can be implemented in a suitable cell host, and the resulting cell can be maintained (e.g., in culture, in the animal) under conditions suitable for expression of the constructs. Appropriate cell hosts can be prokaryotic, including bacterial cells such as .li, .subtilis and/or other suitable bacteria, eukaryotic, such as cells of fungi or yeast (e.g., Pichia pastoris, Aspergillus species, Sacchammyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or other lower eukaryotic cells, and cells of higher eucaryotes such as those from insects (e.g., insect cells Sf9 (WO 94/26087 (O'connor)or animals (e.g., COS cells, such as COS-1 (ATCC (American model collection cultures) Registration # CRL-1650) and COS-7 (ATCC Registration # CRL-1651), Cho (e.g., ATCC Registration # CRL-9096), 293 (ATCC Registration # CRL-1573), HeLa (ATCC Registration # CCL-2), CV1 (ATCC Registration # CCL-70), WOP (Dailey et al., J. Virol. 54:739-749 (1985)), ZTZ, T (Pear et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)), NSO cells, SP2/0, HuT 78 cells, and the like (see, for example, Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1993)).

The invention also VK is uchet way to obtain the merged design of the medicinal product, comprising maintaining a recombinant host cell of the invention under conditions suitable for the expression of the slit structure of the drug. The method may additionally include the extraction or recovery of the merged design of the medicinal product, if desired. In another variant embodiment, the components are merged designs medicines (e.g., dAb that binds human serum albumin and IL-1ra) chemically combine to create a continuous polypeptide chain.

Conjugates

In another aspect, the invention proposed conjugates containing antigennegative fragment of the antibody that binds serum albumin, which is associated with the drug. Such conjugates include conjugates medicines, which contain antigennegative fragment of the antibody that binds serum albumin, to which the drug is covalently linked, and noncovalent drug conjugates, which contain antigennegative fragment of the antibody that binds serum albumin, with which the medicinal product is due ecovalence. Preferably, the conjugates are stable enough, so antigennegative fragment of the antibody that binds serum albumin and drugs the TES tool remain significantly associated (covalently or ecovalence) with each other in vivo (for example, with the introduction of man). Preferably, not more than about 20%, not more than about 15%, not more than about 10%, not more than about 9%, not more than about 8%, not more than about 7%, not more than about 6%, not more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, not more than about 1%, or substantially none of the conjugates do not dissociate or not destroyed to release drug and antigennegative fragment in vivo. For example, the stability conditions in vivo can be conveniently evaluated by incubating the conjugate of a drug or non-covalent conjugate of the drug in 24 hours in serum (e.g., human serum) at 37°C. one example of such a method, equal amounts of the conjugate product and does not conjugated drugs are placed in two different bottle of serum. Half the contents of each vial immediately frozen at -20°C, and the other half is incubated for 24 hours at 37°C. Then all four of the sample can be analyzed by any suitable method such as SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) and/or Western blotting. Western blots can be treated using an antibody which binds carstone tool. All drug strips conjugate medicines will move in accordance with the size of the conjugate of the medicinal product, if not happened dissociation. To assess stability in the conditions of "in vivo" you can use many other suitable methods, for example, analyzing the samples, prepared as described above, appropriate analytical methods, such as chromatography (e.g., gel filtration, ion exchange, reversed-phase chromatography, ELISA, mass spectrometry, and the like.

Conjugates of the medicinal product

In another aspect, the invention proposed conjugate medicines containing antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, and the drug that is covalently associated with the specified antigennegative fragment, provided that the conjugate of a medicinal product is not a single continuous polypeptide chain.

In some embodiments embodiment, the conjugate of the medicinal product contains a variable domain of the heavy chain of immunoglobulin (VH)that has binding specificity in relation to serum albumin, or variable domain of the light chain of immunoglobulin (VL)that has binding specificity from which Oseni serum albumin, and the drug that is covalently associated with the specified VHor VLprovided that the conjugate of a medicinal product is not a single continuous polypeptide chain. Preferably, the conjugate of the medicinal product contains a single VHthat binds serum albumin, or a single VLthat binds serum albumin. In specific embodiments embodiment, the conjugate of the medicinal product contains VKdAb that binds human serum albumin and contains an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In other variants of the embodiment, the conjugate of the medicinal product contains VHdAb that binds human serum albumin and contains an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.

Conjugates of the medicinal product may contain any desired drug and can be obtained by any appropriate means. For example, the drug can be associated with antigennegative fragment of an antibody that binds serum albumin, directly or indirectly through a suitable linker g is upperhouse in one or more positions, such as aminocore, carboxylic, or through amino acid side chains. In one variant embodiment, the conjugate of the medicinal product contains a dAb that binds human serum albumin and the polypeptide drug (e.g., human IL-1ra or a functional variant of human IL-1ra), and aminocore polypeptide drugs (e.g., human IL-1ra or a functional variant of human IL-1ra) is associated with carbonsilicon dAb directly or through a suitable linker group. In another variant embodiment, the conjugate of the medicinal product contains a dAb that binds human serum albumin, and insulinotropic drug (e.g., GLP-1 or an analogue of GLP-1), and aminocore insulinotropic medicinal product is free (i.e. not connected or not connected in conjugate), and carboxylic associated with aminocom.com dAb directly or through a suitable linker group. In other variants of the embodiment, the conjugate of the medicinal product contains a dAb that binds human serum albumin, and two or more different medicines that are covalently linked to dAb. For example, the first drug may be covalently linked (directly or indirectly) from the carboxyl end of the dAb, and the second Lek is rstone tool can be covalently linked (directly or indirectly) with aminocom.com or through the amino group of the side chain (for example, E. the amino group of lysine). In a preferred variant embodiment, aminocore insulinotropic medicines (for example, GLP-1 analogue GLP-1) is free. Such conjugates medicines can be obtained using well-known methods of selective binding. (See, for example, Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).)

You can use a variety of methods of conjugation of drugs with antigennegative fragment of an antibody that has binding specificity in relation to serum albumin. The specific method will depend on the medicinal product, which is subject to conjugation. If desired, linkers that contain terminal functional groups can be used to associate antigennegative fragment and medicines. Typically, the conjugation is carried out in the course of the reaction between a medicinal product which contains the reactive functional group (or modified to contain a reactive functional group)with the linker or directly with antigennegative fragment of an antibody that binds serum albumin. Covalent bonds are formed during a reaction between a medicinal product which contains, or is modified, that is to contain) a chemical group or functional group, which can, under appropriate conditions, to interact with the second chemical group, thus forming a covalent bond. If desired, a suitable reactive chemical group can be added to antigennegative text, or to the linker, using any suitable method. (See, for example, Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).) Many combinations of suitable reactive chemical groups known in this field, for example, the amino group can interact with electrophilic group, such as tosylate, mesilate, the group of halogen (chloro, bromo, fluorescent, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Thiols can interact with maleimido, iodoacetyl, acryllium, pyrimidinylidene, thiol 5-thiol-2-nitrobenzoic acid (TNB-thiol), and the like. Aldehyde functional group can be combined with amino - or gidrodinamicheski molecules, and azide group can interact with trivalent phosphate group for education phosphoramidate or phosphorimetry links. Suitable methods for the introduction of activating groups in the molecule are known in the art (see, for example, Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).)

In some embodiments of the incarnation, antigennegative fragment of the antibody that has binding specification the activity in relation to serum albumin, associated with the medicinal product during the reaction of interaction between the two thiols to disulfide bond formation. In other variants of the embodiment antigennegative fragment of the antibody that has binding specificity in relation to serum albumin, associated with the medicinal product during the reaction of interaction between isothiocyanato group and a primary amine to obtain communication estimacion.

Suitable linker group can be linear or branched and include, for example, tetraethylene glycol, C2-C12alkylene, -NH-(CH2)p-NH - or -(CH2)p-NH- (where R is from one to twelve), -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-, polypeptide chain containing from one to about 100 (preferably from one to about 12 amino acids, and the like.

Non-covalent conjugates of the medicinal product

Some non-covalent linkages (for example, hydrogen bonding forces van der Waals forces) can produce stable, highly specific intermolecular connections. For example, the strength of molecular recognition, achieved through numerous non-covalent links between complementary binding partners, underlie many important biological interactions, such as binding of the enzyme with its the mi substrates, recognition of antigens by antibodies, the binding of ligands with their receptors and stabilization of three-dimensional spatial structure of proteins and peptides. Accordingly, such a weak non-covalent interactions (such as hydrogen binding strength of van der Waals forces, electrostatic interactions, hydrophobic interactions and the like) can be used to associate the drug with antigennegative fragment of an antibody that has binding specificity in relation to serum albumin.

Preferably, the non-covalent bond connecting antigennegative fragment and the drug must be strong enough antigennegative fragment and the drug remained significantly associated with each other in vivo (for example, when administered to a human). Usually, the non-covalent bond connecting antigennegative fragment and the drug has a strength equal to at least about 1010M-1. In preferred embodiments embodiment, the strength of non-covalent connection is at least about 1011M-1at least about 1012M-1at least about 1013M-1at least about 1014M-1or, at least,about 10 15M-1. As is well known, the interaction between Biotin and Avidya and between Biotin and streptavidin are very efficient and stable under various conditions, and as described herein, non-covalent link between Biotin and Avidya or between Biotin and streptavidin can be used to obtain non-covalent conjugate of a medicinal product according to the invention.

Non-covalent linkage can be formed directly between antigennegative fragment of an antibody that has binding specificity in relation to serum albumin, drug, or can be formed between suitable complementary binding partners (e.g., Biotin and Avidya or streptavidin), where one partner is covalently associated with the drug, and the complementary binding partner is covalently bonded with antigennegative fragment. If use of complementary binding partners, one of the binding partners can be covalently linked to drug directly or through a suitable linker group, and the complementary binding partner can be covalently linked to antigennegative fragment of an antibody that binds serum albumin, either directly or through a suitable linker group.

Included is internee binding partners represent pairs of molecules, which selectively communicate with each other. Many complementary binding partners are known in this field, for example, the antibody (or its antigennegative fragment) and his kontny (related) antigen or epitope, enzymes and their substrates and receptors and their ligands. Preferred complementary binding partners are Biotin and avidin, and Biotin and streptavidin.

Direct or indirect covalent binding of the complementary member of the binding pair with antigennegative fragment that has binding specificity in relation to serum albumin, or the medicinal product may be made as described above, for example, by the interaction of complementary binding partner, which contains the reactive functional group (or modified to contain a reactive functional group)with antigennegative fragment of an antibody that binds serum albumin, with or without linker. The specific method will depend on connections (e.g., medicine, complementary binding partner, antigennegative fragment of the antibody that binds serum albumin), which should be konjugierte. If desired, the linkers (for example, homobifunctional the linker is, heterobifunctional linkers)that contain terminal reactive functional groups can be used to associate antigennegative fragment and/or drugs with complementary binding partner. In one variant embodiment, it is possible to use heterobifunctional linker, which contains two different reactive groups. Heterobifunctional linker can be chosen in such a way that one of the reactive groups will interact with antigennegative fragment of an antibody that has binding specificity in relation to serum albumin, or a drug, while the other reactive group will interact with a complementary binding partner. You can use any suitable linker (for example, heterobifunctional linker), and many such linkers are known in this area and is available from commercial sources (e.g., Pierce Biotechnology, Inc., IL).

Compositions and Therapeutic and Diagnostic Methods

The proposed composition containing the composition of the medicinal product according to the invention (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs, including FA the pharmaceutical or physiological compositions (for example, for medical and/or veterinary use). Pharmaceutical or physiological compositions contain one or more compositions of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) and pharmaceutically or physiologically acceptable carrier. Typically, such carriers include aqueous or aqueous-alcoholic solutions, emulsions or suspensions, including saline and/or buffered environment. Parenteral carriers include sodium chloride, dextrose and ringer, dextrose and sodium chloride and ringer's lactate. Suitable physiologically acceptable excipients, if you want to maintain polypeptide complex in suspension, can be chosen from thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates. Intravenous carriers include plasma-substituting solutions and supply and electrolyte infusion solutions, such as those based on dextrose ringer. May also contain preservatives and other additives, such as antimicrobial, antioxidant, chelating agents and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16thEdition).

The composition can contain a desired amount of the composition of the medicinal product (which, for example, the drug conjugate, noncovalent drug conjugate means, the slit structure of the drug). For example, the composition can contain from about 5% to about 99% of the drug conjugate, noncovalent drug conjugate funds or merged design of the medicinal product, by weight. In specific embodiments, embodiment, the composition may contain from about 10% to about 99%, or from about 20% to about 99%, or from about 30% to about 99%, or from about 40% to about 99%, or from about 50% to about 99%, or from about 60% to about 99%, or from about 70% to about 99%, or from about 80% to about 99%, or from about 90% to about 99%, or from about 95% to about 99% of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug), by weight. In one example, the composition is dried by freezing in a high vacuum (freeze-dried).

The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described herein, will generally find use in the prevention, reduction or treatment of vos is Alitalia States (for example, acute and/or chronic inflammatory diseases, such as chronic obstructive pulmonary disease (e.g. chronic bronchitis, chronic obstructive bronchitis, emphysema), allergic hypersensitivity, cancer, bacterial or viral infection, pneumonia, such as bacterial pneumonia (e.g., staphylococcal pneumonia), autoimmune disorders (which include, but are not limited to, type I diabetes, multiple sclerosis, arthritis (e.g. osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, lupus arthritis, spondyloarthropathy, (e.g., ankylosing spondylitis or Bechterew's disease), systemic lupus erythematosus, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis), syndrome behceta and malignant male), endometriosis, psoriasis, adhesions of the abdominal cavity (for example, after surgery of the abdominal cavity), asthma and septic shock. The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described herein, can be used for the prevention, attenuation or treatment of pain, such as chronic or acute traumatic pain, chronic or who stray neuropathic pain, acute or chronic musculoskeletal pain, chronic or acute pain and cancer pain, and the like. The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described in this document, you can also enter for diagnostic purposes.

Malignant neoplasms that can prevent, inhibit or treat by applying the composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described herein, include lymphomas (for example, b-cell lymphoma, acute myeloid lymphoma, jackinsky lymphoma, non-Hodgkin's lymphoma), and myeloma (such as multiple myeloma), lung cancer (e.g. small cell carcinoma of the lung, non-small cell carcinoma of the lung), colorectal cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, cancer breast cancer, ovarian cancer, bladder cancer, leukemia (such as acute myeloblastic leukemia, chronic myeloblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adenocarcinoma, kidney cancer, cancer of the hematopoietic system (e.g., myelodysplastic the syndrome is, myeloproliferative disorders (such as polycythemia Vera, essential (or primary) thrombocythemia, idiopathic myelofibrosis)) and the like.

The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs), described herein, are also suitable for use in the prevention, reduction or treatment of endometriosis, fibrosis, infertility, premature birth, erectile dysfunction, osteoporosis, diabetes (e.g. type II diabetes), impaired growth, HIV infection, respiratory distress syndrome, neoplasms and enuresis.

In a preferred variant embodiment, the present invention relates to the use of compounds in accordance with the invention for the manufacture of drugs for the treatment of hyperglycemia, type 2 diabetes, disorders of glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, (β-cell apoptosis, j3-ce) the first symptom of failure, myocardial infarction, inflammatory bowel syndrome, dyspepsia, cognitive disorders, such as enhancing cognitive functions, neuroprotective actions of atherosclerosis, coronary heart disease and other cardiovascular diseases. Private options waples is ment for these indications, the drug is selected from insulinotropic agent and incretin, glucagon-like peptide-1, GLP-1 peptide, a GLP-1 analog, a GLP-1 derivative, PYY, PYY peptide, PYY analog, PYY derivative, on the Basis 3 Basis-3 peptide, on the Basis of 3 similar, on the Basis of 3 is derived, on the Basis 4 Basis-4 peptide, on the Basis of 4 similar, the Basis-4 derivative, or a combination of two or more of these agents (e.g., GLP-1 and PYY peptide peptide).

In another variant embodiment, the present invention relates to the use of compounds in accordance with the invention for the manufacture of drugs for the treatment of syndrome of the small intestine, inflammatory bowel syndrome or Crohn's disease. In private embodiment variants for these indications, the drug is selected from insulinotropic agent and incretin, glucagon-like peptide-1, GLP-1 peptide, a GLP-1 analog, a GLP-1 derivative, PYY, PYY peptide, PYY analog, PYY derivative, on the Basis 3 Basis-3 peptide, on the Basis of 3 similar, on the Basis of 3 is derived, on the Basis 4 Basis-4 peptide, on the Basis of 4 similar, the Basis-4 derivative, or a combination of two or more of these agents (e.g., GLP-1 peptide and PYY peptide).

In another variant embodiment, the present invention relates to the use of compounds in accordance with the invention for the manufacture of drugs for the treatment of hyperglycemia, type 1 diabetes, Diab is and 2 the type or lack of β-cells. In private embodiment variants for these indications, the drug is selected from insulinotropic agent and incretin, glucagon-like peptide-1, GLP-1 peptide, a GLP-1 analog, a GLP-1 derivative, PYY, PYY peptide, PYY analog, PYY derivative, on the Basis 3 Basis-3 peptide, on the Basis of 3 similar, on the Basis of 3 is derived, on the Basis of 4. On the basis of 4 peptide, on the Basis of 4 similar, the Basis-4 derivative, or a combination of two or more of these agents (e.g., GLP-1 and PYY peptide peptide).

Treatment using the compounds in accordance with the present invention can also be combined with a second or more pharmacologically active compounds, which may or may not be part of a conjugate or merged design drugs. For example, the active agent is selected from antidiabetic agents, anti-obesity, funds, regulating appetite, antihypertensive agents, for the treatment and/or prevention of complications resulting from or associated with diabetes and for the treatment and/or prevention of complications and disorders resulting from or associated with obesity. In the present context, the expression "antidiabetic agent" includes compounds for the treatment and/or prevention of insulin resistance and diseases where insulin resistance is the pathophysiological me what Anisman.

Examples of these pharmacologically active compounds are: Insulin, agonists of GLP-1, sulfonylureas (e.g. tolbutamide, glibenclamide, glipizide and gliclazide), biguanides, such as Metformin, meglitinides, glucosidase inhibitors (e.g., acarbose), glucagon antagonists, inhibitors of DPP-IV (dipeptidylpeptidase-IV)inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, modulatory glucose preparations, thiazolidinediones, such as troglitazone and ciglitazone, compounds modifying the lipid metabolism such as antihyperlipidemic agents, as inhibitors of HMG COA (statins), compounds lowering food intake, RXR agonists and means acting on the ATP-dependent potassium channel of the β-cells (e.g., glyburide, glipizide, gliclazide and Repaglinide); cholestyramine, cholesterol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, nateglinide, Repaglinide; p-blockers, such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE inhibitors (angiotenzinkonvertiruyuschego enzyme), such as benazepril, captopril, enalapril, fosinopril, lisinopril, elatioris, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, as well as α-blockers, such as doxazosin, urapidil, prazosin and terazosin; agonists CART (cocaine - and amphetamine-regulated transcript), antagonists of NPY (neuropeptide Y)antagonists MS (melanocortin 4), antagonists of orexin, agonists of TNF (tumor necrosis factor)agonists, CRF (corticotropin releasing factor)antagonists, CRF BP (corticotropin releasing factor binding protein), agonists of urocortin, P3 agonists, agonists, MSH (melanocyte-stimulating hormone)antagonists sit (melanocyte-concentrating hormone)agonists, CCK (cholecystokinin), inhibitors of reverse capture of serotonin, reuptake inhibitors of serotonin and norepinephrine, a mixture of serotonin and noradrenergic compounds, agonists NT (serotonin)agonists bombezin antagonists Galanina, growth hormone, growth hormone releasing compounds, TRH agonists (tireotropin releasing hormone), modulators of the UCP 2 or 3 (nswazwi protein 2 or 3), leptin agonists, DA agonists (parlodel, depressin), inhibitors of lipase/amylase, modulators RXR (retinoid receptor X), agonists TR (3; antagonists of the histamine H3.

Extra insulin may be in the form of one of the following analogs: Asrv-human insulin, LysB28, Low-human insulin, LysB3 Glu29-human insulin, GlyA21, Agdw, AGD-human insulin and des (B30) human insulin.

Additionally other and the effective drugs include human growth hormone or its analogue, parathyroid hormone or its analogue, a growth factor such as platelet-derived growth factor (PDGF), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), somatomedin, such as insulin growth factor I (IGF-I), insulin growth factor II (IGF-II), erythropoietin (EPO), thrombopoietin (SRW) or angiopoietin, interferon, PUK, urokinase, tissue plasminogen activator (t-PA), an inhibitor of plasminogen activator 1, inhibitor of plasminogen activator 2, von Willebrand factor (von Willebrandt), cytokine, such as interleukin, such as interleukin (IL) 1, IL-1 Ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-20 or IL-21, colony-stimulating factor (CFS), such as GM-CSF, stem cell factor, tumor necrosis factor such as TNF-α, lymphotoxin-α, lymphotoxin-β, CD40L or CD30L, a protease inhibitor such as Aprotinin, human follicle-stimulating hormone or its analogue, an enzyme such as superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, catalase, uricase, bilirubin oxidase, trypsin, papain, alkaline phosphatase, β-glukoronidaza, purine nucleoside phosphorylase or batroxobin, opioid, e.g., endorphins, enkephalins, or opioids not the natural hormone or neuropeptide, is for example, calcitonin, glucagon, gastrin, adrenocorticotropic hormone (ACTH), cholecystokinin, luteinizing hormone, gonadotropin releasing hormone, chorionic gonadotropin, corticotropin-releasing factor, vasopressin, oxytocin, protivodiabeticheskie hormones, thyroid stimulating hormone, tireotropin-releasing hormone, relaxin, prolactin, peptide YY, neuropeptide Y, pancreatic polypeptide, leptin, CART (cocaine - and amphetamine-regulated transcript), CART-related peptide, perilipin, melanocortin (melanocyte-stimulating hormones), such as MS-4, melanin-concentrating hormone, natriuretic peptides, adrenomedullin, endothelin, secretin, Amylin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating polypeptide (RASAR), bombezin, bombezin-like peptides, thymosin, heparinase protein, soluble CD4, hypothalamic releasing factor, melatonin and their equivalents.

The conjugate of a drug or merged design of the medicinal product described in this document, you can also enter for diagnostic purposes or as a radiopharmaceutical tools.

In this application, the term "prevention" includes the introduction of a protective composition before the disease was caused. "Attenuation" refers to the introduction of the composition after zabolevaniya called but before clinical manifestation of the disease. "Treatment" includes the introduction of a protective composition after symptoms become apparent.

Available model systems using animals, which can be used for screening the effectiveness of the compositions of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged structures medicines) in protecting against or treating the disease. Testing methods of systemic lupus erythematosus (SLE) in susceptible mice are known in the field (Knight et al. (1978) J. Exp. Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299: 515). Malignant gravis (MG) have SJL/J female mice by induction of the disease using a soluble AchR protein from another species (Lindstrom et al. (1988) Adv. Immunol., 42: 233). The induce arthritis in susceptible strains of mice by injecting collagen type II (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). The described model, in which the synergistic induce arthritis in susceptible rats by injecting heat shock protein of mycobacteria (Van Eden et al. (1988) Nature, 331: 171). Efficacy for the treatment of osteoarthritis can be assessed in a mouse or rat model, in which induce arthritis intra-articular injection of collagenase (Blom, A.B. et al., Osteoarthritis Cartilage 12:627-635 (2004)). Thyroiditis induce in mice put the m introduction of thyroglobulin, as described (Magap et al. (1980) J. Exp. Med., 152: 1115). Insulin-dependent diabetes mellitus (IDDM) occurs naturally or can be induced in certain strains of mice, such as those described in Kanasawa et al. (1984) Diabetologia, 27: 113. Experimental autoimmune encephalopathy (EAE) in mice and rats serves as a model of multiple sclerosis (MS) person. In this model demyelinate induce disease by injecting myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischerer al., eds., Grune and Stratton, New York, pp.179-213; McFarlin et al. (1973) Science 179: 478 and Satoh et al. (1987) J. Immunol., 138: 179).

The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs) according to the present invention can be applied as a separate input compositions or in combination with other agents. The latter may include a variety of immunotherapy drugs, such as cyclosporine, methotrexate, adriamycin or cisplatin, immunotoxins, and the like. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other means in combination with the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design Le is artenova means) of the present invention or combinations of compositions of medicinal product (for example, the drug conjugates, noncovalent drug conjugates, merged design drugs) in accordance with the present invention containing various drugs.

The composition of the medicinal product (e.g., drug conjugates, noncovalent drug conjugates, merged design drugs) you can enter any individual or entity in accordance with any suitable technique. There are many possible routes of administration, including, for example, oral, dietary, local, transdermal, rectal, parenteral (e.g. intravenous, intraarterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal (into the cavity of the spinal canal), intra-articular injection), and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration, depending on the composition of the medicine and the disease or condition that should be treated. Introduction as assigned, may be local or systemic. The preferred method of administration can vary depending on the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) and status (for example, disease)undergoing treatment. Dosage and frequency of injection will depend on age, sex and condition of the patient, concomitant introduction of other drugs, contraindications, and other parameters that should be taken into account by the Clinician. Enter a therapeutically effective amount of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug). Therapeutically effective amount is an amount sufficient to achieve the desired therapeutic effect in the conditions of introduction.

In a preferred variant embodiment of the invention, pharmaceutical compositions containing GLP-1 drug, or analog or derivative of GLP-1 in accordance with the present invention, it is possible to enter parenterally to patients in need of such treatment. Parenteral administration can be accomplished by subcutaneous, intramuscular or intravenous injection by syringe may prefilled pen. Alternatively, parenteral administration can be accomplished through an infusion pump. An additional possibility is the composition, which may be a powder or a liquid for injection of GLP-1 drugs is whether analogue or derivative of GLP-1 in the form of a nasal or pulmonary spray. As another additional opportunities, GLP-1 drug, or analog or derivative of GLP-1 according to the invention can also enter transdermal, for example, the patch may patch for electrophoresis, or through the mucous membranes, for example, bukalo. In other embodiments, embodiment, the composition is administered orally, e.g. in the form of pills, capsules, drink (e.g., sold as a tonic for loss of body weight for the treatment of obesity).

The composition for parenteral administration of GLP-1 compounds can, for example, be obtained as described in WO 03/002136 (incorporated herein by reference).

Composition for nasal administration of certain peptides can, for example, be obtained as described in European patent No. 272097 (issued by the company Novo Nordisk A/S) or in WO 93/18785 (all incorporated herein by reference).

Herein defined, the term "subject" or "individual" includes animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, Guinea pigs, rats, mice or other bovine, Ovine, equine, canine, feline species, species of rodents or mice.

The composition of the medicinal product (e.g., drug conjugate, noncovalent conjugate of Lekarstvo the th means, fused design drugs) can be entered as a neutral compound or as a salt. Salts of compounds (e.g., compositions drugs, drug conjugates, noncovalent drug conjugates, merged structures medicines), containing the amino group or other basic group can be obtained, for example, interaction with a suitable organic or inorganic acid, such as hydrogen chloride, bromovalerate, acetic acid, Perlina acid and the like. Compounds with Quaternary ammonium group also contain a counterion such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing the group, carboxylic acid or other acidic functional group, can be obtained by reacting with a suitable base, such as hydroxide. Salts of acidic functional groups contain proteotion, such as sodium, potassium and the like.

The invention also offered a kit for use in the introduction of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug) to a subject (e.g. patient), containing the composition of the medicinal product (e.g. the R, the drug conjugate, noncovalent drug conjugate means, the slit structure of the drug), a device for delivery of medication and, perhaps, the instructions for use. The composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug) can be provided in the form of a dosage form, such as a product, dried by freezing in robocom vacuum. In specific embodiments embodiment, a device for delivery of a drug selected from the group consisting of a syringe, an inhaler, a device for intranasal or ocular administration (for example, a spray bottle, dropper bottle for the eyes or nose) and needleless injector.

The composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) according to this invention can be liofilizirovanny for storage or dissolved in a suitable carrier prior to use. You can use any suitable method of freeze-drying (e.g. spray drying, spray drying the layer) and/or methods of dissolution. Experts clear that lyophilization and dissolution can lead to various degrees the Yam loss of antibody activity (for example, in respect of normal immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that the applied amount, you may need to adjust to compensate for loss of activity. In a particular variant embodiment, the invention proposed a composition comprising a lyophilized (freeze dried in high vacuum) the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug), as described herein. Preferably, the lyophilized (freeze dried in high vacuum) the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs) loses no more than about 20%, or not more than about 25%, or no more than about 30%, or not more than about 35%, or no more than about 40%, or not more than about 45%, or no more than about 50% of its activity (e.g. binding activity in relation to serum albumin) during rehydration. The activity represents the amount of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate is about the means fused design drug)that is required for the effect of the composition of the medicinal product before it was lyophilized. For example, the number of conjugate drugs or merged design drugs, necessary to achieve and maintain the desired serum concentration within the desired period of time. The activity of the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug) may be determined using any suitable method, before lyophilization, and the activity can be determined using the same method, after rehydration to determine the magnitude of loss of activity.

The composition containing the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the drug) or a cocktail, you can enter for prophylactic and/or therapeutic applications. In specific therapeutic applications, a quantity sufficient to achieve the desired therapeutic or prophylactic effect, under the conditions of administration, such as at least partial inhibition, attenuation, modulation,destruction or some other measured parameter, population of selected cells, defined as "therapeutically-effective amount or dose". The number needed to achieve this dosage will depend on the severity of the disease and the General state of their own the patient's immune system and overall health, but typically range from about 10 μg/kg to about 80 mg/kg, or from about 0.005-5.0 mg of the conjugate of a drug or merged design drug per kilogram of body weight, and more traditionally applied doses from 0.05 to 2.0 mg/kg/dose. For example, the composition of the medicinal product (for example, fused design drugs, drug conjugate, noncovalent drug conjugate means) according to the invention can be administered daily (e.g., up to four injections per day), every two days, every three days, twice a week, once a week, once every two weeks, once a month or once every two months at a dose of, for example, from about 10 μg/kg to about 80 mg/kg, from about 100 μg/kg to about 80 mg/kg, from about 1 mg/kg to about 80 mg/kg, from about 1 mg/kg to about 70 mg/kg, from about 1 mg/kg to about 60 mg/kg, from about 1 mg/kg to about 50 mg/kg, from about 1 mg/kg to about 40 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 1 mg/is up to about 20 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 10 μg/kg to about 10 mg/kg, from about 10 μg/kg to about 5 mg/kg, from about 10 μg/kg to about 2.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or 10 mg/kg

For prophylactic applications, compositions containing the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate funds merged design drugs or cocktails, you can also enter in similar or slightly smaller doses. The composition comprising the composition of the medicinal product (e.g., drug conjugate, noncovalent drug conjugate means, the slit structure of the medicinal product in accordance with the present invention, can be used for prophylactic and therapeutic application to help you modify, inactivate, destroy, or remove the electoral population of target cells in mammals.

EXAMPLES

Receptor antagonist interleukin 1 (IL1-ra) is an antagonist that blocks the biological activity of IL-1 by konkurentnogo inhibiting the binding of IL-1 receptor of the first type of interleukin-1 (I-1R1). Production of IL-1 is induced in response to stimuli associated with inflammation and mediates various physiological responses, including inflammatory and immunological reactions. IL-1 has different kinds of activity, including the destruction of cartilage tissue and the stimulation of bone resorption. In patients with rheumatoid arthritis (RA) number of locally produced IL-1 is increased, and the levels of natural IL1-ra insufficient to compete with these abnormally increased quantities. There are several types of treatment of RA, including symptom-modifying Antirheumatic drugs (DMARDS)such as methotrexate, and biologic drugs, such as KINERET® (anakinra, Amgen).

KINERET® (anakinra, Amgen) is a recombinant, deglycosylated form receptor antagonist to human interleukin-1, which consists of 153 amino acids and has a molecular mass of 17.3 kDa. (Amino acid sequence KINERET® (anakinra, Amgen) corresponds 152 amino acids in the natural IL-1ra and additional N-terminal methionine). KINERET® (anakinra, Amgen) is indicated for reducing signs and symptoms of moderate to severe rheumatoid arthritis in patients 18 years of age or older who are not helped by therapy with DMARDs. Dosage is a single subcutaneous injection of 100 mg drug one RA is in the day. Tβ1/2makes 4-6 hours, and 71% of patients experiencing adverse reactions at the injection site in 14-28 days.

The inventors have demonstrated that binding of a therapeutic polypeptide with a dAb that binds serum albumin, leads to a compound which (1) has activity similar to the individual a therapeutic polypeptide, and (2) also binds serum albumin. In addition, in the present invention, a method for creating a variant of therapeutic polypeptide having a long half-life in serum. For example, we added a dAb that binds serum albumin, IL1-ra, which leads to a connection that has a longer half-life in serum than IL1-ra separately.

Example 1. The choice tomanovich antibodies that bind mouse, rat and human serum albumin

This example explains a method of making antibodies with a single domain (dAb), directed against serum albumin. Describes the selection of dAbs against mouse serum albumin (MSA), human serum albumin (HSA) and rat serum albumin (RSA).

dAb against mouse serum albumin were selected as described in WO 2004/003019 A2. Used three display library of human antibodies of the bacteriophage. Each of the libraries was based on e is iniciou human frame region for V H(V3-23/DP47 and JH4b) or VK(ø12/o2/DPK9 and Jk1) variety in the side chain encoded by NNK codons that are embedded in the hypervariable segments (CDR1, CDR2 and CDR3).

Library 1 (VH):

Diversity at positions: N, n, N, N, Uw50, N, Na, N, N, N, N, N, N, N.

Library size: 6,2×109

Library 2 (VH):

Diversity at positions: N, n, N, N, Uw50, N, Na, N, N, N, N, N, N, N, N, N, NA, NV.

Library size: 4,3×109

Library 3 (VK):

Diversity at positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96.

Library size: 2×109

Library VHand VKwere pre-selected on the basis of binding to protein a and protein L, respectively, belong to a common ligand, so that the majority of clones in the selected libraries were functional. The size of the libraries shown above, corresponds to the size after pre-selection.

Were performed in two stages of selection for serum albumin, using separately each of the libraries. For each selection the antigen was applied in the form of a coating on immunoprobes (type nunc) in 4 ml of PBS (saline phosphate buffer) at a concentration of 100 μg/ml During the first stage of each of the three libraries was divided separately by the method of panning (sorted) against HSA Sigma) or MSA (Sigma). In the second stage of selection, the phage from each of the six samples selected at the first stage, was sorted against (1) again the same antigen (e.g., 1-stage MSA, 2-stage MSA) and (2) against the reciprocal (corresponding) antigen (for example, 1-stage MSA, 2nd stage HSA), which led eventually to the twelve samples according to the results of the 2nd stage. In each case, after the second stage of selection of 48 clones were tested for binding to HSA and MSA. Soluble fragments dAb received, as described in Harrison et al, Methods Enzymol. 1996; 267: 83-109 for scFv fragments, and kept the standard ELISA Protocol (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133) except that 2% tween-PBS was used as a blocking buffer and associated dAbs were detected using either the protein L-HRP (Sigma) (for VKS)or protein a-HRP (Amersham Pharmacia Biotech) (for VHs).

dAb, which produced a signal above the baseline, indicating binding to MSA, HSA, or both, were tested in ELISA insoluble form on the binding of one of plastic, but all were specific in relation to serum albumin. The clones are then sequenced (see table 1), a finding that was identified 21 unique dAb sequence. The minimum similarity (at the amino acid level) between the selected VKdAb clones were 86,25% ((69/80)×100; the result obtained when all diversified studiessusa different, for example, clones 24 and 34). The minimum similarity between the selected VHdAb clones were 94% ((127/136)×100).

Then, dAbs that bind serum albumin, were tested for their ability to capture biotinylated antigen from solution. Kept the ELISA Protocol (as above), except that the tablet ELISA were coated with 1 μg/ml protein L (clone VK) and 1 μg/ml protein A (for clones VH). Soluble dAb captured from solution, as in the Protocol, and detection was performed using biotinylated MSA or HSA and streptavidin HRP. Biotinylated MSA and HSA prepared in accordance with the manufacturer's instructions, with the aim of achieving an average of 2 betinov molecule serum albumin. Identified twenty-four clone, which was captured biotinylated MSA from solution in ELISA. Two of these clones (clones 2 and 38 below) was also captured biotinylated HSA. Then, dAbs were tested for their ability to bind MSA, applied in the form of a coating on SM chip of a Biacore instrument. Were found eight clones that bound MSA on Biacore.

dAb against human serum albumin and rat serum albumin was selected as previously described for anti-MSA dAbs, except for the following modifications to the Protocol: Phage library of synthetic VHdomains presented with the th library 4G, which is based on human VH3, containing DP47 embryonic gene type and segment JH4. Diversity in the following specific provisions made by mutagenesis (using NNK codons; numbering according to Kabat (The Kabat Database of Sequences of Proteins of Immunological Interest, Kabat Database of Sequences of Proteins representing Immunological Interest)) in CDR1: 30, 31, 33, 35; CDR2: 50, 52, 52a, 53, 55, 56; and CDR3: 4-12 diversified residues, for example N, N, N and N in 4G and N, N, N, N, N, N, Na, 100b, NS, Na, Ne and H100f in 4G n. The last three CDR3 residue are FDY, so CDR3 length varies from 7-15 residues. The library contains >1×1010individual clones.

A subpopulation of VHand VKlibraries preselected binding protein a and protein L, respectively, typical ligands, so that the majority of clones in the libraries coming was functional. The sizes of the libraries listed above, consistent with the size after pre-selection.

Were performed in two stages of selection for the rat and human serum albumin using separate subpopulations VHand VKlibraries. For each selection of antigen either (1) was applied in the form of a coating on immunoprobes (type nunc) in 4 ml of PBS at a concentration of 100 μg/ml or (2) was biomineral and then used for instant selection, the and followed by capture on streptavidin beans (1st stage) and neutravidin beans (2nd stage). (See Table 1 about parts selection strategy applied to isolate each clone.) In each case, after the second stage of selection 24 bacteriophobic clone were tested for binding to HSA or RSA.

If a significant portion of clones in one of the samples was positive in ragovoy ELISA, then the DNA from the sample cloned in expressing vector to obtain soluble dAb and selected individual colonies. Soluble dAb fragments were obtained as described by Harrison et al (Methods Enzymol. 1996; 267:83-109) for scFv fragments, as well as comply with the standard ELISA Protocol (Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133) except that 2% tween-PBS was used as a blocking buffer and associated dAbs were detected using anti-tous-HRP. Clones that were positive in ELISA, and then subjected to screening for binding of MSA, RSA, or HSA, using the device surface plasma resonance BIACORE (Biacore AB). dAbs, which linked MSA, RSA, or HSA, is analyzed next. Then the clones sequenced and identified unique sequences dAb.

Table 1
The Protocol selection for dAb that bind serum albumin
dAbLibrary R1 breedingR2 breedingBinding on Biacore
DOM7r-14G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA
DOM7r-34G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA
DOM7r-44G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA, MSA
DOM7r-54G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA
DOM7r-74G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA, MSA
DOM7r-84G VK10 μg/ml per tube RSA10 μg/ml per tube RSARSA, MSA/td>
DOM7h-14G VK10 μg/ml per tube HSA10 μg/ml per tube HSAHSA
DOM7h-24G VKInstant 100 nm HSASoluble 50 nm HSAHSA
DOM7h-34G VK10 μg/ml per tube HSA10 μg/ml per tube HSA-
DOM7h-44G VK10 μg/ml per tube HSA10 μg/ml per tube HSA-
DOM7h-64G VK
DOM7h-74G VK
DOM7h-84G VKSoluble 200 nm HSASoluble 50 nm RSAHSA, RSA,
MSA
DOM7r-134G VKSoluble 200 nm HSASoluble 50 nm RSARSA, MSA
DOM7r-144G VKSoluble 200 nm HSASoluble 50 nm RSARSA, MSA
DOM7h-214G VH100 m kg/ml HSA per tube100 µg/ml of HSA on the tubeHSA
DOM7h-224G VH100 µg/ml of HSA on the tube100 µg/ml of HSA on the tubeHSA
DOM7h-234G VH100 µg/ml of HSA on the tube100 µg/ml of HSA on the tubeHSA
DOM7h-244G VH100 µg/ml of HSA on the tube100 µg/HSA on tube HSA
DOM7h-254G VH100 µg/ml of HSA on the tube100 µg/ml of HSA on the tubeHSA
DOM7h-264G VH100 µg/ml of HSA on the tube100 µg/ml of HSA on the tubeHSA
DOM7h-274G VH100 µg/ml of HSA on the tube100 µg/ml of HSA on the tubeHSA

dAb, which linked serum albumin on the BIACORE chip (Biacore AB), then analyzed for additional information on the affinity. The analysis was performed using SM chip (karboksimetilirovaniya dextranase matrix), which covered serum albumin. Flow cell 1 was a bare-blocked negative control, flow cell 2 was covered with the help of HSA, the flow cell 3 was covered with the help of RSA and flow cell 4 was coated using MSA. Serum albumin was immobilized in acetate buffer, pH 5.5, using BIACORE computer system to cover Wizard, which was programmed to obtain 500 resonant edit the CI (RUs) covered material. Each interest dAb expressed in periplasmic E. Li in 200 ml, 500 ml and purified from the supernatant using the absorbance of culture medium with protein A-laminar affinity chromatography (Amersham, UK) for VHS and the protein L-agarose affinity chromatography (Affitech, Norway) for VK8, followed by elution with glycine at pH 2.2 and replacing the buffer to PBS. Prepared a series of concentrations of dAb (in the range from 5 nm to 5 μm) diluted in BIACORE HBS-EP buffer and missed the stream along the BIACORE chip.

The affinity (KD) was calculated based on the curves recorder BIACORE by comparing the on-rate and off-rate curves with the curves of the recorder received from concentrations dAb in the field of KD. Identified dAbs with a range of different values of affinity to serum albumin. In the range of 10-100 nm were affinity DOM7h-8 to HSA, DOM7h-2 to HSA and DOM7r-1 to RSA. In the range from 100 nm to 500 nm were affinity DOM7h-7 to HSA, DOM7h-8 of the RSA and DOM7h-26 to HSA. In the range from 500 nm to 5 μm were affinity DOM7h-23 to HSA and DOM7h-1 to HSA. Examples of curves of the recorder presented on Figa-6P.

Example 2. Formatting antibodies against serum albumin in the form of a slit structure with the receptor antagonist IL-1 (IL-1ra)

This example describes a method of obtaining a fused protein containing IL-1ra and dAb that binds serum albumin. There have been two merged designs, one with a dAb N-conco is from IL-1ra (MSA16IL1-ra) and one with a dAb With the end from the IL-1ra (IL1-raMSA 16). Sequence fused structures and vector presented on Figs and 2D. Also got merged control design, which is not connected MSA, and its sequence is presented on Five.

KINERET (anakinra, Amgen) has a short half-life equal to 4-6 hours, and the recommended dosage regimen requires daily injections. This mode leads to side reactions at the injection site in 14-28 days in 71% of cases. Therefore, the form of human IL-1ra, which has a longer half-life in serum, would have benefits and could increase the efficiency and reduce the frequency of dosing. For pharmaceutical drug these two properties are desirable.

Cloning

Briefly, there were developed two multiple cloning site (MCSs), as described in detail below, and inserted in expressing vector with T7 promoter. Were designed restriction sites for insertion ILI-ra, dAb, GAS leader sequence, and linker. One (MCS 1+3) encodes a protein with a dAb N-end from IL-1ra, and the other (MCS 2+4) encodes a protein with a dAb With the end from the IL1-ra.

Clone website 1+3 for the merged design dAblL1-ra

Ndel, insert, Sail, NotI, insert, Xhol, BamHI

gcgcatatgttagtgcgtcgacgtcaaaaggccatagcgggcggccgctgcaggtctcgagtgcgatggatcc

(SEQ ID NO:35)

Clone website 2+4 for the merged design ILI-radAb

Ndel, insert, StUI, Sad, insert, Sail, NotI, TAA TAA BamHI cgcatatgttaagcgaggccttctggagagagctcaggagtgtcgacggacatccagatgacccaggcggccgctaa taaggatccaatgc (SEQ ID NO:36)

GAS leader sequence was then inserted into each vector by splitting MCS using suitable restriction enzymes and ligating the annealed primers encoding the leader sequence. Then similarly inserted linker DNA encoding the linker. DNA encoding IL-1ra, was obtained by PCR (polymerase chain reaction) (using primers designed to add the required restriction sites) of the cDNA clone and inserted into TORO cloning vector. After confirming the correct sequence for sequencing nucleic acids DNA encoding IL-1ra, cut from the TOPO vector and ligated into a vector containing a leader sequence, and linker. Finally, DNA encoding dAb, cut out from the vector expressing dAb, and inserted in the vector processing insert (purified by gel-chromatography) and vector using SaIl/Notl.

Expression and purification

MSA16IL1-ra, IL1-raMSA16 and dummyIL-1ra (idle IL-1ra) expressed in periplasmic E. coli and purified from the supernatant using the absorbance of culture medium with protein L-agarose affinity chromatography (Affitech, Norway) followed by elution with glycine at pH of 2.2. Purified dAbs then analyzed using SDS-PAGE and subsequent staining of Kumasi. For one of the proteins (IL-1raMSA 16) more than 90% of the protein was of the expected size is, and, as a consequence, it was analyzed for the presence of activity without further purification. Other proteins (MSA16IL1-ra and dummyIL-1ra) were contaminated by a band of smaller size and, consequently, were further purified using a liquid ion-exchange chromatography quick resolution (FPLC) on ion-exchange column RESOURSEQ at pH 9. Protein was suirable linear gradient of 0-500 mm NaCl. After SDS-PAGE analysis of the fractions containing the protein of the expected size, combined, received a United faction more than 90% purity. This protein was used for further analysis.

Example 3. The determination of the activity of the merged design dAb IL1-ra in vitro MRC-5, IL-8 analysis

Fused design MSA16IL-1ra were tested for their ability to neutralize the induction of secretion of immunoglobulin IL-8 immunoglobulin IL-1 in MRC-5 cells (ATSC Registration number No. CCL-171; American type culture Collection, Manassas, VA). Method adapted from Akeson, L. et al (1996) Journal of Biological Chemistry 271, 30517-30523, which describes the induction of immunoglobulin IL-8 immunoglobulin IL-1 in HUVEC cells, MRC-5 was used instead of the HUVEC cell line. Briefly, MRC-5 cells, seeded in tablets for micrometrology, incubated overnight in the presence of dAblL-1ra fused proteins or IL-1 hectare of control and IL-1 (100 PG/ml). After incubation, the supernatant was separated from cells by spiritualium and measured the concentration of IL-8 by the sandwich ELISA (R& D Systems).

The activity of IL-1 hectare in fused proteins led to a decrease in the secretion of IL-8. The decrease in the secretion of IL-8 in the activity MSA16IL1-ra merged structure and activity of IL-1raMSA16 fused constructs were compared with the decrease seen when IL-1ra control (recombinant human IL-1 hectare, R&D Systems). Determined the neutralizing dose 50 (ND50each of the tested proteins and presented the results in Table 2.

Table 2
ProteinND50
IL-1ra0.5 nm
MSA16IL-1ra2 nm
IL-1raMSA168 nm

The results demonstrated that IL-1ra remained active as part of a merged design with dAb antibody against serum albumin. Protein MSA16IL-1ra additionally investigated to evaluate its pharmacokinetics (PK study).

Serum albumin, anti-IL-1ra "sandwich" ELISA Three fused design dAb/IL-1ra tested for the ability to bind serum albumin with simultaneous detection using monoclonal anti-IL1-ra antibodies. Studied merged design was a MSA16IL-1ra, IL-1aMSA16 and dummylL-lra. In short, the tablet ELISA was covered during the night a layer of murine serum albumin at a concentration of 10 μg/ml, washed 5 times with 0.05% tween-PBS and then blocked for 1 hour using 4% Marvel PBS (MPBS). After blocking the tablet was washed 5 times with 0.05% tween-PBS and then incubated for 1 hour in the presence of each of the slit structures dAb/IL-1ra, diluted in 4% MPBS. Each slit design incubated at a concentration of 1 μm and at 7 serial 4-fold dilutions (i.e., the decrease in concentrations up to 60 PM). After incubation tablets were washed 5 times with 0.05% tween-PBS and then incubated for 1 hour in the presence of the manufacturer's recommended dilution of rabbit polyclonal antibodies (ab-2573) against the receptor antagonist of human IL-1 (Abeam, UK)diluted in 4% MPBS. After this incubation, the tablets were washed 5 times with 0.05% tween-PBS and then incubated for 1 hour in the presence of a 1/2000 dilution of secondary antibody (anti-rabbit IgG-HRP)diluted in 4% MPBS. After incubation in the presence of the secondary antibody, the tablets were washed 3 times using 0.05% tween-PBS and 2 times using PBS and then showed using 50 μl per well of TMB microwell peroxidase substrate (KPL, MA) and the reaction was stopped using 50 μl per well of HCL. The absorbance was read p and 450 nm.

Both protein MSA16IL-1ra and IL-1raMSA16 were detected at levels more than two times higher than the base level concentration of 1 μm in a "sandwich" ELISA. Protein MSA16IL-1ra was detected at a level two times higher than the basic level or above, at dilutions up to 3.9 nm, while the protein IL-1raMSA16 were detected at levels two times higher than the basic level, only at dilutions up to 500 nm. Linking merged design MSA16IL-1ra serum albumin, as shown, is specific for serum albumin as a control structure (dummyIL-1ra) does not bind serum albumin.

Example 4. Definition of half-life in serum merged structures medicines in the study of RK in mice.

A. Determination in mice half-life in serum MSA-binding fused protein with dAb/HA epitope tag

MSA-binding protein with dAb/HA epitope tag expressed in periplasmic E. coli and purified using the absorbance of culture medium with protein L-agarose affinity chromatography (Affitech, Norway) followed by elution with glycine at pH of 2.2. The half-life in serum fused protein was determined in mice after a single intravenous (IV) injection at a concentration of approximately 1.5 mg/kg animals male strain CD1. Analysis of serum levels was carried out at the same time ELISA using capture goat anti-(Abeam, UK) and detection of the protein L-HRP conjugate (Invitrogen, USA), blocked using 4% Marvel. Washing was performed using 0.05% tween-20, PBS. Standard curves of known concentrations of MSA-binding merged design dAb/HA were established in the presence of 1x mouse serum, in order to ensure comparability with the test samples. Simulation using the one-component model (WinNonlin Software, Pharsight Corp., USA) showed that MSA-binding protein dAb/HA epitope tag was the end-of-phase t1/2 equal to 29.1 per hour, and the area under the curve equal to 559 h-ág/ml of This demonstrated a significant improvement compared with the predicted half-life of individual peptide with an epitope ON the label, which could be so short that would be just a few minutes.

The results of this study with the use of epitope tags as model drugs demonstrate that the in vivo half-life in serum drug can be increased if the drug is manufactured in the form of a merged design drugs or drug conjugate with antigennegative fragment (e.g., dAb) of an antibody that binds serum albumin.

Also evaluated in mice in vivo half-life of the anti-MSA dAbs DOM7m-16 and DOM7m-26, and control dAb that does not tie the MSA provides. Again, DOM7m-16, DOM7m-26 and control dAb contained ON epitope tag, which serves as a model drug (for example, a peptide drug). In this study, dAb control, which does not bind MSA, had an in vivo half-life of 20 minutes, while the in vivo elimination half-life DOM7m-16 and DOM7m-26 were significantly increased. (Fig) further experiments on mice, it was found that DOM7m-16 has an in vivo half-life equal to 29.5 hours.

In another study, the in vivo half-life (t1/2β) DOM7h-8, which contained epitope tag, were evaluated in mice. Modeling using the two-component model (WinNonlin Software, Pharsight Corp., USA) showed that DOM7h-8 had a t1/2p equal to 29.1 per hour.

The results of each of these studies using epitope tags as a model drug (e.g., peptide drugs) demonstrate that in vivo half-life in serum drug can be significantly increased if the drug is manufactured in the form of a merged design drugs or drug conjugate with antigennegative fragment (e.g., dAb) of an antibody that binds serum albumin.

C. Determination in mice half-life in serum MSA binding dAb/L-1ra fused protein

MSA binding dAb/IL-1ra protein (MSA16IL-1ra) expressed in periplasmic E. Li and purified using the absorbance of culture medium with protein L-agarose affinity chromatography (Affitech, Norway) followed by elution with glycine at pH of 2.2. The half-life in serum MSA16IL-1ra (DOM7m-16/IL-1ra), merged structures of IL-1ra with dAb, which does not bind MSA (dummy dAb/IL-1ra), and anti-MSA dAb, merged with epitope tag (DOM7m-16 ON the label), was determined in mice after a single in/injection at a concentration of approximately 1.5 mg/kg animals male strain CD1.

Analysis of serum levels was performed using IL-1ra sandwich ELISA (R&D Systems, USA). Standard curves of known concentrations of dAb/IL-1ra merged structures established in the presence of 1x mouse serum, in order to ensure comparability with the test samples. The simulation was carried out using WinNonlin pharmacokinetic computer program (Pharsight Corp., USA.

It was expected that the merged structure of IL-1ra with anti-MSA dAb would substantially increased half-life in serum in comparison with the control, which was a merged design dAb, not linking MSA, with IL-1ra. Predicted that non-binding MSA control fused design dAb/IL-1ra has a short half-life in serum.

The results of the study are presented in Table 3 and show that merged to the u se of IL-1ra with anti-MSA dAb (DOM7m-16/IL-1ra) had a half-life in serum, which was about 10 times longer than that of the slit structure of IL-1ra with dAb, which does not bind MSA (dummy dAb/IL-1ra). The results also found that I received more than 200-fold improvement (increase) of the area under the curve of the dependence of concentration on time for DOM7m-16/IL-1ra (AUC: 267 hours mcg/ml) compared with the dummy dAb/IL-1ra (AUC: 1.5 hour·ág/ml).

Table 3
AgentThe half-life in serum
DOM7m-16/IL-1ra4.3 hours
dummy dAb/IL-1ra0.4 hours
DOM7m-16 HA tag29 hours

The results of these studies demonstrate that in vivo half-life in serum and AUC drugs can be significantly increased if the drug is manufactured in the form of a merged design drugs or drug conjugate with antigennegative fragment (e.g., dAb) of an antibody that binds serum albumin.

Example 5. Definition rats half-life in serum RSA-binding fused proteins with dAb/HA epitope tag

Anticrimine dAbs against serum album is on expressed with C-terminal TO the labels in periplasm E. coli and purified using the absorbance of culture medium with protein L-agarose affinity chromatography (Affitech, Norway) for VKdAbs and the absorbance of culture medium with protein A-affinity chromatography for VHdAbs, followed by elution with glycine at pH of 2.2. To determine the half-life in serum, groups of 4 rats were injected once/injection at a concentration of 1.5 mg/kg on the basis of DOM7r-27, DOM7r-31, DOM7r-16, DOM7r-3 or control dAb (HEL4), which binds to a foreign antigen. Serum samples were obtained serial blood from the tail vein during the 7-day period and analyzed using a sandwich ELISA using goat anti-(Abeam, Cambridge UK), adsorbed on the tablet ELISA, followed by detection with protein A-HRP conjugate (for VHdAbs) or protein L-HRP conjugate (for VKdAbs). Standard curves of known concentrations of dAb set in the presence of 1x rat serum, in order to ensure comparability with the test samples. Used modeling using the two-component model (with using WinNonlin pharmacokinetic computer program (Pharsight Corp., USA)to calculate t1/2p and the area under the curve (AUC) (table 4).

Table 4
Agent FrameThe affinity (KD) to rat serum albumint1/2βAUC (µg·h/ml)
DOM7r-3VK12 nm13.7 hours224
DOM7C-16VK1 micron34.4 hours170
DOM7r-27VH250 nm14.8 hours78,9
DOM7r-31VH5 µm5,96 hours71,2

The results of this study on rats using epitope tags as a model drug (e.g., peptide drugs) demonstrate that in vivo half-life in serum drug can be significantly increased if the drug is manufactured in the form of a merged design drugs or drug conjugate with antigennegative fragment (e.g., dAb) of an antibody that binds serum albumin.

Prediction of half-life in humans

In vivo half-life of the dAb, the slit structure of a drug or conjugate of a medicinal product in humans can be estimated on the basis of data relative to the half-life obtained on animals, using allometric scaling. Values of log in vivo elimination half-life, defined in 3 animals depicted in the graph relative to the values of the log weight of the animal. Hold the line marked point and use the slope of the curve and cut, cut the curve on the coordinate axis y, to calculate the in vivo half-life in humans, using the formula:

log Y=log(a)+b log(W),

in which Y represents an in vivo half-life in humans, log(a) is a segment, cut off the curve on the coordinate axis, b is the slope of the curve, and W represents the weight of the animal. You can get a line, using the data on the in vivo half-life obtained from animals, which weigh approximately 35 grams (e.g., a mouse), approximately 260 grams (e.g., rats) and approximately 2710 grams. For this calculation, we can assume that the mass of the person is 70,000 grams.

Example 6. The effectiveness of anti-SA dAb/IL-1ra merged design of the medicinal product in the collagen-induced model is rcrit of rheumatoid arthritis in mice

Evaluated the effectiveness of the merged design DOM7m-16/IL-1ra and efficacy of IL-1ra in the recognized model of rheumatoid arthritis in mice (collagen-induced arthritis type II (CIA) in DBA/1 mice). In continuation of the study, mice were kept in a laboratory room in a standard cell type 2, which was placed in the container type Scantainer air, purified using visokoefektivnih filter type HEPA, at 20-24°C with a 12-hour day, 12-hour night cycle. Food (Harlan-Teklad universal diet 2016) and sterilized by ultraviolet water was provided in excess. Mice were placed in a laboratory room at least 7 days before the start of the study, to ensure proper acclimatization.

DBA/1 mice aged 7-8 weeks (obtained from Taconic M & B, Domholtveg, Denmark) was once injectively emulsion Arthrogen-CIA adjuvant and Arthrogen-CIA collagen (both MD biosciences), which was emulsiable in the ratio of 1:1 to obtain a stable emulsion. The emulsion was considered stable when the drop of emulsion added to the chemical glass of water, forming a dense clump. Then the mice were injected with the emulsion.

After twenty-one days after the injection of the emulsion 20 animals with the progression of the arthritic disease were excluded from the study, and the remaining mice were divided into groups of 10 animals each group including the Ala 5 males and 5 females). Mice were treated as shown in Table 5, and all kinds of therapy used in concentrations designed to introduce 10 ml/kg

Table 5
GroupThe treatment
1IL-1ra, 1 mg/kg (intraperitoneal (/b) bolus)
2IL-1ra, 10 mg/kg (W/bolus)
3DOM7m-16/IL-1ra, 1 mg/kg (W/bolus)
4DOM7m-16/IL-1ra, 10 mg/kg (W/bolus)
5ENBREL® (entarecept; Immunex Corporation), 5 mg/kg (W/bolus)
6Saline solution (negative control), 10 ml/kg (W/bolus)
7Dexamethasone (positive control), 0.4 mg/kg (subcutaneous injection)

Clinical indicators of severity of arthritis was registered 3 times a week, beginning with the 21st day 49 day. Mice were subjected to Athanasii for 49 days. Individual mice were subjected to Athanasii earlier, if they were arthritic index, equal to 12 points or more, or had a serious about rezerwuj problems.

For the compilation scale clinical indicators, each limb was assessed as a score in accordance with the criteria listed below, and the scores for all four limbs folded together to get the total score in points for the mouse. This method resulted in the receipt of the indicator scores from 0 to 16 for each mouse. Evaluation criteria scores were: 0 = normal; 1 = weak, but significant redness and swelling of the ankles or wrists, or apparent redness and swelling limited to individual digits, regardless of the number of affected fingers; 2 = moderate redness and swelling of the ankles and wrists; 3 = severe redness and swelling of the entire paw, including the fingers; 4 = maximally inflamed limb with involvement of multiple joints.

Average group arthritic indicators in scores was calculated for each group received the treatment on each day of treatment with the use of clinical indicators in scale from individual mice. Any animals removed from the study for ethical reasons, was assigned the maximum score is 16. The average arthritic group figures in points presented in the form of a graph with respect to time (Fig).

Statistical analysis of the average arthritic group of figures in points on 49 days performed using the Wilcoxon test. E. the statistical analysis detected what two groups that were treated with the help of DOM7m-16/IL-1ra (1 mg/kg or 10 mg/kg (Groups 3 and 4)), has significantly improved arthritic figures in points for 49 day (P<1% and P<0,05% significance levels, respectively) in comparison with the control group treated with saline (Group 6). In contrast, treatment with IL-1ra in the amount of 1 mg/kg (Group 1) did not lead to statistically significant improvement of the arthritic index in points on 49 days, while treatment using IL-1ra in the amount of 10 mg/kg (Group 2) resulted in a significant improvement at P<5% significance level. Treatment ENBREL® (entarecept; Immunex Corporation) (Group 5) resulted in a significant improvement in the arthritic index in points for 49 days at P<10% significance level.

Treatment DOM7m-16/IL-1ra in the dose of 10 mg/kg (Group 4) was effective in improving the arthritic index in points for 49 day (statistically significant at P<0.5% level of significance) compared with standard therapy with ENBREL® (entarecept; Immunex Corporation) in an amount of 5 mg/kg (Group 5). In addition, treatment with the help of DOM7m-16/IL-1ra in a smaller dose of 1 mg/kg (Group 3) was more effective in improving the arthritic index in points on 49 days, than treatment by separate IL-1ra in the same dose (Group 1) (statistically significant at P<10% level significant the spine).

The results show that in certain doses DOM7m-16/IL-1ra was more effective than IL-1ra or ENBREL® (entarecept; Immunex Corporation) in this study. The response to treatment using IL-1ra was, as expected, dose-dependent, and the response to treatment with the help of DOM7m-16/IL-1ra was also dose-dependent. Averages in points during treatment with DOM7m-16/IL-1ra in the amount of 1 mg/kg were consistently lower than the average of the scores obtained by treatment with IL-1ra in the amount of 10 mg/kg These are plotted on a graph of the results (Fig) show that treatment with the help of DOM7m-16/IL-1ra was about 10 times more effective than using IL-1ra in this study.

This higher efficiency DOM7m-16/IL-1ra was observed even though DOM7m-16/IL-1ra protein contains about half the number of IL-1 receptormediated epitopes than IL-1ra, in terms of mass (e.g., 1 mg DOM7m-16/IL-1ra (Molecular mass (Mm) of 31.2 kDa) contains about half the number of IL-1 receptormediated epitopes than 1 mg of IL-1ra (Mm 17,1 kDa).

The results of this study demonstrated that dAb that binds serum albumin, can be combined with IL-1ra (clinically proven therapy for RA), and that the resulting merged design of the medicinal product has properties of a long period of probiogen the serum (provided through dAb), as well as IL-1 receptornegative properties (provided at the expense of IL-1ra). Thanks circulation time in serum merged design drugs, the dose of DOM7m-16/IL-1ra, which was effective in the treatment of CIA, was significantly reduced compared with IL-1ra.

The results of this study demonstrated that, in addition to the advantages of increased half-life and increased AUC, medications manufactured in the form of the slit structures medicines or conjugates of drugs with antigennegative fragment (e.g., dAb) of an antibody that binds serum albumin, are highly effective therapeutic agents that provide advantages over single drug. For example, as demonstrated in the CIA model in mice, low-dose slit design drug was effective and inhibited joint inflammation and damage to joints caused by IL-1, over a longer period of time compared to separate IL-1ra, and also provided greater protection from disease development.

Example 7. Anti-SA dAb/Saporin non-covalent conjugate of the medicinal product

Protein Saporin, inactivating the ribosome (anticancer drug), is extremely is persistent to the action of denaturing agents and proteases and was used as the target of the toxin to T-lymphocytes. Non-covalent conjugate drugs received by connection Saporin and DOM7h-8 through Biotin-streptavidin communication. The results obtained with this non-covalent conjugate drugs, demonstrate that DOM7h-8 retains its binding characteristics of serum albumin in connection with the drug.

A variant of DOM7h-8, called DOM7h-8cys, in which the C-terminal arginine at position 108 (108 amino acid of SEQ ID NO:24) was replaced by a cysteine residue, obtained by expression of a recombinant nucleic acid NV cells. Cells were cultured and induced at 30°C in expressing the system inductance TV readymix (Merck KGa, Germany) for 72 hours prior to extraction of the supernatant by centrifugation. DOM7h-8cys was purified from the supernatant using affinity chromatography on protein L-agarose. Then the ion exchange resin was washed with 10 column volumes of 2×PBS and suirable DOM7h-8cys 0.1 M glycine, pH 2. Suirvey DOM7h-8cys neutralized with the help of 0.2 × volume of Tris pH 8 and concentrated to a concentration of 1 mg/ml (using the hub METHOD with a working volume of 20 ml (Millipore Corp., MA)).

In concentrated DOM7h-8cys replaced the buffer to PBS using NAP5 cialisbuy column (GE Healthcare/Amersham Biosciences, NJ) and determined concentration. dAb was then biomineral (via primary amines, using the EZ-LINK sulfo-MN-1-s-Biotin(Pierce Biotechnology Inc., IL). Biotinylated dAb mixed with substrate streptavidin-saporin in 1:1 molar ratio.

To confirm the formation of the complex dAb/saporin, used a "sandwich" ELISA for detection of intact complexes. Inflicted human serum albumin (HAS) on half of the holes ELISA tablet (Nunc, NY) overnight at a concentration of 10 μg/ml in a volume of 100 μl per well. After incubation over night tablet was washed three times using PBS, 0.05% tween, and then the entire tablet was blocked for 2 hours with 2% PBS. After blocking the tablet was washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of DOM7h-8/saporin non-covalent conjugate, diluted to 0.5 μm in 2% tween-PBS. As a control, on the same tablet ELISA incubated unbound saporin in a concentration of 0.5 μm and unrelated DOM7h-8 in a concentration of 0.5 μm in 2% tween-PBS. Additional monitoring was the same three diluted protein incubated in the wells of ELISA tablet not covered with the help of HSA and blocked with 2% tween. After incubation, the tablet was washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of a 1/2000 dilution of goat anticipatin polyclonal antibodies (Advanced Therapeutic Systems)diluted in 2% tween-PBS. After incubation, the tablet was washed three times with SIP the soup PBS, 0.05% tween, and then incubated for 1 hour in the presence of the secondary detector antibody (1/2000 anticoding Ig HRP conjugate). After incubation, the tablet was washed three times using PBS, 0.05% tween, and once using PBS and abusively shaking on paper. ELISA showed using 100 μl 3,3',5,5'-tetramethylbenzidine as substrate and the reaction stopped with 50 μl 1M chloroethanol acid. The presence of non-covalent conjugates DOM7h-8 and saporin confirmed by comparing OD600conjugate with OD600any of unconjugated parts.

Table 6
DOM7h-8/saporinDOM7h-8 separatelySaporin separately
OD600(the tablet is covered using HAS)0,311to 0.0600,079
OD600(the tablet is blocked with 2% tween-PBS)0,0780,0680,075

The results of this study demonstrated that the drug can be konjugierte with antigennegative fragment antibodies to the e binds serum albumin, and that conjugated antigennegative the fragment retains the binding activity of serum albumin. In addition, due to the stability and strength of the Biotin-streptavidin combination, the results shows that it is possible to obtain covalently linked and ecovalence related conjugates that retain the activity of binding to serum albumin, owned antigennegative fragment of the antibody that binds serum albumin.

Example 8. Anti-SA dAb/Fluorescein conjugate

Fluoresceinisothiocyanate (FITC) can be cross-linked with amino, sulfhydryl, imidazole, tyrosyl or carbonyl groups of the protein. He has a molecular weight of 389 Yes, which is comparable in size with many small molecules drugs. The results obtained with this conjugate, demonstrating that anti-SA dAb supports their binding characteristics of serum albumin in connection with low chemical structure, and show that small molecule drugs can be konjugierte with anti-SA dAbs.

Concentrated DOM7h-8cys was obtained as described in Example 7. In concentrated dAb was replaced with a buffer of 50 mm borate, pH 8 (binding buffer) using NAP5 cialisbuy column (GE Healthcare/Amersham Biosciences, NJ) and concentrated to a concentration of 2.3 mg/ml using a hub METHOD with RA shall by volume of 2 ml (Millipore Corp., MA). FITC (Pierce Biotechnology Inc.) diluted to a concentration of 10 mg/ml in dimethylformamide (DMF) according to the manufacturer's instructions and then mixed with a dAb in binding buffer in a molar ratio of 24:1 FITC:dAb. The reaction was carried out for 30 minutes. At this point, the excess unreacted FITC was removed from the reaction using PDIO cialisbuy column (GE Healthcare/Amersham Biosciences, NJ), which is pre-balanced with the help of PBS, and suirable DOM7h-8cys/ FITC conjugate using PBS.

To confirm that the reaction of the compound FITC/dAb successfully used a "sandwich" ELISA for detection of bound dAb. Inflicted human serum albumin (HSA) on half of the holes ELISA tablet (Nunc, NY) overnight at a concentration of 10 μg/ml in a volume of 100 μl per well. After incubation overnight, the entire tablet was washed three times using PBS, 0.05% tween, and then all wells were blocked for 2 hours with 2% tween-PBS. After blocking the tablet was washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of DOM7h-8cys/FITC, diluted to a concentration of 1 μm in 2% tween-PBS. As a control, on the same tablet ELISA incubated control FITC-coupled antibody at a concentration of 1 μm and unrelated DOM7h-8 at a concentration of 1 μm in 2% tween-PBS. Additional monitoring was the same three diluted protein encubierta the data to the wells of ELISA tablet, not covered with the help of HSA and blocked with 2% tween. After incubation, the tablet was washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of a 1/500 dilution of rat anti-FITC antibody (Serotec)diluted in 2% tween-PBS. After incubation, the tablet was washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of a secondary detection antibody diluted in 2% tween-PBS (1/5000 antikrizisnyi Ig HRP conjugate). After incubation, the tablet was washed three times using PBS, 0.05% tween, and once using PBS and abusively shaking on paper. ELISA showed using 100 μl per well of 3,3',5,5'-tetramethylbenzidine as substrate and the reaction stopped with 50 μl per well of 1 M hydrochloric acid. The presence of conjugates of DOM7h-8 and FITC was confirmed by comparing OD600conjugate with OD600any of unconjugated parts.

Table 7
DOM7h-8/ FITCDOM7h-8 separatelyFITC-coupled antibody (negative control)
OD600(the tablet is covered using HSA)0,3800,042 0,049
OD600(the tablet is blocked with 2% tween-PBS)0,0410,0410,045

Example 9. Anti-SA dAb/peptide conjugates

Many peptides have therapeutic effects. Model peptides with N - or C-terminal cysteine can be connected with dAb against serum albumin.

In this case, will be used four different peptide: peptide 1 YPYDVPDYAKKKKKKC (SEQ ID NO:68); peptide 2 CKKKKKKYPYDVPDYA (SEQ ID NO:69); peptide 3 HHHHHHKKKKKKC (SEQ ID NO:70) and peptide 4: KKKKKKKKKKKK (SEQ ID NO:71). Peptides 1 and 2 comprises a sequence hemagglutinin label (ON-label), and peptides 3 and 4 include the sequence His-tag. Concentrated DOM7h-8cys receive, as described in Example 7.

Concentrated dAb restore using 5 mm dithiothreitol (DTT) and then replace the buffer binding buffer (20 mm BisTris pH 6.5, 5 mm EDTA, 10% glycerol) using a NAP5 dialysis column (GE Healthcare/Amersham Biosciences, NJ). Cysteine block (to prevent the reaction of dAb with each other) using a final concentration of 5 mm dithiodipyridine, which is added to the dAb solution of the initial solution with a concentration of 100 mm dithiodipyridine in DMSO. dAb and dithiodipyridine incubated for a reaction time of linking for 20-30 minutes. Unreacted dithiodipyridine removed using A dialysis column, and dAb elute in binding buffer (20 mm BisTris pH 6.5, 5 mm EDTA, 10% glycerol). The obtained protein is then frozen until needed.

Peptides 1-4 separately dissolved in water at a concentration of 200 μm, restore using 5 mm DTT and then absoluut using NAP5 dialysis column (GE Healthcare/Amersham Biosciences, NJ). Each peptide is then added to the solution recovered and blocked dAb in the ratio of 20:1 for the communication peptide-dAb. To confirm the successful conduct of the reactions of compounds of peptide and dAb, "sandwich" ELISA used for detection of anti-SA dAb/peptide conjugates.

Put human serum albumin by ELISA tablet (Nunc, NY) overnight at a concentration of 10 μg/ml in a volume of 100 μl per well. After incubation over night tablet is washed three times using PBS, 0.05% tween and then blocked for 2 hours using 4% Marvel PBS. After blocking the tablet is washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of DOM7h-8/peptide conjugates diluted to a concentration of 1 μm in 4% Marvel PBS. As a control, on the same tablet ELISA incubated unbound peptide at a concentration of 20 μm and unrelated DOM7h-8 at a concentration of 1 μm in 4% MPBS. After incubation, the tablet is washed three times using PBS, 0.05% tween, and then incubated for 1 hour in the presence of the tvii 1/2000 dilution of goat anti-antibody (Abeam) for peptides 1 and 2 and 1/2000 dilution Ni-NTA-HRP (for peptides 3 and 4), diluted in 4% Marvel PBS. After incubation, the tablet is washed three times using PBS, 0.05% tween, and the wells with goat anti-antibody incubated for 1 hour in the presence of secondary anticoding HRP antibody diluted 1/2000 in 4% MPBS (other wells were blocked for 1 h). After incubation, the tablet is washed three times using PBS, 0.05% tween, and once using PBS and dried by shaking on paper. ELISA are using 3,3',5,5'-tetramethylbenzidine as substrate and the reaction stopped with 1 M chloroethanol acid. The presence of conjugates of DOM7h-8/peptide conjugate is confirmed by comparing OD600conjugate with OD600any of unconjugated parts.

Table 8
Anticancer peptides
Category peptidePeptide sequenceAction/application
LH-RH agonists and antagonistsp-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 SEQ ID NO:89Treatment of malignant diseases that are dependent on sex hormones
Gastrin-releasing peptidep-Glu-Gln-Arg-Leu-Gy-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 SEQ ID NO:90 Small cell carcinoma of lung
Somatostatinp-Ala-Gly-Cys-Lys-Asn-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys SEQ ID NO:91Malignant neoplasms (generic)
GH-RHGln-Trp-Ala-Val-Gly-His-Leu-psi(CH2-NH)-Leu-NH2 (RC-3094) SEQ ID NO:92Glioblastoma, prostate cancer
VEGFArg-Arg-Lys-Arg-Arg-Arg SEQ ID NO:93Human colon cancer
Ala-Thr-Trp-Leu-Pro-Pro-Arg SEQ ID NO:94The proliferation of malignant cells
Arg-Thr-Glu-Leu-Asn-Val-Gly-Ile-Asp-Phe-Asn-Trp-Glu-Tyr-Pro-Ala-Ser-Lys SEQ ID NO:95Proliferation and migration of malignant cells
His-His-Glu-Val-Val-Lys-Phe-Met-Asp-Val-Tyr-Gln SEQ ID NO:96Inhibits the response of endothelial cells
Asn-Ile-Thr-Val-Thr-Leu-Lys-Lys-Phe-Pro-Leu SEQ ID NO:97Angiogenesis inhibitor
EGFCys-His-Ser-Gly-Tyr-Val-Gly-Val-Arg-CysInhibits EGF-OS is consistent cell

SEQ ID NO:98the proliferation
Tyr-Cys-Asp-Gly-Phe-Tyr-Ala-Cys-Tyr-Met-Asp-Val-Nh2 SEQ ID NO:99Binds HER2
IL-6Gly-Gly-Cys-Lys-Leu-Trp-Thr-Ile-Pro-Glu-Cys-Gly-Gly SEQ ID NO: 100Inhibits cell growth
IL-8Ala-Val-Leu-Pro-Arg SEQ ID NO:101Induction of apoptosis and antitumor effect in vivo
PDGFTyr-Gly-Arg-Pro-Arg-Glu-Ser-Gly-Lys-Lys-Arg-Lys-Arg-Lys-Arg-Leu-Lys-Pro-Thr SEQ ID NO:102Inhibits the growth of malignant gliomas
TNFAcCys-Pro-Ser-Glu-Gly-Leu-Cys-NH2 SEQ ID NO:103Inhibits tumor growth
Ac-Cys-Pro-Ser-Glu-Gly-Thr-Pro-Ser-Thr-His-Val-Leu-Cys-NH2 SEQ ID NO:104
Ac-Leu-Ala-Asn-Gly-Val-Glu SEQ ID NO:105
Pro-Gln-Ala-Glu-Gly-Gln-Leu-NH2 SEQ ID NO:106
Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu SEQ ID NO:107
Cyclic Lys-Gly-Asp-Gln-Leu-Ser SEQ ID NO:108
Cyclic Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly SEQ ID NO:109
Alpha-FetoGlu-Met-Thr-Pro-Val-Asn-Pro-GlyInhibits estrogen-
proteinSEQ ID NO:110dependent breast cancer cells
Sialyl-Lewis mimeticIle-Glu-Leu-Leu-Gln-Ala-Arg SEQ ID NO:111Inhibits the colonization of the lung tumor cells
Urokinasetype plasminogen activatorCys-Val-Ser-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-His-Trp-Cys SEQ ID NO:112Antagonist for uPA/uPAR
Phe-X-X-Tyr-Lys-Trp SEQ ID NO:113Antagonist for uPA/uPAR
Lys-Trp-X-X-Ar SEQ ID NO:114Antagonist for uPA/uPAR
Leu-Asn-Phe-Ser-Gln-Tyr-Leu-Trp-Tyr-Thr-NH2 SEQ ID NO:115Antagon the article for uPA/uPAR
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH2 SEQ ID NO:116Inhibits tumor progression and angiogenesis
p53Ac-Met-Pro-Arg-Phe-Met-Asp-Tyr-Trp-Glu-Gly-Leu-Asn-NH2 SEQ ID NO: 117Inhibits Hdm2 and p53 binding
Met-Val-Arg-Arg-Phe-Leu-Val-Thr-Leu-Arg-Ile-Arg-Arg-Ala-Cys-Gly-Pro-Pro-Arg-Val SEQ ID NO:118Prevents widespread p53
Gly-Ser-Arg-Ala-His-Ser-Ser-His-Leu-Lys-Ser-Lys-Gly-Gln-Ser-Thr-Ser-Arg-His-Lys-Lys-Leu SEQ ID NO:119Activates p53
p34cdc2Cys-Ala-Phe-Tyr-Ile SEQ ID NO: 120Inhibits the interaction between P34/P33 and pRb2 and R
Leu-Cys-Ala-Phe-Tyr-Ile-Met-Ala-Lys SEQ ID NO:121
Met-Cys-Ser-Met-Tyr-Gly-lle-Cys-Lys SEQ ID NO:122
Cdk2Tyr-Ser-Phe-Val-His-Gly-Phe-Phe-Asn-Phe-Arg-Val-Ser-Trp-Arg-Glu-Met-Leu-Ala SEQ ID NO:123Inhibits the interaction between Cdk2 and histone H1
p21WAF1Lys-ArgArg-Gln-Thr-Ser-Met-Thr-Ala-Phe-Tyr-His-Ser-Lys-Arg-Arg-Leu-Ile-Phe-Ser SEQ ID NO:124 Induces growth arrest of G1/S
Lys-Arg-Arg-Leu-Ile-Phe-Ser-Lys SEQ ID NO:125
Phe-Leu-Asp-Thr-Leu-Val-Val-Leu-His-Arg SEQ ID NO:126
E2F/DP transcriptionArg-Cys-Val-Arg-Cys-Arg-Phe-Val-Val-Trp-Il-Gly-Leu-Arg-Val-Arg-Cys-Leu-Val SEQ ID NO:127Inhibited E2F function in vitro
Leu-Asn-Trp-l-Trp-l-l-Glu-Val-Leu-Lys-Val-Gln-Lys-Arg-Arg-lle-Tyr-Asp-Ile-Thr-Asn-Val SEQ ID NO:128
Leu-Glu-Gly-Ile-Gln-Leu-lle-Ala-NH2 SEQ ID NO:129
Phe-Trp-Leu-Arg-Phe-Thr SEQ ID NO:130
Trp-Val-Arg-Trp-His-Phe SEQ ID NO:131
Trp-Val-Arg-Trp-His
SEQ ID NO:132
Trp-His-Phe-Ile-Phe-Trp SEQ ID NO:133
Ile-Trp-leu-Ser-Gly-Leu-Ser-Arg-Gly-Val-Trp-Val-Ser-Phe-Pro SEQ ID NO:134
Gly-Ser-Arg-Ile-Leu-Thr-Phe-Arg-Ser-Gly-Ser-Trp-Tyr-Ala-Ser SEQ ID NO:135
Asp-Glu-Leu-Lys-Arg-Ala-Phe-Ala-Ala-Leu-Arg-Asp-Gln-Ile SEQ ID NO:136
l2Lys-Lys-Leu-Ser-Glu-Cys-Leu-Lys-Lys-Arg-Ile-Gly-Asp-Glu-Leu-Asp-Ser SEQ ID NO:137Causes apoptosis in free cell
Gly-Gln-Val-Gly-Arg-Gln-Leu-Ala-Ile-Ile-Gly-Asp-Asp-Ile-Asn-Arg SEQ ID NO:138
Arg-Asn-Ile-Ala-Arg-His-Leu-Ala-Gln-Val-Gly-Asp-Ser-Met-Asp-Arg SEQ ID NO:139
IntegrinsTyr-Ile-Gly-Ser-Arg-NH2 SEQ ID NO:140Inhibit the binding of tumor cells with ECMs
Ac-Tyr-Ile-Gly-Ser-Arg-NH2 SEQ ID NO:141
Ac-Tyr-Ile-Gly-Ser-Arg-NHCH3 SEQ ID NO:142
Ac-Tyr-Ile-Gly-Ser-Arg-N(CH3)2 SEQ ID NO:143
Phe(pNH2)-Ile-Gly-Ser-Arg-NH2 SEQ ID NO:144
Ac-Tyr-Ile-Gly-Ser-Arg-
NHCH(CH3)2 SEQ ID NO:145
CO(Asp-Tyr-Ile-Gly-Ser-Arg-NHPr)2 SEQ ID NO:146
Arg-Gly-Asp SEQ ID NO:147
Tyr-Ile-Gly-Ser-Arg SEQ ID NO:148
Ile-Pro-Cys-Asn-Asn-Lys-Gly-Ala-His-Ser-Val-Gly-Leu-Met-Trp-Trp-Met-Leu-Ala-Arg SEQ ID NO:149
Analogues of angiostatinSer-Pro-His-Arg-Pro-Arg-Phe-Ser-Pro-Ala SEQ ID NO:150
Ser-Pro-His-Ala-His-Gly-Tyr-Ile-Pro-Ser SEQ ID NO:151
Thr-Pro-His-Thr-His-Asn-Arg-Thr-Pro-Glu SEQ ID NO:152
Thr-Pro-His-Arg-His-Gln-Lys-Thr-Pro-Glu SEQ ID NO:153
Glu-Pro-His-Arg-His-Ser-Ile-Phe-Thr-Pro-Glu SEQ ID NO: 154
KatherineAc-Cys-His-Ala-Val-Cys-NH2 SEQ ID NO:155Angiogenesis inhibitor
Histone deacetilazaCys-Glu-Lys-His-Ile-Met-Glu-Lys-Ile-Gln-Gly-Arg-Gly-Asp-Asp-Asp-Asp (SEQ ID NO:156Inhibition of leukemia
MRCys-Thr-Thr-His-Trp-Gly-Phe-Thr-Tumor metastasis
Leu-Cys SEQ ID NO:157

Example 10. Analysis of GLP Composition of the Medicinal product

Efficiency insulinotropic agent can be determined by calculation values AS on the basis of the curve of dependence "dose-effect". Purified plasma membranes from stable transtitional cell lines, UNC-12A (tk-ts13)expressing human GMP-1 receptor, stimulate using GLP-1 and analogues of the peptide, and the generation efficiency of camp (adenosine 3',5'-cyclic monophosphate) was measured using the AlphaScreen™ camp assay kit from Perkin Elmer Life Sciences.

Get a stable transtitional cell line and selected for screening clone with high expressing ability. The cells are then cultured in a 5% CO2in DMEM (Wednesday Needle in the modification of Dulbecco), 5% FCS (feta the other calf serum), 1% Pen/Strep and 0.5 mg/ml G418.

Cells with approximately 80% confluently washed 2X with the help of PBS and harvested using Versene (0.1 M NaCl, 1.5 mm KN2RHO4, 2.7 mm KCl, 8.0 mm PA2NRA4, 1.0 mm EDTA (ethylenediaminetetraacetic acid), 5.6 mm glucose), centrifuged for 5 minutes at 1000 rpm and remove supernatant. An additional stage is conducted under cooling on ice. The cell sediment homogenized using Ultrathurax for 20-30 sec in 10 ml of Buffer 1 (20 mm Na-HEPES (2-[4-(2-hydroxyethyl)-1-piperazine]econsultancy acid), 10 mm EDTA, pH 7.4), centrifuged for 15 minutes at 20,000 rpm and the cell sediment resuspended in 10 ml of Buffer 2 (20 mm Na-HEPES, 0.1 mm EDTA, pH 7.4). The suspension is homogenized for 20 to 30 seconds and centrifuged for 15 minutes at 20000 rpm, Suspension in Buffer 2, homogenization, and centrifugation is repeated once, and membrane resuspended in the Buffer 2, which is ready for further analysis or stored at -80°C.

The analysis of the functional receptor is carried out by measuring the induced peptide generation of camp by using AlphaScreen Technology. The basic principle of AlphaScreen Technology is competition between endogenous and exogenously added camp Biotin-camp. Capture of camp reach through the use of specific antibodies conjugated to acceptor grains. Educated camp calculate and measure the on AlphaFusion Microwell Analyzer. Values IS calculated using a computer program Graph-Pad Prisme.

The stability of the peptide to the destruction of dipeptidyl aminopeptidase IV can be installed in the following analysis of degradation: an Aliquot of peptide incubated at 37°C With an aliquot of the purified dipeptidyl amino peptidases IV within 4-22 hours in a suitable buffer at pH 7-8 (buffer is not albumin). The enzymatic reaction is stopped by the addition triperoxonane acid (TFA), and the decomposition products of the peptide separated and quantitatively determined using HPLC (high performance liquid chromatography) or LC-MS (liquid chromatography with massspectrometric definition) analysis. The mixture is applied on the Bond 300SB-C18 (30 nm pores, 5 μm particles) column, 150×2.1 mm and elute at a rate of 0.5 ml/min using a linear gradient of acetonitrile in 0.1% triperoxonane acid (0% to 100% acetonitrile over 30 min). Peptides and their degradation products can be monitored by their absorbance at 214 nm (peptide bond) or 280 nm (aromatic amino acids) and to quantify the integrated areas of their peaks. The profile decomposition can be installed using LC-MS to determine the mass spectra of the separated peak. The percentage ratio between the intact and the decomposed compound in a given time is used to assess peptide the th DPPIV stability.

The peptide denoted as DPPIV stabilised, if it is 10 times more stable than the natural peptide, based on the percentage of intact compound in a given time. Thus, DPPIV stabilised GLP-1 compound is at least 10 times more stable than GLP-1 (7-37).

Stimulation of adenylate-cyclase activity

BRIN-BD11 cells were seeded in 24-well plates (H/ per well) and cultured for 48 h with prior incubation in medium with the addition of labeled tritium adenine (2 MS) for 16 hours the cells are Then washed twice with cold Hanks buffered saline solution (HBS) and add test solution (400 μl; 37°C). The cells are then treated with various concentrations (10-10-10-5 M) GLP-1 compounds in HBS buffer in the presence of 1 mm IBMX (fosfodiesterazu inhibitor 3-isobutyl-1-methylxanthines) and 5.6 mm glucose (20 min; 37°C). After incubation the test solutions were removed and add 300 ál lyse solution (5% TFA, 3% SDS (sodium dodecyl sulfate), 5 mm unlabeled ATP (adenosine triphosphate) and 300 μm unlabeled cAMP). Dowex and aluminum ion-exchange resin is used to separate labeled with tritium cAMP and labeled with tritium adenine and ATP in the cell lysate as described (Miguel JC, et al. Biochem. Pharmacol. 2003, 65:283).

Insulin secretory responses can be measured in pancreatic β-cells BRIN-BD11 cells. Cells were seeded in 24-is nehalennia tablets at a density of 1×10 5/ well and incubated for accession during the night of cultivation. Immediate investigation of the release of insulin is preceded by a 40-min pre-incubation at 37°C in 1.0 ml of bicarbonate buffer Krebs-ringer's solution (115 mm NaCl, 4,7 mm KCl, 1,28 mm CaCl2·2H2O, 1.2 mm KN2RHO4, 1.2 mm MgSO4·H2O, 10 mm Panso3and 5 g/l bovine serum albumin, pH 7.4) with the addition of 1.1 mm glucose. Test-incubation carried out at 37°C in the presence of 5.6 mm glucose with a range of concentrations of GLP-1 compounds (10-12-10-6 M). After 20 min incubation buffer is removed from each well and aliquots stored at -20°C for measurement of insulin.

Lowering glucose and insulin secretory activity in obese diabetic (ob/ob) mice

The biological activity of GLP-1 compounds in vivo can be assessed in obese diabetic (ob/ob) mice at the age of 12-16 weeks. Animals are placed individually in a room with air conditioning at 22±2°C With 12-hour day: a 12-hour night cycle. Animals in excess of providing clean drinking water and regular access to a standard diet content rodents. Mice subjected to fasting for 18 h and injected intraperitoneally in the amount of 8 ml/kg of body weight saline solution (9 g/l NaCl), glucose alone (18 mmol/kg body weight) or in combination with GLP-1 compound (25 nm/kg body weight). brassy blood collected in chilled fluoride/heparin tubes for microcentrifuge immediately before injection and after 15, 30 and 60 min after injection, and the resulting plasma was stored at -20°C.

Other methods of analysis

The levels of plasma glucose can be set using the Analox glucose analyzer (Hammersmith, London, UK), which use the method of glucose oxidase (Stevens JF, Clin. Chim. Acta 1971, 32:199). Insulin levels can be assessed using radioimmunoassay analysis-loaded dextran coal dust (Flatt PR and Bailey CJ, Diabetologia 1981, 20:573). Differential area under the curve of glucose and insulin in plasma (AUC) can be calculated using GraphPad PRISM version 3.0 (Graphpad Software, San Diego, CA, USA).

The activity of GLP-1 compounds may be part of the composition of the medicinal product according to the present invention provided that the GLP-1 drug is able to bind and induce signalling via GLP-1 receptor. Binding of GLP-1 receptor and signal transduction can be assessed using methods of in vitro assays, such as those described in Examples 2, 3 and 4 of the EP 619322 and Examples 1, 2 and 3 of U.S. Patent No. 5120712, respectively (incorporated herein by reference).

Pharmacokinetic studies can be performed as described in Example 7 of WO 02/46227 (incorporated herein by reference).

Increasing the half-life of GLP-1 derivatives after intravenous or subcutaneous injection

The increase in lane is an ode to the half-life of GLP-1 analogs can be set by monitoring their concentrations in plasma after subcutaneous injection in healthy pigs. For comparison, you can monitor the concentration of plasma GLP-1 (7-37) (natural active form of GLP-1, used as control) after subcutaneous injection.

Compound is dissolved in a carrier suitable for subcutaneous or intravenous administration. Concentration will adjust so that the dosage volume is approximately 1 ml of a Study carried out on 12 dwarf pigs Guttingen male from Ellegaard Gottingen Minipigs ApS. Spend acclimatization for about 10 days before the animals are subjected to study. At the beginning of the acclimatization period, the age dwarf pigs is approximately 5 months, and they weigh in the range of 8-10 kg

The study was conducted in a suitable room vivarium with room temperature set at 21-23°C and relative humidity of approximately 50%. The room lights to ensure a cycle of 12 hours light and 12 hours darkness. Light include from 06.00 to 18.00 hours the Animals are placed in small pens with bedding of straw, six in each pen. Throughout the study the animals have free access to the local drinking water quality, but fast from approximately 16.00 hours on the day before injection to approximately 12 hours after injection. Animals are weighed on arrival and during the injection.

Animals receive a single int is venous or subcutaneous injection. Subcutaneous injection are doing on the right side of the neck approximately 5-7 cm from the ear and 7-9 cm from the middle of the neck. Injections do with limiter on the needle to enter 0.5cm needle. Each test compound is administered to three animals. Each animal received a dose equal to 2 nmol/kg of body weight. Six animals injected during the week, while the six remaining resting.

A full profile of the dependence of concentration on time in the plasma get from each animal. Blood samples are collected according to the following scheme: After intravenous injection: Pre-dose (0), 0,17 (10 minutes), 0,5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 hours after injection. After subcutaneous injection:

Pre-dose(0), 0,5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 hours after injection.

In every moment of the sample taken 2 ml of blood from each animal. Samples of blood taken from the jugular vein. Blood samples collected in test tubes containing buffer for stabilization to prevent enzymatic destruction of GLP-1 analogues. The plasma was immediately transferred to Micronic tube. Approximately 200 μl of plasma is transferred into each Micronic tube. Plasma was stored at -20°C until analysis. Plasma samples will be analyzed by the content of GLP-1 analogues using enzyme immunoassay.

Profiles dependence of concentration on time in the plasma is analyzed by nameparameter Pharma is kineticheskogo method of analysis. In each case, calculate the following pharmacokinetic parameters: AUC, AUC/Dose, AUC%extrapolationWithmax, tmax, λz, t1/2, CL, CL/f, VzVz/fand MRT (mean retention time).

The composition of the invention can also be experienced on the breed pigs Danish Landrace.

Pigs (50% Duroc, 25% of Yorkshire, 25% Danish Landrace, approximately 40 kg) on hunger strike since the beginning of the experiment. Each pig is injected with 0.5 nmol of the tested composition per kg body weight 50 PM isotonic (5 mm phosphate, pH 7.4, with 0.02% Tween®-20 (Merck), 45 mg/ml mannitol (pyrogen-free, Novo Nordisk)). Blood samples are taken from a catheter in the jugular vein. 5 ml blood sample is poured into a chilled glass vials containing 175 μl of the following solution: 0.18 M EDTA, 15000 CE(kallickrein inactivating units)/ml Aprotinin (Novo Nordisk) and 0.30 mm Valine-Pyrrolidide (Novo Nordisk), a pH of 7.4. Over the next 30 min the samples centrifuged for 10 min at 5-6000g. The temperature of the support at 4°C. the Supernatant dispense pipette in a variety of glass bottles and stored at minus 20°C until use.

The concentration of peptides in the plasma set using a "sandwich" ELISA or RIA (radioimmunosorbent analysis) using different mono - or polyclonal antibodies. Selection of antibodies depends on GLP-1 analogue. The time at which maximum concentration in plasma, argyrol in a wide range depending on the specific GLP-1 analogue.

General Protocol analysis for sandwich ELISA in 96-well tablet for micrometrology

The buffer coating (PBS): phosphate buffered saline, pH 7,2

Buffer for washing (PBS-wash): phosphate buffered saline, 0.05 per cent.about. Tween-20, pH 7,2

Analytical buffer (BSA-buffer): phosphate buffered saline solution, 10 g/l bovine serum albumin (Peak 05477), 0,05% vol./about. Tween-20, pH 7,2

Streptavidin buffer: phosphate buffered saline, 0.5 M NaCl, 0,05% vol./about. Tween-20, pH 7,2

Standard: individual compounds in plasma matrix

A-TNP: nonsense antibody

AMDEX: streptavidin-horseradish peroxidase (Amersham RPN4401V)

TMB-substrate: 3,3',5,5'-tetramethylbenzidine (<0.02%), hydrogen peroxide

The analysis is performed in accordance with the following (volume/per well):

1. Spray coated using 100 μl of capture antibody at a concentration of 5 μg/ml in PBS-buffer, incubate over night at 4°C, wash 5x PBS-washing, block, using the last portion of the washing for at least 30 minutes, then empty the tablet.

2. 20 μl sample +100 µl biotinylated detection antibody at a concentration of 1 μg/ml in BSA-buffer with 10 μg/ml A-TNP, incubate for 2 h at room temperature with shaking, rinse 5x PBS-washing, then the empty clause is unset.

3. 100 μl AMDEX 1: 8000 in streptavidin buffer, incubate for 45-60 minutes at room temperature with shaking, rinse 5x PBS-wash, then empty the tablet.

4. 100 ál of TMB-substrate, incubate at room temperature with shaking, stop the reaction by using 100 μl of 4M N3RHO4. Read the absorbance at 450 nm with 620 nm as a comparison. The concentration of the samples can be calculated from standard curves.

General Protocol analysis for RIA

DB-buffer: 80 mm phosphate buffer, 0.1% human serum albumin, 10 mm EDTA, 0.6 mm thiomersal, pH 7.5.

FAM-buffer: 40 mm phosphate buffer, 0.1% human serum albumin, 0.6 mm thiomersal, pH 7.5.

Coal dust: 40 mm phosphate buffer, 0.6 mm thiomersal, 16.7% of bovine plasma, 15 g/l of activated carbon, pH 7.5 (mix the suspension for at least 1 h before use at 4°C)

Standard: individual compounds in plasma matrix

The analysis is performed in test tubes minisorp 12×75 mm (volume/per tube) in accordance with the following:

Coal dust H2O
DB-bufferSampleAntibodyFAMisotopic label
Day 1
Only100 µl
NSB330 ál100 µl
Sample300 ál30 ál100 µl100 µl
Mix, incubate over night at 4°C Day 2
Only 1.5 ml
NSB1.5 ml
Sample1.5 ml

Mix and incubate for 30 min at 4°C. Centrifuge at 3000 rpm for 30 min, and immediately after that move the supernatant in a new tube, close the lid and assess on a gamma counter for 1 minute. The concentration in the samples calculated on the basis of individual standard curves.

GLP-1 radio receptor assay (RRA):

GLP-1 radio receptor analysis is a radiometric legendbase.ui analysis method using LEADseeker radioisotope particles. The analysis is built from fragments of membranes containing GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1, labeled by125125I-labeled GLP-1 will compete for binding to the receptor. When you add LEADseeker particles, they will bind to carbohydrate residues on the fragments and outreach to consumers through WGA-residues. The proximity between125I-molecules and LEADseeker particles causes the emission of light particles. LEADseekr will reflect the outgoing light, and it is reversible correlated with the amount of GLP-1 analogue in the sample.

Reagents and Materials:

Preparation of animal plasma: the Plasma of animals thermally treated for 4 h at 56°C and centrifuged at 10,000 rpm for 10 minutes. After that add Val-Pyr (10 μm) and Aprotinin (500 KIE/ml) and stored at -18°C until use.

GLP-1 analogues: GLP-1 analogues contribute in heat-treated plasma to obtain a dilution series ranging from approximately 1 μm to 1 PM.

GLP-1 RRA analytical buffer: 25 mm Na-HEPES (pH 7.5), 2.5 mm CaCl2, 1 mm MgCl2, 50 mm NaCl, 0.1% ovalbumin, of 0.003% tween-20, 0.005% bacitracin, 0.05% of NaN3.

Suspension GLP-1 receptor: membrane fragments of GLP-1 receptor purified from the kidney cells baby hamster (KSS)expressing human pancreatic GLP-1 receptor. Stored at -80°C until use.

WGA-linked polystyrene LEADseeker radioisotope particles (RPNQ026, Amersham): Particles dissolved in GLP-1 RRA analytical buffer to a concentration of 13.3 mg/ml were Then added, the suspension membrane GLP-1 receptor and incubated at refrigeration (2-8°C) for at least 1 h before use.

Materials

125I-GLP-I (7-36) amide (Novo Nordisk A/S). Kept at -18°C until use.

Ethanol 99.9% vol (De Dansk Sprotfabrikker A/S). Kept at -18°C until use.

Multiscreen® Sob/inert 0.45 μm hydrophobic PTFE (polytetrafluoroethylene) plate (MSRPN0450, Millipore Corp.).

Polypropylene 384-well plates (catalog No. 781075, Greiner Bio-One).

Equipment:

Horizontal mixer for tablet

Rotor centrifuge with a standard tablet for micrometrology and swinging basket

UltrVap, Drydown hub designs (Provair)

LEADseeker® multi-Modal imaging system (Amersham)

Technique:

Sample preparation: Select Multiscreen® Solvinert filter plate on similar chemical resistance to receiving the tablet (i.e. polypropylene tablets) for collecting the filtrate.

Add 150 ál of ice-cold ethanol 99.9% of the empty wells of MultiScreen® Solvinert filter tablet, and then add 50 μl of calibration solution or plasma sample. Place the protective cover on the filter pad and incubate for 15 minutes at 18-22°C on a horizontal mixer for the tablet.

The collected installation of the filter and niemnogo tablet, together with lid, place in the standard rotary installation with standard tablet for micrometrology and swinging basket. The filtrate is collected in the empty holes of the receiving plate at 1500 rpm for 2 minutes. The filtrate is dried to dryness using UltrVap when heated in a current of N2(40°C) for 15 minutes. The dry material was dissolved by adding 100 μl of GLP-1 RRA analytical buffer in each well. This is incubated for 5 minutes on a horizontal mixer.

GLP-1 radio receptor assay: Use the following pipetting scheme and white polystyrene 384-well tablets:

1. 35 μl of GLP-1 RRA analytical buffer.

2. 5 µl of the re-dissolved filtrate.

3. 10 ál125I-GLP-1 (7-36) amide. The original solution was diluted in GLP-1 RRA analytical buffer that will receive 20000 cpm (counts per minute, the number of pulses per minute/per well before using.

4. 15 μl of membrane fragments of GLP-1 receptor (0.5 μg/well), previously applied to the WGA-polystyrene LEADseeker radioisotope particles (0.2 mg/well).

The tablets covered and incubated overnight at 18-22°C. the Light emission from each well detected using multimodal imaging system LEADseeker for 10 minutes.

The stimulation of the formation of camp in the cell line expressing the cloned human GLP-1 recipe is R.

Purified plasma membranes from stable transtitional cell lines, UNC-12A (tk-ts13)expressing human GMP-1 receptor, stimulate using GLP-1 and analogues of the peptide, and the generation efficiency of camp was measured using the AlphaScreen™ camp assay kit from Perkin Elmer Life Sciences.

Get a stable transtitional cell line and selected for screening clone with high expressing ability. Cells were cultured at 5% CO2in DMEM, 5% FCS, 1% Pen/Strep and 0.5 mg/ml G418.

Cells with approximately 80% confluently washed 2X with the help of PBS and harvested using Versene, centrifuged for 5 minutes at 1000 rpm and remove supernatant. An additional stage is conducted under cooling on ice. The cell sediment homogenized using Ultrathurax for 20-30 sec in 10 ml of Buffer 1 (20 mm Na-HEPES, 10 mm EDTA, pH of 7.4), centrifuged 15 min at 20000 rpm and the cell sediment resuspended in 10 ml of Buffer 2 (20 mm Na-HEPES, 0.1 mm EDTA, pH 7,4). The suspension is homogenized for 20 to 30 seconds and centrifuged for 15 minutes at 20000 rpm, Suspension in Buffer 2, homogenization, and centrifugation is repeated once, and membrane resuspended in the Buffer 2, which is ready for further analysis or stored at -80°C. analysis of the functional receptor is performed by the measurement-induced peptide generation of camp when AlphaScreen Technology. The basic principle of AlphaScreen Technology is competition between endogenous and exogenously added camp Biotin-camp. Capture of camp reach through the use of specific antibodies conjugated to acceptor grains. Educated camp calculate and measure on AlphaFusion Microwell Analyzer. Values IS calculated using a computer program Graph-Pad Prisme.

Example 11. Bacterial expression of genetic merged design glucagon-like peptide-1 and IDom7h-8 with GAS leader sequence

GLP-1 (7-37), in which the glutamate at position 9 is replaced by Proline ([Pro9] GLP-1 (7-37)), cloned in the form of a fused design with iDOM7h-8 (P96E mutation according to the Kabat numbering in the CDR3) in pet 12A vector GAS leader sequence (see WO 05/093074). Substitution of GLP-1 glutamate for Proline at 9 position performed in order to give GLP-1 side slit design, the resistance to degradation by cleavage by dipeptidyl peptidase IV (DPPIV) (Brian D. Green et al. (2003) Metabolic Stability, Receptor Binding, camp Generation, Insulin Secretion and Antihyperglycaemic Activity of Novel N-terminal Glu9-substituted Analogues of Glucagon-like-peptide-1: Biol. Chem. (384) 1543-1555). In General, there were three designs, one without the linker, one with PSS amino acids between the [Pro9] GLP-1 (7-37) and iDOM7h-8 and one with PSSGAP amino acids between the [Pro9] GLP-1 (7-37) and iDOM7h-8 (shown in Fig in a bind albumin forms Dom7h-8). Expre is this happened in BL21 DE3 Plys S cells at 30°C for 48 hours using night expressing system autoinduction TV readymix (Novagen) before allocating the supernatant by centrifugation. Fused design [Pro9] GLP-1 (7-37) iDom7h-8 was purified from the supernatant using affinity chromatography on protein L-agarose. Ion-exchange resin was then washed with 10 column volumes of PBS and the bound protein was suirable 0.1 M glycine pH 2. Then put the merged design [Pro9]GLP-1(7-37)-iDom7h-8 in glycine buffer for cation-exchange column (1 ml S-column, GE healthcare), which was previously balanced 20 mm citrate buffer at pH of 6.2. Elution was carried out 0-50% gradient of the same buffer with the addition of 1 M NaCl. Collected fractions with maximum concentrations and the size of the proteins were determined using SDS-PAGE. Fractions with maximum concentrations of the protein of the expected size were combined and replaced the buffer to PBS. The protein identity was confirmed by mass spectrometry analysis and N-terminal sequencing.

Example 12. The determination of the activity of GLP-1 for fused design [Pro9]GLP-1(7-37)-PSSGAP-iDOM7h-8

To confirm that the merged design [Pro9]GLP-1(7-37)-PSSGAP-iDOM7h-8 was active GLP-1, the slit structure was subjected to two different biological methods of analysis. In the first method of analysis, cell line RINm5f rat insulinoma (developed in 1980 Gadzar et al from induced x-ray emission transplantable insulinoma rats) were incubated with various concentrations (from 10 PM to 0.1 MK is) GLP-1 and slit design [Pro 9]GLP-1(7-37)-PSSGAP-iDOM7h-8 for 60 minutes Additionally, as a control was added single analytical quantity Basis-4, GLP-1 analog that is resistant to destruction, dipeptidyl peptidase IV, and a single analytic number only buffer. Although RINm5f cells do not respond to glucose in the processing of nutrient substrates or not stimulants secretion, they are secretory responses, similar to beta-cells. Therefore, to assess the effect of compounds on cell proliferation by measuring the incorporated amounts of 5-bromo-2'-dose irradiation on neurogenesis (BrdU) during DNA synthesis in proliferating cells using the Cell proliferation ELISA system (Amersham, Little Chalfont, UK), see Fig. Applying OD450 to measure the levels of DNA, it was found a dose-dependent increase in the level of DNA simultaneously with the increase in the number of merged design [Pro9]GLP-1(7-37)-PSSGAP-iDOM7h-8 to the concentration of the slit design, equal to 100 nm. GLP-1 also showed the expected dose-dependent response.

In the second method of analysis, RINm5f cells were incubated with different concentrations (from 10 PM to 0.1 μm) GLP-1 and slit design [Pro9]GLP-1(7-37)-PSSGAP-iDOM7h-8 5.6 mm glucose for 60 minutes Additionally, as a control was added single analytical quantity Basis-4, GLP-1 analog that is resistant to destruction DPPIV, and a single analytical if estvo bicarbonate buffer Krebs-ringer (KRB). Insulin secretion was analyzed after incubation for 60 min at 37°C using KRB buffer with the addition of GLP-1, SE or on the Basis of 4. The reaction medium was collected, centrifuged and analyzed the insulin concentration in the supernatant using radioimmunotherapies analysis. The insulin concentration was correlated to the number of cells in each well. The insulin concentrations (expressed in ng/ml/h) was then plotted on the graph relative to the concentration of the compound. Established a dose-dependent increase in the release of insulin in increasing doses merged design [Pro9]GLP-1(7-37)-PSSGAP-iDOM7h-8 to the concentration of the slit design, equal to 10 nm (see Fig). These results are consistent with published data on GLP-1 separately.

Example 13. Bacterial expression of genetic merged design glucagon-like peptide-1 and iDom7h-8 using MRA-T leader sequence

The same 3 designs described in Example 11 (one without the linker, one with PSS amino acids between [Pro9]GLP-l (7-37) and iDOM7h-8 and one with PSSGAP amino acids between [Pro9]GLP-1(1-37) and iDOM7h-8), were made again with MRA-T leader sequence. For clarity, the order of the elements in the design was a MRA-T leader sequence [Pro9]GLP-1 linker (where necessary) and iDom7h-8. Expression occurred is in BL21 DE3 Plys S cells at 25°C for 4 hours in the TV medium, induced using 0.5 mm IPTG (isopropyl-thio-beta-galactoside) when the optical density (OD)equal to 16, before allocating cellular precipitate by centrifugation. Secreted proteins were then allocated processing periplasm. Fused design GLP-1 iDom7h-8 was purified from periplasmic fractions using affinity chromatography on protein L-agarose. Ion-exchange resin was then washed with 10 column volumes of PBS and the bound protein was suirable 0.1 M glycine pH 2. Then put the merged design [Pro9]GLP-1(7-37) in glycine buffer for cation-exchange column (1 ml S-column, GE healthcare), which was previously balanced 20 mm citrate buffer at pH of 6.2. Elution was carried out 0-50% gradient of the same buffer with the addition of 1 M NaCl. In this case, the washing column 20 mm citrate buffer at pH of 6.2 (0% NaCl) led to leaching (appear in the eluate) bands of the expected size, so it was concentrated using 5K vivaspin columns (Vivascience).

Example 14. Expression in Pichia pastoris genetic merged design glucagon-like peptide-1 and iDom7h-8

Fused design [Pro9]GLP-l-PSS-iDOMyh-S (as shown on Fig.16b, but using iDom7h-8) clone in pPICzα vector both separately and with N-terminal EAEA elongation and transformed into Pichia pastoris KM71h. Protein Express (1) using induction methyl alcohol is m for 4 days at 30°C and (2) using induction with methyl alcohol for 2 days at 25°C. The supernatant allocate by centrifugation and check the size of the protein on SDS PAGE gel.

Expect the slit structures are the correct size, according to the results of SDS PAGE analysis, and are active in GLP-1 analysis as described in Example 10 and Example 12.

Example 15. Expression in E. coli glucagon-like peptide-1 and iDom7h-8 in BL21 DE3 cells intracellular

The same slit design, as described in Example 11, clone in rat expressing vector (Novagen), which is designed for expression of proteins in the cytoplasm. It is possible to include in the design proteiny the site of cleavage between subject removal of the N-end and NAR... [Pro9]GLP-1(7-37). This will allow you to break down protein, to provide a fully native N-end. Enzymes that can be used for this purpose include Factor XA, thrombin or DPPI. The protein then Express with a high content in BL21(DE3) cells after IPTG induction, and it accumulates in intracellular inclusion bodies. Intracellular calf separated from BL21 cells and solubilizer in guanidine-HCl. After recovery of intracellular Taurus is subjected to refolding buffer system rolling oxidation-reduction (Buchner, J., Pastan, I. Brinkmann, U. (1992). A method for increasing the yield of properly folded recombinant fusion proteins. Anal. Biochem. 205, 263-2170). After refolding, the protein deleteroute and concentrate on 5K vivaspin column (Vivascience) and purified on an S-to the column (GE healthcare).

Expect the slit structures are the correct size, according to the results of SDS PAGE analysis, and are active in GLP-1 analysis as described in Example 10 and Example 12.

Example 16. Expression in mammalian cells glucagon-like peptide-1 and iDom7h-8

Fused design [Pro9]GLP-1-PSS-DOMYh-S (as shown on Fig.16b) clone in PcDNA(-) vector using murine secretory signal peptide to facilitate secretion of the translated protein into the culture medium. 1 mg DNA get in E. Li using alkaline lysis (set of reagents mega prep, qiagen, CA) and transferout 1.5 l NEC cells cultured in the medium Needle in the modification of Dulbecco (Gibco) for transient expression of the protein. Protein Express by incubating the culture at 37°C for 5 days, and the supernatant (containing the expressed protein) was isolated by centrifugation. Fused design [Pro9]GLP-l-PSS-DOM7h-S clear from periplasmic fraction using affinity chromatography on L-agarose. Ion exchange resin is then washed with 10 column volumes of PBS and bound protein elute with 0.1 M glycine pH 2. Then put protein in glycine buffer for cation-exchange column (1 ml S-column, GE healthcare), which was previously balanced 20 mm citrate buffer at pH of 6.2. The carry out elution 0-50% gradient of the same buffer is with the addition of 1 M NaCl. A protein of the correct size, according to the results of SDS-PAGE gel, and then concentrated using 5K vivaspin columns (Vivascience) and replace the buffer in PBS for biological analysis.

Example 17. Expression in E. Li Peptide YY, merged with DOM7h-8

Peptide YY (3-36) (PYY: ID No. 167 amino acid sequence and ID No. 168 nucleotide sequence IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY) inhibits food intake in humans and has a short half-life from plasma (10-30 min). It is released in response to eating and working through Y2R in the arcuate nucleus for the physiological regulation of food intake. PYY clone in pet GAS vector (WO05093074)adjacent to DOM7h-8 (see Figa and 20b, which shows the peptide-end and N-end from DOM7h-8, respectively). The expression occurs in BL21 DE3 Plys S cells at 25°C for 4 hours in the TV medium, induced using 0.5 mm IPTG before allocating cellular precipitate by centrifugation. Secreted proteins then allocate processing periplasm. Fused design PYY Dom7h-8 clear from periplasmic fractions using affinity chromatography on protein L-agarose. Ion exchange resin is then washed with 10 column volumes of PBS and bound protein elute with 0.1 M glycine pH 2 and cleanse advanced ion exchange. In a purified protein then replace dialysis buffer in PBS, protein concentrate in 5vivaspin column (Vivascience) and subjected to biological analysis to measure the stimulation of the release of camp, as described (Goumain et al. (2001) The Peptide YY-Prefemng Receptor Mediating Inhibition of Small Intestinal Secretion Is a Peripheral 2 Receptor: Pharmacological Evidence and Molecular Cloning: Molecular pharmacology: 60 124-134). Briefly, isolated cells of the intestinal mucus at a concentration of 200 μg protein/ml and incubated under continuous stirring for 45 min at 15°C in 0.5 ml phosphate buffered saline, pH 7.0, containing 1,4% (wt./about.) bovine serum albumin, 0.1% bacitracin and 0.2 mm 3-isobutyl-1-methylxanthines (IBMX), as described (Servin et al. (1989): Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3',5'-monophosphate production in rat small intestine: structural requirements of peptides for interacting with PYY-preferring receptors. Endocrinology 124: 692-700). PYY separately or merged design PYY-Dom7h-8 type together with high-level physiological stimulator of camp in enterocytes (for example, VIP (vasoactive intestinal peptide)). The reaction is initiated by adding cells and stop after 45 min by adding 50 ál of THEM perchloro acid. After centrifugation for 10 min at 4000 g camp, located in the supernatant, treated with succinic acid in obtaining succinyl, and its concentration is measured radioimmunotherapy analysis as described (Laburthe et al., (1982) Alpha-adrenergic inhibition ofcyclic AMP accumulation in epithelial cells isolated fromrat small intestine. Biochim Biophys Acta 721: 101 -108).

Assume that the merged design has the expected size and manifests PYY activity, equivalen the ing nelita control.

Example 18. Expression in E.coli Dom7h-8 Peptide YY, GLP-1, slit design

Fused design [Pro9]GLP-1(7-37)-DOM7h-8-PYY (see Figs) clone in pet GAS vector and then Express as described for Dom7h-8 PYY shown in Example 17. After cleaning the slit design to analyze the biological activity of PYY and GLP-1, following the protocols described in Examples 17 and in Example 12, respectively.

Assume that the slit structures are expected size and show PYY and GLP-1 activity, equivalent nelita control.

The following additional examples, the following notation is used:

DAT 0115, 0116, and 0117 represent a merge of basis with a domain antibody that binds serum albumin (DAT 0115 contains a linker (g4s)3, a DAT 0117 contains helical linker, and 0116 has no linker);

DAT 0120 is a GLP-1 A8G (mutant), merge over helical linker domain antibody that binds serum albumin.

Example 19: the Expression of genetic m GLP-1 (A8G) or Basis 4 and DOM7h-14 AlbudAb:

Or on the basis of 4 or GLP-1 (7-37), where the alanine at position 8 is replaced by a glycine ([Gly8] GLP-1), cloned in fusion with DOM7h-14 (a domain antibody (dAb)that binds serum albumin (AlbudAb), with the amino acid sequence shown below) in the vector PTT-5 (m which can be obtained from the company CNRC, Canada). In each case, GLP-1 or on the Basis of 4 was located at the 5'-end design and dAb on the 3'-end. They received a total of 7 designs (DAT 0114, DAT 0115, DAT 0116, DAT 0117, DAT 0118, DAT 0119, DAT 0120). The linker was either absent or a linker represented gly-ser (G4S)or helical linker (Arai, R., H.Ueda, et al. (2001). "Design of the linkers which effectively separate domains of a bifunctional fusion protein." Protein Eng 14(8): 529-32 .456), or the linker consisted of the second group of GLP-1 and GLP-1 or Basis 4 and dAb. Linkers were included as spacers, in order to spatially separate the GLP-1 or on the basis of 4 from dAb to prevent steric obstacles binding between GLP-1 or Basis-4 and the receptor for GLP-1.

DNA, free of endotoxins, was obtained from E. coli using the alkaline lysis (using a set of Giga for plasmids, free of endotoxins, which can be obtained from Qiagen, CA)and used for transfection of cells NECE (which can be obtained from CNRC, Canada). The transfection was performed in 250 ml/flask of cells NICE with a 1.75×106cells/ml, using 333 μl of 293fectin reagent (Invitrogen) and 250 µg DNA per flask, and the expression was carried out at 30°C for 5 days. The supernatant was collected by centrifugation and purification was carried out by affinity purification on protein L. the Whole lot of protein bound resin was applied on the column and washed in PBS for 10 column volumes. Protein was suirable 50 ml of 0.1 M glycine with rn and neutralized, using Tris with a pH of 8. A protein of the expected size were identified by gel electrophoresis in SDS-PAGE; the dimensions are presented below in Table 9.

Table 9
Molecular weight structures DAT 0114, DAT 0115, DAT 0116, DAT 0117, DAT 0118, DAT 0119, DAT 0120
ProteinThe expected MW
DAT 011418256
DAT 011516896
DAT 011615950
DAT 011719798
DAT 011815936
DAT 011915318
DAT 012018895

Example 20: the Demonstration that the merged structure of GLP-1 and the basis-4 AlbudAb bind serum albumin.

The slit structure of GLP-1 and the basis-4 AlbudAb analyzed using surface plasma resonance (Biacore AB, which can be obtained from GE Healthcare) to obtain information on affinity. Analysis was performed using the chip SM Biacore (karboksimetilirovaniya Dexron as a matrix), to the which was covered with serum albumin. Approximately 1000 resonance units (RUs) of each test serum albumin (serum albumin human, rat and mouse) was immobilized in acetate buffer with pH 5.5. Flow cell 1 Biacore AB is not covered, blocked negative control, flow cell 2 was covered with human serum albumin (HSA) (815 RUs), flow cell 3 was covered with mouse serum albumin (RSA) (Eng 826), and flow cell 4 was covered with mouse serum albumin (MSA) (938 RUs). Each test merged molecule expressed in tissue culture mammals, as described in the example above.

Preparing a range of concentrations merged molecules (in the range from 16 nm to 2 μm) by dilution in buffer BIACORE HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mm EDTA, 0.005% of the surface of the substance P20) and was passed through the BIACORE chip.

The affinity (KD) was calculated on the basis of the BIACORE traces by approximation of the speed curve of the Association and the speed of dissociation to the traces formed by the concentrations of dAb in the field of KD. The values of the affinity (KD) are summarized in the following Table 10.

Table 10
Binding of GLP-1 and the basis-4 AlbudAb with human, mouse and rat serum albumin
/td> Fused design GLP-1 (7-37) A8G, helical linker DOM7h-14Fused design 2xGLP-1 (7-37) A8G DOM7h-14
HSA110 nm150 nm
RSA800 nm700 nm
MSA110 nm130 nm

The above results show that the fused molecules retain the ability to bind with all types of serum albumin, and this indicates that they probably have an increased half-life in vivo.

Example 21: Merged designs GLP-1 and the basis-4 AlbudAb active in the analysis of the binding of the receptor GLP-1 (GLP-1 R-VA):

Fused constructs were subjected to exchange buffer 100 mm NaVI, 20 mm citrate, pH of 6.2. At this time cells SNO 6CRE GLP1R (cells Cho K1 (which can be obtained from the American collection of typical cell cultures. ATSS), stable transfetsirovannyh element of answer 6 of camp, directing reporter gene luciferase and human receptor GLP-1), were sown at 2×105cells/ml in a suspension medium. The suspension culture was maintained for 24 hours. Then the cells were diluted in 15 mm HEPES buffer (obtained from Sigma), steriade is 2 mm L-glutamine (2,5×10 5cells/ml), and distributed in 384-well plates, containing 10 µl/well of the analyzed compounds. After adding a control for the analysis of the tablets were returned to the incubator for 3 h at 37°C and 5% CO2. After incubation, the substrate for luciferase steady-glo (which can be obtained from the company Promega) was added to the wells as described in the set, and tablets hermetically closed self-adhesive covers for tablets (Weber Marking Systems Inc. Cat. No. 607780). The tablets were placed in a reading device (Viewlux, Perkin Elmer) and pre-incubated for 5 minutes, then read fluorescence and put the results on a graph. The compound was analyzed in the concentration range in the presence and absence of 10 μm albumin, which gave the opportunity approximation curve dose-response with albumin and without albumin. Calculated values ECso and summarized in the following Table 11.

Table 11
The activity of the merged structure of GLP-1 and the Basis-4 AlbudAb in the analysis of the binding of the receptor GLP-1 (GLP-1 R-VA)
GLP-1R BA EU50(PM) n=3GLP-1 DITCH(10 μm albumin) EC50(RM) n=2
Fused design on the Basis of 4-(G4S)3-DOM7h-14 8,935
Fused design on the Basis of 4-DOM7h-141272
Fused design on the Basis of the 4 - helical linker - DOM7h-144,315
Fused design GLP-1 A8G - helical linker - DOM7h-1417130
GLP-1 7-362119
On the basis 41,00,82

The above results show that all tested merged molecules retain the efficiency of binding of the receptor GLP-1. These results also demonstrate that this efficiency is maintained in the presence of serum albumin. Therefore, these merged molecules will likely retain the ability to bind the receptor for GLP-1 in vivo.

Example 22: a Demonstration duration DAT 0115 in the mouse model db/db type II diabetes:

The purpose of this study was to determine the duration DAT 0115 on oral glucose tolerance in mice db/db. Animals were sorted by lowering levels of glucose for three days before the ex is eriment, and then they divided the party. Then one animal in each batch were appointed in each of the 26 study groups. This ensured similar average initial level of glucose in each of the study groups.

DAT 0115 (produced in cells NECK and purified as described above) was injected subcutaneously at 1 mg/kg, 0.3 mg/kg or 0.1 mg/kg for 5 h, 24 h, 48 h, 72 h, 96 h and 120 h before oral glucose load. (Not all doses were administered in each moment, see details in the table below). DAT 0115 significantly reduced the AUC of glucose over a 2-hour period of time of oral test glucose tolerance (OGTT) compared with mice db/db treated by the media, in moments of time outside and including 24 h for doses of 0.1 mg/kg and 0.3 mg/kg and outside and including the time point of 72 h for doses of 1 mg/kg on the Basis of 4 entered as a positive control at 42 mg/kg, significantly reduced the AUC of glucose after OGTT in the introduction for 5 h before oral bolus of glucose. The following table 12 shows the percentage reduction in AUC for each of the study groups DAT 0115 compared with the carrier. The asterisk is specified P<0.05 for DAT 0115 compared to media with use of the correction to the frequency of false alerts.

Table 12
Demonstration of the percentage of the reduction in AUC for each of the study groups DAT 0115 compared with the carrier. (The asterisk is specified P<0.05 for DAT 0115 compared to media with use of the correction to the frequency of false detections)
Time OGTT (hours relative to dosing)0.1 mg/kg DAT 01150.3 mg/kg DAT 01151 mg/kg DAT 0115
+560%*Did76%*
+2436%*59%*50%*
+4828%26%37%*
+7216%26%41%*
+96-12%Did12%

Example 23: a Demonstration of the effectiveness of DAT 0115 in the mouse model of obesity induced by diet (DIO):

The purpose of this study was to use an established model power mice (mice with obesity induced by diet)to determine whether the introduction of DAT 0115 on food consumption and, as a result, on body weight. This may have predictive value is e against people. Male mice C57BI/6 (purchased from Taconic) were fattened up to 60% kcal diet with high fat diet for 12 weeks and then transferred in your own vivarium. After arrival, the mice were placed in individual cages on litter Alpha Dry in a room with controlled temperature and humidity (70-72°F (21,11-22,22°C) humidity = 48-50%, the light cycle 5 AM/5 FM). The diet was changed to a diet high in fat 45%, and animals acclimatized for 18 days. Before the introduction of the test compounds to mice were subcutaneously injected with saline for three days and monitored for food intake. The mice were divided into parties and groups, so that body weight and food consumption did not differ between groups and within groups. On the day of the study, groups of 8 mice were injected subcutaneously dose, as described below, using the amount of injection of 5 ml/kg: Three groups were injected dose DAT 0115 (low, medium and high dose), the dose of the molecule negative control (DOM7h-14 AlbudAb, but without conjugate on the Basis 4) and the dose of the positive control of Basis 4.

Table 13
The Protocol for establishing the effectiveness of DAT 0115 in the mouse model of obesity induced by diet (DIO)
GroupInput connectionLevel dose
1Negative control: DOM7h-14 in 100 mm NaCl, 20 mm citrate/sodium citrate pH of 6.21 mg/kg
2On the basis 40.01 mg/kg
3DAT 0115 100 mm NaCl, 20 mm citrate/sodium citrate pH of 6.20.01 mg/kg
4DAT 0115 100 mm NaCl, 20 mm citrate/sodium citrate pH of 6.20.1 mg/kg
5DAT 0115 100 mm NaCl, 20 mm citrate/sodium citrate pH of 6.21 mg/kg

Daily food consumption and body weight were measured daily for 10 days. The results with DAT 0115 showed a dose-dependent decrease in body weight and food intake compared to control DOM7h-14. Therefore concluded that the data from this study on mice support the hypothesis that DAT 0115 must be of good clinical candidate.

While this invention was partially disclosed and described with reference to its preferred variants of the embodiment, the experts in this field it is clear that in the data the m invention it is possible to make various changes in form and details, without going beyond the scope of the invention covered by the appended claims.

1. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin and containing peptide drug and the fragment of the antibody that binds the antigen, where the antigen acts to increase the half-life of the conjugate of a drug or slit structures in vivo, and the antigen is a serum albumin, and where the peptide drug is selected from: glucagon-like peptide-1 (GLP-1) or its analogue or derivative; peptide of Basis 3 or analog or derivative; peptide of Basis 4 or its analogue or derivative; and peptide YY (PYY) or its analogue or derivative.

2. The conjugate of a drug or merged structure according to claim 1, where the peptide drug is a medicinal protein containing the specified drug-related fragment ant the body.

3. The conjugate of a drug or merged structure according to claim 2, where the peptide drug is attached to the antibody fragment via a peptide linker group.

4. The conjugate of a drug or merged structure according to any one of claims 1 to 3, where the antibody fragment is a single variable domain antibody (dAb).

5. The conjugate of a drug or merged structure according to claim 4, where the specified single variable domain immunoglobulin selected from VHVLor VHHdomain.

6. The conjugate of a drug or merged structure according to claim 5, where the peptide GLP-1 contains an amino acid sequence that is homologous to a sequence selected from the group consisting of SEQ ID NO: 157 or 159.

7. The conjugate of a drug or fused design on any of subparagraph 5 or 6, where the peptide GLP-1 contains Gly10, Thr12Asp14, Phe27and Il2.

8. The conjugate of a drug or merged structure according to claim 5, where the analogue of GLP-1 differs from SEQ ID NO: 157 or SEQ ID NO: 159 in no more than 6 amino acids.

9. The conjugate of a drug or merged structure according to claim 1, containing a single variable domain that is specific in relation to serum albumin (SA), which has a dissociation constant (Kd)of 1 nm-500 μm for SA, as set p. the surface plasma resonance.

10. The conjugate of a drug or merged structure according to claim 9, where SA is the specific domain binds SA in the standard ligand-binding assay with an IC50 of 1 nm - 500 μm.

11. The conjugate of a drug or merged structure according to claim 1, having a t1/2 alpha between 1 and 6 hours.

12. The conjugate of a drug or merged structure according to claim 1, having a t1/2 beta between 12 and 60 hours

13. The conjugate of a drug or merged structure according to claim 1, where GLP is a GLP-1 (7-37), GLP-1 (7-36)amide, [Ser8]GLP-1(7-36)amide, [RHS9]GLP-1(7-36), [Pro9]GLP-1(7-37).

14. The conjugate of a drug or fused design on item 13, where GLP has a C-terminal peptide, selected from the list consisting of PSS, PSSGAP or PSSGAPPPS.

15. The conjugate of a drug or merged structure according to claim 1, containing a single protein group.

16. Recombinant nucleic acid encoding a conjugate of a drug or merged structure according to any one of claims 1 to 15.

17. Expressing the vector encoding the conjugate of a drug or slit structure having binding specificity in relation to serum albumin and containing a recombinant nucleic acid according to item 16.

18. A host cell containing a recombinant nucleic acid according to item 16, producing a conjugate of a drug or the cast structure, having binding specificity in relation to serum albumin.

19. A method of obtaining a merged design of a medicinal product, comprising maintaining a host cell according p under conditions suitable for expression of the indicated recombinant nucleic acid, whereby get merged design drugs.

20. A pharmaceutical composition comprising a conjugate of a drug or slit structure having binding specificity in relation to serum albumin, according to any one of claims 1 to 15, are present in therapeutically effective amount, and a physiologically acceptable carrier.

21. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin, according to any one of claims 1 to 15 for the treatment and/or prevention of a condition in a patient, where the condition is selected from the group consisting of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

22. The conjugate of a drug or merged design is AI, having binding specificity in relation to serum albumin, according to any one of claims 1 to 15 for slow or prevent disease progression of type 2 diabetes in a patient.

23. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin, according to any one of claims 1 to 15 to reduce food intake by the patient, decreasing β-cell apoptosis, increasing β-cell function and β-cell mass and/or restore sensitivity to glucose and β-cells in a patient.

24. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin, according to any one of claims 1 to 15 for the treatment and/or prevention of hyperglycemia, type 1 diabetes, type 2 diabetes or deficiency of β-cells in a patient.

25. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin according to claim 1 for the treatment and/or prevention of diabetes.

26. The conjugate of a drug or slit structure having binding specificity in relation to serum albumin according to claim 1 for the treatment and/or prevention of obesity.

27. The conjugate of a drug or slit structure having binding specificity regarding savor the exact albumin, according to claim 1 for reducing food intake by the patient.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL22 specific antibodies. The antibodies are differed by the fact that they are produced of different hybridomas PTA-7312, PTA-7315, and PTA-7319. The PTA-7312 antibody is characterised by the fact that it inhibits STAT3 activation by reaching IC50 in the concentration 0.14 mcg/ml whereas the PTA-7315, PTA-7319 antibodies are characterised by Kd less than 1 nM.

EFFECT: use of the invention can find further application in therapy of the IL22 mediated immune disorders.

24 cl, 41 dwg, 1 tbl, 26 ex

Ox40l antibodies // 2423383

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents OX40L antibodies which contain a Fc-fragment produced from a human body, and do not bind with a complement Clq factor. Besides vectors, cells, methods of producing the antibody, a composition and applications of the antibody for preparing a drug are presented.

EFFECT: antibodies exhibit new and patentable signs, particularly advantage to a patient which suffers inflammatory diseases.

19 cl, 7 tbl, 23 ex

FIELD: medicine.

SUBSTANCE: offered is a fused protein containing from an amino-terminus to carboxyl terminus: binding domain polypeptide which is bound with a biological target molecule; human one-cysteine "link" region IgGl peptide; immunoglobulin heavy chain CH2 constant region polypeptide and immunoglobulin heavy chain CH3 constant region polypeptide. The fused protein represents mixed monomers and dimers, has ADCC, CDC or both cytotoxicities. Also, described are a pharmaceutical composition and a method of treating a B-cell disorder with using the fused protein.

EFFECT: use of the invention can find the further application in medicine in treating B-cell disorders.

26 cl, 26 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of polypeptides with Fc-segment from IgG, which possess increased binding FcRn due to introduction of mutation 308C, 308F, 308W, 308Y or modified binding with introduction of mutation 252Y/308F, 257L/308F, 257L/308Y, 257N/308Y, 279Q/308F, 279Y/308F, ^281S/308F, ^A281S/308Y, 284E/308F, 298A/308F/333A/334A, 308F/332E, 308F/311V, 308F/G385H, 308F/428L, and 308F/434Y. Described is antibody with said mutations in Fc region, as well as application of said Fc regions for obtaining fused protein. Application of the invention provides novel Fc-versions with modified FcRn binding.

EFFECT: possibility of application in medicine for obtaining various constructions, with prolonged time of preservation in blood serum in vivo or, vice versa, in case of therapy with application of radioactive medications, with reduced time of preservation in blood serum in vivo.

14 cl, 44 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.

EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.

6 cl, 5 dwg, 2 tbl, 8 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to immunology and biotechnology. The invention discloses versions of a cytotoxically active CD3-specific binding structure. The structure comprises a first domain specifically binding to human CD3 and an Ig-derived second binding domain which is specific to molecules on the cell surface. The invention describes a coding nucleic acid, a vector for expressing the structure and an eukaryotic cell transformed by the vector. The invention discloses versions of compositions based on the structure for treating, preventing or alleviating various diseases and corresponding methods of treating the diseases. A method of obtaining the structure is disclosed.

EFFECT: use of the invention provides a structure with low immunological potency, which has cytotoxicity comparable to the initial structure, which may find further use in medicine.

60 cl, 18 dwg, 15 tbl, 8 ex

Ox40l antibody // 2395523

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is an antibody which bonds with OX40L and variants of this antibody which contain certain Fc-fragments obtained from the human body and do not bond with the complement factor Clq. The monoclonal antibody is produced by a cell line selected from a group which includes cell lines deposited in the German collection of microorganisms and cell cultures (DSMZ) under inventory numbers No. DSM ACC 2685, DSM ACC 2686, DSM ACC 2688, DSM ACC 2689. The invention also relates to a method of obtaining such antibody, to nucleic acid molecules which code the disclosed antibody. The disclosed antibody has an advantage for patients suffering from inflammatory diseases.

EFFECT: antibody is used in diagnostic composition for detecting OX40L in vitro, in a pharmaceutical composition for preventing and treating inflammatory diseases, as well as in preparing a medicinal agent for preventing and treating inflammatory diseases.

9 cl, 20 dwg, 7 tbl, 23 ex

FIELD: chemistry.

SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.

EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.

7 cl, 12 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: obtained human antibody or its antigen-binding fragment specifically binds tumor necrosis factor hTNFα. The like antibodies show high affinity relative to hTNFα in vitro and in vivo. Antibodies according to the invention are taken as a full-length antibody or its antigen-binding fragment. The antibodies or their fragments are usable for detecting hTNFα and for inhibiting hTNFα activity in human beings suffering from a disorder in the case of which hTNFα activity is harmful.

EFFECT: wide range of applications of high affinity recombinant antibodies to hTNFα or their fragments of low dissociation kinetics.

15 cl, 11 dwg, 17 tbl

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes murine antibody and its humanized variant (CDP870) showing specificity to human tumor necrosis factor-alpha. Amino acid sequence is given in the description. Also, invention describes compounds showing affinity with respect to human tumor necrosis factor-alpha based on humanized antibody wherein lysylmaleimide group bound covalently with one or some methoxypoly(ethylene glycol) molecules by lysyl residue is joined to one of cysteine residues by C-end of heavy chain of the humanized antibody. Invention discloses DNA sequences encoding antibodies showing specificity to human tumor necrosis factor-alpha and variants if expression vectors involving indicated DNAs. Also, invention describes variants of a method for preparing a host-cell using expression vectors and variants of a method for preparing antibodies based on prepared host-cells. Invention discloses therapeutic compositions used in treatment of pathology mediated by tumor necrosis factor-alpha based on antibodies. Invention provides providing antibodies showing high affinity: 0.85 x 10-10 M for murine antibodies and 0.5 x 10-10 M for its humanized variant and low immunogenicity for human for humanized antibodies. Part of patients with improved ACR20 in administration of 5 and 20 mg/kg of CDP870 is 75% and 75% in 8 weeks, respectively. Half-life value of CDP870 in plasma is 14 days.

EFFECT: valuable biological and medicinal properties of antibodies.

58 cl, 24 dwg, 6 tbl, 1 ex

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to a method for preparing protein conjugates. Method involves adding immunoglobulin or physiologically active polypeptide to a single end of nonpeptide polymer having two reactive terminal groups in the presence of reducing agent. From a prepared reaction mixture the complex containing nonpeptide polymer bound with immunoglobulin or physiologically active polypeptide is isolated. Then free reactive terminal group of nonpeptide polymer of the complex is added covalently to immunoglobulin or physiologically active polypeptide in the presence of reducing agent to obtain a protein conjugate. Protein conjugate containing physiologically active polypeptide bound, nonpeptide polymer and immunoglobulin that are added one to another covalently in indicated order is isolated. Invention provides preparing a protein conjugate useful for manufacturing a polypeptide drug owing to its enhanced stability in vivo, prolonged blood half-time index and decreased immunogenic properties.

EFFECT: valuable medicinal properties of conjugate, improved preparing method.

21 cl, 16 dwg, 14 tbl, 12 ex

FIELD: biotechnology, immunology.

SUBSTANCE: invention describes a monoclonal anti-IFNα antibody that binds with the following subtypes of IFNα: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα21 and comprises three CDR-sites of heavy chain. Amino acid is given in the invention description. Invention discloses heavy chain of anti-IFNα antibody or its fragment that comprise indicated CDR-sites also. Invention describes anti-IFNα antibody that comprises at least one light chain and one heavy chain. Invention discloses variants of nucleic acids encoding indicated antibodies and variants of vectors used for expression of nucleic acids, and variants of transformed host-cells. Among expression vectors invention describes also vectors deposited at № 2881 and № 2882 carrying heavy and light chain of antibody, respectively. Invention describes a method for preparing antibody from indicated cells. Invention discloses the murine hybridoma cell line deposited in ATCC at number № РТА-2917, and antibody produced by indicated cell line. Also, invention describes variants of the antibody-base pharmaceutical composition and a method used for diagnosis of autoimmune disease. Also, invention discloses using antibodies in treatment of disease or state associated with enhanced level of IFNα in a patient. Using the invention provides inhibiting biological activity of at least seven human IFNα subtypes simultaneously, namely: IFNα1, IFNα2, IFNα4, IFNα5, IFNα8, IFNα10 and IFNα12 that can be used in diagnosis and therapy of different human diseases mediated by IFNα, such as insulin-dependent diabetes mellitus or erythematosus lupus.

EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, genetic engineering.

SUBSTANCE: invention describes recombinant plasmid DNAs constructed in vitro that comprise artificial genes for light and heavy chains of full-scale human antibody prepared by genetic engineering methods. These genes are created on basis of variable fragments of light and heavy chains of recombinant antibody 1F4 and constant human genes IgG1, cytomegalovirus promoter and polyadenylation site BGH. Plasmids provide biosynthesis of recombinant full-scale human antibodies of class IgG1 in mammalian cells. These antibodies interact specifically with smallpox vaccine virus. The affinity constant for prepared recombinant antibodies is 3.54 x 109 ± 0.38 x 109 M-1. Plasmids are used by combined transfection of human cells HEK 293T. Prepared full-scale recombinant antibody against protein of size 27 kDa of smallpox virus vaccine can be used as a base for creature of pharmaceutical preparations used for diagnosis of some post-vaccine complications caused by smallpox virus vaccine. Also, preparations will comprise decreased therapeutic doses of immunoglobulins that will provide minimal undesirable immune response in patients after administration of the preparation.

EFFECT: valuable medicinal properties of plasmid DNA.

4 cl, 7 dwg, 6 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: bioengineering.

SUBSTANCE: versions of the molecule binding CD45RO and CD45RB, and the anti-CD45RO and anti-CD45RB antibody are invented. In one of versions, the said molecule contains at least one antigen-binding site and includes the subsequently located hypervariable sites CDR1, CDR2 and CDR3. The molecule represents the humanised or monoclonal antibody. CDR1 has the amino acid sequence NYIIH, CDR2 has the amino acid sequence YFNPYNHGTKYNEKFKG and CDR3 has the amino acid sequence SGPYAWFDT. The molecule can additionally contain the subsequently located hypervariable sites CDR1', CDR2' and CDR3'. CDR1' has the amino acid sequence RASQNIGTSIQ, CDR2' has the amino acid sequence SSSESIS and CDR3' has the amino acid sequence QQSNTWPFT. In another version, the molecule contains both heavy and light chains where the amino acid sequences contain the corresponding CDR. The versions of the corresponding coding polynucleotide are disclosed; expression vector and based on it expression system. The host cell is disclosed basing on the expression system. The application of the molecule in treatment of autoimmune diseases, graft rejection, psoriasis, intestine inflammatory disease and allergy is described. The pharmaceutical composition for the said application is disclosed.

EFFECT: enables immunosuppressant induction; inhibiting T-cell response and primary lymphocyte response in mixed lymphocyte culture (MLC); prolongs survival period in mice with severe combined immunodeficiency SCID.

20 cl, 5 dwg, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Claimed is therapeutically active fused protein with reduced immunogenicity. Protein consists of two proteins derived from human proteins connected through the fusion region. Connective region, which covers or surrounds fusion region within the limits from 1 to 25 amino acid residues, contains modification, which removes T-cell epitope, in norm absent in humans. Claimed is application of fused protein for obtaining pharmaceutical composition for tumour treatment. Claimed is nucleic acid coding fused protein. Method of reduction of fused protein immunogenicity by introduction of substitutes of corresponding amino acids is described. Application of the invention allows reducing ability of connective epitope of therapeutically active fused protein to bind with molecules of the main complex of hystocompatibility (MHC) of class II, which finally reduces interaction of epitope with receptors of T-cells and can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

EFFECT: reduction of interaction of epitope with receptors of T-cells, which can find application in medicine for prevention of immunological disorders arising with introduction of therapeutically active protein non-modified in connective region.

23 cl, 12 ex

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns biotechnology. It is described bispecific antibody which binds also the factor of blood coagulation IX or the activated factor of blood coagulation IX, and the factor of blood coagulation X, and functionally replaces the factor of blood coagulation VIII or the activated factor of blood coagulation VIII which strengthens enzymatic reaction. The pharmaceutical composition containing the described antibody is revealed. The present invention can be used as an alternative agent for functional replacement of cofactor which strengthens enzymatic reaction.

EFFECT: creation of bispecific antibody which can replace functional proteins, strengthens enzymatic reaction.

14 cl, 18 dwg, 37 ex

Up!