Monoclonal antibodies against protein rgm a and application thereof

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and immunology. Described are versions of antibodies, binding the GRM molecule, as well as their antigen-binding fragments, amino acid sequences of variable parts of which are presented in the claim materials. Nucleic acid, coding the said antibodies, is presented. Claimed is a method of obtaining the RGM-binding protein, which includes cultivation of a host cell in a culture medium under conditions suitable for obtaining the binding protein, capable of binding with RGM, where the host cell contains an expression vector, containing the separated nucleic acid, coding the said antibody. Described is a pharmaceutical composition for treating a disease, in which the SGM A activity produces a negative impact, which contains a therapeutically efficient quantity of the said antibody and a pharmaceutically acceptable carrier. Claimed is an application of the said antibody for obtaining a medication, used for a) reduction of hRGM A binding with a patient's Neogenin receptor; or b) for reduction of hRGM A binding with BMP-2 and BMP-4 in the patient.

EFFECT: invention makes it possible to obtain antibodies against GRM, which are used for treating diseases, associated with excessive interaction of RGM with the Neogenin receptor, BMP-2 and BMP-4.

13 cl, 16 dwg, 10 tbl, 11 ex

 

The technical FIELD TO WHICH the INVENTION RELATES

In the present application is described RGM A-binding proteins, particularly monoclonal antibodies, and in particular, CDR-grafted, humanized variants that are able to contact RGM A and to prevent binding of RGM proteins with receptor RGM RGM A and other A-binding proteins, and, therefore, to neutralize the function of RGM A. These antibodies can be used to treat certain conditions including, but not limited to, multiple sclerosis, brain injury mammals, spinal cord injury, stroke, neurodegenerative diseases and schizophrenia.

The LEVEL of TECHNOLOGY

Regeneration of axons after injury or after inflammatory lesions or after neurodegenerative diseases of the Central nervous system of mammals (CNS) is almost always impossible; the result depends on the balance between the innate ability of nerve fibers in the Central nervous system to resume growth and any abscopal factors Central nervous system, located at the site of injury or damage that actively prevent the resumption of growth, and, consequently, the ways to regenerate damaged nerve fibers.

It was found that CNS myelin produced by oligodendrocytes, and scar tissue injuries are the most significant inhibitory article is ucture for axon growth during the early stage of damage, due to the collapse of the growth cone and inhibition of neurite growth in vitro and in vivo, which leads to direct inhibition of axon growth. Identified proteins RGM, the main inhibiting factors of CNS myelin and scar tissue (Monnier et al., Nature 419: 392-395, 2002; Schwab et al., Arch. Neurol.62: 1561-8, 2005a; Schwab et al. Eur. J. Neurosci. 21: 1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006; for reviews, see: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006; Yamashita et al. Curr. Opin. Neurobiol. 17: 29-34, 2007). Proteins RGM stimulated in sections destruction or damage in people who died from brain trauma or ischemic stroke (Schwab et al., Arch. Neurol. 62: 1561-8, 2005a) and stimulated in rats with spinal cord injury (Schwab et al. Eur. J. Neurosci. 21: 1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006, in respect of the review see: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006; Yamashita et al. Curr. Opin. Neurobiol. 17: 29-34, 2007). In addition, the first results using clinical samples of patients with multiple sclerosis and healthy individuals made it possible to suggest that RGM A person stimulated in the cerebrospinal fluid of patients suffering from MS (data not shown).

To assess regeneration-stimulating effect of RGM A-specific polyclonal antibodies, these antibodies were injected at moderate severe model of spinal cord injury, where approximately 60% of the spinal cord at the thoracic level 9/10 were dissected. G is ecologicheskoe the study found, what is the damage caused to rupture all dorsal and lateral fibers corticospinal way. RGM A-specific polyclonal antibody, administered locally by the pump within two weeks, induced backside of the regeneration of damaged nerve fibers (Hata et al., J. Cell Biol. 173:47-58, 2006).

Hundreds of nerve fibers located outside the area of damage, and the long fibers were regenerating more than 10 mm beyond this damage while regenerating fibers were not detected distal relative to this damage in the treated control animal antibody. Functional recovery anti-RGM A-treated rats was significantly improved in comparison with the processed control antibody rats with spinal cord damage, thus confirming that RGM is A potent inhibitor of neuroregenerative and valuable target to stimulate recovery in the testimony, characterized by axonal damage or damage to the nerve fibers. (Hata et al., J. Cell Biol. 173:47-58, 2006; Kyoto et al. Brain Res. 1186: 74-86, 2007). In addition, the neutralization of the protein RGM A blocking function polyclonal antibody not only stimulated the growth of damaged nerve fibers in rats with spinal cord injury, but also increased the education of their synapse, thus making possible the correction reposteria damaged neural circuits. (Kyoto et al. Brain Res. 1186: 74-86, 2007).

The familyrgm-geneincludes three different gene, two of which,rgmaandbexpressed in the CNS of mammals, giving rise to proteins RGM A and RGM B, while the third member,rgm c, is expressed in the periphery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006), where RGM C plays an important role in iron metabolism.In vitro, RGM A inhibits neurite outgrowth by binding to Neogene, which was identified as the receptor RGM (Ms. Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62, 2004). Neogene was first described as retranslateui protein (Keino-Masu et al. Cell, 87(2):175-85, 1996). This discovery is important as it has been reported that the binding of Netrin-1 and Neogynona or its cognate receptor DCC (deleterows in colorectal cancer) rather stimulate, but did not inhibit neurite outgrowth (Braisted et al. J. Neurosci. 20: 5792-801, 2000). Thus, blocking RGM runs A RGM-mediated growth inhibition, providing a binding neogynona with its stimulating neurite outgrowth ligand Netrino. Based on these observations, we can make the assumption that the neutralization of RGM A superior neutralization neogynona in models of spinal cord injured person. In addition to the binding of RGM Neogene induction and inhibition of neurite growth, binding of RGM A or B with morphogenetic proteins bone BMP-2 and BMP-4 can inhibit successful narora is enerali and functional recovery (Mueller et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 361: 1513-29, 2006).

In this area there is a need for improved antibodies, capable of binding RGM A, preferably, a monoclonal antibody that blocks RGM A and prevents interaction RGM A and its receptor and/or binding proteins, i.e. Neogynona and BMP-2, BMP-4.

The present application relates (a) to obtain a neutralizing monoclonal antibodies against RGM A, which selectively inhibits the binding of RGM A and receptor, Neogynona and morphogenetic proteins bone 2 and 4 (BMP-2, BMP-4), and (b) to obtain a neutralizing monoclonal antibodies against RGM A, which selectively inhibits the binding of RGM A morphogenetic proteins bone 2 and 4 (BMP-2, BMP-4). I believe that these neutralizing monoclonal antibodies of the present invention stimulate the growth of the diseased or damaged nerve fibers and the formation of functional synapses regenerating nerve fibers, as one of the neutralizing monoclonal antibodies of the present invention, apparently, converts the inhibitory nature of RGM a condition in which nerve cells tend to migrate and grow on RGM And the substrate, and not on a permissive substrate, such as Collagen I. in Addition, the antibody is able to induce long-regeneration modelin vivodamage to the optic nerve of rats, and increased the calls for myelination damaged and regenerating nerve fibers.

Thus, a proposal was made that neutralizing monoclonal antibodies of the present invention stimulate neuronal regeneration and growth of damaged or destroyed neural connections in the damaged or inflamed CNS of an individual, such as multiple sclerosis, after acute spinal cord injury, brain injury or neurodegenerative diseases, such as Huntington's chorea, Parkinson's disease, Alzheimer's disease.

The INVENTION

According to one aspect of the present invention relates to a binding protein that dissociates from RGM A person (hRGM A) with a value of KDequal to 1×10-7M or less, and the rate constant for the dissociation of koffequal to 1×10-2with-1or less, both constants determined by the method of resonance of the surface plasmons.

According to another aspect of the invention relates to a binding protein, such as binding to a protein having the above kinetic properties that is associated with RGM A person and neutralizes the activity inhibitory to the growth of neurites RGM A person, as defined by the standard in vitro assays, for example, analysis of neural growth Ntera shown in example 3 below.

The present invention also relates to a binding protein, as op is Adelino above, having at least one of the following additional functional characteristics:

ability to bind RGM A rat,

ability to bind RGM C man and

ability to bind RGM C rats.

In particular, described herein binding protein modulates the ability of RGM to contact at least one of its receptors.

Such a binding protein, in particular, is associated with receptornegative domain RGM A person. Were identified by N - and C-terminal receptornegative domains. Specific variants binding proteins according to the invention are able to bind with N-terminal receptornegative domain RGM A, as shown by inhibition of binding of N-terminal fragment of hRGM A, for example, 47-168, and receptor molecules, such as Neogene and BMP-4. The indicated N-terminal fragment of hRGM A may have an overall length from about 30 to about 150 amino acid residues, or from about 30 to about 122 amino acid residues. As a non-limiting example, you can specify the Fragment 0 (corresponding to N-terminal residues 47-168) hRGM A, as described herein, or any shorter receptornegative fragment.

In particular, the binding protein modulates, preferably inhibits, at least one of the following interactions:

the binding of RGM A person with BMP-4,

linking hRGM Neogene man,

linking hRGM C Neogene man,

the binding of RGM A person with BMP-2 people.

According to one of specific embodiments, a binding protein, as defined herein, is a humanized antibody.

The above-described binding protein may have antigennegative domain and the specified antigennegative domain capable of binding an epitope of a molecule RGM and specified antigennegative domain contains at least one CDR that contains the amino acid sequence selected from the group consisting of

GTTPDY(SEQ ID NO: 59),
FQATHDPLT(SEQ ID NO: 62),
ARRNEYYGSSFFDY(SEQ ID NO: 65),
LQGYIPPRT(SEQ ID NO: 68), and

modified amino acid sequence of CDR, the sequence of which at least 50% identical to one of the specified sequences. In another embodiment, the invention relates to a binding protein that contains antigennegative domain and the specified binding domain SPO is Aubin to bind the epitope molecules RGM, moreover, the specified antigennegative domain contains at least one CDR that contains the amino acid sequence selected from the group consisting of:

GTTPDY(SEQ ID NO: 59),
FQATHDPLT(SEQ ID NO: 62),
ARRNEYYGSSFFDY(SEQ ID NO: 65),
LQGYIPPRT(SEQ ID NO: 68), and

modified amino acid sequence of CDR, the sequence of which at least 50% identical to, for example, at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identical to one of these two sequences.

For example, the binding protein may contain two of these CDR, for example, SEQ ID NO: 59 and 62; or SEQ ID NO: 65 and 68; where at least one of the CDR can be modified and have a sequence that is at least 50%, e.g. at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identical to one of these two sequences.

The specified binding protein may also contain at least one CDR that contains the amino acid sequence selected from the group consisting of SEQ ID NO: 57, 58, 60, 61, 63, 64, 66, 67 and modified amino acid serial is Inesta CDR, which at least 50%, e.g. at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identical to one of these two sequences.

Another variant of implementation refers to a binding protein, wherein said at least one CDR contains an amino acid sequence selected from the group consisting of:

SEQ ID NO:57Residues 31-35 SEQ ID NO:34
SEQ ID NO:58Residues 50-66 SEQ ID NO:34
SEQ ID NO:59Residues 99-104 SEQ ID NO:34
SEQ ID NO:60Residues 24-39 SEQ ID NO:10
SEQ ID NO:61Residues 55-61 SEQ ID NO:10
SEQ ID NO:62Residues 94-102 SEQ ID NO:10
SEQ ID NO:63Residues 31-35 SEQ ID NO:55
SEQ ID NO:64Residues 50-66 SEQ ID NO:55
SEQ ID NO:65The remains 97-110 SEQ ID NO:55

SEQ ID NO:66Residues 24-34 SEQ ID NO:56
SEQ ID NO:67Residues 50-56 SEQ ID NO:56
SEQ ID NO:68Residues 89-97 SEQ ID NO:56

In one particular embodiments, the specified binding protein comprises at least 3 CDRs selected from a set of CDRs of the variable domain, consisting of:

Set VH 5F9
VH 5F9 CDR-H1Residues 31-35 SEQ ID NO:34SEQ ID NO:57
VH 5F9 CDR-H2Residues 50-66 SEQ ID NO:34SEQ ID NO:58
VH 5F9 CDR-H3Residues 99-104 SEQ ID NO:34SEQ ID NO:59
Set VL 5F9
VL 5F9 CDR-L1Residues 24-39 SEQ ID NO:10SEQ ID NO:60
VL 5F9 CDR-L2Residues 55-61 SEQ ID NO:10/td> SEQ ID NO:61
VL 5F9 CDR-L3Residues 94-102 SEQ ID NO:10SEQ ID NO:62
Set VH 8D1
VH 8D1 CDR-H1Residues 31-35 SEQ ID NO:55SEQ ID NO:63
VH 8D1 CDR-H2Residues 50-66 SEQ ID NO:55SEQ ID NO:64
VH 8D1 CDR-H3The remains 97-110 SEQ ID NO:55SEQ ID NO:65
Set VL 8D1
VL 8D1 CDR-L1Residues 24-34 SEQ ID NO:56SEQ ID NO:66
VL 8D1 CDR-L2Residues 50-56 SEQ ID NO:56SEQ ID NO:67
VL 8D1 CDR-L3Residues 89-97 SEQ ID NO:57SEQ ID NO:68

or set the variable domains, in which at least one of these 3 CDR is a modified amino acid sequence of CDR that is at least 50%, e.g. at least 55, 60, 65, 70,75, 80, 85, 90, 95% identical to the original sequence.

In particular, each of the above modifications can be obtained by the addition, deletion or, in particular, the replacement of one amino acid or more amino acids, or combinations thereof.

In another variant implementation, the binding protein comprises at least two sets of CDR variable domains.

In particular, said at least two CDR set selected from the group consisting of:

VH 5F9-set & VL 5F9-set; and

VH 8D1-set & VL 8D1-set.

Binding protein of the present invention further comprises acceptor frame area of the person.

Specified acceptor frame area of a person may contain at least one amino acid sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.

Binding protein according to the invention may in particular contain at least one of the sets of frame sequences selected from the group consisting of sets:

(1)Set VH3-48 (SEQ ID NO:15, 16 and 17)
Set VH3-33 (SEQ ID NO:21, 22 and 23)
Set VH3-23 (SEQ ID NO:24, 25 and 26)

each of which is combined with additional frame sequence selected from the

JH3 (SEQ ID NO:18),

JH4 (SEQ ID NO:19),

JH6 (SEQ ID NO:20);

or

(2)selected from the group consisting of the sets
A18-set: (SEQ ID NO: 27, 28 and 29)
A17-set: (SEQ ID NO: 31, 32 and 33)

each of which can be combined with additional frame sequence selected from the group consisting of JK2 (SEQ ID NO:2).

In accordance with the specific variant implementation, the binding protein of any option, described above, contains at least one CDR-grafted variable domain of the heavy chain selected from SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42 and 43; and/or at least one CDR-grafted variable domain light chain selected from SEQ ID nos: 44, 45 and 46.

More specifically, the binding protein according to the invention contains a combination of two variable domains, where the two variable domains have amino acid sequences selected from:

SEQ ID NO: 35 & 44; 36 & 44; 37 & 44; 38 & 44; 39 & 44; 40 & 44; 41 & 44; 42 & 44;43 & 44;

SEQ ID NO: 35 & 45; 36 & 45; 37 & 45; 38 & 45; 39 & 45; 40 & 45; 41 & 45; 42 & 45; 43 & 45;

SEQ ID NO: 35 & 46; 36 & 6; 37 & 46; 38 & 46; 39 & 46; 40 & 46; 41 & 46; 42 & 46; 43 & 46;

In another embodiment according to the invention, the specified acceptor frame section human binding protein contains at least one amino acid replacement frame section at a key residue, and the specified key residue selected from the group consisting of:

residue adjacent to a CDR;

the remainder of the site of glycosylation;

rare residue;

residue capable of interacting with an epitope RGM;

residue capable of interacting with a CDR;

canonical residue;

contact residue between the variable region of the heavy chain and the variable region of light chain;

balance, in the Vernier zone;

The N-terminal residue capable of forming Pyroglutamate, and

balance in the overlapping region CDR1 variable regions of the heavy chain according to Hotie and the first frame section of the heavy chain according to Kabat.

In particular, these key residues selected from the group consisting of

(position in the sequence of the heavy chain) 1, 5, 37, 48, 49, 88, 98

(position in the sequence of the light chain): 2, 4, 41, 51.

In one of specific embodiments binding protein according to the invention is a consensus variable domain of a human or contains a consensus human variable domain.

In accordance with another variant of the westline binding protein according to the invention, specified acceptor wireframe plot of man contains at least one amino acid replacement frame region, where the amino acid sequence of frame section at least 65%, such as at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%identical to the sequence specified acceptor frame area of human rights and contains at least 70 amino acid residues, for example at least 75, 80 or 85 residues identical to the specified acceptor frame area of the person.

In accordance with a specific embodiment, the binding protein according to the invention contains at least one variable domain with mutated frame section having the amino acid sequence selected from the group consisting of:

SEQ ID NO: 47, 48, 49, 50; (VH domain),

and/or selected from the group consisting of:

SEQ ID NO: 51, 52, 53 and 54 (VL domain).

In particular, the binding protein contains two optional mutated in wireframe plot variable domains, where the two variable domains have amino acid sequences selected from the group consisting of:

SEQ ID NO: 47 & 44; 47 & 45; 47 & 46; 47 & 51; 47 & 52; 47 & 53; 47 & 54;

SEQ ID NO: 48 & 44; 48 & 45; 48 & 46; 48 & 51; 48 & 52; 48 & 53; 48 & 54;

SEQ ID NO: 49 & 44; 49 & 45; 49 & 46; 49 & 51; 49 & 52; 49 & 53; 49 & 54;

SEQ ID NO: 50 & 44; 50 ≈ 45; 50 & 46; 50 & 51; 50 & 52; 50 & 53; 50 & 54;

Binding proteins according to the invention described in the present description, is able to bind at least one target selected from RGM molecules.

In particular, proteins are able to bind RGM A person and, optionally, one additional molecule RGM person or abacadabra monkey, rat, chicken, frog and fish.

For example, proteins can contact RGM A rat RGM C person and/or RGM C rats.

In particular, the binding protein according to the invention capable of modulating, in particular, is able to neutralize or inhibit the biological function of a target selected from RGM molecules defined above.

In particular, the binding protein according to the invention modulates, in particular, inhibits the ability of RGM to contact at least one of its receptors, for example, Neogene and BMP such as BMP-2 and BMP-4.

For example, the binding protein modulates, in particular, reduces and preferably inhibits at least one of the following interactions:

the binding of RGM A person with BMP-4,

linking hRGM Neogene man,

linking hRGM C Neogene man,

the binding of RGM A person with BMP-2 people.

Binding proteins with different combinations of functional traits and, therefore, to show the different functionality of the basic characteristics, as described in the present description, are within the scope of the present invention. Non-limiting examples of such characteristics are shown in the list below.

Features
Sign12345678910111213141516171819202122
The binding of RGM A person++++++++++++ ++++++++++
The binding of RGM A rat+-+-++-+-+-+-+-+-+-++
The binding of RGM With person+----+--++--+ +--++--++
The binding of RGM With rat+---------++++----++++
Inhibition of binding of hRGM Neogene man+--++++++++++ +++++++++
Inhibition of binding of hRGM With Neogene man+----+--------++++++++
Inhibition of binding of hRGM A BMP-2++++++------- ---------
Inhibition of binding of hRGM A BMP-4 person++++++----------------

For example, feature 1 is found in the case of antibody 5F9 described in the present invention, and its derivatives described in the present description.

For example, feature 2 is found in the case of antibodies 8D1, described in the present invention, and its derivatives described in the present description.

In particular, the binding protein according to the invention is able to inhibit at least od the biological activity of RGM, in particular, RGM A, where specified RGM A is selected from RGM A person, abacadabra monkey, rat, chicken, frog and fish.

In accordance with another embodiment, the binding protein according to the invention has one or more of the following kinetic characteristics:

(a) the rate constant of Association (Kon) for the specified target selected from the group consisting of: at least about 102M-1c-1; at least about 103M-1c-1; at least about 104M-1c-1; at least about 105M-1c-1; at least about 106M-1c-1and at least approximately 107M-1c-1as measured by the method of resonance of the surface plasmons;

(b) the rate constant of dissociation (koff) for the specified target selected from the group consisting of: at most about 10-2with-1; at most 10-3with-1; at most about 10-4with-1; at most 10-5with-1; and at most about 10-6with-1as measured by the method of surface plasmon resonance; or

(c) a dissociation constant (KD) for the specified target, selected from the group consisting of: at most about 10- M; at most about 10-8M; at most about 10-9M; at most about 10-10M; at most about 10-11M; at most about 10-12M and at most 10-13M.

In accordance with an additional aspect, the invention relates to the structure of the antibody containing the above-described binding protein, and this design contains antibodies linker polypeptide, or a constant domain of immunoglobulin.

This design antibody or binding protein according to the invention can be selected from the group consisting of: molecules of the immunoglobulin, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, gumanitarnogo antibodies, Fab, Fab', F(ab')2, Fv, disulphide-linked Fv, scFv, single domain antibodies, diately, multispecific antibodies, antibodies with dual specificity, with two immunoglobulin variable domains and especifismo antibodies.

The design of the antibodies according to the invention the specified binding protein contains the constant domain of the heavy chain of an antibody selected from the group consisting of:

constant domain of human IgM,

constant domain of human IgG1,

constant domain of human IgG2,

constant domain of human IgG3,

constant domain of IgG4 man

constant domain of human IgE,

constant domain of human IgD,

constant domain of human IgA1,

constant domain of human IgA2,

constant domain IgY man and

the corresponding mutated constant domains.

In particular, the design of the antibodies according to the invention contains the constant domain of the immunoglobulin with the amino acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 13 and 14.

According to another aspect of the present invention relates to conjugate antibodies that are described in the present description the design of the antibodies, with the indicated antibody further comprises a means selected from the group consisting of: molecules immunoadhesin, renderer, a therapeutic agent and a cytotoxic tools, and each tool is conjugated, e.g., covalently linked, with the specified binding protein.

For example, the specified tool is the renderer is selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and Biotin. In particular, the specified renderer is a radioactive label selected from the group consisting of:3H,14C,35S90Y99Tc111In125I131 I177Lu,166Ho and153Sm.

For example, the specified agent is a therapeutic or cytotoxic agent selected from the group consisting of: antimetabolite, an alkylating tools, antibiotic, growth factor, cytokine, antiangiogenic funds antimitoticescoy tools, anthracycline, toxin and apoptotic tools.

In accordance with another embodiment, the specified binding protein according to the invention, as described in the present description, has the character of glycosylation characteristic of the man.

In addition, binding proteins, the structure of antibodies and conjugate antibodies according to the invention can be in the form of crystal (in crystalline form), preferably with biological activity.

In particular, the crystal is a pharmaceutical crystal not containing media controlled release. Thanks a specified crystalline form of the binding protein, antibody or conjugate antibodies may have a longer half-life in vivo than the corresponding soluble copy.

In another aspect the invention relates to a selected nucleic acid that encodes the amino acid sequence of the binding protein, the amino acid sequence of construction antibodies and amino acid sequence of alnost conjugate antibody as described in the present description.

The present invention also relates to a vector containing a selected nucleic acid, as described in the present description. In particular, the vector is selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and a Shader with a.

The invention also relates to the cell host containing the vector. In particular, indicated a host cell is a prokaryotic cell, for example, E. coli; or eukaryotic cell, and may be selected from the group consisting of cells unicellular organism, animal cells, plant cells and fungal cells. In particular, the specified eukaryotic cell is an animal cell selected from the group consisting of: mammalian cells, cells of birds and insect cells. Preferably, the specified a host cell selected from HEK cells, CHO cells, COS cells and yeast cells. The yeast cell may be Saccharomyces cerevisiae, insect cell may be a cell Sf9.

The invention also relates to a method for producing a protein capable of binding RGM, involving the cultivation of the host cell, as defined in the present description, in a culture medium under conditions sufficient to obtain a binding protein capable of binding RGM.

The invention also relates to a protein obtained in accordance with the specified method.

The invention also relative who are getting ready for the song to release binding protein, moreover, this composition contains

(a) composition, where this composition contains crystallized product is a protein defined in the present description, and an ingredient; and

(b) at least one polymeric carrier.

The specified polymer carrier may be a polymer selected from the group consisting of: poly(acrylic acid), poly(cyanoacrylates), poly(amino acids), poly(anhydrides), poly(depsipeptide), poly(esters), poly(lactic acid), poly(copolymer of lactic and glycolic acid) or PLGA, poly(b-hydroxybutyrate), poly(caprolactone), poly(dioxanone); poly(ethylene glycol), poly((hydroxypropyl)methacrylate, poly[(organo)phosphazene], poly(arteparon), poly(vinyl alcohol), poly(vinylpyrrolidone), copolymers of maleic anhydride and alkylvinyl ether, pluronics polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosinolates, sulfated polysaccharides, their mixtures and copolymers.

The specified ingredient may be selected from the group consisting of albumin, sucrose, trehalose, lactate, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethyleneglycol and polyethylene glycol.

According to another aspect, the invention relates to a method of treatment of a mammal, providing adieu introduction to the mammal an effective amount of the composition, defined in the present description.

According to another aspect, the invention relates to pharmaceutical compositions containing the product (in particular, the binding protein construct or conjugate described above) and a pharmaceutically acceptable carrier.

Specified pharmaceutically acceptable carrier can function as an adjuvant, which can be used to increase the absorption or dispersion of the specified binding protein.

For example, the specified adjuvant is hyaluronidase.

In accordance with another embodiment, the specified pharmaceutical preparation further comprises at least one additional therapeutic agent for the treatment of disorders in which the activity of RGM has a negative effect. For example, the tool selected from the group consisting of a therapeutic agent, renderer, cytotoxic tools, angiogenesis inhibitors; kinase inhibitors; antagonists of molecules costimulation; blockers of adhesion molecules; anticytokine-antibody or functional fragment; methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an Antirheumatic agents; muscle relaxant, a narcotic, non-steroidal anti-inflammatory drugs (NSAI), analgesic funds anesthetic remedies, sedatives, local anesthetic, a neuromuscular blocker, an antimicrobial agent, an antipsoriatic drugs, steroid, anabolic steroid, an erythropoietin, an immunization funds, immunoglobulin, an immunosuppressive funds, growth hormone, tools, hormone replacement therapy, radiopharmaceutical, an antidepressant, antipsychotics, stimulant, anti-asthma drugs, beta-agonists, inhalation steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

The present invention also relates to a method of reducing the activity of RGM A person involving contacting RGM A person at least one product (in particular, binding protein, construct or conjugate described in the present description above), so that decreases at least one activity of RGM A person.

The present invention also relates to a method for reducing the binding of hRGM a receptor by Neogene the individual, if necessary, providing the stage of introduction of the individual product according to the invention (in particular, binding protein, designs or conjugate described in the present description above).

The present invention also relates to a method smart the decision is binding hRGM A morphogenetic protein bone 2 and/or morphogenetic protein bone 4 (BMP-2 and BMP-4) the individual optionally, providing the stage of introduction of the individual product according to the invention (in particular, binding protein, designs or conjugate described in the present description above).

The present invention also relates to a method of treating disorders associated with the activity of RGM A the individual to which the stages of introducing one or in combination with other therapeutic means of the product according to the invention (in particular, binding protein, designs or conjugate described in the present description above).

The present invention also relates to a method of reducing the activity of RGM A an individual suffering from a disorder in which the activity of RGM A negative impact, implying the individual product according to the invention (in particular, binding protein, designs or conjugate described in the present description above), single or in combination with other therapeutic agents.

The breach preferably includes neurological diseases selected from the group consisting of amyotrophic lateral sclerosis, brachial plexus injuries, brain damage, including traumatic brain damage, cerebral palsy, disease, Guillain Barre, leukodystrophies, multiple sclerosis, post-vaccination, polio is Aelita, spina bifida (spina bifida), spinal cord injury, spinal muscle atrophy, spinal tumors, stroke, transverse myelitis; dementia, senile dementia, mild cognitive impairment associated with Alzheimer's dementia, Horai Huntington, tardive dyskinesia, hyperkineses, mania, Parkinson's disease, syndrome Steele-Richardson, down syndrome, severe pseudoparallelism gravis, nerve injury, vascular amyloidosis, cerebral hemorrhage (bleeding I in the brain with amyloidosis, inflammation of the brain, acute disorders with confusion, amyotrophic lateral sclerosis, glaucoma and Alzheimer's disease.

Other specific variants according to the invention are defined below:

the selected binding protein that specifically interacts with at least one epitope of the protein hRGM A;

specified isolated protein which is a neutralizing monoclonal antibody or its antigennegative fragment;

specified antigennegative fragment containing VH domain and VL;

specified neutralizing antibody that reduces the ability of hRGM A contact with its receptor;

specified neutralizing antibody capable of inhibiting the biological activity of hRGM A;

the specified antibody that recognizes the receptor RGM A, selected from a receptor RGM A person, sobakovodov what's monkeys rat, chicken, frog and fish;

the specified antibody that recognizes a protein RGM A, amino acid sequence which is 90% homologous to amino acid sequence SEQ ID NO:2;

the specified antibody, where RGM protein encodes A nucleic acid that is 90% homologous nucleic acid sequence SEQ ID NO:1;

the specified antibody that is at least 90% identical to the amino acid sequence with a sequence containing the variable region of the heavy chain (VH region)containing the sequence of SEQ ID NO:9 or 34, or humanitarian, optional, optional mutated version of the specified region VH;

the specified antibody that is at least 90% identical to the amino acid sequence with a sequence containing the variable region of light chain (VL region), containing the sequence of SEQ ID NO:10, or humanitarian, optional, optional mutated version of the specified area VL,

the specified antibody that binds to hRGM A, where the antibody is glycosylated;

the indicated antibody or antigennegative fragment, where the aforementioned antibody or antigennegative fragment is a murine antibody, a humanized antibody, a fully human antibody, chimeric antibody, antigennegative of the piece is ω gumanitarnogo antibodies or antigennegative fragment of a chimeric antibody;

the indicated antibody or antigennegative fragment, where the aforementioned antibody or antigennegative fragment is antigennegative fragment selected from the group consisting of a Fab fragment, Fab'fragment, F(ab')2fragment and an Fv fragment;

the specified monoclonal antibody that specifically binds at least one epitope hRGM A, where the specified monoclonal antibody is a monoclonal antibody secretively hybridoma cell line described in the present description;

the specified monoclonal antibody binding which leads to inactivation of interaction hRGM And its receptor;

specified hybridoma cell line that produces a monoclonal antibody that specifically binds at least one epitope hRGM A;

specified hybridoma cell line, where hybridoma selected from the group consisting of hybridoma human, mouse, rat, sheep, pigs, cattle, goats and horses;

the specified monoclonal antibody binding which leads to inactivation of hRGM A;

specified hybridoma cell line that produces a monoclonal antibody that specifically binds at least one epitope hRGM A;

specified hybridoma cell line, where hybridoma selected from the group SOS is oasa from hybridoma person, mice, rats, sheep, pigs, cattle, goats and horses;

the specified monoclonal neutralizing antibody or antigennegative fragment having at least one characteristic selected from the group consisting of:

a) binding RGM A mammal with an affinity in the range of nm or less;

b) functional counteract the activity of RGM A in vitro analysis of neurite growth with efficiency µm, nm or less;

c) induction in vivo sprouting (growth of nerve fibers) in the model crushing the optic nerve.

d) induction in vivo sprouting in the model spinal cord injury;

e) facilitate in vivo experimental spinal cord injury by strengthening the regenerative growth of damaged nerve fibers or

f) facilitate in vivo experimental spinal cord injury by increased formation of synapses;

the selected nucleic acid encoding the indicated monoclonal neutralizing antibody or antigennegative fragment;

a vector containing the specified selected nucleic acid;

the specified vector selected from the group consisting of pcDNA; pTT; pTT3; pEFBOS; pBV; pJV; pHybE and a Shader with a;

a host cell transformed by the specified vector, where a host cell selected from the group consisting of cells unicellular organism, animal cells, plant cells and gabneulebi;

specified a host cell, where the animal cell is a cell of a mammal selected from the group consisting of HEK293, CHO and COS;

a host cell transformed by the vector, where the host-cell is a eukaryotic cell;

a method of obtaining a binding protein that binds hRGM A, providing for the cultivation of the host cell in a culture medium under conditions sufficient to obtain a binding protein that binds hRGM A;

the pharmaceutical composition containing the specified monoclonal antibody or antigennegative part and a pharmaceutically acceptable carrier;

a method of reducing binding of hRGM a receptor by Neogene the individual, if necessary, providing the stage of introduction of the individual specified antibody;

a method of reducing binding of hRGM A morphogenetic protein bone 2 and morphogenetic protein bone 4 (BMP-2 and BMP-4) the individual, if necessary, providing the stage of introduction of the individual specified antibody;

a method of treating disorders associated with the activity of RGM A individual to which the stages of the introduction of the specified antibodies, single or in combination with other therapies;

a method of reducing the activity of RGM A an individual suffering from a disorder in which the activity of RGM has A negative impact, OEM home button Flex cable is trevose introduction to the individual specified antibodies, one or in combination with other therapies;

the specified antibody containing at least one VH region containing the amino acid sequence selected from SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42 and 43;

the specified antibody containing at least one VL region containing the amino acid sequence selected from SEQ ID nos: 44, 45 and 46;

the specified antibody, optionally modified 1-5 mutations in the sequence of VH or VL;

the specified antibody, where these mutations selected from reverse mutations wireframe plot and mutations Vernier and VH/VL-adjacent residues.

Any indication of SEQ ID NO: 34 or any reference to SEQ ID NO: 34, described herein, also refers to SEQ ID NO:9.

BRIEF DESCRIPTION of FIGURES

Figure 1A shows the binding of monoclonal antibodies with hRGM A in ELISA analysis.

The figure 1B shows the binding of monoclonal antibodies with hRGM A, expressed in HEK 293 cells.

Figure 1C depicts the binding of monoclonal antibodies with RGM A rat, expressed in HEK 293 cells.

The figure 2 shows the binding of full-RGM Neogene. MAB 5F9 inhibits the binding of full-size fc-associated hRGM Neogene.

The figure 3 depicts the binding of full-RGM A and BMP-4. MAB 5F9 inhibits the binding of fc-associated fragment (47-422) full-hRGM A BMP.

The figure 4 shows binding fragment 0 RGM A and BMP-4. MAB 5F9 inhibits the binding of fc-associated fragment 0 (47-168) with BMP-4.

The figure 5 shows the binding of full-RGM A and BMP-2. MAB 5F9 inhibits the binding of fc-associated fragment of a full-sized hRGM A (47-422) with BMP-2.

The figure 6 presents the Association of microphotographs showing mAb5F9-neutralizing fragment RGM A in the analysis of neurite growth NTera cells. MAB 5F9 neutralizes the inhibitory growth activity of fc-conjugated, strong inhibitory fragment of hRGM A in the analysis of neurite growth with units Ntera person. A. the Control culture, the growth of neurons Ntera on laminin, B. on the substrate laminin-fragment hRGM A (47-168), C - E. on the substrate laminin-fragment hRGM A (47-168) in the presence of 0.1 μg/ml of MAB 5F9 (C), 1 μg/ml of MAB 5F9 (d), 10 μg/ml MAB 5F9 (E.).

The figure 7 shows the quantitative analysis of the results of the analysis of NTera 2. MAB 5F9 will neutralize dependent dose-dependent manner inhibitory growth activity of fc-conjugated, strong inhibitory fragment of hRGM A (fragment 0, 47-168) in the analysis of neurite growth with units Ntera person.

The figure 8 shows the quantitative analysis of test strips SH-SY5Y. MAB 5F9 will neutralize the repulsion induced by bands consisting of full-RGM A human neural cells SH-SY5Y man in striped coatings and membranes. In the absence of MAB 5F9 (A) or in the presence of disciplnary MAB neurons SH-SY5Y prefer to avoid RGM A-bands. This behavior turns increasing concentrations of MAB 5F9. (B-D). When higher concentrations of MAB (10 μg/ml) (E), neuronal SH-SY5Y find a strong preference for bands RGM And in comparison with the bands of Collagen I.

Figure 9 summarizes the quantitative analysis of the characteristics of binding of mABs 5F9 and 8D1. MAB 5F9 and 8D1 evaluated in the analysis of the binding of hRGM A - Neogene, hRGM A - BMP-2 and hRGM A - BMP-4 at various concentrations.

The figure 10 shows the neutralizing activity against chemoattractive activity of humanized antibody 5F9 (h5F9.21, h5F9.23, h5F9.25) in the analysis of chemotaxis SH-SY5Y.

Figure 11 shows in vivo neuroregenerative activity local application 5F9 on the model of the optic nerve of the animal. Local application of MAB 5F9 will neutralize RGM A and stimulates the regenerative growth of damaged axons of the optic nerve in the model crushing the optic nerve of the animal (rats). In the treated 5F9 animals (A), many GAP-43 positive fibers are extended off-site crushing in contrast to the control MAB 8D1 (B), which is not associated with RGM A rat.

Figures 12A and 12B shows a quantitative analysis of the local application 5F9 in the model damage to the optic nerve of the animal. (A) 5F9, but not control MAB 8D1, significantly increased the number of regenerating liver GAP-43-positive fibers. Significantly more of filaments is (p< 0,05) was observed in animals treated with 5F9, at distances of 200 μm, 400 μm and 600 μm, and at 1200 μm fiber was found only in 5F9-treated animals but not in control animals. (B) 5F9 significantly increased GAP-43-positive area in the area of optic nerve damage in comparison with the control antibody 8D1 and control carrier SFR. The area of regenerative growth (GAP-43-positive area) was measured using the software Axiovision software (Zeiss).

The figure 13 shows the in vivo neuroregenerative activity system application 5F9 on the model of the optic nerve of the animal. Animals were treated 5F9 on day 0 and day 21 with 2 mg/kg and 10 mg/kg, respectively. The antibody or the media was injected intraperitoneally or intravenously. Shows the merged image of the optic nerves of rats. In the treated 5F9 animals (A), numerous GAP-43-fiber lengthened off-site destruction in contrast to the control animals treated SFR (B). Plot crushing localized in the left edge and regenerating fibers stained with antibody against GAP-43. Numerous fibers are observed in the upper and lower edge of the optic nerve in the treated 5F9 animals, but not in the treated SFR animals.

In figure 14A and figure 14B shows the quantitative analysis of system use 5F9 in the Delhi damage to the optic nerve of the animal.

The figure 15 shows the activity of re-myelination (remyelination) in vivo systemic use 5F9 on the model of the optic nerve of the animal. Animals were treated 5F9 on day 0 and day 21 with 2 mg/kg and 10 mg/kg, respectively. The antibody or the media was injected intraperitoneally or intravenously. Shows the merged image of the optic nerves of rats. The myelination visualize using antibodies directed against the marker of myelin, the basal of myelin protein MBP. Lots of crushing are located in the middle of the treated nerves and this zone is free in the treated carrier control animals (A and B). In the treated 5F9 animals (C and D), there are numerous MBP-positive structures in the Central zone (Central crushing) of the optic nerves.

The figure 16 shows the quantitative effect of remyelination systemic use 5F9 on the model of the optic nerve of the animal.

DETAILED DESCRIPTION

The present invention relates to RGM A-binding proteins, more specifically, to a monoclonal RGM A-antibodies, particularly humanized monoclonal RGM A-antibodies, or their antigennegative parts that connect RGM A. Various aspects of this application relate to antibodies and fragments of antibodies and their pharmaceutical compositions, and so is e to nucleic acids, recombinant expressing vectors and cells-owners to obtain such antibodies and fragments. Uses of antibodies according to the invention for the detection of RGM A person; to neutralize RGM person and/or activity of RGM A human, in vivo or in vitro, and for the regulation of gene expression is also included in the present invention.

1. Common definitions

If there is no other definition, scientific and technical terms used in the present invention, will have values that are known in the art of ordinary skill in this field. Value and volume of these terms is clear, however, in the case of any uncertainty, the definitions provided in the present description should be considered in the first place compared to a dictionary or other definitions. In addition, unless the context otherwise requires, words in the singular shall include the plural, and the terms in the plural include the singular. In this application, the use of the word "or" means "and/or"if no other instructions. In addition, the use of the term "comprising", as well as other forms, such as "includes" and "included"are not limiting. Terms such as "element" or "component"include both elements and components containing one unit and elements and components that soda is subject to more than one subunit, if no other instructions.

Generally, the nomenclature and methods used in relation to the culture of cells and tissues, molecular biology, immunology, Microbiology, genetics and chemistry of proteins and nucleic acids and hybridization used in the present description, are well known and commonly used in this field. Methods according to the invention is usually carried out according to conventional methods well known in the field and described in various General and more specific references that are cited and described in the description, unless otherwise specified. Enzymatic reactions and purification techniques are carried out in accordance with the descriptions of the manufacturer, as is usually done in this area, or as described in the present description. Laboratory procedures and methods of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry used in the present description and nomenclature used in connection with them, are well known and are commonly used in this field. Standard methods are used for chemical syntheses, chemical analyses, pharmaceutical means of obtaining the compositions and delivery and treatment.

For clarity of the present invention, the selected terms are defined below.

The term "polypeptide" from OSISA in the context of the present description to a polymer chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also belong to the polymer chain of amino acids. The term "polypeptide" includes natural or synthetic proteins, protein fragments and polypeptide analogs of a protein sequence. The polypeptide may be Monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide, which due to its origin or source is not associated with naturally occurring components that surround it in its natural state; essentially free from other proteins of the same species; is expressed by the cell of another type or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is natural, will be "isolated” from its natural components. Protein can also be obtained essentially free of its natural components through the allocation using protein purification are well known in this field.

The term "selection" refers in the context of the present description to a method for producing a chemical molecule such as a polypeptide that is essentially free from natural components by allocating, for example, using the methods of isdi proteins, well known in this field.

The term "RGM A man" (abbreviated in this description as hRGM A)relating to connected with glycosylphosphatidylinositol (gpi) glycoprotein with 450 amino acids, was first described as a repellent of neurite growth or inhibitor of neurite growth during development of topographic projections (processes) (Stahl et al. Neuron 5: 735-43, 1990; Mueller, in Molecular Basis of Axon Growth and Nerve Pattern Formation, Edited by H. Fujisawa, Japan Scientific Societies Press, 215-229, 1997). Family genesrgmincludes three different gene, two of them,rgm aandbexpressed in the CNS of mammals, while the third member,rgm c, is expressed in the periphery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361:1513-29, 2006), where it plays an important role in iron metabolism. Proteins RGM people have a sequence identity of 43% to 50%; the homology of the amino acids RGM A man and rat is equal to 89%. Proteins RGM person does not have significant homology with the sequence of other known proteins. They are Proline rich proteins containing the RGD region, and have structural homology with a domain of factor a Background of Villebranda and split at the N-terminal amino acid 168 unknown protease with the formation of functionally active proteins (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361: 1513-29, 2006).

In vitro RGM A inhibits the growth of neurite (axon) when picomolar concentrations by binding with Neogene,which was identified as the receptor RGM (Ms. Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62,2004). Neogene was first described as retranslateui protein (Keino-Masu et al. Cell, 87(2):175-85, 1996), but its affinity towards Netrin (KD2 nm) is orders of magnitude lower than for RGM (KD0.2 nm) (Ms. Rajagopalan et al. Nat Cell Biol.: 6(8), 756-62, 2004). This is an important discovery, as it has been reported that the binding of Netrin-1 Neogene or its cognate receptor DCC (deletionism in colorectal cancer) encouraged, not inhibited neurite outgrowth (Braisted et al. J. Neurosci. 20: 5792-801, 2000).

Along with the binding of RGM Neogene and induction of inhibition of neurite growth, binding of RGM A or B with morphogenetic proteins bone BMP-2 and BMP-4 could represent another obstacle to successful neuroregeneration and functional recovery (Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci. 361:1513-29, 2006). It was reported that both classes of proteins (Neogene and BMP) transducer inhibiting neurite outgrowth signal RGM through A completely different and independent ways transduction of signals. Typically, the expression of these proteins BMP is relatively low in most areas of the Central nervous system of adults, but it was reported that it rapidly increases in the expression and accumulation of some BMPs (e.g., BMP-2, BMP-6, BMP-7) in response to injury and stroke (Lai et al., Neuroreport 8: 2691-94, 1997; Martinez et al. Brain Res. 894: 1-11, 2001; Hall and Miller, J. Neurosci. Res. 76: 1-8, 2004; Setoguchi et al., Exp. Neurol. 189: 33-44, 2004) in Addition, in models of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), BMP-4, BMP-6 and BMP-7 increases were regulated in the spinal cord of a mouse (Ara et al., J. Neurosci. Res. 86: 125-35, 2008). It was reported that BMP-2 inhibited neurite outgrowth by binding to cell surface RGM A, BMP-receptors I and II and the direct activation of LIM-kinase (Matsuura et al. Biochem Biophys Res Commun., 360: 868-73, 2007), and, therefore, believe that blocking the interaction of RGM A-BMP-2 will further increase functional recovery after damage to the Central nervous system.

As mentioned above, in rats with spinal cord injury and people with brain damage were found massive accumulation of cellular RGM in the damage site, and the nature of the staining RGM A activity in rats at the site of spinal injury is very similar to the staining of pan RGM-antibody in humans, which suggests that a large part of the pan RGM-staining in humans is associated with localization RGM A and not with localization RGM B (Schwab et al., Arch. Neurol.62: 1561-8, 2005a; Schwab et al. Eur. J. Neurosci. 21:1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006). In the brain of a healthy person, pan RGM-staining (RGM immunoreactivity A & B) were detected on the fibers of the white matter, oligodendrocytes, perikaryon few neurons, some vascular smooth muscle and the few endothelial cells. Staining of astrocytes was observed. The nature of OCD is shivani RGM in the brain of adults is very similar to the nature of the staining, observed in the spinal cord of adult rats (Schwab et al. Eur. J. Neurosci. 21:1569-76, 2005 b; Hata et al. J. Cell Biol. 173:47-58, 2006).

On the basis of the accumulation of RGM A to areas of damage in the damage in the brain and spinal cord due to inhibiting the cell growth of neurites activity, suggest that this protein exerts inhibitory neurite outgrowth activity, and its neutralization by antibodies or their antigennegative fragment that binds at least one epitope RGM And man, can lead to improved re-growth of damaged nerve fibers and increased functional recovery in the testimony, characterized by damage to nerve fibers and accumulation of RGM.

Unless otherwise indicated, the term "RGM A" also includes molecules RGM A, selected or derived from other species, for example, rodents such as mice or rats; specifically obtained from rats molecule referred to in the present description "RGM A rat".

TABLE 1
The LIST of SEQUENCES of MOLECULES, SIMILAR TO RGM A
ProteinID sequenceDescription
hRGM ASEQ ID NO: 2Protein sequence of alnost RGM A person
SEQ ID NO: 1The nucleotide sequence of RGM A person
hRGM ASEQ ID NO: 4Protein sequence RGM A-fc man
SEQ ID NO: 3The nucleotide sequence of RGM A-fc man
hRGM ASEQ ID NO: 6Protein sequence hRGM A-light chain-fc man
SEQ ID NO: 5The nucleotide sequence of hRGM A-light chain-fc man
RGM A ratSEQ ID NO: 8Protein sequence RGM A rat
SEQ ID NO: 7The nucleotide sequence of RGM A rat

"Biological activity" refers in the context of the present description to all inherent biological properties of RGM A, defined in the present description.

The terms "specific binding" or "specifically binding"in the context of the present description, when referring to the interaction of antibodies, b is the left main coronary artery or peptide with a second chemical molecule, mean that this interaction depends on the presence of a particular structure (e.g., “antigenic determinant” or "epitope", as defined below) on this chemical molecule; for example, the antibody recognizes a specific structure of a protein and binds to a specific protein structure, and no protein at all. If the antibody is specific against epitope "A", the presence of molecules containing epitope A (or free, unlabeled (A), in a reaction containing labeled "A" and the antibody will reduce the amount of labeled And bound to this antibody.

The term "antibody”in the context of the present description, in the broadest sense refers to any immunoglobulin molecule (Ig), consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative, which retain the basic characteristics of the binding epitope of the Ig molecule. Such mutant, variant or derivative variants are known in this field. They are not limiting the invention of the options described below. They say that antibody "capable of binding" a molecule if it is capable of specifically reacting with this molecule with binding means that a given molecule with the antibody.

The term "monoclonal antibody" refers in the context of the present description is of a preparation of antibody molecules, which have a common amino acid sequence of the heavy chain and the total amino acid sequence of the light chain, in contrast to drugs “polyclonal” antibodies that contain a mixture of different antibodies. Monoclonal antibodies can be obtained several new technologies, such as phage, bacterial, yeast or ribosomal display, as well as classical methods, examples of which are derived from hybrid antibodies (e.g., antibody, Sekretareva hybridomas obtained hybridoma technology, such as the standard hybridoma technology of Kohler and Milstein hybridoma methodology ((1975) Nature 256:495-497).

In the full-size antibody, each heavy chain comprises variable region heavy chain (abbreviated in this description as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CH3. Each light chain consists of a variable region light chain (abbreviated in this description as LCVR or VL) and a constant region of the light chain. The constant region of the light chain consists of one domain, CL. These VH - and VL-region can be further subdivided into the field of hypervariability called complementarity-defined regions (CDR), interspersed with regions that are more conservative, called the data frame sections (FR). Each VH and VL is composed of three CDRs and four FR located from aminobenzo to carboxilic in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Of the immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The term "antigennegative part" or "antigennegative fragment" of an antibody (or simply "antibody" or "antibody fragment"), in the context of the present description, refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen (for example, hRGM A). It was shown that antigennegative function of an antibody can perform the fragments of full-length antibodies. Such antibodies can also be bispecific, doubly-specific or multispecificity formats; specific can be contacted with two or more different antigens. Examples of binding fragments encompassed by the term "antigennegative part of the antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL domains, VH, CL and CH1; (ii) F(ab')2-fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of antibodies with a single shoulder, (v) a dAb fragment (Ward et al., (1989) Nature 41:544-546, Winter et al., PCT publication WO 90/05144 A1 included in the present description by reference), which contains a single variable domain; and (vi) selected complementarity determining region (CDR). In addition, although these two domains Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be retrieved as a single protein chain in which the VL - and VH-region are connected with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). It is assumed that such single-chain antibodies are also included in the term "antigennegative part of the antibody. Other forms of single-chain antibodies, such as diamela, are also included in the scope of this term. Diately are divalent, bespecifically antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a short linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to connect with complementary domains of another chain and creating two antigenspecific site (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci USA 90:6444-6448; Poljak, RJ., et al. (1994) Structure 2:1121-123). Such antigennegative part known in this field (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Veriag. New York. 790 pp. (ISBN 3-540-41354-5).

The term "construction antibody" refers in the context of the present description to a polypeptide that contains one or more antigenspecific parts according to the invention, associated with the linker polypeptide, or a constant domain of immunoglobulin. Linker polypeptides contain two amino acid residue or more amino acid residues, connected by peptide bonds and are used to associate one or more antigenspecific parts. Such linker polypeptides are well known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Constant domain of immunoglobulin called the constant domain of the heavy chain or the constant domain of the light chain. Amino acid sequence of the constant domain of the heavy chain and the constant domain of the light chain of human IgG is known in this field and are presented in table 2.

TABLE 2
The sequence of the CONSTANT DOMAIN of the HEAVY CHAIN AND the CONSTANT domain of the LIGHT chain of HUMAN IgG
ProteinID sequence Sequence
123456789012345678901234567890
The constant region of the gamma 1 IgSEQ ID NO:11ASTKGPSVFFLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Mutant constant region of the gamma 1 IgSEQ ID NO:12ASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Constant region Ig KappaSEQ ID NO:13TVAAPSVFIFPPSDEQLKSGTASVVCLLNN
FYPREAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC
Constant region Ig lambdaSEQ ID NO:14QPKAAPSVTLFPPSSEELQANKATLVCLIS
DFYPGAVTVAWKADSSPVKAGVETTTPSKQ
SNNKYAASSYLSLTPEQWKSHRSYSCQVTH
EGSTVEKTVAPTECS

In addition, antitail his antigennegative part can be part of larger molecules immunoadhesin, formed by covalent or non-covalent coupling of the antibodies or portions of antibodies with one or more other proteins or peptides. Examples of such molecules immunoadhesin include the use of cow's field of streptavidin to get tetramer scFv molecules (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine labels to obtain bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058). Part of the antibodies, such as Fab and F(ab')2fragments can be derived from a full-sized antibodies using conventional methods, such as cleavage by papain or pepsin, respectively, full-size antibodies. Furthermore, antibodies, part of the antibody and molecules of immunoadhesin can be obtained using standard methods of recombinant DNA described in the present description.

"Isolated antibody", in the context of the present description, is an antibody that is essentially free of other antibodies having different antigenic specificities (e.g., the selected antibody that specifically binds to hRGM A, is essentially free of antibodies that specifically bind antigens other than hRGM A). However, isolated antibody, which is specific binding is seen with hRGM A, may have cross-reactivity to other antigens, such as molecules RGM A variety of other species. In addition, the selected antibody can be essentially free of other cellular material and/or other chemicals.

The term "human antibody", in the context of the present invention includes antibodies having variable and constant region derived from immunoglobulin sequences of the germline of the person. These human antibodies of the invention may include amino acid residues not encoded by the sequences of immunoglobulin germ line person (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", in the context of the present description, does not include antibodies in which CDR sequences derived from other species of mammal, such as a mouse, have been grafted on to a frame sequence of a human.

The term "recombinant human antibody", in the context of the present invention includes all human antibodies that receive, Express or secrete recombinant means, such as antibodies expressed using a recombinant expressing vector, transfitsirovannykh in cell x is zaina (described additionally below) antibodies isolated from a recombinant, combinatorial libraries of human antibodies (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H. and W.E. Highsmith, (2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21: 371-378), antibodies isolated from an animal (e.g. a mouse)that is transgenic in terms of genes of the human immunoglobulin (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green, L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little, M. et al (2000) Immunology Today 21: 364-370), or antibodies obtained, expressed, created or selected by any other means that involve splicing of immunoglobulin gene sequences to different DNA sequences. Such recombinant human antibodies have variable and constant region derived from immunoglobulin sequences of the germline of the person. However, in some embodiments, the implementation of such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animal, transgenic regarding sequences Ig human somatic mutagenesis in vivo), and, consequently, the amino acid sequence of the VH - and VL-regions of these recombinant antibodies are sequences that, while derived from sequences of the VH and VL sequences of the germline of the man and kindred, can not exist in nature in the repertoire (spectrum) germline human antibodies in vivo.

The term "chimeric antibody" refers to antibodies that contain sequences of variable region of the heavy chain and light chain of one type and sequence of the constant region of another kind, such as antibodies having a variable region of the heavy chain and light chain of the mouse associated with constant regions of human rights. This chimeric antibody can be obtained recombinant molecular biological methods or can be obtained by physical conjugation of these fields.

The term "CDR-grafted antibody" refers to antibodies that contain sequences of variable region of the heavy chain and light chain of the same species, but in which the sequences of one or more of these CDR region of VH and/or VL replaced with CDR sequences of another species, such as antibodies having a variable region of the heavy chain and light chain of the mouse, in which one or more CDRs of the mouse (e.g., CDR3) were replaced with CDR sequences of a person.

The terms "Kabat numbering", "definitions of Kabat and tagging in Kabat" used in the present description are interchangeable. These terms, which are well known in this field, refer to the system of numbering amino acid OST the Cove, which are more variable (i.e. hypervariable)than other amino acid residues in the variable regions of the heavy chain and light chain of the antibody or its antigennegative part (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the variable regions of the heavy chain, this hypervariable region is in the range of positions of the amino acids 31-35 for CDR1, the provisions of amino acids 50-65 for CDR2, and the provisions of amino acids 95-102 for CDR3. For the variable region of the light chain, this hypervariable region is in the range of positions of the amino acids 24-34 for CDR1, the provisions of amino acids 50-56 for CDR2, and the provisions of amino acids 89-97 for CDR3.

In the context of the present description, the terms "acceptor" and "acceptor antibody" refers to a sequence of the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence of one or more frame sections. In some embodiments, the implementation, the term "acceptor" refers to the amino acid sequence or nucleic acid sequence providing or encoding the constant region (constant region). In one the m of the embodiment, the term "acceptor" refers to the amino acid sequence or nucleic acid sequence providing or encoding one or more frame sections and a constant region (constant regions). In one specific embodiment, the term "acceptor" refers to the amino acid sequence or nucleic acid sequence of human antibodies that provides or encodes at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence of one or more frame regions. According to this variant implementation, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5 or at least 10 amino acid residues, which do not occur in one or more specific provisions of the human antibodies. The acceptor framework region and/or acceptor constant region can be derived from the antibody gene germ line gene matured antibodies, antibodies (e.g. antibodies, are well known in this field, antibodies in the development or antibodies, commercially available).

In the context of the present description, the term "CDR" refers to the complementarity determining about the Asti in variable sequences of the antibodies. There are three CDRs in each of the variable regions of the heavy chain and light chain, representing CDR1, CDR2 and CDR3, for each of these variable regions. The term "set CDR" refers in this context to a group of three CDRs that are in a single variable region capable of binding an antigen. The exact boundaries of these CDR was defined differently in accordance with different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)), provides not only an unambiguous numbering system residues, which can be used in any variable regions of antibodies, but also provides precise boundaries of the residues that define these three CDR. These CDR's may be CDR according to Kabat. In article Chothia et al. (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that some of the Subpart in the CDR according to the Kabat accept almost identical conformation of the peptide skeleton, despite the large diversity at the level of amino acid sequence. These subparts were named L1, L2 and L3 and H1, H2 and H3, where "L" and "H" indicate the region of the light chain and heavy chain, respectively. These areas may be called the CDR Chotia, which have boundaries that overlap with the Kabat CDR. Other boundaries that define CDR, were described by Padlan (FASEB J. 9:pages 133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other determine the value of the CDR regions may also not be strictly obey one of these systems, but nevertheless, will overlap with the Kabat CDR, although they may be shortened or lengthened in the light of predictions or experimental discoveries that specific residues or groups of residues or even all CDR does not impact significantly on binding to the antigen. Used in this description, the methods can use CDR, defined in accordance with any one of these systems, although the preferred embodiments of use CDRs defined according to Kabat or Hatia.

In the context of the present description, the term "canonical" balance refers to the residue in a CDR or framework region that defines a specific canonical CDR structure defined Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both articles included in the present description by reference). According to Chothia et al., the critical part of the CDR of many antibodies have an almost identical conformation of the peptide skeleton of these CDR despite the great diversity at the level of amino acid sequence. Each canonical structure specifies the first set of torsion angles of the peptide backbone for interconnecting segment of amino acid residues that form a loop.

In the context of the present description, the terms "donor" and "donor antibody" refers to an antibody that provides one or more CDR. In a preferred embodiment, done by the means, donor antibody is an antibody of a species different from the species from which the antibody from which you obtained or produced frame area. In the context of gumanitarnogo antibodies, the term "donor antibody" refers to an antibody is not the person providing one or more CDR.

In the context of the present description, the term "frame area" or "frame sequence" refers to the remaining sequences of the variable regions minus these CDR (i.e. after subtracting CDR). Because the exact definition of the CDR sequences can be determined by different systems, the value of the frame sequence is subject to different interpretations. These six CDRs (CDR-L1, -L2 and-L3 light chain and CDR-H1, -H2 and-H3 of the heavy chain) also divide the frame region on the light chain and heavy chain of four Subplate (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without a precise definition of specific cobblestay as FR1, FR2, FR3 or FR4, frame region called by other authors, represents the combined FR in the variable regions of a single natural-occurring chain of immunoglobulin. In the context of the present description, one FR is one of the four cobblestay and FR plural present two or more of the four cobblestay constituting a frame area.

In this area known acceptor sequence of the heavy chain and light chain of a human. In one embodiment, the invention acceptor sequence of the heavy chain and light chain of a human selected from the sequences described in table 3 and table 4. In these tables describe the various combinations regarding frame sequence FR1-FR4 person.

TABLE 3
ACCEPTOR SEQUENCE of the HEAVY CHAIN of HUMAN
SEQ ID No.Region proteinSequence
12345678901234567890123456789012
15VH3-48/JH3 FR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS
16VH3-48/JH3 FR2WVRQAPGKGLEWVS
17VH3-48/JH3 FR3RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR
18VH3-48/JH3 FR4WGQGTMVTVSS
15VH3-48/JH4 FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
16VH3-48/JH4 FR2WVRQAPGKGLEWVS
17VH3-48/JH4 FR3RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR
19VH3-48/JH4 FR4WGQGTLVTVSS

15VH3-48/JH6 FR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS
16VH3-48/JH6 FR2WVRQAPGKGLEWVS
17VH3-48/JH6 FR3RFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR
20VH3-48/JH6 FR4WGQGTTVTVSS
21VH3-33/JH3 FR1QVQLVESGGGVVQPGRSLRLSCAASGFTFS
22VH3-33/JH3 FR2WVRQAPGKGLEWVA
23VH3-33/JH3 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
18VH3-33/JH3 FR4WGQGTMVTVSS
21 VH3-33/JH4 FR1QVQLVESGGGVVQPGRSLRLSCAASGFTFS
22VH3-33/JH4 FR2WVRQAPGKGLEWVA
23VH3-33/JH4 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
19VH3-33/JH4 FR4WGQGTLVTVSS
21VH3-33/JH6 FR1QVQLVESGGGVVQPGRSLRLSCAASGFTFS
22VH3-33/JH6 FR2WVRQAPGKGLEWVA
23VH3-33/JH6 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
20VH3-33/JH6 FR4WGQGTTVTVSS
24VH3-23/JH3 FR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS
25VH3-23/JH3 FR2WVRQAPGKGLEWVS
26VH3-23/JH3 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
18VH3-23/JH3 FR4WGQGTMVTVSS
24VH3-23/JH4 FR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS
25VH3-23/JH4 FR2WVRQAPGKGLEWVS
26VH3-23/JH4 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
19VH3-23/JH4 FR4WGQGTLVTVSS
24VH3-23/JH6 FR1EVQLLESGGGLVQPGGSLRLSCAASGFTFS

25VH3-23/JH6 FR2WVRQAPGKGLEWVS
26VH3-23/JH6 FR3RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
20VH3-23/JH6 FR4WGQGTTVTVSS
TABLE 4
ACCEPTOR sequence of the LIGHT chain of the HUMAN
SEQ ID No.Region proteinSequence
12345678901234567890123456789012
27A18JK2 FR1 DIVMTQTPLSLSVTPGQPASISC
28A18/JK2 FR2WYLQKPGQSPQLLIY
29A18/JK2 FR3GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
30A18/JK2 FR4FGQGTKLEIKR
31A17/JK2 FR1DVVMTQSPLSLPVTLGQPASISC
32A17/JK2 FR2WFQQRPGQSPRRLIY
33A17/JK2 FR3GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
30A17/JK2 FR4FGQGTKLEIKR

In the context of the present description, the term "gene antibody germ line" or "fragment of a gene" refers to a sequence of immunoglobulin encoded by non-lymphoid cells that were not subjected to a maturation process that leads to genetic rearrangeable and mutation for expression of specific immunoglobulin. (See, for example, Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)). One of the benefits of various options for the implementation of the present invention, oznachaet from the recognition that what genes antibody germline more likely than genes matured antibodies retain the essential structure of the amino acid sequences characteristic of individuals in these species, therefore, less likely to be recognized as genes from alien source when used therapeutically in these types.

In the context of the present description, the term "key" residues refers to specific residues in the variable regions, which have a greater influence on the binding specificity and/or affinity of antibodies, in particular, gumanitarnogo antibodies. The key residue includes, but is not limited to, one or more of the following residues: the residue that is adjacent to a CDR, a potential glycosylation site (may be a website or N-or O-glycosylation), a rare residue, a residue capable of interacting with antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between the variable region of the heavy chain and the variable region of the light chain, and the balance in the Vernier zone, and the remainder in the overlapping region CDR1 variable regions of the heavy chain of Hatia and the first frame section of the heavy chain according to Kabat.

The term "humanitariannet antibody" generally refers to antibodies that contain sequences of variable regions is agelou and light chain type, non-human (e.g., a mouse), but in which at least a portion of the VH and/or VL sequence has been modified so that it has become more "human-like", i.e. more similar to the variable sequences of the germline of the person. One type gumanitarnogo antibody is a CDR-grafted antibody wherein the CDR sequence of the person entered in the sequence of VH and VL to replace the corresponding CDR sequences are not human.

In particular, the term "humanitariannet antibody" in the context of the present description refers to an antibody or a variant, derivative, analog or fragment, which immunospecificity associated with interest antigen and which contain framework (FR) region having essentially the amino acid sequence of human antibodies, and complementarity determining region (CDR)having essentially the amino acid sequence of the antibodies, non-human. In the context of the present description, the term "essentially" in the context of CDR refers to a CDR having the amino acid sequence, at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of CDR of the antibody is human. Humanitariannet antibody contains essentially all of at least one, typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv)in which all or substantially all of the CDR regions correspond to the CDR regions of the immunoglobulin is not human (i.e. donor antibody)and all or essentially all of the framed areas are framed regions of the consensus sequence of human immunoglobulin. Preferably, humanitariannet antibody contains at least a portion of constant region of immunoglobulin (Fc), typically part of a constant region of human immunoglobulin. In some embodiments, implementation, humanitariannet antibody containing light chain and at least the variable domain of the heavy chain. This antibody may also include region CH1, hinge region, region, CH2, CH3 and CH4 of the heavy chain. In some embodiments, implementation, humanitariannet antibody contains only humanitarian light chain. In some embodiments, implementation, humanitariannet antibody contains only humanitarian heavy chain. In specific embodiments, implementation, humanitariannet antibody contains only humanitarianly variable domain light chain and/or humanitarian heavy chain.

Humanitariannet antibody may be selected from any class of immunoglobulins, including Chi is Le IgY, IgM, IgG, IgD, IgA and IgE, and any isotype, including, without limitation, IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Humanitariannet antibody may contain sequences from more than one class or isotype, and a particular constant domains can be selected to optimize desired effector functions using methods well known in the field.

Frame and CDR region gumanitarnogo antibodies should not exactly match the original (parent) sequences, for example, CDR or the consensus skeleton plot donor antibodies may be mutagenicity replacement, insertion and/or deletion of at least one amino acid residue so that the residue CDR or framework residue at that site does not correspond to any donor antibody, no consensus frame section. However, in the preferred embodiment, such mutations will not be extensive. Usually, at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% of the residues gumanitarnogo antibodies will correspond to residues in the original sequences of the FR and CDR. In the context of the present description, the term "consensus wireframe plot" refers to the frame region in the consensus sequence of the immunoglobulin. In the ontext of the present description, the term "consensus sequence of immunoglobulin" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences of immunoglobulins (See, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany, 1987). In the family of immunoglobulins, each position in the consensus sequence are occupied by the amino acid, occurring most often in this position in this family. If two amino acids are found with the same frequency, any of which can be included in the consensus sequence.

In the context of the present description, a "Vernier" called Sobibor frame residues that can adjust the structure of the CDR and to fine-tune the matching antigen, as described in article Foote and Winter (1992, J. Mol. Biol. 224:487-499), which is incorporated into this description by reference). The remains of the Vernier zone form a layer, the underlying CDR, and can affect the structure of the CDR and the affinity of this antibody.

The term "inhibition of binding" RGM with one of its receptors used in this description, includes partial (for example, approximately 20%, 40%, 60%, 80%, 85%, 90%, 95% or more) or full reduction of the binding activity of a specified receptor. Specified "inhibition of binding can be determined using any suitable the way, available in this field, preferably in any way, in the present description as an example, such as, for example, analyses based binding ELISA.

In the context of the present description, the term "neutralizing" refers to neutralize the biological activity of the protein target with the specific binding binding protein protein target. Neutralization may be the result of different ways of binding the specified binding protein with the target. For example, the neutralization may be due to binding of the binding protein in the target area, which does not affect the binding of the receptor molecule target. Alternatively, binding of the binding protein can lead to blockade of binding of this receptor to the target, and this blockade eventually neutralizes the activity of the protein target. According to this invention, can meet each of these different mechanisms. Preferably the neutralizing binding protein is a neutralizing antibody, the binding of which with hRGM A leads to neutralize the biological activity of hRGM A. Preferably this neutralizing binding protein binds hRGM A and reduces the biological activity of hRGM A at least approximately 20%, 40%, 60%, 80%, 85% or more. Neutralization of biological activity of hRGM A neutralizing binding Bel is ω can be assessed by measuring one or more indicators of biological activity of hRGM A, well known in this field. For example, neutralization of hRGM A pay inhibition in the analysis of neural growth in Ntera (see example 3 below). Analysis of neurite growth Ntera determines the inhibition of neurite growth. In the absence of inhibition of the protein or fragment RGM A and in the presence of stimulating neurite outgrowth substrate laminin neural aggregates NTera find extensive and dense network of growing neurites. RGM A or fragments of RGM A inhibit neurite outgrowth, resulting in reduced length and reduced numbers of neurites. Blocking the function of RGM A antagonists or MAB, such as mAb 5F9 neutralized the inhibitory neurite outgrowth activity of strong fragment of the light chain (amino acids 47-168) fc-conjugated hRGM A protein RGM A man in the analysis of neurite growth with aggregates of differentiated neurons NTera person, leading to a strong increase in the length and number of neurites.

"Neutralizing monoclonal antibody” refers in the context of the present description to the drug molecules are antibodies, which after binding to the specific antigen capable of competing for binding and to inhibit binding of the natural ligand for the specified antigen. In one specific embodiment of this application neutralizing antibodies of the present invention is able to compete with RGM A for binding to Neogene and/or with BMP-2 and/or the BMP-4 and prevent the biological activity or function RGM A. In particular, the neutralizing antibodies of the present invention is able to contact RGM A and to prevent binding with Neogene and/or with BMP-2 and/or BMP-4 and prevent the biological activity or function RGM A. the Term "activity" includes such activities as the specificity/affinity of binding of antibodies against the antigen, for example, anti-hRGM A-antibody, which binds to the antigen RGM A, and/or neutralizing effect of the antibodies, for example anti-hRGM A-antibody binding with hRGM A inhibits the biological activity of hRGM A, for example as defined in the analysis of binding hRGM A-Neogynona, the analysis of binding hRGM A-BMP-2 or the analysis of binding hRGM A-BMP-4, as described below in the experimental section.

The biological activity of RGM A can be described as regulation of cell migration. A particular example of cell migration is the growth of neurites, which are delayed or inhibited protein RGM A. in Addition, it was shown that RGM proteins modulate the activity of BMP proteins. Published in the present description, the examples describe synergistic synergistic activity of RGM proteins in BMP-path, on the one hand, and the inhibitory activity of RGM proteins in BMP-path, which is important for regulation of iron metabolism and regeneration of bone and cartilage and in the Central nervous system for remyelination and regeneration.

The term "epitope" or "antigenic determine what the" includes any polypeptide determinant, can specifically bind to an immunoglobulin or T-cell receptor. In some embodiments, implementation, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and, in some embodiments, the implementation may have specific three dimensional structural characteristics and/or characteristics of the specific charge. The epitope is a region of the antigen that is bound by antibody. In some embodiments, implementation, saying that the antibody specifically binds the antigen, mainly when it learns its antigenicity in a complex mixture of proteins and/or macromolecules.

The term "surface plasmon resonance" refers in the context of the present description to the optical phenomenon that allows you to analyze biospecific interaction real-time detection of changes in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jόnsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jόnsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term "kon" in the context of the present description refers to the rate constant for Association of the antibodies is and with the antigen to form a complex of the antibody/antigen, as is well known in this field.

The term "koff" applies in the context of the present description to the rate constant for dissociation of antibodies from complex antibody/antigen, as is well known in this field.

The term "KD” applies in the context of the present description to the dissociation constant of the specific interaction of the antibody/antigen, as is well known in this field.

The term "labeled binding protein" refers in the context of the present description to a protein with the included label that provides an identification of the binding protein. Preferably, the label is detected marker, for example, the incorporation of radioactively labeled amino acids or accession to the biotinylated polypeptide of the parts of the molecule that can be detected labeled Avidya (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following labels: radioisotopes or radionuclides (e.g.,3H,14C,35S90Y99Tc111In125I131I177Lu,166Ho or153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, the Christmas phosphatase); chemiluminescent markers; biotinylated groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., pairs of sequences latinboy zipper, binding sites for secondary antibodies, metallovedeniye domains, epitope tags); and magnetic agents, such as gadolinium chelates.

The term "conjugate antibody" refers to a binding protein, such as antibody chemically linked with the second chemical molecules, such as therapeutic or cytotoxic agent. The term "agent" is used in this description to refer to chemical compounds, mixtures of chemical compounds, a biological macromolecule, or an extract made from biological materials. Preferably, these therapeutic or cytotoxic tools include, but are not limited to, pertussis toxin, Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogues or homologues.

The terms "crystal" and "kristallizuetsya" include this aspect the antibody or antigennegative h the STI, which exists in the form of a crystal. The crystals are a form of solid state of matter, which differs from other forms, such as amorphous solid state or liquid state. The crystals consist of regular, repeating, three-dimensional ordered rows of atoms, ions, molecules (e.g. proteins, such as antibodies or molecular complexes (e.g., complexes of antigen/antibody). These three-dimensional ordered rows arranged in accordance with a specific mathematical relationships, which are well understood in this area. Fundamental element, or structure member, which is repeated in the crystal, called the asymmetric unit. The repetition of this asymmetric link in a location that is subject specific, well-defined crystallographic symmetry, provides a "unit cell" of the crystal. The repetition of this unit cell through regular broadcasts (transfers) in all three dimensions provides crystal. Cm. Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999).

The term "polynucleotide" in the context of the present description refers to a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides, or modified formulable type of nucleotide. The term includes single-stranded and double-stranded forms of DNA, but is preferably double-stranded DNA.

The term "isolated polynucleotide" in the context of the present description to identify polynucleotide (e.g., of genomic, cDNA or polynucleotide synthetic origin or some combination thereof), which, owing to its origin as a "selected polynucleotide": not associated with all polynucleotides or part of polynucleotide that this "dedicated polynucleotide” found in nature; is functionally associated with polynucleotide that is not associated in nature; or does not occur in nature as part of a larger sequence.

The term "vector"in the context of the present description, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. One type of vector is a "plasmid", which is a circular double-stranded loop of DNA, which can be legirovanyh additional DNA segments. Another type of vector is a viral vector, and in this viral genome can be legirovanyh additional DNA segments. Certain vectors are capable of Autonomous replication in a cell host, in which they are administered (e.g., bacterial vectors having a bacterial site initiation R is plicatio (origin of replication), and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) can be integrated into the genome of the host cell, and thereby are replicated along with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors are referred to as "recombinant expressing vectors" (or simply "expressing vectors"). Usually, expressing vectors that are applicable in the methods of recombinant DNA, are often in the form of plasmids. In this description, the terms "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, it is expected that this invention includes such other forms expressing vectors as viral vectors (e.g., retroviruses are defective replication, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "functionally linked" refers to the immediate neighborhood in which the components described are in a relationship that allows them to function in the intended way. The regulatory sequence functionally linked” to the coding sequence are ligated so that the expression of this coding consequently the STI is achieved under conditions compatible with these regulatory sequences. "Functionally related sequences include regulatory sequences of the expression, which are contiguous with the gene of interest and a regulatory sequence for the expression of that act in TRANS-position or at a distance to regulate gene of interest. The term "regulatory sequence expression" refers in the context of the present description to a polynucleotide sequence that is necessary to implement the expression and processing of coding sequences to which they legirovanyh. Regulatory sequence for expression include appropriate sequence initiation, transcription termination, promoter and enhancer sequences; efficient signal processing RNA, such as splicing signals, polyadenylation; sequences that stabilize cytoplasmic mRNA; sequences that enhance the efficiency of translation (i.e. a consensus Kozak sequence); sequences that enhance protein stability; and when it is desirable, sequences that enhance protein secretion. The nature of such regulatory sequences differs depending on the organization the ISM of the host; in prokaryotes, such regulatory sequences generally include promoter, the binding site of the ribosome and the sequence termination of transcription. It is assumed that the term "regulatory sequence" includes components whose presence is important for the expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and sequences of merger partner.

"Transformation", as defined in the present description, refers to any process by which exogenous DNA enters the cell host. Transformation can occur under natural or artificial conditions using various methods, well known in this field. The transformation can be based on any known method for introducing foreign nucleic acids sequences in prokaryotic or eukaryotic cell host. This method is chosen based on the subject of transformation of the host cell and may include, without limitation, viral infection, electroporation, lipofection and the bombarding particles. Such "transformed” cells include stably transformed cells in which the introduced DNA is capable of replication either as Autonomous plasmids can replicate the, either as part of the chromosome of the host. They also include cells that transtorno Express insertional DNA or RNA for limited periods of time.

The term "recombinant a host cell" (or simply "a host cell"refers in the context of the present description to the cage, which was introduced exogenous DNA. It should be understood that such terms refer not only to the specific of the considered cell, but also to the progeny of such cells. Because certain modifications may occur in succeeding generations due to mutations or environmental influences, such descendants may actually not be identical to the parent cell, but are still included in the scope of the term "a host cell" in the context of the present description. Preferably the cell hosts include prokaryotic and eukaryotic cells selected from any of the kingdoms of plants or animals. Preferred eukaryotic cells include cells of unicellular organisms (protists), the cells of fungi, plants and animals. Most preferably the cell hosts include, but are not limited to, prokaryotic cell line E. coli, lines of mammalian cells CHO, HEK 293 and COS; the line of insect cells Sf9 cells and fungi Saccharomyces cerevisiae.

For recombinant DNA, synthesis of oligonucleotide and tissue culture, transformez and (for example, electroporation, lipofection) can be used in a standardized way. Enzymatic reactions and purification techniques can be performed in accordance with the descriptions of the manufacturer or in accordance with how they are usually performed in the field or as described in the present description. Previous methods and procedures can usually be performed in accordance with conventional methods, well known in this field, described in various General and more specific references that are cited and discussed in this description. See, for example, the manual Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated into this description by reference for any purpose.

The term "transgenic organism", known in the field and used in the present description, refers to an organism having cells that contain a transgene, where the transgene is introduced into the body (or the body of the ancestor of this organism), expresses the polypeptide is not expressed in nature in the body. "Transgene" is a DNA construct that is stable and functionally integrated into the host cell genome, from which it develops a transgenic organism, and which controls the expression of the product encoded by the gene in one or more cell types or tissues of this transgenic organisms is A.

The terms "regulate" and "modulate" are used interchangeably and, in the context of the present description, refers to a change or shift activity of interest molecules (e.g., biological activity of hRGM A). Modulation may be an increase or decrease in the magnitude of a certain activity or function of interest of the molecule. Examples of the activities and functions of the molecules include, but are not limited to, the characteristics of the binding, enzymatic activity, activation of cellular receptors and transduction of signals.

Accordingly, the term "modulator" refers in the context of the present description, the connection is able to change or shift the activity or function of interest molecules (e.g., biological activity of hRGM A). For example, the modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of this activity or function observed in the absence of the modulator. The term "agonist" refers in the context of the present description to the modulator, which, upon contact with interest the molecule causes an increase in the value of this activity or function observed in the absence of the agonist. Specific interest agonists may include, but are not limited to, polypeptides hRGMA or polypeptides, nucleic acids, carbohydrates, or any other molecules which bind with hRGM A. the Term "antagonist" refers in the context of the present description to the modulator, which, upon contact with interest the molecule, causes a decrease in the activity or function of the molecule observed in the absence of the antagonist. Examples of antagonists include, but are not limited to, proteins, peptides, antibodies, Patiala, carbohydrates or small organic molecules. Patiala described, for example, in WO01/83525.

Specific interest antagonists include antagonists that block or modulate the biological or immunological activity of hRGM A. Antagonists hRGM A may include, but are not limited to, proteins, nucleic acids, carbohydrates, or other molecules that are associated with hRGM A, such as monoclonal antibodies that interact with the molecule RGM A. it Should be noted that the interaction with RGM A can result in binding and neutralizing other ligands/components of cell membranes and can be useful for additive or synergistic functioning against numerous diseases.

In the context of the present description, the term "effective amount" refers to the amount of therapeutic agent that is sufficient to reduce or sublinearity and/or duration of a violation or one or more symptoms of the disorders, prevent the recurrence, development, beginning the progression of one or more symptoms associated with the disorder, the detection of violations or increase or improve the prophylactic or therapeutic effects of another therapeutic agent (e.g., prophylactic or therapeutic agent).

The term "sample" is used in the context of the present description in its broadest sense. "Biological sample"in the context of the present description includes, but is not limited to, any quantity of a substance from a living being or formerly living creature. Such beings include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such substances include, but are not limited to, blood, serum, urine, synovial fluid, cells, organs, tissue, bone marrow, lymph nodes and spleen.

2. Polypeptides that bind hRGM A

The basic version of this application includes the selected proteins or polypeptides that specifically bind at least one epitope of the protein RGM A. Selected proteins or polypeptides that specifically bind at least one epitope of the protein RGM A, is able to inhibit the binding of RGM A and receptor, Neogynona and/or morphogenetic proteins bone 2 and 4 (BMP-2, BMP-4).

The most is the preferred option of this application includes antibodies, associated with the RGM A or its antihistamine parts or pieces.

Preferably, the anti-RGM A-antibodies of the present invention exhibit high ability to reduce or neutralize the activity of RGM A, for example, estimated by any of several in vitro and in vivo, are known in this area or are described below.

Most preferably the present application includes a neutralizing monoclonal antibodies against RGM A, which selectively prevents the binding of RGM A and receptor, Neogynona and morphogenetic proteins bone 2 and 4 (BMP-2, BMP-4), and the generation of neutralizing monoclonal antibodies against RGM A, which selectively prevents the binding of RGM A, it can be used morphogenetic proteins bone 2 and 4 (BMP-2, BMP-4).

Preferably, these neutralizing monoclonal antibody of this application is a human antibody or humanitariannet antibody. The term "human antibody" refers to antibodies having variable and constant regions corresponding to the sequences of immunoglobulin germline person, or derived from them (for example, see Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). However, human antibodies of this application may include amino acid residues not encoded by the sequences of immunol Bulina germ line person (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.

In a different implementation, the antibody is a recombinant antibody or a monoclonal antibody. The most preferred neutralizing antibodies of this application called mAb5F9 and mAb8D1 and their functional fragments, and it is assumed that other antibodies and functional fragments of the antibodies with equivalent properties relative mAb5F9 and mAb8D1, such as high-affinity binding to RGM A low dissociation kinetics and high neutralizing capacity, are part of this invention. The affinity of binding and dissociation rate of an anti-RGM A-antibodies of this application in relation to the immunogenic polypeptide RGM A or its fragment can be determined by any method known in this field. For example, the affinity of binding can be measured using a competitive ELISA assays, RIA, BIAcore or KinExA technology. The dissociation rate can also be measured using BIAcore or KinExA technology. The affinity of binding and dissociation rate measured by surface plasmon resonance using, for example, BIAcore.

One of the preferred monoclonal antibodies of the present invention, the antibody mAb5F9 has at least 90% identity Amin is acid sequence with a sequence containing the variable region of the heavy chain (VH-region)containing the sequence of SEQ ID NO:9 or 34, and the variable region of light chain (VL-region)containing the sequence of SEQ ID NO:10.

It is also expected that the selected monoclonal antibodies that interact with RGM A to this application may be glycosylated binding protein, in which the antibody or antigennegative part contains one or more carbohydrate residues. Getting occur in vivo protein may be subjected to additional processing, known as post-translational modification. In particular, sugar (glucosamine) residues may be added to the enzymatic process known as glycosylation. The obtained proteins bearing covalently linked oligosaccharide side chains, known as glycosylated proteins or glycoproteins. Glycosylation of proteins is dependent on the amino acid sequence of the protein of interest, as well as from the host cell, in which this protein is expressed. Different organisms can produce various enzymes of glycosylation (e.g., glycosyltransferases and glycosidase) and have a variety of available substrates (nucleotidase). Due to these factors, the nature of the glycosylation of proteins and the composition helicoiling residues may vary in C the dependence on system-master, in which is expressed a specific protein. Glucosamine remains applicable in this invention may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably, this glycosylated binding protein contains such glucosimine remains that the nature of glycosylation is the nature of the glycosylation of human nature.

Antibodies of this application contain a constant region of the heavy chain, such as a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, IgY or IgD. In addition, this antibody can contain a constant region of light chain, or a constant region of light chain Kappa or constant region light chain lambda. Alternatively, part of the antibody can be, for example, Fab-fragment or single-chain Fv fragment. Replacement of amino acid residues in the Fc portion to change the effector functions of antibodies are known in the art (Winter, et al. U.S. patent No. 5648260; 5624821). Fc part of the antibody mediates several important effector functions, for example, induction of cytokines, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC) and the half-life/speed clearance of antibodies and complexes antiheretical. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases can be neoba atelinae or even harmful, depending on therapeutic purpose. Some isotypes of human IgG, particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγR and complement C1q, respectively. Neonatal Fc receptor (FcRn) are critical determinants of the half-life of antibodies in the bloodstream. In another embodiment, at least one amino acid residue is replaced in the constant region of the antibody, for example, the Fc region of the antibody, so that the effector functions of antibodies are modified.

3. Generation of anti-hRGM A-antibodies

3.1. A common part

Antibodies of this application can be generated by immunizing a suitable host (e.g., vertebrates, including humans, mice, rats, sheep, goats, pigs, cattle, horses, reptiles, fish, amphibians, and bird eggs, amphibians and fish). To generate antibodies of this application of the owner are subjected to immunization immunogenic polypeptide RGM A or its fragment according to the invention. The term "immunization" refers in this description to a method of delivering an antigen to the immune repertoire (spectrum), which is natural, not genetically modified organism or transgenic organism, including transgenic organism, modified to represent the artificial immune repertoire of a person. Similarly, immunogenic preparation is a composition of antigen which contains adjuvants or other supplements that could increase the immunogenicity of the antigen.

Immunization of animals can be performed by any method known in this field. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods of immunization of animals (not humans), such as mice, rats, sheep, goats, pigs, cattle and horses, well known in this field. See, for example, Harlow and Lane, and U.S. Patent No. 5994619. In one preferred embodiment, the antigen RGM A set with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete adjuvant's adjuvant, RIBI (muramyldipeptide) or ISCOM (immunostimulating complexes). Such adjuvants can protect the polypeptide from the quick dispersion of opinion in the local floor, or they may contain substances that stimulate the secretion of host factors that are chemotactic towards macrophages and other immune system components. Preferably, when the introduction of the polypeptide, the immunization scheme will include two or more injections of the polypeptide, distributed over several weeks.

It is assumed that the animal host are subjected to immunization with the antigen associated with the cell membrane of intact or damaged cells, and antibodies of this application identific the shape of the binding immunogenic polypeptide according to the invention. After immunization of the host animal with the antigen, antibodies can be obtained from the animal. This animal by krovoisliania or killing receive containing antibody serum. This serum can be used in the form in which it is received, or these antibodies can be purified from the serum. Serum or immunoglobulins, thus obtained, are polyclonal, therefore, have a heterogeneous set of properties.

3.2. Monoclonal anti-RGM A-antibodies produced using hybridoma technology

Monoclonal antibodies can be obtained using a wide variety of ways known in this area, including hybridoma, recombinant technology and the technology of phage display or a combination of both. For example, monoclonal antibodies can be produced using hybridoma methods, including methods known in this field and are described, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (these references are incorporated by reference in their entirety). The term "monoclonal antibody" in the context of the present description is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibodies is about" refers to the antibody, which is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the way in which it is received.

Methods of obtaining and screening for specific antibodies using hybridoma technology are routine and well known in this field. In one embodiment, the present invention provides methods of generating monoclonal antibodies as well as antibodies obtained by this method comprising culturing hybridoma cells secreting the antibody according to the invention, where, preferably, hybridoma generate the fusion of splenocytes isolated from a mouse immunized with an antigen according to the invention with myeloma cells and then screening hybridomas obtained from this fusion, hybridoma clones that secrete an antibody able to bind a polypeptide according to the invention. Briefly, mice can immunotherapeutic antigen RGM A. In the preferred embodiment, the antigen RGM A set with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete adjuvant's adjuvant, RIBI (muramyldipeptide) or ISCOM (immunostimulating complexes). Such adjuvants can protect the polypeptide from the quick dispersion of opinion in the local floor, or they may contain substances which cat is which stimulate the secretion of host factors which are chemotactic towards macrophages and other immune system components. Preferably, when the introduction of the polypeptide, the immunization scheme will include two or more injections of the polypeptide, distributed over several weeks.

After detecting the immune response, for example, detection of antibodies specific against the antigen RGM A, in the serum of the mouse, move the mouse spleen and isolated splenocytes. Then splenocytes drained well-known methods with any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridoma select and clone by limiting dilution. Then hybridoma clones analyzed by methods known in this field, for cells that secrete antibodies capable of binding RGM A. By immunization of mice positive hybridoma clones can be generated ascitic fluid, which typically contains high levels of antibodies.

In another embodiment, from that immunized animal can be obtained by producing the antibody immortalized hybridoma. After immunization the animal is killed and spleen cells merge with termed by the myeloma cells, also well known in this field. See, for example, Harlow and Lane, supra. Predpochtitelno embodiment, these myeloma cells do not secrete immunoglobulin polypeptides ("non-secretory cell line). After merging and selection using antibiotics these hybridoma subjected to screening using RGM A or part thereof, or cells expressing RGM A. In the preferred embodiment, perform the initial screening using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA. An example of a screening ELISA is provided in WO 00/37504 included in the present description by reference.

Producing anti-RGM A-antibody hybridoma select, clone and subjected to additional skrining for desirable characteristics, including strong growth in hybrid, high production of antibodies and the desired characteristics of the antibodies discussed additionally below. Hybridoma can grow and proliferate in vivo in syngeneic animals, animals that are deprived of the immune system, for example, “Nude” mice or in cell cultures in vitro. The methods of selection, cloning and propagation of hybridomas is well known qualified in this area specialists.

In a preferred embodiment, hybridoma are mouse hybridomas, as described above. In another preferred embodiment, hybridoma get in species other than human and mouse, so the x as rats, sheep, pigs, goats, cattle or horses. In another embodiment, hybridoma are human hybridomas, in which " non-secretory myeloma human fused with a human cell expressing an anti-RGM A-antibody.

Antibody fragments which recognize specific epitopes may be generated by known methods. For example, Fab fragments and F(ab')2according to the invention can be obtained by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2-fragments). F(ab')2fragments contain the variable region, constant region light chain and SN domain of the heavy chain.

3.3. Anti-RGM A-monoclonal antibodies obtained using SLAM

In another aspect of the invention, recombinant antibodies generated from separate lymphocytes using procedures known in this field of fashion with selected antibodies by lymphocytes (SLAM), as described in U.S. Patent No. 5627052, PCT Publication WO 92/02551 and Babcock, J.S. et al. (1996) Proc. Natl. Acad. Sci USA 93:7843-7848. In this method, single cells secreting interest antibodies, e.g., lymphocytes derived from any one of the immunized animals described above, is subjected to screening using antigenspecific is yoga hemolytic analysis plaques where the antigen RGM A, subunit RGM A or its fragment associated with sheep red blood cells using a linker, such as Biotin, and used to identify single cells that secrete antibodies specific against RGM A. After identification of interest secreting antibody cells cDNA variable region of the heavy chain and light chain release from these cells using PCR with reverse transcriptase and these variable regions can then be expressed, in the context of appropriate constant regions of immunoglobulin (e.g., constant regions of human immunoglobulin)in the cells of the host mammal, such as COS cells or CHO. Cell owners, transfetsirovannyh these amplificatoare sequences of immunoglobulin derived from the selected in vivo lymphocytes, can then be subjected to further analysis and selection in vitro, for example, by means of panning transfected cells to select cells expressing antibodies to RGM A. These amplificatoare sequence of the immunoglobulin can then be manipulated in vitro, such as methods of affinity maturation in vitro, such as the methods described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3.4. Monoclonal anti-RGM A-antibodies produced using transgenic animals

In another embodiment of the invention, antibodies obtained by immunization of an animal (not human)that contains multiple loci or all of the loci of the immunoglobulin, antigen RGM A. In the preferred embodiment, this animal (not human) is transgenic XENOMOUSE mouse, obtained by genetic engineering of the mouse strain, which contains large fragments of the immunoglobulin loci of human rights and is defective in the production of antibodies mouse. See, for example, Green et al. Nature Genetics 7:13-21 (1994) and U.S. Patent№ 5916771, 5939598, 5985615, 5998209, 6075181, 6091001, 6114598 and 6130364. See, also WO 91/10741, published July 25, 1991, WO 94/02602, published February 3, 1994, WO 96/34096 and WO 96/33735, both published October 31, 1996, WO 98/16654, published April 23, 1998, WO 98/24893, published June 11, 1998, WO 98/50433 published November 12, 1998, WO 99/45031, published September 10, 1999, WO 99/53049, published October 21, 1999, WO 00 09560, published on 24 February 2000 and WO 00/037504, published June 29, 2000. Transgenic XENOMOUSE mouse produces a similar repertoire of the adult human repertoire of fully human antibodies and generates antigen-specific mAb person. Transgenic XENOMOUSE mouse contains approximately 80% of the repertoire of human antibodies by introducing in the amount of mega digested YAC fragments of the heavy chain loci of the human x lo the moustache light chain germline configuration. Cm. Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), the descriptions of which are incorporated into this description by reference.

3.5. Monoclonal anti-RGM A-antibodies, obtained from libraries of recombinant antibodies

Howin vitrocan also be used to generate antibodies according to the invention, in which a library of antibodies are screened to identify antibodies having the desired binding specificity. Methods for screening libraries of recombinant antibodies is well known in this area and include the methods described in, for example, Ladner et al. U.S. patent No. 5223409; Kang et al. PCT publication no WO 92/18619; Dower et al. PCT publication no WO 91/17271; Winter et al. Publication no WO 92/20791; Markland et al. PCT publication no WO 92/15679; Breitling et al. PCT publication no WO 93/01288; McCafferty et al. PCT publication no WO 92/01047; Garrard et al. PCT publication no WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the Publication of the patent application U.S. 20030186374 and PCT Publication no WO 97/29131, the contents of which are incorporated into this description by reference.

Library of recombinant antibodies can be obtained from indev is Yes, immunized RGM A or part RGM A. alternatively, a library of recombinant antibodies may occur from untreated individual, i.e. the individual who has not been immunized RGM A, for example, a library of human antibodies from individual-a person who has not been immunized RGM A person. Antibodies according to the invention is chosen by screening libraries of recombinant antibodies by peptide containing RGM A person, for selection by means of antibodies that recognize RGM A. Methods for such screening and selection are well known in this field, such as the methods described in the references in the previous paragraph. To select antibodies of the invention having specific affinity binding in respect of hRGM A, such as antibodies, which dissociate from RGM A person with a specific constant speed koffmay be used are known in this field the method of surface plasmon resonance for the selection of antibodies with the desired rate constant koff.For selection of antibodies according to the invention having a particular neutralizing activity against hRGM A, such as antibodies with specific IC50can be used standard methods known in this field, to assess the inhibition activity hRGM A.

In one aspect, this invention relates to the selected antibody or antigennegative the part, which bind RGM A person. Preferably, the antibody is a neutralizing antibody. In a different implementation, the antibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies according to this invention can also be generated using various methods phage display technique, known in the field. In the methods of phage display, functional domains of antibodies are presented on the surface of phage particles which carry the coding them polynucleotide sequence. In particular, such phage can be used to represent antigenspecific domains expressed from a repertoire or the combined libraries of antibodies (e.g., human or mouse). Phage expressing antigennegative domain that binds an antigen, can be selected or identified with the use of an antigen, e.g., using labeled antigen or antigen bound to a solid surface or bead or attached to a solid surface or bead. Phage used in these methods is typically filamentous phage including binding domains fd and M13, expressed from phage with Fab domains, Fv, or stabilized by disulfide Fv antibodies, fused with a protein or gene III or gene VIII phage. An example of the methods of phage display, which can be used to generate antibodies of the present invention, include examples of the methods described in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT Publication No. PCT/GB91/01134; PCT Publications no WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent№ 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743 and 5969108; these references are included in the present description by reference in their entirety.

As described in the above references, after selection of phage encoding region of the antibody from the phage can be isolated and used to generate full-size antibodies, including human antibodies, or any other desired antigennegative fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in more detail below. For example, can also be used ways recombinant obtain Fab fragments, Fab' and F(ab')2using methods known in this field, such as the methods described in PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (these references are included in the present description p the tool reference in their entirety). Examples of methods that can be used to produce single-chain Fv antibodies, include the methods described in U.S. Patent 4946778 and 5258498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993) and Skerra et al., Science 240:1038-1040 (1988).

Alternative screening libraries of recombinant antibodies by phage display, other methodologies known in the field for screening large combinatorial libraries can be used to identify antibodies according to the invention with dual specificity. One type of alternative expression systems is the type in which a library of recombinant antibodies Express in the form of mergers RNA-protein as described in PCT Publication no WO 98/31700 (Szostak and Roberts and Roberts, R.W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, creates a covalent fusion between mRNA and peptide or protein that it encodes, by in vitro translation of synthetic mRNAs that are puromycin, peptidyl-acceptor antibiotic, at their 3'end. Thus, specific mRNAs may be enriched from a complex mixture of mRNAs (e.g., combinatorial libraries) based on the properties of the encoded peptide or protein, for example, the antibodies or portion thereof, such as the binding of this antibody or part thereof with a double antigen specificity. Nucleic acid sequences encoding ant the body or parts thereof, retrieved from screening such libraries may be expressed by recombinant methods as described above (for example, in mammalian cells the host), and, in addition, can be subjected to additional maturation affinity or additional rounds of screening mergers mRNA-peptide, which were introduced mutations selected in the source sequence (original selected sequence), or other methods of affinity maturation in vitro of recombinant antibodies described above.

In another approach, antibodies of the present invention can be also generated using yeast display, known in this area. Methods yeast display, use genetic methods to attach the domains of antibodies to the cell wall of yeast and view them on the surface of yeast. In particular, such yeast can be used to represent antigenspecific domains expressed from a repertoire or combinatorial libraries of antibodies (e.g., human or mouse). Examples of methods yeast display, which can be used to generate antibodies of the present invention include the methods described Wittrup, et al. U.S. patent No. 6699658 included in the present description by reference.

4. Getting specific recombinant RGM A-antibodies from which retenu

Antibodies of this invention can be obtained by any of several methods known in this field. For example, can be used for expression of the host cells, where expressing vector (expressing vectors)encoding the heavy and light chains, transferout in cell host by standard methods. It is assumed that various forms of the term "transfection" includes a large variety of ways, usually used for introducing exogenous DNA into a prokaryotic or eukaryotic cell host, for example, electroporation, precipitation of calcium phosphate, transfection using DEAE-dextran and the like, Although it is possible to Express the antibodies of the invention in any prokaryotic or eukaryotic cells, it is preferable for the expression of antibodies in eukaryotic cells-the owners and the most preferred is the expression in the cells of the host mammal, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells, collect and secrete a properly laid and immunologically active antibody.

Preferred cells of mammalian hosts for expression of recombinant antibodies according to the invention include cells of Chinese hamster ovary cells Cho (including cells dhfr-CHO, described in Ulaub and Chasin, (1980) Proc. Natl. Acad. Sci USA 77:4216-4220, used with a DHFR-breeding marker, for example, as described in R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. With the introduction of expressing recombinant vectors encoding antibody genes, in cells of the host mammal, antibodies are produced by culturing the host cells for a period of time sufficient for expression of the antibody in the cells of the host, or, more preferably, secretion of the antibody into the culture medium which produces the cells of the host. Antibodies can be extracted from the culture medium using standard methods of protein purification.

Cell host can also be used to obtain functional fragments of antibodies, such as Fab fragments or scFv molecules. It will be clear that variations of the above procedure are within the scope of this invention. For example, it may be desirable transfection of the host cell DNA encoding functional fragments or light chain and/or heavy chain antibodies according to the invention. Recombinant DNA technology can also be used to remove some or all of the DNA that encodes any of the light chain and the heavy chain or both chains, which is not required for binding interest antigens. Molecules, expresser is defended from such truncated DNA molecules, also included in the antibody according to the invention. In addition, can be obtained bifunctional antibodies, in which one heavy chain and one light chain are the chains of the antibody according to the invention and the other heavy chain and the other light chain are specific against the antigen, other than interest antigens, resulting in the crosslinking of antibodies according to the invention with a second antibody standard chemical methods of stitching.

In one preferred system for recombinant expression of the antibodies or antigennegative part according to the invention, expressing recombinant vector encoding both the heavy chain of the antibody and the light chain of the antibody is introduced into cells dhfr-CHO mediated by calcium phosphate transfection. This recombinant expressing the vector of each of the genes of the heavy and light chain antibody functionally linked to regulatory elements of the CMV enhancer/AdMLP promoter to run high levels of transcription of these genes. This recombinant expressing the vector also carries the gene for DHFR, which allows for selection of CHO cells that were transliterowany this vector, using methotrexate selection/amplification. Selected transformed cell hosts cultivate opportunities for the expression of heavy and light chains of the antibody and of the cult of the social environment to extract the intact antibody. To obtain recombinant expressing vector, transfection of host cells, selection of transformants, culturing host cells and extraction of this antibody from the culture medium using standard methods of molecular biology. In addition, this invention provides a method for the synthesis of recombinant antibodies according to the invention by culturing a host cell according to the invention in a suitable culture medium until it is synthesized recombinant antibody according to the invention. The method may further include the allocation of this recombinant antibodies from the culture medium.

4.1. Anti-RGM A-antibodies

Table 5 is a list of amino acid sequences of regions VH and VL preferred anti-hRGM A-antibodies according to the invention.

TABLE 5
LIST of AMINO acid SEQUENCES of REGIONS VH And VL of ANTI-hRGM A-ANTIBODY 5F9 AND 8D1
SEQ ID No.Region proteinSequence
123456789012345678901234567890
34 VH 5F9EVQLVESGGGLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSS
57VH 5F9 CDR-H1Remains
31-35
SEQ ID NO:34
NYGMN
58VH 5F9 CDR-H2Remains
50-66
SEQ ID NO:34
MIYYDSSEKHYADSVKG
59VH 5F9 CDR-H3Remains
99-104
SEQ ID NO:34
GTTPDY
10VL 5F9DVVLTQTPVSLSVTLGDQASMSCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIKR
60VL 5F9 CDR-L1Remains
24-39
SEQ ID NO:10
RSSQSLEYSDGYTFLE

61VL 5F9 CDR-L2Remains
55-61
SEQ ID NO:10
EVSNRFS
62VL 5F9 CDR-L3 Remains
94-102
SEQ ID NO:10
FQATHDPLT
55VH 8D1EVQLQQSGPELVKPGTSVKMSCKTSGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVSS
63VH 8D1 CDR-H1Remains
31-35
SEQ ID NO:55
SYVMH
64VH 8D1 CDR-H2Remains
50-66
SEQ ID NO:55
YIIPYNDNTKYNEKFKG
65VH 8D1 CDR-H3Remains
97-110
SEQ ID NO:55
ARRNEYYGSSFFDY
56VL 8D1DIQMTQSPASLSASLEEIVTITCQASQDIDNYLAWYHQKPGKSPRLLIYGATNLADGVPSRFSGSRSGTQFSLKINRLQIEDLGIYYCLQGYIPPRTFGGGTKLELKR
66VL 8D1 CDR-L1Remains
24-34
SEQ ID NO:56
QASQDIDNYLA
67VL 8D1 CDR-L2Remains
50-56
SEQ ID NO:56
GATNLAD
/p>

68VL 8D1 CDR-L3Remains
89-97
SEQ ID NO:56
LQGYIPPRT

Above the selected CDR sequences of anti-RGM A-antibodies form a new family of RGM A-binding proteins identified in accordance with this invention. To generate and to select the CDR according to the invention, with the preferred RGM A binding and/or neutralizing activity against hRGM A, can be used standard methods known in this field for generating binding proteins of the present invention and evaluation of binding of RGM A and/or neutralizing characteristics of this binding protein, including, but not limited to, the methods specifically described in the present description.

4.2. Chimeric anti-RGM A-antibodies

A chimeric antibody is a molecule in which different portions of antibodies derived from different animal species, such as antibodies having a variable region derived from mouse monoclonal antibodies, and a constant region of human immunoglobulin. Methods of obtaining chimeric antibodies are known in this field. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent No. 5807715; 4816567 and 4816397, which included the present description by reference in their entirety. Additionally, there may be used methods developed for "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454, which is incorporated into this description by reference in their entirety) by splicing the genes from a molecule of antibody mouse to the appropriate antigen specificity together with genes from a molecule of the human antibodies of the appropriate biological activity.

In one embodiment, chimeric antibodies according to the invention is obtained by replacing the constant region of the heavy chain of monoclonal mouse antibodies against RGM A human antibodies described in the present description, a constant region of human IgG1.

4.3. CDR-grafted anti-RGM A-antibodies

CDR-grafted antibody according to the invention contain a sequence of variable regions of heavy and light chains of human antibodies in which one or more CDR regions VHand/or VLreplaced with CDR sequences of the antibodies are not human, such as mouse antibodies according to the invention. Frame sequence from any human antibodies can serve as a template for transplantation CDR. However, a direct replacement chain for this frame area often leads to some loss of binding affinity against this antigen. The more homologous human antibody is in regard to the similar antibody mouse the less likely is the possibility that the Association CDR mouse with the frame section will introduce disturbances in these CDR, which can reduce the affinity. Thus, preferably, the flexible frame area of the person who is selected to replace the variable frame section mouse has at least 65% sequence identity with the variable frame section antibodies mouse. More preferably, the variable regions of human and mouse separately from these CDRs have at least 70% sequence identity. Even more preferably, the variable regions of human and mouse separately from these CDRs have at least 75% sequence identity. Most preferably, the variable regions of human and mouse separately from these CDRs have at least 80% sequence identity. Methods of obtaining CDR-grafted antibodies known in the art (Jones et al., Nature 321:522-525 (1986); U.S. Patent No. 5225539). In one specific embodiment, this invention provides a CDR-grafted antibodies with chains VHand/or VLdescribed in table 6.

TABLE 6:
CDR-GRAFTED ANTIBODIES
SEQ ID No.Region protein Sequence
123456789012345678901234567890
35
(15)
(16)
(17)
(18)
VH 5F9.1-GL
(VH3-48/JH3 FR1)
(VH3-48/JH3 FR2)
(VH3-48/JH3 FR3)
(VH3-48/JH3 FR4)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGTTPDYWGQGTMVTVSS

36
(15)
(16)
(17)
(19)
VH 5F9.2-GL
(VH3-48/JH4 FR1)
(VH3-48/JH4 FR2)
(VH3-48/JH4 FR3)
(VH3-48/JH4 FR4)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGTTPDYWGQGTLVTVSS
37
(15)
(16)
(17)
(20)
VH 5F9.3-GL
(VH3-48/JH6 FR1)
(VH3-48/JH6 FR2)
(VH3-48/JH6 FR3)
(VH3-48/JH6 FR4)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGTTPDYWGQGTTVTVSS
38
(21)
(22)
(23)
(18)
VH 5F9.4-GL
(VH3-33/JH3 FR1)
(VH3-33/JH3 FR2)
(VH3-33/JH3 FR3)
(VH3-33/JH3 FR4)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTMVTVSS
39
(21)
(22)
(23) (19)
VH 5F9.5GL
(VH3-33/JH4 FR1)
(VH3-33/JH4 FR2)
(VH3-33/JH4 FR3)
(VH3-33/JH4 FR4)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTLVTVSS
40
(21)
(22)
(23) (20)
VH 5F9.6-GL
(VH3-33/JH6 FR1)
(VH3-33/JH6 FR2)
(VH3-33/JH6 FR3)
(VH3-33/JH6 FR4)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTTVTVSS

41
(24)
(25)
(26)
(18)
VH 5F9.7-GL
(VH3-23/JH3 FR1)
(VH3-23/JH3 FR2)
(VH3-23/JH3 FR3)
(VH3-23/JH3 FR4)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
42
(24)
(25)
(26) (19)
VH 5F9.8-GL
(VH3-23/JH4 FR1)
(VH3-23/JH4 FR2)
(VH3-23/JH4 FR3)
(VH3-23/JH4 FR4)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTLVTVSS
43
(24)
(25)
(26) (20)
VH 5F9.9-GL
(VH3-23/JH6 FR1)
(VH3-23/JH6 FR2)
(VH3-23/JH6 FR3)
(VH3-23/JH6 FR4)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTTVTVSS
44
(27)
(28)
(29)
(30)
VL 5F9.1-GL
(A18/JK2 FR1)
(A18/JK2 FR2)
(A18/JK2 FR3)
(A18/JK2 FR4)
DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
45
(31)
(32)
(33)
(30)
VL 5F9.2-GL
(A17/JK2 FR1)
(A17/JK2 FR2)
(A17/JK2 FR3)
(A17/JK2 FR4)
DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

46
(31)
(28)
(29)
(30)
VL 5F9.3-GL
(A17/JK2 FR1)
(A18/JK2 FR2)
(A18/JK2 FR3)
(A18/JK2 FR4)
DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

CDR sequences derived from mAb 5F9 bold, reference is also made to a particular frame of the sequence (FR1-FR4) by specifying the corresponding SEQ ID NO: (see tables 3 and 4)

4.4. Humanized anti-RGM A-antibodies

Humanized antibodies are antibody molecules from antibody type is not human, which binds the desired antigen having one or more complementarity determining regions (CDR) of the form not of a man and a frame region of the molecules of the immunoglobulin. The known sequence of human Ig described, for example, in www.ncbi.nlm.nih.gov/etrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/.about.pedro/research_tools.html; www.mgen.uni-heidelberg.de/SD/IT/IT.html; www.whfreeman.com/immunology/CH-05/kuby05.htm; www.library.thinkquest.org/12429/lmmune/Antibody.html; www.hhmi.org/grants/lectures/1996/vlab/; www.path.cam.ac.uk/.about.mrcZ/m-ikeimages.html; www.antibodyresource.com/; mcb.harvard.edu/BioLinks/lmmuno - logy.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html; www.biotech.ufl.edu/.about.hcl/; www.pebio.com/pa/340913/340913 .html-; www.nal.usda.gov/awic/pubs/antibody/; www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html; www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html; www.biotech.ufl.edu/.about.fccl/protocol.html; www.isac-net.org/sites_geo.html; aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html; baserv.uci.kun.nl/.about.jraats/linksl.html; www.recab.uni - hd.de/immuno.bme.nwu.edu/; www.mrc-cpe.cam.ac.uk/imt-doc/pu - blic/INTRO.html; www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/; www.biochem.ucl.ac.uk/.about. martin/abs/index.html; antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html; www.unizh.ch/.about.honegger/AHOsem-inar/Slide01.html; www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm; www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.cryst.bioc.cam. ac.uk/.about.fmolina/Web-pages/Pept/spottech.html; www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html. Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1983), each of which are incorporated fully by reference. Such input sequences can be used to reduce immunogenicity or decrease, increase or modification of the binding affinity, the velocity of the Association, the rate of dissociation, avidity, specificity, time-life, or any other suitable characteristics, as is well known in this field./p>

The frame remains in the frame areas of a person can be replaced by the corresponding residue of the antibody-donor CDR to change, preferably improve, antigen binding. These frame replacement identify ways well known in this field, for example, by modeling of the interactions of the CDR and framework residues to identify the frame of residues important for antigen binding and the identification of unusual frame residues in specific positions. (See, for example, Queen et al., U.S. patent No. 5585089; Riechmann et al., Nature 332:323 (1988), which is incorporated into this description by reference in their entirety). Three-dimensional models of immunoglobulin are generally available and known to a skilled in this area specialists. Available computer programs that illustreret and show the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these displays allows you to analyze the likely role of these residues in the functioning of such the candidate immunoglobulin sequence, i.e., to analyze the remains, which affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the consensus and input (imported) sequences and the joint is tive, so get the desired characteristics of the antibodies, such as increased affinity against the target antigen (antigen-targets). Usually, these CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies can be humaniterian using methods known in this field, such as, but not limited to, the methods described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT Publication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592106; EP 519596, EP 239400, U.S. Patents№ 5565332, 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567, each of these references is hereby incorporated into this description by reference in full, including cited in these references.

5. Additional variants of the antibodies according to the invention

5.1. Merged antibodies and immunoadhesins

This application also describes a fused antibody or immunoadhesin, which can be obtained and which contains all of RGM A-antibody or part of RGM A-antibodies of the present invention associated with another polypeptide. In some embodiments, the implementation only variable region RGM A-antibodies associated with the polypeptide. In other embodiments, implementation, VH-domain RGM A-antibodies of this application is related to the first polypeptide, while the VL domain of the antibody is linked to a second polypeptide that associates with the first polypeptide in such a way that it allows the VH and VL domains can interact with one another with formation of antigennegative site. In other embodiments, implementation, VH domain is separated from the VL-domain linker, which allows the VH and VL domains can interact with each other (see section below under the heading single-Chain Antibodies). Then the antibody VH-linker-VL associated with interest polypeptide. It merged antibody applied in the direction of the polypeptide in a cell or tissue that expresses RGM A. interest the polypeptide may be a therapeutic agent such as a toxin, or may be a diagnostic tool, such as an enzyme, which can be easily visualized, such as horseradish peroxidase. In addition, can be created and merged antibodies in which two (or more) single-chain antibodies are associated with each other. Applicable, if desirable is the creation of a divalent or polyvalent antibodies on a single polypeptide chain or if it is desirable creating especifismo antibodies.

One option provides a labeled binding protein, in which the antibody or part of an antibody of this application derivative or linked to another functional molecule (e.g., another peptide or protein). For example, a labeled binding protein of this application can be obtained functional binding antibodies or parts of antibodies of this application (chemical coupling, genetic fusion, non-covalent Association or otherwise) with one or more other molecular constituents such as nucleic acid, another antibody (e.g., bespecifically antibody or diatel), the detected agent, cytotoxic agent, a pharmaceutical agent and/or a protein or peptide that can mediate Association of the antibody or part of an antibody with another molecule (such as cortical area (core) streptavidin or polyhistidine label)).

Applicable detected agents, which can be derivativea antibody or part of an antibody of this application, include fluorescent compounds. Examples of fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonate, phycoerythrin etc. Antibody can also be derivative detectivesyme enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, etc. by derivatization of the antibody detektivami enzyme, it will be detected by adding additional reagents that the enzyme uses to education detective the second reaction product. For example, when there is horseradish peroxidase as program agent, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detektivami. The antibody can also be derivative nucleic acid, Biotin, and detected through indirect measurement of the binding avidin or streptavidin.

5.2. Single-chain antibodies

This proposal includes a single-chain antibody (scFv)that binds immunogenic RGM A according to the invention. To obtain scFv VH and V-coding DNA is functionally linked to DNA encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser), so these VH and VL sequences can be expressed in the form of a contiguous single-chain protein, with the VL and VH regions joined this flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 30 Nature (1990) 34 8:552-554). This is a single-chain antibody can be monovalent, if you use only a single VH and VL, bivalent, if you use two VH and VL, or polyvalent, if you use more than two VH and VL. Two of these scFv fragments linked through the linker, called "diatesom", and this form is also included in the invention.

5.3. Bespecifically antibodies

This application VK is uchet next bespecifically antibody or antigennegative fragment, in which one specificity specificity is against immunogenic RGM A polypeptide of this application. For example, can be generated bespecifically antibody that specifically binds to the immunogenic polypeptide RGM A according to the invention through one binding domain and the second molecule through a second binding domain. In addition, can be generated single-chain antibody containing more than one VH and VL, which binds specifically with the immunogenic polypeptide according to the invention and with another molecule, which is associated with the weakening mediated by myelin collapse of the growth cone and inhibition of the growth of neurites (axons) and sprouting (growth of nerve fibers). Such bespecifically antibodies may be generated using methods that are well known, for example, Fanger et al. Immunol Methods 4:72-81 (1994) and Wright and Harris, 20 (supra).

In some embodiments, implementation, bespecifically antibodies obtained using one or more variable regions of the antibodies according to the invention. In another embodiment, this bespecifically antibody is obtained using one or more CDR regions from the indicated antibodies.

5.4. Derivationally and labeled antibodies

The antibody or antigennegative fragment of this application can be derivativea or tie the us with another molecule (for example, another peptide or protein). Typically, the antibody or antigennegative fragment derivatized so that the derivatization or this tagging will not have a negative effect on the binding of immunogenic polypeptide according to the invention.

For example, the antibody or part of an antibody of this application can be functionally linked (by chemical coupling, genetic fusion, non-covalent Association or otherwise) with one or more other molecular constituents such as nucleic acid, another antibody (e.g., bespecifically antibody or diatel), the detected agent, cytotoxic agent, a pharmaceutical agent and/or a protein or peptide that can mediate Association of the antibody or part of an antibody with another molecule (such as an area of bark (core) streptavidin or polyhistidine label)). In addition, the antibody or antigennegative part can be part of a larger molecule immunoadhesin, formed by covalent or non-covalent coupling of the antibodies or portions of antibodies with one or more other proteins or peptides. Examples of such molecules immunoadhesin include the use of the medullary region (cortex) of streptavidin to get tetramer scFv molecules (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, Marche the nogo peptide and C-terminal polyhistidine labels to obtain bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058). Part of the antibodies, such as Fab and F(ab')2fragments can be derived from a full-sized antibodies using conventional methods, such as cleavage by papain or pepsin, respectively, full-size antibodies. Furthermore, antibodies, part of the antibody and molecules of immunoadhesin can be obtained using standard methods of recombinant DNA.

Derivativenote antibody can be obtained by crosslinking of two or more antibodies (of the same type or different types, for example, to create bespecifically antibodies). Suitable cross-linking linkers include linkers that are heterobifunctional having two different reactive groups separated by an appropriate spacer (e.g., complex ether m-maleodoranti-N-hydroxysuccinimide), or homophonically linkers (e.g., disuccinimidyl). Such linkers are available from Pierce Chemical Company, Rockford, III.

Derivativenote antibody may also be labeled antibody. For example, agents detection, which can be derivativea antibody or part of an antibody according to the invention are fluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonate, phycoerythrin, lanthanide phosphors and the like, the Antibody may is also be labeled with enzymes, applicable for detection, such horseradish peroxidase, galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. options that aim detektivami enzyme, the antibody is detected by adding additional reagents that the enzyme uses to education program reaction product. For example, when there is horseradish peroxidase as program agent, add hydrogen peroxide, diaminobenzidin. The antibody can also be labeled with Biotin, and detected through indirect measurement of the binding avidin or streptavidin. The antibody can also be tagged with a pre-defined polypeptide epitope recognized by a secondary reporter (e.g., a pair of sequences latinboy zipper, binding sites for secondary antibodies, metallovedeniye domains, epitope tags (tags)). RGM A-antibody or antigennegative fragment can be labeled radioactively labeled amino acid. This radioactive label can be used both for diagnostic and for therapeutic purposes. Radioactively labeled RGM A-antibody can be used diagnostically, for example, to determine the levels of RGM receptor A in the individual. In addition, radioactively labeled RGM A-antibody can be used terap whitesky for the treatment of spinal cord injury.

Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides15N35S90Y99Tc111In125I131I177Lu,166Ho,153Sm. RGM A-antibody or antigennegative fragment can also be derivativea chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups can be applied to improve the biological characteristics of the antibodies, for example, to increase half-life in serum or to increase the binding fabric. In addition, the label for the polypeptides can include a nucleic acid, e.g. DNA detection using PCR or increasing gene expression or siRNA for suppression of gene expression in RGM A-bearing cells or tissues.

The class and subclass of RGM A-antibodies can be determined using any method known in this field. Usually, the class and subclass of antibodies can be determined using antibodies that are specific against a particular class and subclass of antibodies. Such antibodies are commercially available. The class and subclass of antibodies can be determined using ELISA, Western blotting, and other methods. Alternatively, the class and subclass can be determined by sequencing all or part of constantinpole heavy and/or light chains of these antibodies, by comparing their amino acid posledovatelnosti with known amino acid sequences of different classes and subclasses of immunoglobulins and the definition of the class and subclass of the antibody.

5.5. Immunoglobulins with dual variable domain

Binding proteins or antibodies with dual variable domain (DVD), used in the present description, are binding proteins that contain two or more antigenspecific site and are multivalent proteins, for example, multivalent binding proteins, for example, divalent and tetravalent. The term "multivalent binding protein” is used in this application to denote binding protein containing two or more antigenspecific sites. This multivalent binding protein is preferably designed in such a way that it has two or more antigenspecific sites and is usually not found in nature antibody. The term "multispecificity binding protein" refers to a binding protein capable of binding two or more targets, or not to associate. These DVDs can be monospecific, i.e. able to bind one antigen, or multispecificity, i.e. capable of binding two or more antigens. Connecting DVD-proteins containing two heavy polypeptide the second circuit DVD and two light polypeptide chains DVD, called DVD-Ig. Each half-DVD-Ig contains DVD polypeptide heavy chain DVD polypeptide light chains and two antigenspecific site. Each binding site contains a variable domain of the heavy chain variable domain and a light chain, i.e. has a total of 6 CDR, involved in the binding to the antigen, one antigennegative website. Connecting DVD-proteins and methods of obtaining binding DVD proteins described in Application for U.S. patent No. 11/507050 and incorporated into the present description by reference. It is assumed that the present invention includes connecting a DVD protein containing proteins that can bind RGM A. Preferably, this connecting DVD-protein capable of binding RGM A and the second target. This second target is selected from the group consisting of MAB anti-inflammatory activities (IL-1, IL-6, IL-8, IL-11, IL-12, IL-17, IL-18, IL-23, TNF alpha/beta, IFN-beta, gamma, LIF, OSM, CNTF, PF-4, alkaline protein of platelets (PBP), NAP-2, beta-TG, MIP-1, MCP2/3, RANTES, lymphotactin), mediating the transport of proteins (receptor insulin receptor transferrin, thrombin receptor, leptin receptor, LDL receptor), other neurodegenerative MAB (NgR, Lingo, p75, CSPG (for example, NG-2, Maracana, brevican, versican, Arcana) hyaluronic acid, mAG, tenascin, NI-35 AND NI-250, IMP, perlecan, Maracana, phosphacan, nogo-A, OMGP, Sema4D, Sema 3A, afrina B3, afrina A2, afrina A5 MAG, EphA4, plexin B1, TROY, wnts, ryk rec, BMP 2, BMP-4, BMP-7), neuroprotective who's activities MAB (EGF, EGFR, Sema 3), anti-amyloid remedy beta MAB (for example, m266, 3D6 (bapineuzumab), anti-globular MAB 7C6), localized in the Central nervous system receptors and transporters (serotonin receptors, dopamine receptors, DAT, Asc-1, GlyT1).

5.6. Antibodies with dual specificity

This application also describes a technology of antibodies with dual specificity". Antibodies with dual specificity can serve as agonists, antagonists, or both in various combinations. Antibodies with dual specificity are antibodies in which the VH-chain binds to the first antigen and a VL-chain binds to the second antigen, as shown in the examples WO2008082651.

5.7. Kristallizuetsya antibodies

Another variant of this application provides kristallizuetsya binding protein. The term "kristallizuetsya" applies in this contexte to the antibody or antigennegative parts that exist in the form of a crystal. The crystals are a form of solid state of matter, which differs from other forms, such as amorphous solid state or liquid state. The crystals consist of regular, repeating, three-dimensional ordered rows of atoms, ions, molecules (e.g. proteins, such as antibodies or molecular complexes (e.g., complexes of antigen/antibody). These three-dimensional ordered series is built with the availa able scientific C with a specific mathematical equations, which are well known in this field. A fundamental unit, or elementary link of molecules that are repeated in the crystal, called the asymmetric unit. The repetition of the asymmetric unit in the crystal in a location that corresponds to a specific, well-defined crystallographic symmetry, provides a “unit cell” of the crystal. Repeat this cell of the crystal through regular broadcasts (transfers) in all three directions provides crystal. Cm. Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nded., pp. 201-16, Oxford University Press, New York, New York, (1999).

Preferably, the present application describes a full-sized crystals RGM A-antibodies and their fragments, described in the present description, and finished formulations and compositions containing such crystals. In one embodiment, the crystallized binding protein has a greater half-life in vivo than the soluble copy of this binding protein. In another embodiment, this binding protein retains biological activity after crystallization.

Kristallizuetsya binding protein according to the invention can be obtained in accordance with methods known in this field, such as the methods described in WO 02072636 included in the present description by reference.

HIV antibodies

Another variant implementation of the invention provides glycosylated binding protein, where the antibody or antigennegative part contains one or more carbohydrate residues. The products formed in vivo protein may be subjected to additional processing, known as post-translational modification. In particular, sugar (glucosamine) residues may be added to the enzymatic process known as glycosylation. The obtained proteins bearing covalently linked oligosaccharide side chains, known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain and a variable domain. Carbohydrate residues in the Fc domain having an important effect on the effector function of the Fc domain with minimal effect on the binding of the antigen or the half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on antigennegative activity of this antibody. Glycosylation in the variable domain may have a negative effect on the binding affinity of antibodies, probably due to spatial difficulties (physical barrier) (Co, M.S., et al., Mol. Immunol. (1993) 30:1361-1367), or cause uvelichenie the affinity against this antigen (Wallick, S.C., et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J.(1991) 10:2717 2723).

One aspect of the present invention is aimed at obtaining mutants, glycosylation site, which was mutated O - or N-linked glycosylation site binding protein. Qualified in this field specialist can generate such mutants using standard well-known technologies. The mutant glycosylation sites that retain biological activity but have increased or decreased binding activity are the following object of the present invention.

In another embodiment, the glycosylation of the antibody or its antigennegative part according to the invention is modified. For example, can be obtained glycosylamine antibody (i.e. antibody devoid of glycosylation). Glycosylation can be altered, for example, to increase the affinity of this antibody against the antigen. Such carbohydrate modifications can be performed, for example, by changing one or more glycosylation sites in the sequence of this antibody. For example, can be obtained one or more amino acid substitutions that lead to the elimination of one or more sites of glycosylation variable regions for elimination through this glycosylation in sa is the same. This glycosylamine can increase the affinity of this antibody against the antigen. This approach is described in more detail in PCT Publication WO2003016466A2 and U.S. Patent No. 5714350 and 6350861, all references included in the present description by reference in full.

Additionally or alternatively, it may be obtained a modified antibody according to the invention, which has an altered type of glycosylation, which is hypoparathyroidism antibody having reduced the number fucosamine residues or an antibody having increased the number of dividing in half GlcNAc structures. It is shown that such modified distribution (patterns) glycosylation increase ADCC-the ability of the antibodies. Such carbohydrate modifications can be effected, for example, expression of this antibody in the cell host with a modified apparatus glycosylation. Cells with the modified apparatus of glycosylation have been described in this area and can be used as host cells for the expression in them of recombinant antibodies according to the invention to obtain by means of this antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and European Patent No: EP 1176195; PCT Publication WO 03/035835; WO 99/5434280, all these references are included in the present description by reference is their entirety.

Glycosylation of proteins is dependent on the amino acid sequence of the protein of interest, as well as from the host cell, in which this protein is expressed. Different organisms can produce various enzymes of glycosylation (e.g., glycosyltransferases and glycosidase) and have a variety of available substrates (nucleotidase). Due to these factors, the nature of the glycosylation of proteins and the composition helicoiling residues may vary depending on the system host, which is expressed specific protein. Glucosamine remains applicable in the invention, can include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably, this glycosylated binding protein contains such glucosimine remains that the nature of glycosylation is the nature of the glycosylation of human nature.

Qualified in this field specialist known that different glycosylation of proteins can lead to different characteristics of proteins. For example, the effectiveness of therapeutic protein derived from a microorganism host such as yeast, and glycosylated using endogenous pathway of yeast, can be reduced in comparison with the efficiency of the same Bel is a, expressed in mammal cells, such as cell line Cho. These glycoproteins may also be immunogenic in humans and discover a reduced life time in vivo after injection. Specific receptors in humans and other animals can learn specific glycosamine residues and to stimulate rapid clearance of this protein from the bloodstream. Other negative effects may include changes in protein folding, solubility, susceptibility to proteases, directed migration, transport, compartmentalization, secretion, recognition of other proteins or factors of antigenicity or allergenicity. Thus, the practitioner may prefer a therapeutic protein with a specific composition and nature of glycosylation, e.g., composition and distribution of glycosylation identical or at least similar, with the composition and distribution of glycosylation produced in human cells or in species-specific cells to the intended individual animal.

The expression of glycosylated proteins, non-glycosylated proteins of the host cell, can be achieved by genetic modification of the host cell for the expression of heterologous enzymes of glycosylation. Using methods known in this field, the practitioner can generate antibodies or anti-Christ. envasive part, showing glycosylation of proteins person. For example, the yeast strains were genetically modified to Express not naturally occurring glycosylation enzymes, so glycosylated proteins (glycoproteins)produced by these strains of yeast, find glycosylation of proteins with identical glycosylation of proteins of animal cells, particularly human cells (patent Application U.S. 20040018590 and 20020137134 and PCT Publication WO2005100584 A2).

In addition, qualified in this field specialist will be clear that the protein of interest can be expressed using the library of host cells, genetically engineered for the expression of various enzymes of glycosylation, so that cells are the owners, members of this library, produce a protein of interest with a specific new distributions glycosylation. Preferably, a protein that has specifically selected the new distribution of glycosylation shows improved or modified biological properties.

5.9. Antiidiotypic antibodies

In addition to these binding proteins, the present invention also relates to antiidiotypic (anti-Id) antibody specific for such binding proteins according to the invention. Anti-Id antibody is the antibody that is the same is no unique determinants, usually associated with antigennegative region of another antibody. This anti-Id can be obtained by immunization of an animal of this binding protein or CDR-containing region. Immunized animal will recognize idiotypical determinants of the immunizing antibody and to answer them and to produce anti-Id antibody. Anti-Id antibody can also be used as an immunogen for the induction of an immune response in yet another animal with obtaining the so-called anti-anti-Id antibodies.

6. Use of antibodies

Due to their ability to contact RGM A human neutralizing antibodies of the present invention, or parts thereof, can be used to detect RGM A person (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA) or immunohistochemistry tissue. This application provides a method for detecting RGM A person in a biological sample, involving contacting a biological sample with the antibody or part of an antibody according to the invention and detecting either the antibody (or parts of antibodies)associated with RGM A person or unbound antibody (or parts of antibodies) to detect through this RGM A person in biological the th sample. This antibody is directly or indirectly mark detektivami substance to facilitate detection of the bound or unbound antibody. Suitable detected substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable complexes prosthetic groups include streptavidin/Biotin and avidin/Biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorofluorescein, ancillary or phycoerythrin; examples of the fluorescent substances include luminal and examples of suitable radioactive material include3H,14C,35S90Y99Tc111In125I131I177Lu,166Ho,153Sm.

Antibodies and portions of the antibodies of this application preferably is able to neutralize the activity of RGM A person both in vitro and in vivo. Thus, such antibodies, and portions of the antibodies according to the invention can be used to inhibit the binding of RGM A to its receptor by Neogene, with BMP-2 and BM-4, and, consequently, to inhibit the resulting activity.

In another embodiment, the crust is Asa application provides a method of reducing the activity of RGM A the individual, mainly in individuals suffering from diseases or disorders in which the activity of RGM A negative impact. This application provides methods of reducing the activity of RGM A an individual suffering from such diseases or disorders, prevention of binding of RGM A and receptor, Neogynona, and/or with BMP-2 and/or BM-4, by use of the monoclonal antibodies of this application. Antibodies of the present invention, in particular, humanized antibodies described in the present description, can be administered to the individual-to man for therapeutic purposes. In addition, the antibodies of this application can be administered to the mammal, not the person expressing RGM A, with which this antibody is able to bind, for veterinary purposes or animal used as a model for human disease. With regard to the latter application, such animal models may be applicable for the evaluation of therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time schemes introduction).

In the context of the present description, the term "a disorder in which the activity of RGM A negative impact"includes diseases and other disorders in which, as shown, the presence of RGM A or its resulting activity of the individual suffering from this disorder is, or allegedly is sustained fashion is responsible for the pathophysiology of this violation, or is a factor that contributes to the deterioration of this violation. Thus, I believe that the disorder in which the activity of RGM A negative impact, is a disorder in which the decrease in the activity of RGM A reduces symptoms and/or progression of this violation. Not limiting the invention, examples of disorders that can be treated with antibodies of the invention include disorders discussed in the section below relating to the pharmaceutical compositions of the antibodies according to the invention.

It is recognized that RGM A plays an important role in the pathology associated with various diseases, including neurological diseases associated with neurodegeneration or inhibition neuroregenerative processes leading to paralysis. It includes dementia, senile dementia, mild cognitive disorder, dementia associated with Alzheimer's disease, horey of Hungtington, late dyskinesia, hyperkinesia, mania, Parkinson's disease, syndrome Steele-Richardson, down syndrome, severe pseudoparalysis the gravis, nerve damage, vascular amyloidosis, cerebral hemorrhage I with amyloidosis, inflammation of the brain, hereditary ataxia, acute violation with disorientation in time and space, glaucoma, Alzheimer's disease, lateral AMI the trophic sclerosis, brachial plexus injury, brain injury, including traumatic brain injury, cerebral palsy, a disease Guillain-Barre, leukodystrophies, multiple sclerosis, post-vaccination polio, Spina Bifida (spina bifida, spinal cord injury, atrophy of the muscles of the spine, tumors of the spine, stroke, and transverse myelitis.

In addition, as discussed earlier, between any of the above partners can use DVD immunoglobulins, or antibodies with dual specificity. Such antibody preparations, as described above, may be applicable for the treatment of Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, multiple sclerosis, peripheral nerve injuries, schizophrenia, depression, anxiety, and any plasticity and growth of neurites and is associated with neurotoxicity disease, quoted above.

Antibodies of this application can also be combined with peptides, making it possible transmembrane transfer to enable targeting of intracellular proteins target. Such peptide sequences may include, but are not limited to,tat, antennapediapolyalanine, some antimicrobial peptides. Such peptides may allow transfer across membranes, including the Isla cell plasma membrane, but also epithelial and endothelial membranes, including the blood-brain barrier, the intestinal mucosa, brain shell and others.

The antibody or part of an antibody of this application may also be administered with one or more additional agents with small molecules that are applicable in the treatment of disorders involving the activity of RGM A, as discussed in the previous paragraphs. It should be understood that the antibodies of this application or antihistamie parts can be used separately or in combination with additional means, for example, therapeutic tool, with this additional tool is selected qualified for the intended goal for him. For example, this option can be a therapeutic tool, recognized this area as a tool that is applicable for the treatment of a disease or condition which is subject to treatment with an antibody of the present invention. This additional means may also be a tool that gives the best indication of this therapeutic compositions, for example, by means of which affects the viscosity of this composition.

7. The pharmaceutical composition

This invention also provides a pharmaceutical composition comprising the antibody or antigenspecific according to the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the antibodies according to the invention is applicable, but not limited to, diagnosis, detection or monitoring violations in the prevention, treatment, control, or reduction of the intensity of violations or one or more symptoms and/or research. In a specific embodiment, the composition comprises one or more antibodies according to the invention. In another embodiment, this pharmaceutical composition comprises one or more antibodies according to the invention and one or more prophylactic or therapeutic agents other than antibodies of the invention, for the treatment of disorders in which the activity of RGM A negative impact. Preferably, it is known that these prophylactic or therapeutic agent are applicable or have been, or are currently in use in the prevention, treatment, control, or reduction of the intensity of violations or one or more of its symptoms. In accordance with these variants of implementation, this composition may further contain a carrier, diluent or excipient.

Antibodies and part of the antibodies according to the invention can be incorporated into pharmaceutical compositions suitable for administration to the individual. Usually, this pharmaceutical composition of the ash is incorporated an antibody or part of an antibody according to the invention and a pharmaceutically acceptable carrier. In the context of the present description, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delaying absorption agents and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more components from water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases it will be preferred inclusion isotonic agents, for example, sugars, polyalcohol, such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as moisturizing or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibodies or portions of antibodies.

There are various delivery systems, and they can be used for introducing one or more antibodies according to the invention or a combination of one or more antibodies according to the invention and a prophylactic agent or therapeutic agent, applicable to the prevention, monitoring, treatment or reduction of the intensity drug the decision or one or more symptoms for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells that can Express the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), the construction of a nucleic acid as part of a retroviral or other vector, etc. Ways of introducing preventive or therapeutic agent according to the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural introduction, intratumoral introduction, and introduction to the mucous membrane (for example, the introduction in the nose and in the mouth). In addition, can be used pulmonary introduction, for example, using the inhaler or nebulizer and composition with aerosol agent. See, for example, U.S. Patents№ 6019968, 5985320, 5985309, 5934272, 5874064, 5855913, 5290540 and 4880078; and PCT Publication no WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, all of which are included in the present description by reference in their entirety. In one embodiment, the antibody according to the invention, a combined therapeutic agent or composition according to the invention is administered using the technology of pulmonary drug delivery Alkermes AIR® (Alkermes, Inc., Cambridge, Mass.). In one specific embodiment, the implementation of the Oia, preventive or therapeutic agent according to the invention is administered intramuscularly, intravenously, into the tumour, oral, intranasal, pulmonary, by or subcutaneously. These preventive or therapeutic agent can be administered in any convenient way, for example, by infusion or bolus injection, by absorption through epithelial or skin-mucous lining (for example, through the mucous membrane of the mouth, rectally or through the intestinal mucous membrane, etc. and can be administered together with other biologically active agents. The administration can be systemic or local.

In a specific embodiment, it may be desired prophylactic or therapeutic agents of the invention locally to the area in need of treatment; this may be achieved, for example, without limitation, local infusion, by injection, or by means of an implant, with the specified implant is porous or non-porous material, including membranes and matrices, such as membrane from Celestica, polymers, fibrous matrices (e.g., Tisseel®), or collagen matrices. In one embodiment, an effective amount of one or more antibodies, antagonists according to the invention is administered locally to the affected area of the individual for the prevention, treatment, control and/or humanitarianintervention disorders or symptom. In another embodiment, an effective amount of one or more antibodies, antagonists according to the invention is administered locally to the affected area in combination with an effective amount of one or more therapeutic agents (e.g., one or more prophylactic or therapeutic agents), other than the antibody according to the invention for the prevention, treatment, control and/or reduce the intensity of violations or one or more of its symptoms.

In another embodiment, it is a preventive or therapeutic agent can be delivered in a controlled or sustained release. In one embodiment, to achieve controlled or sustained release of therapeutic agents according to the invention can be used in the pump (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, to achieve controlled or sustained release of therapeutic agents according to the invention can be used polymeric materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During t al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105); U.S. Patent No. 5679377; U.S. Patent No. 5916597; U.S. Patent No. 5912015; U.S. Patent No. 5989463; U.S. Patent No. 5128326; PCT Publication no WO 99/15154 and PCT Publication no WO 99/20253. Examples of polymers used in the form prolonged release include, but are not limited to, poly(2-hydroxyethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid)copolymer(ethylene-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), copolymers (lactides and glycolide) (PLGA) and polyarteritis. In one preferred embodiment, the polymer used in the form of prolonged action, is inert, free from leaching of impurities, stable during storage, sterile, and biodegradiruemym. In another embodiment, a controlled or sustained release can be placed near a preventive or therapeutic target that allows you to enter only a small fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

System of controlled release described in the review Langer (1990, Science 249:1527-1533). Any method known to the skilled in this field specialist, can be used to receive the composition sustained release, containing one or more therapeutic agents according to the invention. Cm. for example, U.S. Patent No. 4526938, PCT Publication WO 91/05548, PCT Publication WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", Radiotherapy &Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854 and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of these references is hereby incorporated into this description by reference in its full scope.

In one specific embodiment where the composition according to the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of the encoded it preventive or therapeutic agent by constructing it as part of a suitable expressing vector nucleic acid and inserting it so that it becomes intracellular, for example, using a retroviral vector (see U.S. Patent No. 4980286), or direct injection or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or receptors on the cell surface or transiillumination or the introduction of its in connection with homeobox-like peptide, which, as you know, included in the kernel (see Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for the expression of homologous recombination.

The pharmaceutical composition according to the invention is prepared in such a way that it is compatible with the intended method of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g. intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), cutaneous (e.g., local), transmucosal and rectal administration. In one particular embodiment, this composition is prepared in accordance with routine procedures as pharmaceutical compositions adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration people. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers agent and a local anesthetic such as lidocaine to ease pain at the injection site.

If the composition of the invention must be administered topically, the compositions can be prepared in the form of ointment, cream, transdermal patch, lotion, gel, shampoo, pray, aerosol, solution, emulsion, or other form well-known to the skilled in this field specialist. See, for example, Remington''s Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For unfit for aerosols local dosage forms typically use a viscous - semi-solid or solid form, containing a carrier or one or more excipients that are compatible with the local application and having a dynamic viscosity preferably greater than the viscosity of water. Suitable forms include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments (liquid ointment), medicinal ointments and the like, which, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing various properties such as, for example, the osmotic pressure. Other suitable dosage forms for local administration include spray aerosol preparations in which the active ingredient, preferably in combination with a solid or liquid inert carrier, Packed in a mixture with a pressurized volatile (e.g., gaseous) a propellant, such as freon, or a compressible bottle. If desirable, to pharmaceutical compositions and dosage forms of m which may be added moisturizers or wetting means. Examples of such optional ingredients are well known in this field.

If the method according to the invention provides for intranasal introduction of the composition, this composition can be prepared in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agent for application in accordance with this invention can be conveniently delivered in the form of an aerosol spray from pressurized packs or nebulizer, with the use of a suitable propellant (e.g., DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the unit dose can be determined by providing a valve to deliver a measured quantity. For use in inhaler or insufflator may be prepared with capsules and cartridges (cartridge) (consisting, for example, from gelatin)containing powdery mixture of the compounds being considered and a suitable powder base, such as lactose or starch.

If the method according to the invention provides for oral administration, the compositions can be prepared for oral administration in the form of tablets, capsules, starch wafers (capsules), gel gelatin capsules, the solution is in, suspensions, etc. Tablets or capsules can be prepared with conventional methods with pharmaceutically acceptable excipients such as binding agents (e.g., pre-klasterizovannykh corn starch, polyvinylpyrrolidone or hypromellose); fillers (e.g. lactose, microcrystalline cellulose or calcium phosphate); lubricating agents (e.g. magnesium stearate, talc or silica); dezintegriruetsja agents (e.g., potato starch or glycolate sodium starch) or moisturizing agents (e.g. sodium lauryl sulphate). These pills can have a coating applied by methods well known in the field. Liquid preparations for oral administration can be (but are not limited to the form of solutions, syrups or suspensions, or they can be presented as a dry product for restore water or other suitable vehicle before use. Such liquid preparations can be prepared in conventional ways with pharmaceutically acceptable additives such as suspendresume agents (e.g., syrup of sorbitol, cellulose derivatives or hydrogenated suitable for food fats); emulsifying agents (e.g. lecithin or Arabian gum); non-aqueous vehicles (e.g. almond oil, oil is Fira, ethyl alcohol or fractionated vegetable oils), preservatives (e.g. methyl or propyl-p-hydroxybenzoate or sorbic acid). These preparations may also contain optional buffer salts, flavoring, coloring and sweetening agents. Preparations for oral administration can be suitably prepared for slow release, controlled release or sustained release of a prophylactic or therapeutic agent (prophylactic or therapeutic agents).

The method according to the invention may include pulmonary introduction, for example, using the inhaler or nebulizer, the composition including aerosol forming agent. See, for example, U.S. Patents№ 6019968, 5985320, 5985309, 5934272, 5874064, 5855913, 5290540 and 4880078; and PCT Publication no WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346 and WO 99/66903, each of these references is hereby incorporated into this description by reference in its full. In one specific embodiment, the antibody according to the invention, the tool combination therapy and/or composition according to the invention is administered using the technology of pulmonary drug delivery Alkermes AIR® (Alkermes, Inc., Cambridge, Mass.).

The method according to the invention may include the introduction of a composition prepared for parenteral administration by injection (for example the EP, bolus injection or continuous infusion). Compositions for injection can be prepared in the form of a uniform (standard) dose (e.g., in ampoules or mnogochasovykh containers with an added preservative. These songs can have such forms as suspensions, solutions or emulsions in oily or aqueous carriers can contain contributing to the preparation of agents, such as suspendida, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form to restore a suitable carrier (e.g. sterile pyrogen-free water) before use. The methods according to the invention can additionally provide for the introduction of the compositions prepared in the form of depot drugs. Such forms of long-term action can be introduced by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, these compositions can be, for example, prepared with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins or sparingly soluble derivatives (e.g., with a sparingly soluble salt).

The methods of the invention provide for compositions prepared as neutral or salt forms. Pharmaceutically priemel the appropriate salts include salts, formed with anions such as anions derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc. and salts formed with cations such as the cations derived from sodium, potassium, ammonium, calcium, ferric hydroxides, Isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Usually, the ingredients of the compositions are supplied either separately or mixed together in the form of a uniform (standard) dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the method of administration is by infusion, may be provided ampoule of sterile water for injection, so that these ingredients can be mixed prior to introduction.

In particular, this invention also provides a package of one or more prophylactic or therapeutic agents or pharmaceutical compositions according to the invention in a sealed container such as an ampoule or sachet indicating the quantity of this tool. In one embodiment, one or more prophylactic or therapeutic agents or pharmaceutical compositions according to the invention are supplied as a dry sterilized of lyophilizers the frame powder or water-free concentrate in a hermetically sealed container and can be restored (for example, water or saline) to the appropriate concentration for administration to the individual. Preferably, one or more prophylactic or therapeutic agents or pharmaceutical compositions according to the invention are supplied as a dry sterilized lyophilized powder in a hermetically sealed container at a uniform dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg. These lyophilized prophylactic or therapeutic agents or pharmaceutical compositions according to the invention should be stored at 2°C - 8°C in their original container and these prophylactic or therapeutic agents or pharmaceutical compositions according to the invention should be administered within 1 week, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours, or within 1 hour after recovery. In an alternative embodiment, one or more prophylactic or therapeutic agents or pharmaceutical compositions according to the invention are supplied in liquid form in a hermetically sealed container indicating the amount the VA and the concentration of this tool. Preferably, the liquid form of the input composition is supplied in an airtight container at at least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml This liquid form should be stored at 2°C - 8°C in its original container.

Antibodies and part of the antibodies according to the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. Preferably, these antibodies or parts of antibodies prepared in the form of an injection solution containing 0.1-250 mg/ml antibody. This injection solution may consist of a liquid or lyophilized dosage forms in a bottle made of quartz or amber vials or pre-filled syringe. The buffer may be L-histidine (1-50 mm), optimally 5-10 mm, at pH 5.0-7.0 (optimal pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. To modify the toxicity of this solution can be used sodium chloride at a concentration of 0-300 mm (preferably 150 mm for liquid dosage forms). For freeze-dried Lakers the public forms may be included cryoprotectants, mostly 0-10% sucrose (optimally 0.5 to 1.0%). Other suitable cryoprotectants include trehalose and lactose. For a lyophilized dosage form can include fillers, mainly 1-10% mannitol (preferably 2-4%). As for liquid and lyophilized dosage forms can be used stabilizers, mainly 1-50 mm L-methionine (preferably 5-10 mm). Other suitable fillers include glycine, arginine, and can be included as 0-0,05% Polysorbate-80 (optimally 0,005-0,01%). Additional surfactants include, but are not limited to, Polysorbate 20 and surfactants BRIJ. Pharmaceutical composition comprising antibodies, and portions of the antibodies according to the invention, prepared in the form of an injectable solution for parenteral administration may additionally include the agent, the applicable adjuvant, such as agents used to increase the absorption or dispersion of a therapeutic protein (e.g., antibodies). Especially applicable adjuvant is a hyaluronidase, such as Hylenex® (recombinant hyaluronidase human). The addition of hyaluronidase in the injection solution improves the bioavailability to humans after parenteral administration, in particular, subcutaneous injection. It also allows you to use higher is their volumes for the sites of injection (i.e. exceeding 1 ml) with less pain and less discomfort and get the minimum number of reactions at the injection site (see WO2004078140, US2006104968 included in the present description by reference).

The composition of the invention can be in various forms. They include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic application. Typical preferred compositions are in the form of injection or infusion solutions, such as compositions similar to the compositions used for passive immunization of humans with other antibodies. The preferred method of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In one preferred embodiment, this antibody is administered by intravenous infusion or injection. In another preferred embodiment, this antibody is administered by intramuscular or subcutaneous injection.

Usually, therapeutic compositions must be sterile and stable under the conditions of preparation and storage. This composition can be made is in the form of a solution, the microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating this active compounds (i.e. antibodies or parts of antibodies) in the required amount in an appropriate solvent with one ingredient or combination of ingredients enumerated above, if required, followed by sterilization using a sterilizing filter. Generally, dispersions are prepared by incorporating this active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from the ingredients listed above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and spray drying, which give a powder of the active ingredient plus any additional desired ingredient from its pre-filtered through a sterilizing filter solution. Should the fluidity of the solution can be maintained, for example, using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by using surfactants. Prolonged absorption of injectable compositions mo which should be implemented by inclusion in the composition of the agent, which delays absorption, for example, monostearate salts and gelatin.

Antibodies and portions of the antibodies of this invention can be administered in a variety of ways known in this area, although for many therapeutic applications, the preferred method/method injection is a subcutaneous injection, intravenous injection or infusion. As will be clear to the skilled in this field specialist, this method and/or method of introduction will vary depending on the desired results. In some embodiments, the implementation, the active compound can be prepared with a carrier that will protect the connection from the quick release, for example, in the form of controlled release, including implants, transdermal patches, microencapsulation delivery system. Can be used biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. Many methods of cooking such ready-made forms are patented or generally known qualified in this area specialists. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In some embodiments, implementation, antibody or part of an antibody according to the invention can enter the change oral, for example, with an inert diluent or assimilated edible carrier. This compound (and other ingredients, if desired) may also be enclosed in a gelatin capsule with a hard or soft shell, compressed into tablets, or incorporated directly into the diet of the individual. For oral therapeutic administration, these compounds may be incorporated with excipients and used in the form suitable for swallowing tablets, transbukkalno tablets, pastilles, capsules, elixirs, suspensions, syrups, wafers and the like For the introduction of compounds according to the invention in another way than parenteral administration, it may be necessary to cover the connection material to prevent its inactivation or joint introduction this connection with such material.

In these compositions may also include additional active compounds. In some embodiments, implementation, antibody or part of an antibody according to the invention is prepared jointly and/or administered in conjunction with one or more additional therapeutic tools that are applicable for the treatment of disorders in which the activity of RGM A negative impact. For example, anti-RGM A-antibody or part of an antibody according to the invention can be jointly prepared and/or co-introduced with one or more complement the elegance antibodies, which bind other targets (e.g., antibodies that bind cytokines or that bind cell surface molecules). In addition, one or more antibodies according to the invention can be used in combination with two or more previous therapies. Such combination therapy can be advantageous to use a lower dose of an administered therapeutic agents, thus avoiding possible toxicities or complications associated with different monoterpene.

In some embodiments, implementation, antibody to RGM A or its fragment is associated with prolonging the half-life of the carrier, known in this area. Such media include, but are not limited to, the Fc-domain, polyethylene glycol and dextran. Such carriers are described, for example, in the Application U.S. No. 09/428082 and published PCT Application no WO 99/25044, which is incorporated into this description by reference for any purpose.

In one specific embodiment, introducing the nucleic acid sequences containing the nucleotide sequence encoding the antibody according to the invention or another prophylactic or therapeutic agent according to the invention for treating, preventing, controlling or reducing the intensity of violations or one or more symptoms through GE therapy. Gene therapy is called therapy performed by the administration to an individual, expressed or can be expressed nucleic acid. In this embodiment of the present invention, these nucleic acids produce coded antibody or preventive or therapeutic agent according to the invention, which mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in this field, can be used in accordance with this invention. In respect of reviews of methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the field of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993) and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy are described in US20050042664 A1, which is incorporated into this description by reference.

RGM plays A critical role in the pathology associated with various defined above diseases. It has been described that RGM A and RGM proteins are raising way adjustable in the lesions in people suffering from traumatic damage is possible brain (Schwab et al., Arch. Neurol. 62:1561-8, 2005a), in areas of partial shade and bark damaged by the stroke of the human brain (Schwab et al., Arch. Neurol. 62:1561-8, 2005a), in the substance of Negro patients suffering from Parkinson's disease (Bossers et al. Brain Pathology, vol. 19:91-107, 2008). Thus, RGM A-antibodies are suitable agents for a combination therapy of intracerebral stroke, traumatic brain damage, Alzheimer's disease and other neurodegenerative disorders of the human nervous system. In the case of patients with stroke treatment within the first three hours consists of delivery of tissue plasminogen activator for lysis of blood clots (Liang et al. Arch. Neurol. 65:1429-33, 2008), and such treatment could in principle be combined with delivery RGM A-antibodies, which provides an approach using different treatment and much more advanced window treatment. In the case of Alzheimer's disease, combining drugs with RGM A-antibodies is possible with an approved amplifiers cognitive abilities, Donepezil, Memantine, and this approach can significantly slow progressive neuropathology. Intranasal delivery of insulin has a positive effect on the attention and memory (Hanson and Frey, BMC Neurosci. 9:S5, 2008) and is a feasible way of introduction to RGM A-antibodies that can bypass the blood-brain defence is R. In patients with Parkinson's disease (PD), the current treatment is based primarily on the dopaminergic agents, such levodopa, the prodrug of dopamine (Khor and Hsu, Curr. Clin. Pharmacol. 2:234-43, 2007), ropinirole, neorganicheskoy the dopamine agonist (Jost et al. J. Neurol. 255 Suppl. 5:60-63, 2008), monoamine oxidase inhibitors In Rasagiline and Selegiline (Elmer and Bertoni, Expert Opin. Pharmacother. 9:2759-72, 2008). Despite their favorable action in the event of early and moderate Parkinson's disease (PD), none of these drugs are not able to prevent the progressive degeneration of the substance of the Negro and associated subcortical and cortical areas of the brain, and therefore, combination therapy with stimulating the regeneration of RGM A-antibodies could slow down the process of this disease.

Any neuroprotective agent, whether antioxidant, acceptors radicals, an anticonvulsant drug such as Phenytoin, or drug Erythropoietin, is suitable for combination therapy with Pro-regenerative RGM A-antibodies, extending through this is usually a very short window of therapeutic treatment of neuroprotective agents.

Antibodies and part of the antibodies according to the invention can be used to treat people suffering from such diseases.

It should be clear that h is of antibodies according to the invention or their antigennegative parts can be used separately or in combination with additional means, for example, therapeutic tool, with this additional tool is selected qualified for its intended purpose. For example, the additional agent can be a therapeutic tool, which is recognized in the field as an effective means to treat the disease or condition to be treated by the antibody of the present invention. This option can also be a tool that attaches to a preferred feature of this therapeutic compositions, for example, a tool that affects the viscosity of this composition.

In addition, it should be clear that the combinations that should be included in this invention is a combination, applicable for the intended goals for them. The agents below are illustrative for this purpose, but are not intended to be limiting. Combinations, which are part according to the invention can be antibodies of the present invention and at least one additional agent selected from the lists below. The combination may also include more than one additional funds, for example, two or three additional funds, if this combination is such that it can perform its intended function.

Non-limiting examples of therapeutic agents DL is multiple sclerosis, which antibody or part of an antibody according to the invention can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-B1b (BETASERON; Chiron/Berlex); interferon α-n3) (Interferon Sciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferon β1A-IF (Serono/lnhale Therapeutics), Peginterferon α2b (Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; cladribine; antibodies to other cytokines person, growth factors and their receptors or antagonists of other cytokines human growth factors and their receptors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies according to the invention or their antigennegative part can be combined with antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Antibodies according to the invention or their antigennegative parts can also be combined with means, such as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate-mofetil, Leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, inhibitors of complement, adrenergic agents, agents, to the which inhibit signaling of proinflammatory cytokines, such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38 or inhibitors of MAP-kinase)inhibitors of IL-1β-converting enzyme, TACE inhibitors, inhibitors of signal transmission T cells, such as kinase inhibitors, inhibitors of metalloproteinases, sulfasalazin, azathioprine, 6-mercaptopurine, inhibitors of angiotensin converting enzyme, soluble receptors, cytokines and their derivatives (e.g., soluble receptor p55 or p75 TNF, sIL-1RI, SIL-1RII, sIL-6R) and antiinflammatory cytokines (such as IL-4, IL-10, IL-13 and TGFβ).

Preferred examples of therapeutic agents for multiple sclerosis with which it can be combined antibody or antigennegative part, include interferon-β, for example, IFNβ1 and IFNβ1b; Copaxone, corticosteroids, caspase inhibitors, for example inhibitors of caspase-1 inhibitors, IL-1, TNF inhibitors, and antibodies to CD40 ligand and CD80.

Antibodies according to the invention or their antigennegative parts can also be combined with means, such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, Fampridine, glatiramer acetate, natalizumab, cannabidiol and immunogen NNSO3, ABR-215062, AnergiX.MS antagonists of receptors chemokines, BBR-2778, calkulin, CPI-1189, LEM (encapsulated in the liposome mitoxantrone), THCCBD (agonist cannabinoid) MBP-8298, mesopram (PDE4 inhibitor), MNA-715, anti-IL-6-receptor antibody, it AWACS, pirfenidone-allotrope 1258 (RDP-1258), STNF-R1, talampanel, teriflunomide, TGF-beta2, diplomatic, antagonists of VLA-4 (for example, TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists, agonists of IL-4.

The pharmaceutical compositions according to the invention may include a "therapeutically effective amount" or "prophylactically effective amount" of an antibody or part of an antibody according to the invention. "Therapeutically effective amount" is the amount effective, when necessary doses and within the required time periods to achieve the desired therapeutic result. A therapeutically effective amount of the antibodies or parts of antibodies can be determined by a qualified in this field specialist and may vary in accordance with factors such as the disease state, age, sex and weight of the individual and the ability of the antibodies or parts of antibodies to induce a desired response in the individual. Therapeutically effective amount is the amount in which therapeutically favorable action exceed any toxic or harmful effects of the antibodies or portions of antibodies. "Prophylactically effective amount" is the amount effective, when necessary doses and within the required time periods to achieve the desire is wow preventive result. Typically, since a prophylactic dose is used in individuals prior to or at early stages of disease, the prophylactically effective amount will be less than therapeutically effective amount.

Scheme of doses can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, you can enter a single bolus may be administered, several divided doses over time or the dose may be proportionally reduced or increased in the case of urgent need of therapeutic situation. Particularly preferred is the preparation of parenteral compositions in the form of a dose at once for ease of administration and uniformity of dosage. Form a uniform (standard) dose (dose per intake) is called to physically discrete units suitable as a uniform (standard) doses for the treated individuals, mammals; each uniform (standard) dose contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in consideration of the required pharmaceutical carrier. Specification in respect of a uniform (standard) forms according to the invention is dictated by (a) the unique characteristics of the active compounds and to skretny therapeutic or prophylactic effect, to be achieved, and (b) the disadvantages inherent in this field of compounding such an active compound, the effect on the sensitivity of the individual, and depends directly on them.

Exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the antibodies or parts of antibodies according to the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg it Should be noted that the magnitude of the dose may vary depending on the type and severity of the condition, the symptoms of which should be reduced. In addition, it should be clear that for any particular individual, a particular scheme of doses should be adjusted over time in accordance with the needs of the individual and the professional opinion of the person introducing these songs or controlling the introduction of these compositions, and that the dose ranges set in the present description are only exemplary and are not intended to limit the scope or practice of the claimed composition.

For the skilled in this field specialists will be easily understood that other suitable modifications and adaptations of the methods described in the present description of the invention are obvious and can be obtained by using appropriate equivalents without deviating from the scope of posovremeni or described in the present description of the options. After a detailed description of this invention, this invention will be best understood by reference to the following examples, which are included only for illustration purposes and are not intended to limit the invention.

EXAMPLES

Methods

The following methods describe in detail the experimental procedures used in the Examples section below.

(i) ELISA tablets for direct binding covered hRGM A (R&D) at a concentration of 2 μg/ml in carbonate buffer. Then the wells were blocked with 2% blocking solution (Bio-Rad) for 1 hour at room temperature. Biotinylated antibodies were serially diluted with dilution factor of 1:5 in 0.1% BSA/SFR along the plate and incubated for 1 hour at room temperature. The detecting reagent was a dilution of 1:10000 streptavidin-HRP in 0.1% BSA/SFR. Detection was performed with TMB reagent, which was stopped 2 h H2SO4and read OD at 450 nm.

(ii) FACS-analysis. Stable transfectants of HEK293 cells, sverkhekspressiya hRGM A, or BAF3 cells, sverkhekspressiya ratRGM A, were subjected to staining of the unlabeled MAB 5F9 or 8D1 for more than 15 minutes at 4°C in 0.1% buffered BSA/SFR. Detection was performed with the antibody RE mouse IgG against rats.

(iii) Solid phase ELISA assays for the evaluation of MAB 5F9 in the analysis of the binding of hRGM A - Neogene.

Tablets ELISA (Measurement Plate Cert. Maxi Sorb. F96 UNC 439454) were coated for 1 hour at 37°C concentration of 2.5 µg/ml extracellular domain of His-tagged protein Neogynona person (the concentration of the original solution: 30 mg/ml). After inhibition, unbound Neogene was removed in 3 separate stages of leaching using SFR containing 0.02% tween-20. The block is covered with Neogene tablets was performed by adding 200 μl per well of blocking solution of 3% bovine serum albumin (BSA), SFR, tween-20 (0,02%). After inhibition for 1 hour at 37°C, this blocking solution was removed and added fragments of RGM A or a full-sized protein conjugated with fc-label (tag) human, with antibody or no antibody. In some embodiments, the implementation of the antibody pre-incubated with fc-conjugated protein hRGM a for 1 hour at room temperature. Covered Neogene the plates were incubated with hRGM a with an antibody or no antibody for 1 hour at 37°C. After 3 washing stages SFR-tween 20 (0.02%), plates were incubated with labeled Biotin antibody against the fc (1 mg/ml, diluted 1:200 in SFR containing 0.6% BSA, 0.02% tween-20), catalog numbers Jackson ImmunoResearch: 709-065-149, for 1 hour at 37°C. Unbound antibody was removed 3 stages of leaching using SFR/tween-20 (0,02%). To visualize the labeled Biotin fc-antibody was added to the complex, consisting of a Strap is avidin-peroxidase (Roche, cat.# 11089153001)diluted 1:5000 SFR, with 0.6% BSA, 0.02% tween-20, followed by inhibition at 37°C for 1 hour. Unbound peroxidase complex was removed in 3 successive stages of leaching (SFR-tween 20 (0.02 percent) before adding peroxidase substrate (Measurement Pure TMB, Pierce # 34021). The reaction of the substrate was stopped after 1-30 minutes after it is added to the wells using a 2.5 M H2SO4. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos.

(iv) Solid phase ELISA assays for the evaluation of MAB 5F9 in the analysis of the binding of hRGM A - BMP-4.

Tablets ELISA (Measurement Plate Cert. Maxi Sorb. F96 NUNC, 439454) were coated for 1 hour at 37°C with a solution containing a concentration of 2.5 μg/ml recombinant protein BMP-4 (R&D Systems, # 314-BP, Lot # BEM316061). After incubation, unbound BMP-4 was deleted in 3 separate stages of leaching using SFR containing 0.02% tween-20. The block is covered with BMP-4 tablets was performed by adding 200 μl per well of blocking solution of 3% bovine serum albumin (BSA), SFR, Tween-20 (0,02%). After incubation for 1 hour at 37°C, this blocking solution was removed and added fragments of RGM A or a full-sized protein conjugated with fc-label (tag) human, with antibody or no antibody. In some embodiments, the implementation of the antibody pre-incubated with fc-conjugase is these proteins hRGM a for 1 hour at room temperature. Covered with BMP-4 the plates were incubated with hRGM a with an antibody or no antibody for 1 hour at 37°C. After 3 washing stages SFR-tween 20 (0.02%), plates were incubated with labeled Biotin antibody against the fc (1 mg/ml, diluted 1:200 in SFR containing 0.6% BSA, 0.02% tween-20), catalog numbers Jackson ImmunoResearch: 709-065-149, for 1 hour at 37°C. Unbound antibody was removed 3 stages of leaching using SFR/tween-20 (0,02%). To visualize the labeled Biotin fc-antibody was added to the complex, consisting of a Streptavidin-peroxidase (Roche, cat.# 11089153001)diluted 1:5000 SFR, with 0.6% BSA, 0.02% tween-20, followed by inhibition at 37°C for 1 hour. Unbound peroxidase complex was removed in 3 successive stages of leaching (SFR-tween 20 (0.02 percent) before adding peroxidase substrate (Measurement Pure TMB, Pierce # 34021). The reaction of the substrate was stopped after 1-30 minutes after it is added to the wells using a 2.5 M H2SO4. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos.

(v) Solid phase ELISA assays for the evaluation of MAB 5F9 in the analysis of the binding of hRGM A - BMP-2.

Tablets ELISA (Measurement Plate Cert. Maxi Sorb. F96 NUNC, 439454) were coated for 1 hour at 37°C with a solution containing a concentration of 2.5 μg/ml recombinant protein BMP-2 (R&D Systems, # 355-BM, Lot # MSA04). After incubation, unbound BMP-2 was removed 3 from the preliminary stages of leaching using SFR, containing 0.02% tween-20. The block is covered with BMP-4 tablets was performed by adding 200 μl per well of blocking solution of 3% bovine serum albumin (BSA), SFR, Tween-20 (0,02%). After incubation for 1 hour at 37°C, this blocking solution was removed and added fragments of RGM A or a full-sized protein conjugated with fc-label (tag) human, with antibody or no antibody. In some embodiments, the implementation of the antibody pre-incubated with fc-conjugated protein hRGM a for 1 hour at room temperature. Covered with BMP-2 plates were incubated with hRGM a with an antibody or no antibody for 1 hour at 37°C. After 3 washing stages SFR-tween 20 (0.02%), plates were incubated with labeled Biotin antibody against the fc (1 mg/ml, diluted 1:200 in SFR containing 0.6% BSA, 0.02% tween-20), catalog numbers Jackson ImmunoResearch: 709-065-149, for 1 hour at 37°C. Unbound antibody was removed 3 stages of leaching using SFR/tween-20 (0,02%). To visualize the labeled Biotin fc-antibody was added to the complex, consisting of a Streptavidin-peroxidase (Roche, cat.# 11089153001)diluted 1:5000 SFR, with 0.6% BSA, 0.02% tween-20, followed by inhibition at 37°C for 1 hour. Unbound peroxidase complex was removed in 3 successive stages of leaching (SFR-tween 20 (0.02 percent) before adding peroxidase substrate (Pue Measurement TMB, Pierce # 34021). The reaction of the substrate was stopped after 1-30 minutes after it is added to the wells using a 2.5 M H2SO4. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos.

(vi) cell Culture Ntera-2

Cells human Ntera-2 was obtained from the German Collection of Microorganisms and Cell Cultures (DMSZ, Braunschweig). Frozen original solutions undifferentiated cells Ntera-2 were thawed in DMEM containing 10% fetal calf serum (FCS; JRH Bioscience, Kansas, USA) and 5% horse serum (HS; Sigma, Germany). Cells were grown in flasks for cultivation (Greiner, Germany), until they reached confluently 80%.

For differentiation of neuronal cells Ntera-2 were sown at a density of 2.5×106cells/175 cm2in the medium for differentiation (DMEM containing 10% FCS, 5% HS, 1% penicillin-streptomycin, retinoic acid, 10 μm). Cells were differentiated for 3 weeks and the medium was replaced twice a week.

After differentiation, the cells were separated using trypsin-EDTA and were divided at a ratio of 1:6. After 48 hours of neural cells were separated gently from the underlying cells. Shifted cells transferred to aggregation in the new environment in the new shake flasks for cultivation (Corning, USA). Differentiated cells Ntera-2 was given to aggregated into homogeneous horizontal is the shaking off at 37°C for 24 hours in basal medium for neurons (Neurobasal) (Gibco), supplemented with B27 (Gibco), glutamine (Gibco) and penicillin-streptomycin. Units Ntera-2 were sown at a density of approximately 20-30 units on top of the glass in a 24-hole cups. Covered pre-polylysine cover glasses coated with laminin (20 μg/ml, Sigma) and recombinant fc-associated fragment No. 786 (amino acids 47-168) RGM A person at a concentration of 10 μg/ml After inoculation, the culture was treated with MAB 5F9 added at three different concentrations (0.1 µg/ml; 1 μg/ml; 10 μg/ml) to the culture medium and further incubated for 24 hours at 37°C in basal medium for neurons (Neurobasal). Then the aggregates were fixed in 4% paraformaldehyde (2 hours, room temperature) and increased permeability of the cells by the addition of 0.1% Triton X-100 in SFR (20 minutes, room temperature). For fluorescent staining of culture blocked SFR containing 1% BSA, for 1 hour at room temperature. After blocking Ntera cells were incubated with monoclonal antibody mouse against isotype 3 β-tubulin (clone SDL3D10, Sigma # T8660) for 2 hours at room temperature. Unbound antibody was removed 3 different stages of leaching (5-15 min each) and Ntera cells were incubated with Cy-3-conjugated donkey antibody against mouse Ig. (Jackson ImmunoResearch Lot 62597), diluted 1:350-fold in SFR/0.5% BSA and 0.5 μg/ml of bisbenzimide. After incubi the cation within 1 hour of culture were washed 3 times to remove unbound secondary antibody. For fluorescence microscopy, the cover glass was poured in Fluoromount G (Southern Biotech, Eching).

Image units Ntera-2 were obtained using a fluorescent microscope Zeiss Axiovert 200 and neurite outgrowth of these cultures were automatically analyzed using the native system image acquisition and analysis system. Automatic analysis of neurite growth were performed using Image Pro Plus 4.5 and statistical analysis of these data was performed using Graph Pad Prism 4. The growth of neurites were normalized relative to control cultures grown in the absence of fragment #786 RGM A person.

(vii) Culture SH-SY5Y

Cells SH-SY5Y (ATCC, CRL-2266) are the human neuroblastoma cell derived from a metastatic brain tumor. These cells were grown in a medium consisting of 50% balanced salt solution Earl (Invitrogen Life Technologies, Cat. # 24010-043) and 50% F12 (Ham) nutrient mixture + GlutaMAX-1 (Invitrogen Life Technologies, Cat. # 31765-027). This medium additionally supplemented V / V heat inactivated 10% fetal calf serum (FCS, JRH Biosciences, Kansas Cat. # 12107-1000M), 1% NEAA (solution MEM nonessential amino acids (Sigma-Aldrich Cat.# M 1745) and 1% penicillin (10000 u/ml)/streptomycin (10000 ág/ml) (Invitrogen Life Technologies, Cat. # 15140-122). To stimulate the differentiation of neurons and neural growth projections, the cells SH-SY5Y cultured in the medium supplemented with 10 μm retino the howling acid (RA, Sigma-Aldrich Cat. # R2625-050MG)), within a few days. Differentiated cells SH-SY5Y were grown in flasks for tissue culture and were removed by careful trypsinization and were sown on glass cover glass, covered with a striped pattern (pattern) RGM protein A or its fragment and Collagen I.

(viii) Obtaining striped top glass

A modified version of the analysis bands on cover glasses were done slightly different way, as described previously (Knoell et al. Nature Protocols 2:1216 - 1224, 2007) and are summarized below.

Sterile silicon matrices to obtain strips purified proteins were pressed on the surface of Petri dishes with the rough side facing upwards. Washed with ethanol, a clean cover glass was put on this matrix and the corners of the matrix marked with a ballpoint pen on the reverse side of the cover glass. Matrix carrying this cover glass, gently turned upside down with the cover glass facing the bottom of the Petri dishes. Fc-conjugated full-inhibitory antibody RGM A or fc fragments or recombinant RGM A person (R&D Systems Cat. # 2459 RM) was mixed with 10 μl antimisting FITC-labeled antibodies (Fab-specific goat antibodies against mouse IgG, Sigma-Aldrich Cat. # F-4018) to render the strips RGM A. using a Hamilton syringe, 50 μl of RGM A - FITC antibodies carefully in what was aziraphale through the input channel. Excess liquid was left this matrix via the output channel and removed tissue Kleenex. After incubation of the matrix glass cover at 37°C for 2 hours, the first covering solution (containing RGM A), was washed with 200 ál SFR. At the next stage, covering the glass with strips of RGM was transferred into A 24-hole Cup, covered with 500 μl of Collagen I (Collagen tail of the rat, Becton Dickinson Biosciences Cat. # 354236), to fill in the empty spaces between the bars RGM A and incubated at 37°C for 2 hours. Finally got a picture of alternating bands of RGM A and Collagen on top of the glass. After incubation, unbound Collagen I was washed three separate stages of leaching using SPR and differentiated cells SH-SY5Y were sown on these cover glasses. Incubation of cells SH-SY5Y this with figure (pattern) substrate kept at 37°C for 20-24 hours in the presence or in the absence of monoclonal antibodies directed against RGM A person.

For the analysis of immunofluorescence assay cells were fixed in 4% paraformaldehyde for 2 hours at room temperature or over night at 4°C and the permeability of the cells was increased by incubation with SFR, containing 0.1% Triton X-100, within 10-20 minutes at room temperature. After blocking with 3% BSA for 60 minutes, cells were incubated with primary Academy is a body (a monoclonal antibody against clone isotype 3 β-tubulin SDL 3D10, Sigma-Aldrich Cat. # T8660) for 2 hours at room temperature and after several stages of leaching with secondary antibody (donkey artemisinin antibody Cy-3 Jacksonlmmuno Research Lot:62597)diluted in SFR with 0.1% BSA, for 1 hour. Kernel contrasted with the use of bisbenzimide H33258 (Riedel-De-Haen, Cat. # A-0207). Finally, the cells were embedded in Fluoromount G (Southern Biotechnology Associates Inc.: Cat. # 010001). Cells were analyzed using fluorescence Axioplan2 microscope (Zeiss).

(ix) Construction and expression of recombinant anti-RGM A-antibodies

DNA encoding cDNA-fragments of variable regions of the heavy chain of the antibody rat monoclonal antibodies against against RGM A person 5F9 and 8D1, cloned in expressing the pHybE vector containing the constant region of human IgG1, which contains 2 amino acid mutations in the hinge region, homologous recombination in bacteria. These mutations are substitution of leucine to alanine at positions 234 and 235 (EU numbering, Lund et al., 1991, J. Immunol., 147:2657). Variable region light chain of a monoclonal antibody 5F9 and 8D1 cloned in the pHybE vector containing the constant region of Kappa man. Examples of vectors pHyb-E include pHybE-hCk and pHybE-hCg1,z,non-a (see Application for U.S. patent No. 61/021282). Full-size antibodies transtorno expressed in 293E cells by cotransfection chimeric cDNA of the heavy chain and light chain, legirovannykh in the expression p is asmide pHybE. Supernatant cells containing the recombinant antibody was purified by chromatography on Protein a-Sepharose and bound antibody was suirable the addition of acid buffer. Antibodies neutralized and were dialyzed in SFR. Then the purified monoclonal antibodies against RGM A person tested for their ability to bind RGM A, using ELISA as described in Example 1, and competitive ELISA described in Example 7.

Example 1:The generation of monoclonal antibodies against human RGMA

Monoclonal antibodies rat versus human RGMA was prepared as follows:

Example 1A:Immunization of rats with antigen RGMA person

Twenty-five micrograms of recombinant purified human RGMA (R&D Systems Cat#2459-RM lot MRH02511A)mixed with complete adjuvant's adjuvant (Sigma)were subcutaneously injected with four 6-8-week-old rats Harlan Sprague Dawley on day 1. On days 21, 42 and 63, twenty-five micrograms of recombinant purified human RGMA, mixed with incomplete adjuvant's adjuvant (Sigma)were subcutaneously injected with the same 4 rats Harlan Sprague Dawley. Day 144 day or 165 rats were intravenously injected with 10 micrograms of recombinant purified human RGMA.

Example 1B:Generating hybridoma

Splenocytes obtained from the immunized rats described in example A, was merged with SP2/O cells at a ratio of 2:1 in accordance with the lips of blenny way described in Kohler, G. and Milstein 1975, Nature, 256:495, to generate hybridomas. Products mergers were sown in selective medium containing azaserine and gipoksantin, in 96-well tablets at a density of 1.5×105the spleen cells per well. After seven to ten days after the fusion, was observed macroscopic hybridoma colonies. Supernatant from each well containing colonies of hybridomas were tested by direct ELISA (see example 2) the presence of antibody to human RGMA. ELISA-positive cell lines tested in FACS against stably transfected HEK293 cells expressing human RGMA or rats. Line hybridoma cells rats were then tested in a direct ELISA for cross-reactivity with mouse RGMA and ELISA binding with fused protein HuRGMA 47-168.

Table 7
Binding of rat monoclonal antibodies against RGMA
NameDirect ELISA rHuRGMAFACS HEK293 - rhRGMADirect ELISA rRatEGMADirect ELISA hRGMA 47-168/HulgGFc
ML68-8D1YesYesNoYes

ML69-5F9YesYesYesYes

Example 2. Direct ELISA binding of mAb 5F9 and 8D1.

As shown in figure 1A, mAb 5F9 and 8D1 contact hRGM A similar titers as described above in section (i). It was also shown that mAb 5F9 associated with mouse RGM A in ELISA, whereas 8D1 not able to connect with mouse RGM A (data not shown). Figure 1B shows that mAb 5F9 and 8D1 contact HEK293 cells, overexpression hRGM A FACS. Figure 1C shows that 5F9, but not 8D1, was able to bind cells BAF3, sverkhekspressiya rat RGM A, FACS. FACS was performed as described in section (ii).

To assess the binding of mAb 5F9 in competitive assays, the binding of hRGM A-Neogene, used solid-phase ELISA assays. Prepared tablets ELISA and used them as described in section (iii) of this application. hRGM A was added at a concentration of 0.5 μg/ml antibody 5F9 for 1 hour at 37°C. mAb 5F9 used in the following concentrations: 1,25 µg/ml; 0,63 mg/ml; 0,32 mg/ml; 0.16 µg/ml; 0.08 µg/ml; 0.04 µg/ml, 0.02 μg/ml to 0.01 μg/ml Binding hRGM A visualized using labeled Biotin anti-fc antibodies and complex streptavidin-peroxidase. The tablets were analyzed (definition OD) at a wavelength of 450 nm using fotomat is and Anthos. As shown in figure 2, the three highest concentrations of antibodies inhibited dependent dose-dependent manner the binding of full-RGM A person with Neogene.

Solid phase ELISA assays were used to assess mAb 5F9 in competitive assays, the binding of hRGM A - BMP-4. Prepared tablets ELISA and used them as described in section (iv) of this application. hRGM A was added at a concentration of 0.5 μg/ml antibody 5F9 for 1 hour at 37°C. mAb 5F9 used in the following concentrations: 1,25 µg/ml; 0,63 mg/ml; 0,32 mg/ml; 0.16 µg/ml; 0.08 µg/ml; 0.04 µg/ml, 0.02 μg/ml to 0.01 μg/ml Binding hRGM A visualized using labeled Biotin anti-fc antibodies and complex streptavidin-peroxidase. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos. As shown in figure 3, the four highest concentrations of antibodies inhibited dependent dose-dependent manner the binding of full-RGM A person with BMP-4.

Solid phase ELISA assays were used to assess the inhibition of binding of mAb 5F9 fragment 0 (47-168) hRGM A BMP-4. Tablets ELISA covered for 1 hour at 37°C. concentration of 2.5 μg/ml recombinant protein BMP-4 people. Light chain hRGM A (fragment 0, 47-168) was added at a concentration of 0.5 μg/ml antibody 5F9 for 1 hour at 37°C. mAb 5F9 used in the following concentrations: 1,25 µg/ml; 0,63 mg/ml of 0.32 µg/is l; 0.16 µg/ml; 0.08 µg/ml; 0.04 µg/ml, 0.02 μg/ml to 0.01 μg/ml Binding hRGM A visualized using labeled Biotin anti-fc antibodies and complex streptavidin-peroxidase. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos. Figure 4 depicts the antibody concentration of 1.25 μl/ml, 0,63 µl/ml and 0.32 μg/ml, inhibiting dependent dose-dependent manner the binding of the light chain RGM A person with BMP-4.

Solid phase ELISA assays were used to assess mAb 5F9 in competitive assays, the binding of hRGM A - BMP-2. Prepared tablets ELISA and used them as described in section (v) of this application. Full hRGM A was added at a concentration of 0.5 μg/ml antibody 5F9 for 1 hour at 37°C. mAb 5F9 used in the following concentrations: 5 mg/ml; 2.5 µg/ml to 1.25 mg/ml; 0,63 mg/ml; 0,32 mg/ml; 0.16 µg/ml Binding hRGM A visualized using labeled Biotin anti-fc antibodies and complex streptavidin-peroxidase. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos. Figure 5 depicts the antibody concentration 5 ug/ml, 2.5 μg/ml to 1.25 mg/ml, to 0.63 μg/ml, inhibiting the binding of full-RGM A person with BMP-2.

Solid phase ELISA assays were used to assess mAb 5F9 and 8D1 in the analysis of the binding of hRGM A - Neogene, hRGM A - BMP-2 and hRGM A - BMP-4 (figure 9). As described, the Board is s ELISA covered for 1 hour at 37°C. concentration of 2.5 µg/ml extracellular domain of His-tagged protein Neogynona person or 2.5 µg/ml BMP-2 or BMP-4. Added full-size fc-conjugated hRGM A concentration of 0.5 μg/ml antibody for 1 hour at 37°C. mAb 5F9 and 8D1 used at the following concentrations: 5 mg/ml; 2.5 µg/ml to 1.25 mg/ml; 0,63 mg/ml; 0,32 mg/ml; 0.16 µg/ml; 0.08 µg/ml Binding hRGM A visualized using labeled Biotin anti-fc antibodies and complex streptavidin-peroxidase. The tablets were analyzed (definition OD) at a wavelength of 450 nm using a photometer Anthos. As shown in figure 9, the rat monoclonal antibody 8D1 inhibits or reduces the binding of RGM A person with BMP-2 and BMP-4, but not able to inhibit its binding to Neogene.

Example 3. The activity of mAb 5F9 in the analysis of neurite growth with aggregates of differentiated neurons Ntera person

The Ntera cells were obtained and cultured as described in section (vi) Methods of this application. mAb 5F9 neutralized the inhibitory neurite outgrowth activity strong fc-conjugated light chain (amino acids 47-168) protein RGM A man in the analysis of neurite growth with aggregates of differentiated neurons Ntera person. As shown in figure 6, in the absence of inhibitory RGM A protein or fragment in the presence of stimulating frost of neurite substrate laminin, neural aggregates Ntera find extensive and dense network of growing neurites (A). Figure 6 also shows th the presence of light chain hRGM And dramatically reduces the number, the density and length of neurites NTera, confirming the strong inhibitory activity of this fragment hRGM A. Few neurites, leaving the unit are short and strongly associated (In). Parts C-E of figure 6 show that increasing concentrations of mAb 5F9 added to the cultures, neutralized or derepressible dependent dose-dependent manner inhibitory neurite outgrowth activity of the fragment of the light chain hRGM A. With increasing concentrations of mAb, the growth of neural units Ntera completely destroyed, despite the presence of inhibitor RGM A (C: 0.1 µg/ml of mAb 5F9; D: 1 µg/ml of mAb 5F9; E: 10 µg/ml of mAb 5F9).

Quantitative analysis of the neutralizing activity of mAb 5F9 in the analysis of neurite growth with units of human Ntera performed to test strong fc-conjugated inhibitory fragment of the light chain (amino acids 47-168) RGM protein A person. The growth of these crops is automatically analyzed for the presence of aggregates, colored bisbenzimide, and then photographed. This staining was marked only the units, but not growing neurites. However, they were stained with antibody to β3-tubulin and labeled with fluorophore secondary antibody. The growth of neurites automatically determined by calculating the index of neurite growth, index, determined by subtracting the area of cell bodies (protoplasts) of painted β3-tubulin area of this unit and its you who Tupou. Then this factor is divided by the area of the cell bodies, as described in Lingor et al. J. Neurochem. 103:181-189, 2007. Figure 7 shows that mAb 5F9 dependent on the dose follows (0,1-10,0 g) neutralized the inhibitory neurite outgrowth activity of fc-conjugated, strong inhibitory fragment of hRGM A (fragment 0, 47-168; 10 µg) in the analysis of neurite growth with units Ntera person.

Example 4. The activity of mAb 5F9 in the bands hRGM A/Collagen I

Cells SH-SY5Y cultured and used as described in section (vii) of this application. Cover with strips of RGM A and Collagen I on glass were prepared as described in section (viii) of this application. Coating with alternating bands of hRGM A/Collagen I and Collagen I received for these experiments in accordance with the Protocol described in the literature (Knoell et al. Nature Protocols 2: 1216-1224, 2007). In the absence of mAb 5F9 (A), neural cells SH-SY5Y find a clear preference for bands of Collagen I with more than 90% of these cells preferring band I Collagen bands hRGM A. With increasing concentrations of mAb 5F9 neural cells SH-SY5Y prefer bands hRGM A bands of Collagen I (B-E). At the highest used concentration of mAb (E)), the neuronal SH-SY5Y find a strong preference for bands hRGM A in comparison with the bands of Collagen I (see figure 8). This can be interpreted as a unique characteristic of mAb 5F9, as it piratelaurabird nature RGM A attractive activity. In the presence of increasing concentrations 5F9, neural cells prefer to megarobot and grow on RGM A substrate, and not on a permissive substrate, such as Collagen I. This unikalny sign has never been described before for monoclonal antibodies.

Example 5: Construction of CDR-grafted antibodies

Using standard methods, well known in this field, CDR-sequence chains of VH and VL of a monoclonal antibody 5F9 (see table 5 above) transplanted in different acceptor sequence heavy and light chain of a human. Based on comparisons of sequences of the VH and VL sequences of VH and VL of a monoclonal antibody 5F9 of the present invention are selected following a known sequence of a person:

a) VH3-48, VH3-33, and VH3-23, and the connecting sequence hJH3, hJH4 and hJH6 to construct acceptor sequences of the heavy chain (in accordance with the above table 3);

b) A17 and A18, and hJK2 to construct acceptor sequence light chain (in accordance with the above table 4).

Transplantation of the corresponding CDR of VH and VL 5F9 in these acceptor sequences were obtained following CDR-grafted, humanized, modified sequences of VH and VL (see also table 6 above): VH 5F9.1-GL, VH 5F9.2-GL, VH 59.3-GL, VH 5F9.4-GL, VH 5F9.5-GL, VH 5F9.6-GL, VH 5F9.7-GL, and VH 5F9.8-GL; VL 5F9.1-GL, VL 5F9.2-GL and VL 5F9.3-GL.

Example 6: construction of the frame of reverse mutations in CDR-grafted antibodies

To generate frame reverse mutations gumanitarnogo antibodies, mutations introduced in the sequence of CDR-grafted antibodies obtained in accordance with example 5, by de novo synthesis of this variable domain and/or using mutagenic primers and PCR, and methods that are well known in this field. Various combinations of reverse mutations and other mutations design for each of the CDR grafts as follows.

For heavy chain VH 5F9.1-GL, VH 5F9.2-GL, and VH 5F9.3-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: V37→I, V48→I, S49→G and/or R98→K.

For heavy chain VH 5F9.4-GL, VH 5F9.5-GL, and VH 5F9.6-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: V37→I, V48→I, A49→G, R98→K.

For heavy chain VH 5F9.7-GL, VH 5F9.8-GL, and VH 5F9.9-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: V37→I, V48→I, S49→G.

Additional mutations include the following:

for heavy chain VH 5F9.1-GL, VH 5F9.2-GL, and VH 5F9.3-GL: D88→A,

for heavy chain VH 5F9.4-GL, VH 5F9.5-GL, and VH 5F9.6-GL: Q1→E and

for heavy chain VH 5F9.7-GL, VH 5F9.8-GL, and VH 5F9.9-GL: L5→V.

For easy price and VL 5F9.1-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: I2→V, M4→L, Y41→F.

For light chain VL 5F9.2-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: M4→L, R51→L.

For light chain VL 5F9.3-GL one or more of the following related area with Vernier and VH/VL residues mutate back as follows: M4→L, Y41→F.

Example 7: Construction and expression of recombinant humanized anti-RGMA antibodies

Expressing the pHybE vector carrying the heavy and light chains containing frame reverse mutation, was cotranslationally cells 293-6E for transient obtain full humanized antibodies, as described in (ix) above. Mutations were introduced in the sequence of CDR-grafted antibodies obtained in accordance with example 5, by de novo synthesis of this variable domain and/or using mutagenic primers and PCR and by methods well known in the field. Amino acid sequence regions VH and VL humanized antibodies described in table 8.

Specifically, for heavy chain:

VH 5F9.1, VH 5F9.5 and VH 5F9.9 contain VH 5F9.4-GL with mutation Q1→E.

VH 5F9.2, VH 5F9.6, VH 5F9.10, VH 5F9.19, VH 5F9.20, VH 5F9.21 and VH 5F9.22 contain VH 5F9.4-GL with mutation Q1→E and the following reverse mutations adjacent to the area Vernier and VH/VL residues: V37→I, V48→I, A49→G, R98→K.

VH 5F9.3, VH 5F9.7 and VH 5F9.11 contain VH 5F9.7-GL with mutation L5→V VH 5F9.4, VH 5F9.8, VH 5F9.12, VH 5F9.23, VH 5F9.24, VH 5F9.25 and VH 5F.26 contain VH 5F9.7-GL with mutation L5→V and the following reverse mutations adjacent to the area Vernier and VH/VL residues: V37→I, V48→I, S49→G.

For light chain:

VL 5F9.1, VL 5F9.2, VL 5F9.3 and VL 5F9.4 are identical to the VL 5F9.1-GL.

VL 5F9.5, VL 5F9.6, VL 5F9.7 and VL 5F9.8 are identical to the VL 5F9.2-GL.

VL 5F9.9, VL 5F9.10, VL 5F9.11 and VL 5F9.12 are identical to the VL 5F9.3-GL.

VL 5F9.19 and VL 5F9.23 contain VL 5F9.2-GL with the following reverse mutations adjacent to the area Vernier and VH/VL residues: M4→L, R51→L.

VL 5F9.20 and VL 5F9.24 contain VL 5F9.2-GL with the next reverse mutation adjacent to the area Vernier and VH/VL residue: M4→L.

VL 5F9.21 AND VL 5F9.25 contain VL 5F9.3-GL with the following reverse mutations adjacent to the area Vernier and VH/VL residues: M4→L, Y41→F.

VL 5F9.22 and VL 5F9.26 contain VL 5F9.3-GL with the next reverse mutation adjacent to the area Vernier and VH/VL residue: M4→L.

TABLE 8
EXPRESSION of HUMANIZED ANTIBODIES
SEQ ID No.Region proteinSequence
123456789012345678901234567890
47VH h5F9.1EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTMVTVSS
44VL h5F9.1DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYFLE WYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

48VH h5F9.2EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
44VL h5F9.2DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
49VH h5F9.3EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
44VL h5F9.3DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
50VH h5F9.4EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
44VL h5F9.4DIVMTQTPLSLSVTPGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

47 VH h5F9.5EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTMVTVSS
45VL h5F9.5DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
48VH h5F9.6EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
45VL h5F9.6DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
49VH h5F9.7EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
45VL h5F9.7DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

50VH h5F9.8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
45 VL h5F9.8DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
47VH h5F9.9EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVAMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTTPDYWGQGTMVTVSS
46VL h5F9.9DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
48VH h5F9.10EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
46VL h5F9.10DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

49VH h5F9.11EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWVSMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
46VL h5F9.11DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
50 VH h5F9.12EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
46VL h5F9.12DVVMTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
48VH h5F9.19EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
51VL h5F9.19DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

48VH h5F9.20EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
52VL h5F9.20DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
48VH h5F9.21EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
53 VL h5F9.21DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
48VH h5F9.22EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
54VL h5F9.22DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

50VH h5F9.23EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
51VL h5F9.23DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
50VH h5F9.24EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
52VL h5F9.24DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFQQRPGQSPRRLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR
50 VH h5F9.25EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
53VL h5F9.25DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

50VH h5F9.26EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMNWIRQAPGKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGTTPDYWGQGTMVTVSS
54VL h5F9.26DVVLTQSPLSLPVTLGQPASISCRSSQSLEYSDGYTFLEWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQATHDPLTFGQGTKLEIKR

Example 8: Characterization of humanized antibody 5F9 using competitive ELISA

Tablets ELISA (Costar 3369) were coated overnight at 4°C with 50 µl per well of 0.25 µg/ml hRGMA in 0.2 M sodium carbonate-bicarbonate buffer, pH of 9.4, washed with wash buffer (SFR containing 0.1% tween-20) and blocked for 1 hour at room temperature with 200 µl per well of 2% fat-free dried milk in SFR. After washing with washing buffer was added in duplicate a mixture of biotinylated chimeric antibody 5F9 (final concentration of 0.1 μg/ml) and unlabeled competitive TEC is-antibodies (starting at a final concentration of 50 mg/ml and serially diluted in 5-fold) in 50 µl per well of ELISA buffer. After incubation of these plates for 1 hour at room temperature and washing with wash buffer, bound antibodies were detected using 100 μl per well of a dilution of 1:10000 conjugated with HRP streptavidin (Fitzgerald) in ELISA buffer. After incubation for 1 hour at room temperature and washing with wash buffer, color development was performed by adding 100 µl per well of buffer TMB (Zymed). After incubation for 15 minutes at room temperature, color development was stopped by adding 50 μl per well of 1 n hydrochloric acid. The optical density was read at 490 nm.

Table 9 shows the values of the IC50humanized antibody 5F9 obtained using the computer program GraphPad Prism (GraphPad Software Inc., San Diego, CA).

h5F9.20
Table 9
The value of the IC50humanized antibody 5F9 in competitive ELISA assays
AntibodyIC50(ág/ml)AntibodyIC50(ág/ml)
h5F9.1>10h5F9.19N/A
h5F9.2>10>2,0
h5F9.3>10h5F9.210,60
h5F9.4>10h5F9.22>2,0
h5F9.5>10h5F9.230,55
h5F9.6>10h5F9.241,32
h5F9.7>10h5F9.250,66
h5F9.8>10h5F9.26>2,0
h5F9.9>10
h5F9.10>10
h5F9.11>10
h5F9.12>10

Example 9: determination of the affinity of chimeric and humanized antibodies using BIACORE technology

The BIACORE analysis (Biacore, Inc, Piscataway, NJ) determines the affinity of antibodies kinetic measurements of rate constants of Association, the rate of dissociation. The binding of the antibody with purified recombinant RGM A person was determined by measurements based on surface plasmon resonance, instrument Biacore® 3000 (Biacore® AB, Uppsala, Sweden) using bandwidth HBS-EP (10 mm HEPES [pH to 7.4], 150 mm NaCl, 3 mm EDTA and 0.005% surfactant P20) at 25°C. All chemicals were obtained from Biacore® AB (Uppsala, Sweden). Approximately 5000 units EN goat antibodies against human IgG, (Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, IL)diluted in 10 mm sodium acetate (pH 4.5) was immobilized directly through biosensor chip SM category, sufficient for research, using the standard set for linking amine in accordance with the instructions and procedures of the manufacturer at 25 μg/ml of Unreacted part of the molecules on the biosensor surface was blocked by ethanolamine. As the surface reactions used the surface modified carboxymethylcysteine in flow cell 2 and 4. Unmodified carboxymethylated without goat antibodies against human IgG use the Wali in the flow cell 1 and 3 as the reference surface. Purified antibodies were diluted in HEPES buffered saline for grip when passing through the surface reaction, specific against goat antibodies against human IgG. Human antibodies that are subject to seizure as a ligand (25 μg/ml), were injected with the matrices reactions to a flow rate of 5 μl/min rate constants of Association and dissociation, Kon(units M-1with-1and Koff(units with-1), was determined with the continuous speed of the current 25 ál/MiG. Rate constants were obtained by performing kinetic measurements at ten different concentrations of antigen in the range from 0.30 to 50 nm. For kinetic analysis, equations of velocities derived from the model binding 1:1 Langmuir customized both phases of the Association and dissociation of all eight injections (using analysis of global adjustment) using the Biaevaluation program 4.0.1. Then the equilibrium dissociation constant (unit M) the reaction between humanitarianism antibodies and purified recombinant RGM A person was calculated from the kinetic rate constants using the formula: KD= koff/kon.

Table 10
The affinity of chimeric and humanized monoclonal antibodies against RGM A
the name kon1/(M.C)koff(1/s)KD(nm)
chimeric 5F9the 7.65×105of 2.36×10-33,09
h5F9.213,55×1052,69×10-3to 7.59
h5F9.235,07×105of 2.21×10-34,37
h5F9.255,70×1053,29×10-35,78

Example 10: Humanized antibody 5F9 neutralize chemoattractive activity of RGM A man in the analysis of chemotaxis neural cells SH-SY5Y

Analysis of chemotaxis measures the behavior of cells in response to diffundere factors that can be chemoattracted or chemoattractive activity. RGM A was described as a protein that acts as in membrane-bound (dependent on the contact repulsion), and soluble diffundiruet form (chemoattractive) and, therefore, were evaluated in the analysis of chemotaxis hRGM A. For this is purpose used RGM A-sensitive cells SH-SY5Y human neuroblastoma, bearing RGM-receptor Neogene (Schaffar et al. J. Neurochemistry: 107:418-431, 2008). Cells SH-SY5Y were grown in an environment of balanced salt solution Earl/F12 (EBSS/F12), supplemented with 10% fetal calf serum and 1% nonessential amino acids (MEM-NEAA). For the induction of neurite growth of cells cultured in the medium supplemented with 10 μm retinoic acid (RA). After 5-6 hours, the cells were trypsinization and considered for planting in 24-hole camera Boyden (BD Falcon 351185, HTS Multiwell System). 500 ál of cell suspension (corresponding to 1×105cells) were added into the inner circle of each well. This inner circle is separated from the larger outer circle of each well PET membrane with a pore diameter of 8 μm. 600 μl medium +/- RGM A +/- antibodies have pietravalle in the outer circle, and these cells were cultured in multilaunch chambers of Boyden over night at 37°C. After incubation the medium aspirated and replaced with fixative (2% paraformaldehyde). Fixation was continued for 2 hours at room temperature and after several stages of washing SFR performed increased permeability using SFR, containing 0.1% Triton X-100 (15 min, RT). Cell staining was performed by incubating them for 1 hour in the dark in a solution of Alexa Fluor 488 Phalloidin 1:100 (Invitrogen A12379) and bisbenzimide (H332456) 1:100. After 2 stages of washing SFR, culture was poured SFR, closed up parafilm and kept in the dark is for those analysis using a fluorescent microscope (Zeiss Axiovert).

In the absence of hRGM A cells migrate through the pores of the membranes and can be counted after fixation and staining. Consider only those cells that are attached to the bottom of the membrane, since these cells migrated through the membrane PET. Cells on the upper side of this membrane can be carefully removed before fixation. This analysis of chemotaxis proved that the presence of hRGM A significantly reduced number of cells SH-SY5Y migrating through the membrane, more than 80%. Rat monoclonal antibody 5F9, chimeric human-rat antibody 5F9, but not izotopicheskii similar control rat monoclonal antibody (p21), partially or fully neutralized chemoattractive activity at 10 µg/ml, which was manifested in the form of large quantities of cells found at the bottom of this membrane (figure 10).

Example 11: 5F9 induces the regeneration of crushed, damaged axons of the optic nerve in a rat model of optic nerve damage

Model crushing the optic nerve or optic nerve) provides an animal model for testing various substances, which stimulate the regeneration of the optic nerve fibers and reduce the massive cell death of retinal ganglion.

The experiments were performed in adult male rats Sprague Dawley and Wistar male rats, recip is the R from Charles River (D) Laboratories (Germany). Animals kept in separate cells during the cycle of light/dark 12:12 hours with access to feed and water ad libitum. Crushing the optic nerve is performed only in the left eye with the help of minimal surgery of the anterior chamber of the eyeball. This method is minimally invasive and damaging the optic nerve and was developed by the authors according to the invention in accordance with surgical methods for the anterior part of the optic nerve of the man. Before the procedure the procedure and during the procedure the operation anaesthetize animals inhalation anesthesia using Sevoflurane (Abbott GmbH Co. & KG, Delkenheim, Germany) and fixed on the operating table by means of the jaw clamp and adhesive tape for limbs. The drop in body temperature to prevent placing animals on a heating pad-pillow. For crushing surgery optic nerve of the rat in the anterior chamber of the eyeball, the left eye is gently released from the ligaments and connective tissue. As a first stage, carry out the surgical incision (2-3 mm) of adjacent tissue in the outer corner of the eye. Then expose the optic nerve by using a pair of tweezers to offset the side of the eye muscles and tear glands to save them. In the next stage, meninges open longitudinally using microsonic to expose the optic nerve.

Local antibodies and buffer

After damage by crushing the optic nerve of male rats Sprague Dawley processed locally antibody 5F9 (n = 10 animals), control antibody 8D1 (n = 10 animals) or control vehicle SFR (n = 10 animals). The experimenters were not known groups of different processing (blind experiment). For local application of antibodies, small pieces of absorbable gelatin sponge (length: 2.5 mm, width: 2.5 mm, height: 2.5 mm) was soaked 20 μl of the antibody solution 10 mg/ml or 20 ml SPR and placed directly next to the site of optic nerve damage. After minimal invasive surgery and application of antibodies, animals were placed on paper towels in a clean cage that is installed on the heater (heating device) for controlling the temperature of the body, until they began to move. This eye put ointment that contains an antibiotic (Gentamytrex, Dr. Mann Pharma) in order to avoid bacterial infection and drying of the sclera. Intraperitoneally injected carprofen (Rimadyl 5 mg/kg, Pfizer GmbH, Karlsruhe) for the treatment of postoperative pain immediately after surgery and then twice a day for 3-day period. Animals were monitored and controlled regularly for several hours immediately after surgery and during the following days, to be sure that all of these animals survived and recovered from anesthesia and surgery. After 5 weeks after surgery and application of antibodies/media, animals were anestesiologi excessive dose Marcarena (40-60 mg/kg) and were perfesional the injection of a 4% solution of paraformaldehyde in the heart. The optic nerves were isolated and transferred into a 4% solution of paraformaldehyde and left for 1 hour at room temperature to ensure proper fixation of the tissue. After postfixation, the optic nerve was kept overnight in 30% sucrose solution (4°C). The next day, the optic nerves were embedded in Tissue Tek, frozen and did longitudinal sections with a thickness of 16 μm using a Cryostat.

For immunoablative (immune mark) sections of optic nerves were fixed with cold acetone (10 min), washed 3x (5 minutes) buffered by Tricom solution (TBS, Fluka 93312) and blocked and their permeability increased with the use of TBS containing 5% bovine serum albumin and 1% Triton X-100 (30 minutes) at room temperature). The remaining BSA and detergent was removed 2 separate what Tamiami rinse (5 minutes each) with TBS. Slices were incubated for 1 hour at room temperature with polyclonal rabbit antibody against anti-GAP-43 antibody (Abeam, ab 7562)diluted 1:100 in 5% solution of BSA/TBS. After 3 stages of washing TBS, 0.1% tween, the sections were incubated for 1 hour at room temperature with Alexa Fluor 488-conjugated goat secondary antibody against rabbit IgG (Molecular Probes A11034)diluted 1:1000 in 5% BSA/TBS, containing a dilution of 1:100 bisbenzimide (H33258, 50 µg/ml)to visualize cell nuclei. Before pouring, the stained sections were washed 3 times with TBS, 0.1% Tween (each stage is 5 minutes) and distilled water. The sections were embedded in Fluoromount G, covered with cover glass and kept in the dark for microscopic documentation.

Obtained using a fluorescent microscope Zeiss image (figure 11) stained longitudinal sections were stored using the software Zeiss Axiovison. Individual pictures of each nerve was set for analysis using the Photoshop Image Analysis (Adobe). Quantitative analysis was performed in two different ways using a combination of images of these optic nerves. GAP-43-positive area in each plot damage was measured using Axiovision (figure 12B). Regardless of this, the first quantitative analysis of individual regenerating fibers (GAP-43-positive is e) considered in 4 different zones: 0-200 μm, 200-400 μm, 400-600 μm and 600-1200 μm off-site crushing. Data analysis and statistical evaluation of data was performed using the software Graphpad Prism (figure 12A).

Systemic antibodies and buffer

For systemic delivery of antibodies male Wistar rats were treated systemically (intraperitoneal, ip) or intravenously (iv) antibody 5F9 (n = 10 animals) or control vehicle SFR (n = 10 animals). Animals were injected with two times and the injection was performed on day 0, shortly after the crushing of the nerve and at day 21 after crushing. Dose provided antibodies were equal to 2 mg/kg on day 0 and 10 mg/kg / day 21. Animals were killed after five weeks after injury by crushing and fabric selection, preparation, slicing, staining and quantitative analysis were performed as described above. As before, the experimenters were not aware of these two different groups of treatment. Combined images of the optic nerves of rats are shown in figure 13. In the treated 5F9 animals (A), many GAP-43 positive fibers stretched off-site crushing in contrast to the control animals treated SFR (IN). Plot crushing is located on the left border and regenerative fibers stained with antibody to GAP-43. Numerous fibers are observed on the top and bottom edge of sitelog the nerve in 5F9-treated animals, but not in treated SFR animals.

5F9, but not control-the media SFR significantly increased the number of regenerating liver GAP-43-positive fibers. Significantly more fibers (p < 0,001) were found in animals treated with 5F9, at distances of 300 μm to 1800 μm than in the treated carrier animals. Animals were treated 5F9 at day 0 and day 21 with 2 mg/kg and 10 mg/kg, respectively. The antibody or the media was injected intraperitoneally or intravenously. Data from the analysis of 9 animals per group. Analyzed the 3 series cryostatic slices per animal (figure 14A).

In the second embodiment, male Wistar rats were treated after damage to the optic nerve systemically (iv) antibody 5F9 (n = 10 animals), control antibody 8D1(n = 10 animals) or control vehicle SFR (n = 10 animals). Rats were injected with once weekly 2 mg/kg of antibody, administered iv, and the injection was started immediately after crushing the optic nerve. All rats received 4 injections and animals were Manasarovar after 5 weeks after the crushing of the nerve. The experimenters were not known variants (blind experiment), and the processing of tissue and quantitative analysis were performed as described previously. 5F9, but not control-the media SFR, significantly increased the number of regenerating liver GAP-43-positive fibers. Significantly more fibers (p < 0,001)were found in animals processed 5F9, at distances of 200 microns to 1400 microns, than in the treated carrier or control animals. Animals were treated iv once a week for 4 weeks, starting on day 0, antibody 5F9 (2 mg/kg per dose), control antibody 8D1 (2 mg/kg per dose) or SFR (figure 14C).

Example 11: 5F9 induces remyelinization crushed axons of the optic nerve in a rat model of optic nerve damage

Marker for oligodendrocytes and myelin is basal myelin protein (MBP). To answer the question whether there are any differences in the remyelination in different groups of processing, used antibody directed against MBP. For visualization process of remyelination, sections of the optic nerve of animals treated systematically recorded cooled (-20°C) acetone (10 minutes), washed 3x (5 minutes) buffered by Tricom saline (TBS, Fluka 93312) and blocked and increased permeability of TBS containing 5% bovine serum albumin and 1% Triton X-100 (30 minutes), at room temperature). The remaining BSA and detergent was removed 2 separate stages rinse (5 minutes each) with TBS. Slices were incubated for 3 hours or over night at 4°C With polyclonal rabbit antibody against anti-MBP antibodies (Abeam, ab 2404), diluted 1:100 in 5% solution of BSA/TBS. After 3 stages of washing TBS, 0.1% tween, slicers, and who was kopirovali for 1 hour at room temperature with Alexa Fluor 488-conjugated goat secondary antibody against rabbit IgG (Molecular Probes A11034), diluted 1:1000 in 5% BSA/TBS, containing a dilution of 1:100 bisbenzimide (H33258, 50 µg/ml)to visualize cell nuclei. Before pouring, the stained sections were washed 3 times with TBS, 0.1% Tween (each stage is 5 minutes) and distilled water. The sections were embedded in Fluoromount G, covered with cover glass and kept in the dark for microscopic documentation.

Obtained using a fluorescent microscope Zeiss image stained longitudinal sections were stored using the software Zeiss Axiovison. Individual pictures of each nerve was set for analysis using the Photoshop Image Analysis (Adobe). Quantitative analysis was performed in two different ways using the merged images of these optic nerves. MBP-positive area in each plot damage was measured using Axiovision. Data analysis and statistical evaluation of data was performed using the software Graphpad Prism.

Animals were treated 5F9 at day 0 and day 21 with 2 mg/kg and 10 mg/kg, respectively. The antibody or the media was injected intraperitoneally or intravenously. Got merged image of the optic nerves of rats.

The myelination visualized using antibodies directed against the marker of myelin, the basal of myelin protein MBP. Lots crushing p is sporogony in the middle of these combined nerves, and this zone is free in the treated carrier control animals (a and b). In the treated antibody 5F9 animals (C and D)observed a number of MBP-positive structures in this middle zone (Central crushing) of the optic nerves (figure 15).

The myelination visualized using antibodies directed against the marker of myelin, the basal of myelin protein MBP. Square MBP was measured using Zeiss Axiovison. M1 and M2 are the two independent measurements and M is the average measured MPB-positive area. 5F9 increases significantly (p<0.001 in comparison with control carrier) MBP-area crushing the optic nerve (3.5-fold) (figure 16).

1. The selected monoclonal antibody, containing antigennegative domain where the specified antibody capable of binding an epitope of a molecule RGM specified antigennegative domain contains a set of CDRs of the variable domain, consisting of:
a) VH 5F9 CDR-H1 sequence of SEQ ID NO:57, VH5F9 CDR-H2 sequence of SEQ ID NO:58 and VH5F9 CDR-H3 of the sequence SEQ ID NO:59; and
b) VL5F9 CDR-L1 sequence of SEQ ID NO:60, VL5F9 CDR-L2 sequence of SEQ ID NO:61 and VL5F9 CDR-L3 sequence of SEQ ID NO:62.

2. The antibody according to claim 1, additionally containing acceptor framework region of a person where the specified acceptor framework region of human content is t at least one amino acid sequence, selected from the group consisting of SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.

3. The antibody according to claim 1, containing the variable domain of the heavy chain selected from SEQ ID NO:35, 36, 37, 38, 39, 40, 41, 42 and 43; and/or variable domain of the light chain selected from SEQ ID nos:44, 45 and 46.

4. The antibody according to claim 1, containing the variable domain of the heavy chain with the amino acid sequence SEQ ID NO:34 and a light chain with the amino acid sequence SEQ ID NO:10.

5. The selected monoclonal antibody, which is capable of binding an epitope of a molecule RGM containing:
(a) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:44;
(b) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:44;
(c) variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:44;
(d) variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:44;
(e) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and the variable region is easily the chain with the amino acid sequence SEQ ID NO:45;
(f) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:45;
(g) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:45;
(h) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:45;
(i) variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:46;
(j) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:46;
(k) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:46;
(l) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:46;
(m) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with aminokislot the th sequence of SEQ ID NO:51;
(n) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:52;
(o) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:53;
(p) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:48 and a variable region light chain with the amino acid sequence SEQ ID NO:54;
(q) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:51;
(r) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:52;
(s) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:53; or
(t) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:50 and the variable region of the light chain with the amino acid sequence SEQ ID NO:54.

6. The selected monoclonal antibody, which is capable of binding an epitope of a molecule RGM containing:
(a) the variable region of the heavy the Loy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:51;
(b) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:52;
(c) variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:53;
(d) variable region of the heavy chain with the amino acid sequence SEQ ID NO:47 and a variable region light chain with the amino acid sequence SEQ ID NO:54;
(e) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:51;
(f) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:52;
(g) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:53; or
(h) the variable region of the heavy chain with the amino acid sequence SEQ ID NO:49 and a variable region light chain with the amino acid sequence SEQ ID NO:54.

7. Antigennegative fragment selected monoclonal antibody according to any one of claims 1 to 6.

8. The selected nucleic acid to drousha the antibody molecule according to any one of claims 1 to 6.

9. The method of obtaining protein, the ability to communicate with RGM, comprising culturing the host cell in a culture medium under conditions suitable for obtaining a binding protein capable of contact RGM, where a host cell contains the expression vector containing the selected nucleic acid encoding the antibody molecule according to any one of claims 1 to 6.

10. Pharmaceutical composition for treating diseases in which the activity of RGM And has a negative effect, containing a therapeutically effective amount of the antibody according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

11. The use of antibodies according to any one of claims 1 to 6 to obtain medicines used to a) reduce binding hRGM And receptor Neogenin patient; or (b) to reduce binding hRGM And bone morphogenetic protein-2 and bone morphogenetic protein-4 (BMP-2 and BMP-4) in the patient.

12. The use of antibodies according to any one of claims 1 to 6 for receiving drugs to reduce the activity of RGM And in a patient suffering from a disease in which activity of RGM And is pathological.

13. The application indicated in paragraph 12, where the disease includes neurological diseases selected from the group including amyotrophic lateral sclerosis, brachial plexus injury, brain damage, including traumat the economic damage to the brain, cerebral palsy, a disease Guillain-Barre, leukodystrophy, multiple sclerosis, post-vaccination, poliomyelitis, spina bifida, spinal cord injury, spinal muscle atrophy, spinal tumors, stroke, transverse myelitis; dementia, senile dementia, cognitive impairment moderate degree of dementia associated with Alzheimer's disease, horey Huntington, late dyskinesia, hyperkinesia, mania, Parkinson's disease, syndrome Steele-Richardson, down syndrome, severe pseudoparalysis the gravis, nerve injury, vascular amyloidosis, brain hemorrhage I with amyloidosis, inflammation of the brain, acute violation confusion, amyotrophic lateral sclerosis, glaucoma and Alzheimer's disease.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to biochemistry. A method of immunoassay of human protein CXCL1 is described. Human CXCL1 or its fragment is measured in a sample with application of two or more types of monoclonal antibodies to human CXCL1 or their fragments. Each of two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identifies any of regions of a sequence of amino acid sequences, represented in SEQ ID NO:1-3, which represent partial sequences of an amino acid sequence, constituting human protein CXCL1. Two or more types of the monoclonal antibodies to human CXCL1 or their fragments specifically identify regions of the sequence, different from each other. Claimed are the monoclonal antibodies or their fragments, each of which specifically identifies any region of the amino acid sequence, represented in SEQ ID NO:1-3, and has a new amino acid sequence.

EFFECT: invention makes it possible to determine human protein CXCL1 with high sensitivity.

15 cl, 9 dwg, 1 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: compounds can be applied for treatment of oncologic and autoimmune diseases. Invention also characterises method of obtaining conjugates, pharmaceutical composition and medication, which contains modified proteins. In general formulae 1 or 2 , R1 is selected from the group representing (CH3)2N-,

R2 is selected from the group representing where R3 as terminal substituent represents -NH2, or and R4 represents H or C1-C3alkyl.

EFFECT: novel compounds possess affinity for CD16a receptor.

18 cl, 20 dwg, 3 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented: a method for tumour cell growth inhibition in an individual and a method for immune response enhancement in an individual involving the introduction of a PD-1 monoclonal antibody and a CTLA-4 10D1 monoclonal antibody to the individual. The PD-1 monoclonal antibody has the following properties: it binds to human PD-1 having the value KD equal to 1×10-8 M or less; however it binds neither to human CD28, nor to CTLA-4, nor to ICOS; it is able to enhance the T-cell proliferation in the mixed lymphocyte reaction (MLR) analysis; it is able to enhance the gamma interferon production in the MLR analysis; it is able to enhance the interleukine-2 (IL-2) secretion in the MLR analysis.

EFFECT: invention provides a synergic effect when using the above antibodies in a combination.

4 cl, 54 dwg, 7 tbl, 25 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to biotechnology and represents a polypeptide construction for treatment, prevention and relief of disorders, associated with an adhesion of platelets and platelet-mediated aggregation or its dysfunction, which includes one or more single-domain antibodies, aimed against the von Willebrand factor (vWF), and one or more single-domain antibodies aimed against serum albumen (SA). The invention also relates to nucleic acid, coding such polypeptide construction, to compositions, containing the said construction, and to its application for obtaining medications for prevention, treatment and relief of the said disorders.

EFFECT: claimed invention makes it possible to extend an assortment of medications for treatment, prevention and relief of disorders, associated with the platelet adhesion and platelet-associated aggregation or its dysfunction.

15 cl, 30 dwg, 32 tbl, 69 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and biotechnology. There are presented versions of nucleic acids each of which codes a heavy-chain amino acid sequence of immunoglobulin IgG1. The above chain contains glycine-lysine dipeptide coded by ggaaaa, ggcaaa or gggaaa codon at the C terminal of the CH3 domain. There are described: a plasmid coding a heavy chain of immunoglobulin; version cells providing immunoglobulin IgG1 expression; a method for producing immunoglobulin in mammalian cells; a method for improving immunoglobulin expression in the mammalian cells; - using the versions of a nucleic acid.

EFFECT: using the invention provides preventing the by-product expression of weight 80 kDa that can find application in producing immunoglubulins.

18 cl, 7 dwg, 3 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to recovered monoclonal antibodies, particularly CDR-grafted humanised antibodies binding to an epitope of human RAGE molecule, and particularly possess an ability to inhibit RAGE binding to various ligands. The invention also refers to a method for preparing the above antibodies, a recovered nucleic acid coding them, an expression vector, a host cell and a pharmaceutical composition.

EFFECT: invention provides treating the diseases or disorders associated with advanced glycation end product (RAGE) receptor, including Alzheimer's disease effectively.

16 cl, 13 dwg, 10 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an IL-1β-binding antibody or its IL-1β-binding fragment containing V heavy and light chain regions. The above antibody binds to human IL-1β with dissociation constant less than 1pM. Versions of the antibody are described. There are disclosed corresponding coding nucleic acids (NA), as well as: a NA passage vector to a host cell, the host cell producing a coded polypeptide. What is described is using the antibody for preparing the other format of the above antibody: "camel-like", VHH antibody, nanobody. What is disclosed is a pharmaceutical composition for treating or preventing an IL-1β-related disease in a mammal on the basis of the antibody, as well as a method of treating or preventing the IL-1β-related disease in a mammal.

EFFECT: using the invention provides the novel IL-1β-specific antibodies with high IL-1β affinity that can find application in medicine for preventing, treating the diseases mediated by IL-1β activity.

39 cl, 20 dwg, 6 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are versions of an isolated monoclonal antibody, specific to hGM-CSF, where each version is characterised by a heavy and light chain. Each of the versions is characterised by the fact that it contains six appropriate CDR. Described are: a pharmaceutical composition, and a set, representing medication, based on the antibody application. Disclosed are: a coding isolated nucleic acid, an expression vector, containing it, and a vector-carrying host cell, used for the antibody obtaining. Described is a method of obtaining the antibody with the cell application.

EFFECT: claimed inventions can be applied for treating disease or disorder, associated with superexpression of hGM-CSF.

25 cl, 9 dwg, 14 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: present invention refers to biotechnology and medicine. What is presented is a method for generating an antibody and its functional fragments against a tumour antigen expressed on the tumour surface resistant to at least one anti-tumour compound by applying ground homogenate, and/or suspension, and/or cell lysate originated from the same tumour for immunisation. There are also disclosed using the method according to the invention for producing the monoclonal antibodies and their functional fragments, the monoclonal antibodies produced by the method, nucleic acids coding them, an expression vector, a host cell, and a method for preparing the antibody with using them, as well as hybridomes secreting these antibodies and their functional fragments for preparing a drug, the anti-tumour composition and using it as a drug.

EFFECT: invention can find further application in therapy of resistant tumours.

42 cl, 7 dwg, 4 ex, 6 tbl

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

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