# Patent

## Title
Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use

## Abstract

The invention concerns the identification and use of neogenin receptor-binding domains of members of the repulsive guidance molecule (RGM) protein family as well as polypeptide fragments derived therefrom. The inventive domains, i.e. peptide fragments are suited as agents for the active or passive immunization of individuals as well as diagnostic and therapeutic agents for use with diseases or pathological conditions in whose origin or progression, a member of the RGM family and a cellular receptor assigned to this molecule are involved. The invention also concerns monoclonal and polyclonal antibodies directed against the inventive binding domains and against the polypeptides derived therefrom, and to method for producing the inventive domains, polypeptides and antibodies.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a U.S. national stage entry of International Patent Application No. PCT/EP2006/009497, filed on Sep. 29, 2006, which claims priority to U.S. patent application No. 60/722,565, filed on Oct. 1, 2005, and European Patent Application No. 05021451.9, filed on Sep. 30, 2005, the entire contents of all of which are fully incorporated herein by reference.


The present invention relates to the identification and use of the receptor-binding domain of members of the repulsive guidance molecule (RGM) protein family, and to polypeptide fragments derived therefrom. The domains and peptide fragments according to the invention are suitable as agents for active or passive immunization of individuals and as diagnostic and therapeutic agents for use for diseases or pathological conditions in whose development or progression a member of the RGM family and a cellular receptor assigned to this molecule is involved. The invention additionally relates to monoclonal and polyclonal antibodies against the binding domains according to the invention and against the polypeptides derived therefrom, and to methods for preparing the domains, polypeptides and antibodies according to the invention.
PRIOR ART
The function of the members of the RGM protein family was described for the first time by Monnier, P. P. et al., Nature, 419, pp. 392-395, 2002. This family includes three members disclosed to date, which are referred to as RGM A, RGM B (also referred to as DRAGON) and RGM C (also referred to as hemojuvelin) (Niederkofler V. et al., J. Neurosci. 24, 808-18, 2004). These are glycoproteins which are bound to the plasma membrane via a lipid anchor (glycosylphosphatidylinositol anchor=GPI anchor). The members of this protein family do not have any extensive sequence homology to other proteins, and structural features which have been identified are substantially the following regions: an N-terminal signal peptide; an RGD sequence; a proteolytic cleavage site around the amino acid sequence GDPH; a structural homolog of von Willebrand factor domain (vWF D); a hydrophobic sequence in the vicinity of the C terminus and a C-terminal GPI anchor consensus sequence (cf. also FIG. 2).
In humans, the coding sequences for RGM A are located on chromosome 15, for RGM B on chromosome 5 and for RGM C on chromosome 1. A characteristic expression pattern is to be observed. RGM A and B are expressed in particular in adult brain and spinal cord, RGM C is expressed in particular in skeletal muscle, liver and myocardium, and RGM B is additionally expressed in cartilage tissue.
RGM proteins were originally identified as candidate proteins playing an important part in the formation of topographic neuronal projections (Stahl B. et al., Neuron 5:p 735-43, 1990; Mueller B. K. et al., Curr. Biol. 6, pp. 1497-1502, 1996; Mueller B. K in Molecular Basis of Axon Growth and Nerve Pattern Formation, Edited by H. Fujisawa, Japan Scientific Societies Press, 215-229, 1997). Their ability to act in a repulsive or inhibitory manner on growing nerve fibers was a decisive functional feature which played an important part in their isolation, cloning and characterization. The activity was readily detectable in simple cellular assay systems. RGM proteins have an inhibitory or repulsive effect in two different cellular assays. In the collapse assay, RGM proteins are added to growing nerve fibers. The binding of RGM to the RGM receptor induces a vigorous response involving all the membranous elements of the neuronal growth cone. The original extended hand-like growth cone is converted thereby into a thin thread. In the presence of RGM, the nerve fibers remain inhibited, retract greatly and are no longer able to continue their growth.
RGM proteins exert part of their effect by binding to the RGM receptor neogenin (Rajagopalan S. et al., Nat Cell Biol. 6, pp. 756-62, 2004). Neogenin is closely related to the DCC receptor (deleted in colorectal cancer). Both receptors are members of the immunoglobulin superfamily and have an extracellular, a transmembrane and an intracellular domain. Both have been described as receptors for a further ligand, netrin-1, but only neogenin, not DCC, binds RGM proteins. The extracellular domains of these receptors consist of 4 immunoglobulin-like domains followed by 6 fibronectin repeat domains.
The function of RGM A in the nervous system is understood best, and it is particularly noteworthy for its effect of inhibiting at very low concentrations the growth of nerve fibers. Injury to the central nervous system in adult humans and in adult rats leads to an accumulation of RGM proteins at the site of the lesion (Schwab J. M. et al., Arch. Neurol. in press, 2005; Schwab J. M. et al., Eur. J. Neurosci. 21:p. 387-98, 2005). Renewed outgrowth of the injured nerve fibers is prevented thereby, and permanent, more or less severe functional deficits occur, depending on the location of the site of the lesion. This inhibitory activity of RGM for nerve fiber growth is mediated by binding to the neogenin receptor (Rajagopalan S. et al., loc. cit.). However, the same receptor mediates, via binding of netrin-1, also a contrary effect stimulating nerve fiber growth.
Very recent results indicate that RGM proteins also fulfill important tasks in the central and peripheral nervous system, in regulating iron metabolism, in neoplastic diseases, in inflammatory processes and in the formation of bone and cartilage tissue.
Based on the observation that RGM A, B and C bind with high affinity to the neogenin receptor, the object of the present invention was to characterize in detail the functional binding domain of these molecules, especially of RGM A. This would make it possible to generate antibodies against the domain and against active RGM peptides which are derived therefrom and which with high probability neutralize the inhibitory activity of RGM and which might thereby stimulate the regeneration or renewed outgrowth of nerve fibers. In addition, binding domains or active RGM peptides derived therefrom could be provided per se as therapeutic effective agents.
BRIEF DESCRIPTION OF THE INVENTION
The above object has surprisingly been achieved by isolating and characterizing the binding domain of human RGM proteins, especially RGM A, and active polypeptide fragments thereof.



DESCRIPTION OF THE FIGURES
FIG. 1 shows the sequence alignment of the human forms of RGM A (GenBank #NP—064596.1) RGM B (GenBank #NP—001012779) and RGM C (GenBank #NP—998818.1).
FIG. 2 shows in diagrammatic representation the structure of RGM molecules. Between the N-terminal signal peptide and the C-terminal GPI anchor there are shown the RGD sequence, the von Willebrand factor domain (vWF D) and a hydrophobic sequence in the region of the C terminus in front of the anchor region. The binding domain region of interest according to the invention is presumed to be between vWFD and hydrophobic region. Below the diagram are the corresponding amino acid positions for human RGM A, the proteolytic cleavage site between amino acids 168 and 169.
FIG. 3A shows by means of fluorescence micrographs the influence of various RGM A peptide fragments on nerve fiber growth of rat neuronal cells. Whereas peptide 1 shows inhibitory activity at a concentration of 10 μg/ml, peptide 4 is inactive at the same concentration. Corresponding control mixtures with buffer or PBS is shown for comparison. FIG. 3B shows a bar diagram of measured axon growth indices (a measure of the area covered by axons in relation to the cell aggregate size; mean and standard deviation is shown) to illustrate the concentration-dependence of the axon growth-inhibitory activity of the RGM A peptide 1 of the invention. The corresponding bar diagram for the RGM A peptide 4 is shown in FIG. 3C.
FIG. 4A depicts fluorescence micrographs of human neuronal NTera cells which illustrate the influence of RGM A peptide 1 and peptide 4 (each in a concentration of 30 μg/ml) on nerve fiber growth of these cells. As comparison with the likewise depicted control image (incubation with PBS instead of peptide) shows, only peptide 1 has an inhibitory effect on nerve fiber growth. FIG. 4B depicts the correspondingly measured axon growth indices for test series with peptide 1, peptide 4 and control (PBS) (upper diagram: individual measurements and mean, lower diagram: bar diagram with mean and standard deviation).
FIG. 5A shows the results of an analysis of RGM A fragments in NTera nerve fiber growth assay. Both the RGM A fragments 2 (218-284) and 6 (168-422) were added in a concentration of 90 nM to NTera neurons. Both fragments strongly inhibit the nerve fiber growth of the NTera neurons. ***=p<0.001 significance versus PBS control.
FIG. 5B shows fluorescence micrographs of the assay results on RGM A fragments in the NTera nerve fiber growth assay. The RGM A fragments 2 (218-284), 3 (266-335), 4 (316-386) and 6 (168-422) were pipeted in the stated concentrations to NTera neurons. The active, meaning inhibitory for axon growth, fragments were 2, 3 and 6, but fragment 4 was inactive.
FIG. 5C shows further results of the analysis of RGM A fragments in the NTera nerve fiber growth assay. Both the RGM A fragments 3 (286-335) and 6 (168-422) were added in a concentration of 6 μg/ml to NTera neurons. Both fragments strongly inhibit nerve fiber growth of the NTera neurons. **=p<0.01 significance versus buffer control, ***=p<0.001 significance versus buffer control.
FIG. 6A shows the result of the analysis of RGM A peptide 1 and two peptides partly overlapping therewith (Down-1 and Up-1) in the Ntera nerve fiber growth assay. The peptides were added in a concentration of 10 μg/ml or 30 μg/ml to NTera aggregates. 24-36 hours later, the cultures were fixed, stained and analyzed. All 3 RGM A peptides were active in a concentration of 30 μg/ml and inhibited nerve fiber growth highly significantly.
FIG. 6B shows the result of the analysis of three further RGM A peptides in the Ntera nerve fiber growth assay. The RGM A peptides were added in a concentration of 10 μg/ml or 30 μg/ml to NTera aggregates. 24-36 hours later, the cultures were fixed, stained and analyzed. All 3 RGM A peptides (5-Ak, 6-Ak, 7-Ak) were inactive in a concentration of 10 μg/ml; only peptides 7-Ak and 6-Ak showed at the higher concentrations a distinct trend in the direction of inhibition of nerve fiber growth.
FIG. 7A shows the result of the RGM A—neogenin binding assay. In this assay, the polyclonal antibodies directed against the peptide Down-1 were very potent and blocked even at the lowest concentrations (Down 1 8641 & Down 1 8640) the interaction of the RGM A ligand with its receptor, the neogenin protein. A control antibody (rabbit control) had no effect, whereas a polyclonal antibody commercially available from R & D Systems (PAB anti RGM) likewise blocked the RGM A—neogenin interaction, although substantially less efficiently.
FIG. 7B shows how the Down-1 specific polyclonal antibody neutralizes the inhibitory activity of the potent RGM A fragment 2 in the Ntera nerve fiber growth assay. NTera cultures were treated in the outgrowth assay with PBS, fragment 2 (2 μg/ml) or with fragment 2 (2 μg/ml) and simultaneously with polyclonal antibody Down-1 (0.6 μg/ml).



DETAILED DESCRIPTION OF THE INVENTION
I. Explanations of General Terms
“Receptors” designate in the context of the present invention in particular surface molecules which are bound to a cell membrane and which are able to interact with a, for example soluble, ligand and, as a consequence of this interaction, may induce a signal which is directed for example into the interior of the cell, or a signal cascade (also referred to as signaling).
“Ligand” designates a natural, i.e. formed in vivo or artificially generated, low or high molecular weight binding partner for a “receptor”. The ligand is preferably able to move freely in the extracellular environment.
“Immunogen” designates a peptide fragment of the invention in glycosylated or unglycosylated form which is suitable for inducing the formation of antibodies against the immunogen. Binding of the immunogen (as hapten) to a macromolecular carrier may be advantageous where appropriate.
“Epitope” or antigenic determinant designates the region determining the antibody specificity of an antigen, such as, for example, of a protein. If this epitope is newly formed in a section of the protein or expressed on the accessible molecule surface, for example through external influences such as, for example, an interaction of a protein with a ligand, the term used is “neoepitope”.
A “domain” of a protein or antibody designates a complex structure which is formed by alpha-helix and/or beta-pleated sheet elements and is demarcated within the protein.
Unless stated otherwise, the term “RGM protein of the invention” encompasses both the neogenin-binding domain and polypeptides derived therefrom of a member of the family of RGM molecules, especially RGM A, B and C. Also encompassed in particular are functional polypeptides “with inhibitory activity”.
“Inhibiting” polypeptides or polypeptides “having inhibitory activity” are those which reduce or completely inhibit nerve cell growth in a nerve cell growth assay described herein.
Unless stated otherwise, “RGM” stands for RGM A, B and C, especially RGM A.
“Neogenin” or “neogenin receptor” are synonymous terms.
II. Specific Aspects of the Invention
A first aspect of the invention relates to receptor-binding, especially neogenin receptor-binding, domains of the repulsive guidance molecule (RGM) in glycosylated or especially unglycosylated form, preferably derived from RGM from mammals such as, for example, human, rat or mouse, or poultry, such as, for example, chicken.
A preferred embodiment relates to neogenin receptor-binding domains derived from a human RGM A as shown in SEQ ID NO: 2, human RGM B as shown in SEQ ID NO: 4 or human RGM C as shown in SEQ ID NO: 6. In this connection, the binding domains comprise in particular an amino acid sequence with a length of up to 150, such as, for example, up to 125, up to 100, up to 80, up to 30, or up to 20, such as, for example, 10 to 20, such as, for example, 11, 12, 13, 14, 15, 16, 17, 18 or 19, consecutive amino acid residues from an amino acid sequence region of RGM, specifically C-terminal relative to the RGD cleavage site and N-terminal relative to the GPI anchor region of RGM such as, in particular, RGM A.
The invention relates in particular to those neogenin receptor-binding domains which are characterized by the following partial sequence as shown in SEQ ID NO: 7:









GX1X2VEX3X4AX5YIGTTX6X7X8RQ






in which X1 to X8 are any amino acid residues.

In particular therein
X1 is Gln, H is or Asn;
X2 is His or Tyr
X3 is Ile or Met
X4 is Gln or His
X5 is Lys, Arg or Ala
X6 is Ile or Val
X7 is Val, Phe or Ile and
X8 is Val or Ile.
Specific examples which should be mentioned are:









GQHVEIQAKYIGTTIVVRQ (SEQ ID NO: 8)



 



GHYVEMHARYIGTTVFVRQ (SEQ ID NO: 9)



 



GNHVEIQAAYIGTTIIIRQ (SEQ ID NO: 10)






Examples of neogenin receptor-binding domains include an amino acid sequence from amino acid position 200-350 as shown in SEQ ID NO: 2, from amino acid position 200-330 as shown in SEQ ID NO: 4, or from amino acid position 180-350 as shown in SEQ ID NO: 6, or functional neogenin receptor-binding fragments thereof.
Such domains or fragments include in particular:
amino acid positions 200-325, 200-300, 200-285, 250-285 or 260-285 or 260-280 or the fragments 215-340, 260-340, 250-300, 260-291, 210-260 or 290-350 as shown in SEQ ID NO: 2; or
the sequence fragments of SEQ ID NO: 4 and 6 which can be derived from a sequence alignment of SEQ ID NO: 2 with SEQ ID NO: 4 and 6 (cf. appended FIG. 1) and which correspond to the amino acid positions detailed above of SEQ ID NO: 2; and
amino acid positions 200-300, 280-340, 240-300, 260-300 or 240-280 as shown in SEQ ID NO: 4 or
amino acid positions 200-350, 200-320, 220-310, 250-290 or 260-280 as shown in SEQ ID NO: 6.
Further specific examples of binding domains suitable according to the invention are:
KITEKVSGQHVEIQAKYIGTTIWRQVGRYLT (SEQ ID NO: 23) derived from RGM A;
RIVERESGHYVEMHARYIGTTVFVRQVGRYLT (SEQ ID NO: 24) derived from RGM B;
SIQTANPGNHVEIQAAYIGTTIIIRQTAGQLS (SEQ ID NO: 25) derived from RGM C.
KITEKVSGQHVEIQAK (SEQ ID NO: 26) derived from RGM A;
YIGTTIWRQVGRYLT (SEQ ID NO: 27) derived from RGM A;
WNAVEDWDSQGLYLC (SEQ ID NO: 28) derived from RGM A;
TIIFKNFQECVDQKVYQA (SEQ ID NO: 29) derived from RGM A;
RIVERESGHY VEMHAR (SEQ ID NO: 31) derived from RGM B;
YIGTTVFVRQ VGRYLT (SEQ ID NO: 32) derived from RGM B;
SIQTANPGNH VEIQAA (SEQ ID NO: 33) derived from RGM C; and
YIGTTIIIRQ TAGQLS (SEQ ID NO: 34) derived from RGM C.
Further examples of neogenin receptor-binding domains or binding fragments thereof include at least 10, such as, for example, 10-30, 10-25, 10-20 or 10-15, such as, in particular, 10, 11, 12, 13, 14, or 15, consecutive amino acid residues from one of the abovementioned peptides or from the sequence region from position 260 to 291 or 267 to 285 as shown in SEQ ID NO: 2, from the sequence region from position 260 to 325 as shown in SEQ ID NO: 4 or from the sequence region from position 250 to 300 as shown in SEQ ID NO: 6.
A further aspect of the invention relates to antigenic polypeptide fragments of the neogenin receptor-binding domains as defined above. The invention relates in particular to those antigenic polypeptide fragments which can be used to produce immunoglobulin molecules and modulate, in particular partly or completely antagonize, the binding of RGM to the neogenin receptor. Mention may be made for example of those antigenic polypeptide fragments which include at least 10, such as, for example, 10-30, 10-25, 10-20 or 10-15, such as, in particular, 10, 11, 12, 13, 14, or 15, consecutive amino acid residues of a peptide as shown in SEQ ID NO: 7, 8, 9, 10, 23 to 29 or 31 to 34.
A further aspect of the invention relates to the use of a neogenin receptor-binding domain as defined above or of a polypeptide fragment as defined above for producing a polyclonal antiserum or monoclonal antibody against RGM, where the antiserum or the antibody modulates, preferably partly or completely antagonizes, in particular the binding of RGM to the neogenin receptor.
The invention also relates to polyclonal antisera or monoclonal antibodies against RGM as defined above for use in diagnosis or therapy.
The invention also relates to the use of a polyclonal antiserum or monoclonal antibody of the invention for producing a pharmaceutical composition for the diagnosis or therapy of diseases and disease stages which are mediated by an interaction of the neogenin receptor with RGM or an RGM fragment. These diseases or disease stages are selected in particular from:

    a) mechanical injuries of the skull, brain and spinal cord,
    b) chronic disorders such as neurodegenerative, inflammatory and autoimmune diseases,
    c) impairments of neuronal regeneration, of axonal sprouting, of axonal extension and of neuronal plasticity,
    d) neoplastic diseases and tumor metastasis.


The invention further relates to the use of a neogenin receptor-binding domain as defined above or of a polypeptide fragment as defined above for producing a composition for the diagnosis or therapy of diseases or disease stages which are mediated by an impaired interaction of RGM or an RGM fragment with the associated receptor (such as, for example, the neogenin receptor). These diseases or disease stages are selected in particular from:

    a) altered axonogenesis processes associated with psychotic disorders and chronic states of pain caused by excessive axon sprouting and/or pathological synaptogenesis.
    b) disorders associated with an impaired iron metabolism, in particular juvenile hemochromatosis
    c) disorders associated with an impaired bone growth
    d) disorders associated with degenerative cartilage changes
    e) disorders associated with damage to intervertebral disks and vertebrae
    f) disorders associated with deregulated, uncontrolled cell migration processes.


A further aspect of the invention relates to the use of a neogenin receptor-binding domain as defined above or of a polypeptide fragment as defined above as target for detecting or for identifying RGM-binding ligands.
Another aspect of the invention relates to the use of a neogenin receptor-binding domain as defined above or of a polypeptide fragment as defined above as immunogen for active or passive immunization.
The invention also relates to polyclonal antisera obtainable by immunization of a mammal with an antigenic amount of a neogenin receptor-binding domain as defined above or of a polypeptide fragment as defined above; and monoclonal antibodies against a neogenin receptor-binding domain as defined above or against a polypeptide fragment as defined above, and antigen-binding fragments thereof, where appropriate in humanized form.
The invention also relates to pharmaceutical compositions comprising in a pharmaceutically suitable carrier at least one active ingredient selected from:

    a) a neogenin receptor-binding domain as defined above or a polypeptide fragment as defined above or
    b) monoclonal or polyclonal antibodies as defined above.


Pharmaceutical compositions of this type are used in particular for intrathecal, intravenous, subcutaneous, oral or parenteral, nasal and inhalational administration.
The invention further relates to an expression vector comprising at least one coding nucleic acid sequence for a neogenin receptor-binding domain as defined above or for a polypeptide fragment as defined above, operatively linked to at least one regulatory nucleic acid sequence.
The invention further relates to:

    
    
        recombinant microorganisms which harbor at least one vector as defined above.
        hybridomal cell lines which produce a monoclonal antibody as defined above.
        methods for producing a neogenin receptor-binding domain as defined above or a polypeptide fragment as defined above, where a recombinant microorganism as defined above is cultured, and the produced protein product is isolated from the culture. In this case a hybridoma cell line as defined above is cultured, and the produced protein product is isolated from the culture.
    
    


III. Further Information for Implementation of the Invention
1. Polypeptides
The invention relates in particular to binding domains of proteins of the RGM family and peptide fragments derived from these domains. Whereas RGM A and its binding domain and fragments derived therefrom were investigated in particular according to the invention, the invention also relates to corresponding domains and fragments of homologous proteins, such as, in particular, homologous members of the RGM family, such as, in particular, RGM B and RGM C.
“Functional equivalents” or analogs of the specifically disclosed RGM domains or polypeptides are in the context of the present invention polypeptides which differ therefrom, such as, for example, those having a degree of homology of less than 100% to the neogenin binding domains of proteins as shown in SEQ ID NO: 2, 4 or 6, but which still possess the desired biological activity. In particular, they should be capable of binding to the neogenin receptor and/or show an inhibitory effect in a nerve fiber growth assay described herein and moreover inhibit partly or completely, statistically significantly (p<=0.05), nerve fiber growth.
“Functional equivalents” mean according to the invention in particular mutants which have in at least one of the sequence positions of the abovementioned specific sequences an amino acid which differs from that specifically mentioned, but nevertheless have one of the biological activities mentioned herein. “Functional equivalents” thus comprise the mutants obtainable by one or more amino acid additions, substitutions, deletions and/or inversions, it being possible for said changes to occur in any sequence position as long as they lead to a mutant having the profile of properties according to the invention. Functional equivalence exists in particular also when there is a qualitative agreement between the mutant and unmodified polypeptide in the reactivity pattern, i.e. for example identical biological effects are to be observed but differ greatly in the level of expression. Examples of suitable substitutions of amino acid residues are the following:














Original residue
Examples of substitution









Ala
Ser



Arg
Lys



Asn
Gln; His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu; Val



Leu
Ile; Val



Lys
Arg; Gln; Glu



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










“Functional equivalents” in the above sense are also precursors of the polypeptides described, and functional derivatives and salts of the polypeptides. The term “salts” means both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention. Salts of carboxyl groups can be prepared in a manner known per se and comprise inorganic salts such as, for example, sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases such as, for example, amines, such as triethanolamine, arginine, lysine, piperidine and the like. Acid addition salts such as, for example, salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid are likewise an aspect of the invention. “Functional derivatives” of polypeptides of the invention can likewise be prepared on functional amino acid side groups or on their N- or C-terminal end with the aid of known techniques. Derivatives of these types comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
“Functional equivalents” of course also comprise polypeptides obtainable from other organisms, and naturally occurring variants. For example, areas of homologous sequence regions can be found by sequence comparison, and equivalent enzymes can be established on the basis of the specific requirements of the invention.
“Functional equivalents” are moreover fusion proteins having one of the abovementioned polypeptide sequences or functional equivalents derived therefrom, and at least one further heterologous sequence functionally different therefrom in functional N- or C-terminal linkage (i.e. with negligible mutual functional impairment of the portions of the fusion proteins). Nonlimiting examples of such heterologous sequences are, for example, enzymes and immunoglobulins.
“Functional equivalents” also comprised by the invention are homologs of the specifically disclosed proteins and peptides. These have at least 40%, or at least 50%, or at least 60%, such as, for example, at least 75%, or in particular at least 85%, such as, for example, 90%, 95% or 99%, homology to one of the specifically disclosed sequences, calculated, for example, by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. A percentage homology of a homologous polypeptide of the invention means in particular the percentage identity of the amino acid residue based on the complete length of one of the amino acid sequences specifically described herein.
A “derived” amino acid sequence means according to the invention, unless indicated otherwise, a sequence which has an identity of at least 80% or at least 90%, in particular 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%, with the initial sequence.
“Identity” between two sequences means identity of the amino acid residues over the complete length of the sequence in each case, such as, for example, the identity calculated by comparison with the aid of the Vector NTI Suite 7.1 Software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1), setting the following parameters:
Multiple alignment parameter:
















Gap opening penalty
10



Gap extension penalty
10



Gap separation penalty range
 8



Gap separation penalty
off



% identity for alignment delay
40



Residue specific gaps
off



Hydrophilic residue gap
off



Transition weighing
 0










Pairwise alignment parameter:
















FAST algorithm
on



K-tuple size
1



Gap penalty
3



Window size
5



Number of best diagonals
5










For further illustration of homologs included according to the invention, reference is made to the following table which shows the percentage agreement of RGM B and C with RGM A for the full-length protein and for certain partial sequence regions which are of particular interest according to the invention.














RGM A - RGM B
RGM A - RGM C




















Full-length proteins
51%2)3)
47%



AA1) 200-350
62%2)
55%



AA 200-325
61%
55%



AA 200-300
66%
60%



AA 200-285
65%
66%



AA 250-285
58%
61%



AA 260-285
57%
78%







1)Amino acids residue, indication of position based on RGM A sequence (SEQ ID NO: 2)



2)Amino acid identity of the full-length proteins and of the peptide fragments in percent



3)Method: BLAST 2 SEQUENCES VERSION BLASTP 2.2.5 [Nov. 16, 2002] Matrix: Blosum62 (gap open: 11 gap extension: 1)






In the case where protein glycosylation is possible, equivalents of the invention comprise proteins of the type designated above in deglycosylated or glycosylated form and modified forms obtainable by altering the glycosylation pattern.
Homologs of the peptides of the invention can be identified by screening combinatorial libraries of mutants such as, for example, truncation mutants. For example, it is possible to generate a variegated library of peptide variants by combinatorial mutagenesis at the nucleic acid level, such as, for example, by enzymatic ligation of a mixture of synthetic oligonucleotides. There is a large number of methods which can be used to produce libraries of potential homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector. The use of a degenerate set of genes makes it possible to provide all sequences which encode the desired set of potential protein sequences in one mixture. Methods for synthesizing degenerate oligonucleotides are known to the skilled worker (e.g. Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477).
2. Nucleic Acids
The invention further relates to the coding nucleic acid sequences for the RGM binding domains and polypeptides described above, such as, in particular, as shown in SEQ ID NO: 1, 3 and 5, and nucleic acid sequences or partial sequences derived therefrom.
All nucleic acid sequences of the invention (single- and double-stranded DNA and RNA sequences, such as, for example, cDNA and mRNA) can be prepared in a manner known per se by chemical synthesis from the nucleotide units, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. Chemical synthesis of oligonucleotides can take place for example in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). Addition of synthetic oligonucleotides and filling in of gaps using the Klenow fragment of DNA polymerase and ligation reactions, and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
A “derived” nucleic acid sequence means according to the invention, unless indicated otherwise, a sequence which has an identity of at least 80% or at least 90%, in particular 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%, with the initial sequence.
“Identity” between two nucleic acids means the identity of the nucleotides over the complete length of the nucleic acid in each case, in particular the identity which is obtained by comparison with the aid of the Vector NTI Suite 7.1 Software from Informax (USA) using the Clustal method (see above).
The invention also relates to nucleic acid sequences coding for one of the above peptides and their functional equivalents, which can be obtained for example by use of artificial nucleotide analogs.
The invention relates both to isolated nucleic acid molecules which code for peptides of the invention or biologically active segments thereof, and nucleic acid fragments which can be used for example as hybridization probes or primers for identifying or amplifying coding nucleic acids of the invention.
The nucleic acid molecules of the invention may additionally comprise untranslated sequences from the 3′ and/or 5′ end of the coding region of the gene.
An “isolated” nucleic acid molecule is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid and may moreover be substantially free of other cellular material or culture medium if it is prepared by recombinant techniques, or free of chemical precursors or other chemicals if it is synthesized chemically.
A nucleic acid molecule of the invention can be isolated by means of standard techniques of molecular biology and the sequence information provided by the invention. For example, cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a segment thereof as hybridization probe and standard hybridization techniques (as described for example in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). It is moreover possible to isolate a nucleic acid molecule comprising one of the sequences of the invention or a segment thereof by polymerase chain reaction using the oligonucleotide primers constructed on the basis of this sequence. The nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis. The oligonucleotides of the invention can also be prepared by standard synthesis methods, e.g. using an automatic DNA synthesizer.
The invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences, or a segment thereof.
The nucleotide sequences of the invention make it possible to produce probes and primers which can be used for identifying and/or cloning homologous sequences in other cell types and organisms. Such probes and primers usually comprise a nucleotide sequence region which hybridizes under stringent conditions to at least about 12, preferably at least about 25, such as, for example, about 40, 50 or 75, consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or of a corresponding antisense strand.
Further nucleic acid sequences of the invention are derived from coding sequences for the RGM domains and peptides of the invention and differ therefrom by addition, substitution, insertion or deletion of one or more nucleotides, but still code for peptides having the desired profile of properties.
The invention also comprises nucleic acid sequences which comprise so-called silent mutations, or are modified in accordance with the codon usage of a specific original or host organism compared with a specifically mentioned sequence, as well as naturally occurring variants such as, for example, splice variants or allelic variants, thereof. Sequences obtainable by conservative nucleotide substitutions (i.e. the relevant amino acid is replaced by an amino acid of the same charge, size, polarity and/or solubility) are likewise an aspect.
The invention also relates to the molecules derived from the specifically disclosed nucleic acids through sequence polymorphisms. These genetic polymorphisms may exist because of the natural variation between individuals within a population. These natural variations normally result in a variance of from 1 to 5% in the nucleotide sequence of a gene.
The invention further also comprises nucleic acid sequences which hybridize with the abovementioned coding sequences or are complementary thereto. These polynucleotides can be found by screening genomic or cDNA libraries and if appropriate be amplified therefrom by means of PCR with suitable primers, and subsequently isolated for example with suitable probes. A further possibility is the transformation of suitable microorganisms with polynucleotides or vectors of the invention, to multiply the microorganisms and thus the polynucleotides and subsequently to isolate them. An additional possibility is to synthesize polynucleotides of the invention also by a chemical route.
The property of being able to “hybridize” onto polynucleotides means the ability of a polynucleotide or oligonucleotide to bind under stringent conditions to an almost complementary sequence, while there are nonspecific bindings between non-complementary partners under these conditions. For this purpose, the sequences should be 70-100%, in particular 90-100%, such as, for example, 95%, 96%, 97%, 98%, or 99%, complementary. The property of complementary sequences being able to bind specifically to one another is made use of, for example, in the Northern or Southern blotting technique or in the primer binding in PCR or RT-PCR. Oligonucleotides with a length of 30 base pairs or more are normally employed for this purpose. Stringent conditions mean, for example, in the Northern blotting technique the use of a washing solution, for example 0.1×SSC buffer with 0.1% SDS (20×SSC: 3M NaCl, 0.3M Na citrate, pH 7.0), at 50-70° C., preferably 60-65° C., for eluting nonspecifically hybridized cDNA probes or oligonucleotides. In this case, as mentioned above, only nucleic acids with a high degree of complementarity remain bound to one another. The setting up of stringent conditions is known to the skilled worker and is described for example in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A further aspect of the invention relates to antisense nucleic acids. This comprises a nucleotide sequence which is complementary to a coding sense nucleic acid. The antisense nucleic acid may be complementary to the entire coding strand or only to a section thereof. In a further embodiment, the antisense nucleic acid molecule is antisense to a noncoding region of the coding strand of a nucleotide sequence. The term “non-coding region” relates to the sequence sections which are referred to as 5′- and 3′-untranslated regions.
An antisense oligonucleotide may be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides long. An antisense nucleic acid of the invention can be constructed by chemical synthesis and enzymatic ligation reactions using methods known in the art. An antisense nucleic acid can be synthesized chemically, using naturally occurring nucleotides or variously modified nucleotides which are configured so that they increase the biological stability of the molecules or increase the physical stability of the duplex formed between the antisense and sense nucleic acids. Examples which can be used are phosphorothioate derivatives and acridine-substituted nucleotides. Examples of modified nucleotides which can be used for generating the antisense nucleic acid are, inter alia, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueuosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl-queuosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w and 2,6-diaminopurine. The antisense nucleic acid may also be produced biologically by using an expression vector into which a nucleic acid has been subcloned in the antisense direction.
3. Expression Constructs and Vectors
The invention additionally relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for an RGM protein of the invention or functional equivalent or immunoglobulin, and to vectors comprising at least one of these expression constructs.
Such constructs of the invention preferably comprise a promoter 5′-upstream from the particular coding sequence, and a terminator sequence 3′-downstream, and, if appropriate, other usual regulatory elements, in particular each operatively linked to the coding sequence. “Operative linkage” means the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, other regulatory elements in such a way that each of the regulatory elements is able to comply with its function as intended for expression of the coding sequence. Examples of sequences which can be operatively linked are targeting sequences and enhancers, polyadenylation signals and the like. Other regulatory elements comprise selectable markers, amplification signals, origins of replication and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
In addition to the artificial regulatory sequences it is possible for the natural regulatory sequence still to be present in front of the actual structural gene. This natural regulation can, if appropriate, be switched off by genetic modification, and expression of the genes can be increased or decreased. The gene construct can, however, also have a simpler structure, that is to say no additional regulatory signals are inserted in front of the structural gene, and the natural promoter with its regulation is not deleted. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place, and gene expression is enhanced or diminished. The nucleic acid sequences may be present in one or more copies in the gene construct.
Examples of promoters which can be used are: cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, lambda-PR or lambda-PL promoter, which are advantageously used in Gram-negative bacteria; and the Gram-positive promoters amy and SPO2, the yeast promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH or the plant promoters CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, not or the ubiquitin or phaseolin promoter. The use of inducible promoters is particularly preferred, such as, for example, light- and, in particular, temperature-inducible promoters such as the PrPl promoter. It is possible in principle for all natural promoters with their regulatory sequences to be used. In addition, it is also possible advantageously to use synthetic promoters.
Said regulatory sequences are intended to make specific expression of the nucleic acid sequences and protein expression possible. This may mean, for example, depending on the host organism, that the gene is expressed or overexpressed only after induction or that it is immediately expressed and/or overexpressed.
The regulatory sequences or factors may moreover preferably influence positively, and thus increase or reduce, expression. Thus, enhancement of the regulatory elements can take place advantageously at the level of transcription by using strong transcription signals such as promoters and/or enhancers. However, it is also possible to enhance translation by, for example, improving the stability of the mRNA.
An expression cassette is produced by fusing a suitable promoter to a suitable coding nucleotide sequence and to a terminator signal or polyadenylation signal. Conventional techniques of recombination and cloning are used for this purpose, as described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector, which makes optimal expression of the genes in the host possible. Vectors are well known to the skilled worker and can be found, for example, in “Cloning Vectors” (Pouwels P. H. et al., eds, Elsevier, Amsterdam-New York-Oxford, 1985). Vectors also mean not only plasmids but also all other vectors known to the skilled worker, such as, for example, phages, viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors may undergo autonomous replication in the host organism or chromosomal replication.
Examples of suitable expression vectors which may be mentioned are:
Conventional fusion expression vectors such as pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway, N.J.), with which respectively glutathione S-transferase (GST), maltose E-binding protein and protein A are fused to the recombinant target protein.
Nonfusion protein expression vectors such as pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Yeast expression vector for expression in the yeast S. cerevisiae, such as pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for constructing vectors suitable for use in other fungi such as filamentous fungi comprise those which are described in detail in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy et al., eds, pp. 1-28, Cambridge University Press: Cambridge.
Baculovirus vectors which are available for expression of proteins in cultured insect cells (for example Sf9 cells) comprise the pAc series (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170:31-39).
Plant expression vectors such as those described in detail in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binary vectors with selectable markers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “Binary Agrobacterium vectors for plant transformation”, Nucl. Acids Res. 12:8711-8721.
Mammalian expression vectors such as pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).
Further suitable expression systems for prokaryotic and eukaryotic cells are described in chapters 16 and 17 of Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
4. Recombinant Host Organisms:
The vectors of the invention can be used to produce recombinant organisms which are transformed, for example, with at least one vector of the invention and can be employed for producing the domains or polypeptides of the invention. The recombinant constructs of the invention described above are advantageously introduced and expressed in a suitable host system. Cloning and transfection methods familiar to the skilled worker, such as, for example, coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring about expression of said nucleic acids in the particular expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., eds, Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Suitable host organisms are in principle all organisms which enable expression of the nucleic acids of the invention, their allelic variants, their functional equivalents or derivatives. Host organisms mean, for example, bacteria, fungi, yeasts, plant or animal cells. Preferred organisms are bacteria, such as those of the genera Escherichia, such as, for example, Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells from animals or plants, for example Sf9, CHO or HEK293 cells.
Successfully transformed organisms can be selected through marker genes which are likewise present in the vector or in the expression cassette. Examples of such marker genes are genes for antibiotic resistance and for enzymes which catalyze a color-forming reaction which causes staining of the transformed cell. These can then be selected by automatic cell sorting. Microorganisms which have been successfully transformed with a vector and harbor an appropriate antibiotic resistance gene (for example G418 or hygromycin) can be selected by appropriate antibiotic-containing media or nutrient media. Marker proteins present on the surface of the cell can be used for selection by means of affinity chromatography.
If desired, the gene product can also be expressed in transgenic organisms such as transgenic animals such as, in particular, mice, sheep or transgenic plants.
The invention further relates to methods for the recombinant production of RGM domains or polypeptides of the invention or functional, biologically active fragments thereof, wherein a peptide-producing recombinant host organism is cultured, expression of the polypeptides is induced if appropriate, and they are isolated from the culture. The peptides can also be produced on the industrial scale in this way if desired.
The recombinant host can be cultured and fermented by known methods. Bacteria can be grown, for example, in TB or LB medium and at a temperature of 20 to 40° C. and a pH of from 6 to 9. Details of suitable culturing conditions are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
If the polypeptides are not secreted into the culture medium, the cells are then disrupted and the product is obtained from the lysate by known protein isolation methods. The cells may alternatively be disrupted by high-frequency ultrasound, by high pressure, such as, for example, in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of a plurality of the methods mentioned.
The peptides can be purified by known chromatographic methods such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and by other usual methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T. G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
It is particularly advantageous for isolation of the recombinant peptide to use vector systems or oligonucleotides which extend the cDNA by particular nucleotide sequences and thus code for modified polypeptides or fusion proteins which serve, for example, for simpler purification. Suitable modifications of this type are, for example, so-called tags which act as anchors, such as, for example, the modification known as hexa-histidine anchor, or epitopes which can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can be used to attach the peptides to a solid support, such as, for example, a polymer matrix, which can, for example, be packed into a chromatography column, or can be used on a microtiter plate or another support.
These anchors can at the same time also be used for recognition of the peptides. It is also possible to use for recognition of the peptides conventional markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive labels, alone or in combination with the anchors for derivatizing the peptides.
5. Immunoglobulins
5.1 Definition
The present invention relates to monoclonal or polyclonal antibodies which bind specifically to an RGM protein of the invention or derivative/equivalent thereof, i.e. antibodies having specificity for an RGM protein of the invention or derivative/equivalent thereof. The present invention also relates to parts of these antibodies, especially antigen-binding parts thereof, i.e. antibody fragments which bind an RGM protein of the invention or a derivative/equivalent thereof.
The antibody of the invention is preferably chosen so that it has particular binding kinetics (e.g. high affinity, little dissociation, low off rate (koff), strong neutralizing activity) for the specific binding to RGM protein of the invention or derivate/equivalent thereof.
Thus, antibodies with an affinity for the RGM protein of the invention or derivative/equivalent thereof in the region of KD=10−6-10−12 M can be provided.
According to a further aspect, the antibodies of the invention can be chosen so that they bind the RGM protein or derivative/equivalent thereof with a koff rate constant of 0.1 s−1 or less.
The antibodies are preferably isolated antibodies. According to a further aspect, the antibodies are neutralizing antibodies. The antibodies of the invention include in particular monoclonal and recombinant antibodies. The antibodies of the invention may comprise an amino acid sequence which derives completely from a single species, and thus may be for example a human antibody or a mouse antibody. According to further embodiments, the antibody may be a chimeric antibody or a CDR graft antibody or another type of humanized antibody.
The term “antibody” is intended to refer to immunoglobulin molecules which are formed from 4 polypeptide chains, two heavy (H) chains and two light (L) chains. The chains are usually linked together by disulfide bonds. Every heavy chain is composed of a variable region of the heavy chain (abbreviated here to HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain is formed from three domains CH1, CH2 and CH3. Each light chain is composed of a variable region of the light chain (abbreviated here to LCVR or VL) and a constant region of the light chain. The constant region of the light chain is formed from a CL domain. The VH and VL regions may be further divided into hypervariable regions which are referred to as complementarity-determining regions (CDR) and are interspersed with more conserved regions which are referred to as framework regions (FR). Each VH and VL region is formed from three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The term “antigen-binding part” of an antibody (or simply “antibody part”) refers to one or more fragments of an antibody having specificity for an RGM protein of the invention or derivative/equivalent thereof, the fragment(s) still having the ability to bind specifically the RGM protein or derivative/equivalent thereof. It has been shown that the antigen-binding function of an antibody can be undertaken by fragments of a complete antibody. Examples of binding fragments include within the meaning of the term “antigen-binding part” of an antibody (i) an Fab fragment, i.e. a monovalent fragment composed of the VL, VH, CL and CH1 domains; (ii) an F(ab′)2 fragment, i.e. a bivalent fragment which comprises two Fab fragments linked together by a disulfide bridge in the hinge region; (iii) an Fd fragment which is composed of the VH and CH1 domains; (iv) an Fv fragment which is composed of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a VH domain or VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolated complementarity-determining region (CDR). Although the two domains of the Fv fragment, namely VL and VH, are encoded by separate genes they can furthermore be connected together by a synthetic linker by use of recombinant methods, whereby they can be produced as a single protein chain in which the VL and VH regions are present together in order to form 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. Nat. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term “antigen-binding part” of an antibody. Other types of single-chain antibodies such as diabodies likewise belong thereto. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but with use of a linker which is too short for the two domains to be present together on the same chain, the domains thus being forced to pair with complementary domains of another chain and to form two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
A further possibility is for an antibody or antigen-binding part thereof to be part of a larger immunoadhesion molecule which is formed by covalent or non-covalent association of the antibody or antibody part with one or more further proteins or peptides. Such immunoadhesion molecules involve the use of the streptavidin core region in order to produce a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cysteine residue, of a marker peptide and of a C-terminal polyhistidine tag in order to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).
Antibody parts, such as Fab and F(ab′)2 fragments, can be produced from whole antibodies by using conventional techniques such as digestion with papain or pepsin. It is additionally possible to obtain antibodies, antibody parts and immunoadhesion molecules by using standardized recombinant DNA techniques. An “isolated antibody having specificity for an RGM protein of the invention or derivative/equivalent thereof” describes an antibody which has specificity for an RGM protein of the invention or derivative/equivalent thereof and which is substantially free of other antibodies having different antigen specificities.
The term “neutralizing antibody” describes an antibody whose binding to a particular antigen leads to inhibition of the biological activity of the antigen. This inhibition of the biological activity of the antigen can be assessed by measuring one or more indicators of the biological activity of the antigen, using a suitable in vitro or in vivo assay.
The term “monoclonal antibody” describes an antibody which is derived from a hybridoma (e.g. an antibody which is secreted by a hybridoma produced by means of hybridoma technology such as the standardized hybridoma methods of Köhler and Milstein). An antibody derived from a hybridoma and having specificity for an RGM protein of the invention or derivative/equivalent thereof is therefore referred to as a monoclonal antibody.
The term “recombinant antibody” describes antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed by use of a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes (see, for example, Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are combined with other DNA sequences. Recombinant antibodies include, for example, chimeric, CDR graft and humanized antibodies.
The term “human antibody” describes antibodies whose variable and constant regions correspond to immunoglobulin sequences of the human germline, as described for example by Kabat et al. (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), or are derived therefrom. The human antibodies of the invention may, however, comprise amino acid residues which are not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and especially in CDR3. Recombinant human antibodies of the invention have variable regions and may also comprise constant regions derived from immunoglobulin sequences of the human germline (see 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). According to particular embodiments, such recombinant human antibodies are, however, subjected to an in vitro mutagenesis (or to a somatic in vivo mutagenesis if an animal which is transgenic due to human Ig sequences is used), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which, although they are related to VH and VL sequences of the human germline or are derived therefrom, do not naturally exist within the human antibody germline repertoire in vivo. According to particular embodiments, such recombinant antibodies are the result of a selective mutagenesis or back-mutation, or both.
The term “back-mutation” refers to a method in which some or all of the somatically mutated amino acids of a human antibody are replaced by the corresponding germline residues of a homologous germline antibody sequence. The sequences of the heavy and light chain of a human antibody of the invention are separately compared with the germline sequences in the VBASE database in order to identify the sequences with the greatest homology. Deviations in the human antibody of the invention are returned to the germline sequence by mutation at defined nucleotide positions which encode such deviant amino acids. The direct or indirect significance of each amino acid, identified in this way as candidate for back-mutation, for antigen binding was to be investigated, and an amino acid which, after mutation, impairs a desirable property of the human antibody was not to be included in the eventual human antibody. In order to minimize the number of amino acids for a back-mutation, it is possible to leave unchanged those amino acid positions which, although deviating from the closest germine sequence, are identical to the corresponding amino acid sequence of a second germline sequence, provided that the second germine sequence is identical and colinear with the sequence of the human antibody of the invention in at least 10 and preferably in 12 amino acids on both sides of the amino acid in question. Back-mutations can be undertaken at any stage in antibody optimization.
The term “chimeric antibody” comprises antibodies in which individual parts of the molecule are derived from different species. Thus, chimeric antibodies are, without being restricted thereto, for example antibodies which comprise sequences for the variable region of the heavy and light chain from one species, but in which the sequences of one or more of the CDR regions from VH and/or VL are replaced by CDR sequences of another species. The variable regions in such antibodies may have mouse heavy and light chains in which one or more of the mouse CDRs (e.g. CDR3) are replaced by human CDR sequences.
The term “humanized antibody” describes antibodies which comprise sequences of the variable region of heavy and light chain from a non-human species (e.g. mouse, rat, rabbit, chicken, camelid, goat), but in which at least one part of the VH and/or VL sequence has been modified in order to be “more human-like”, i.e. be like variable sequences of the human germline. One type of humanized antibody is a CDR graft antibody in which human CDR sequences are inserted into non-human VH and VL sequences in order to replace the corresponding non-human CDR sequences.
A method for measuring the binding kinetics of an antibody is based on so-called surface plasmon resonance. The term “surface plasmon resonance” refers to an optical phenomenon with which it is possible to analyze biospecific interactions by detecting changes in protein concentrations with a biosensor matrix, using for example the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jönsson, U. et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, 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 “Koff” describes the off rate constant for dissociation of an antibody from the antibody/antigen complex.
The term “Kd” describes the dissociation constant of a particular antibody-antigen interaction.
The binding affinity of the antibodies of the invention can be assessed by using standardized in vitro immunoassays such as ELISA or BIAcore analyses.
5.2 Production of Immunoglobulins
5.2.1 Production of Polyclonal Antibodies
The present invention relates to polyclonal antibodies against RGM domains and polypeptides of the invention and the production thereof.
For this purpose, a host is immunized with at least one RGM protein of the invention or derivative/equivalent thereof; and an antibody-containing serum of the host which is formed in response to the immunization is obtained.
If the RGM polypeptides to be used have only weak or no immunogenicity, their immunogenicity can be increased by coupling them to carriers, preferably a carrier protein such as keyhole limpet hemocyanin (KLH), Limulus Polyphenus hemocyanin (LPH), bovine serum albumin (BSA) or ovalbumin (OVA). A number of coupling possibilities are available to the skilled worker and are generally known. An expedient possibility is, for example, reaction with glutaraldehyde, for example by incubation of RGM protein with a suitable peptide or peptide mixture in water or an aqueous solvent. This reaction can conveniently be carried out at ambient temperature, usually meaning room temperature. However, cooling or gentle heating may also be expedient. The reaction usually leads to the desired result within a few hours, and a reaction time of, for example, 2 h is in the normal range. The glutaraldehyde concentration is usually in the ppm to % range, expediently from 10 ppm to 1%, preferably from 100 ppm to 0.5%. Optimization of the reaction parameters is within the scope of the skilled worker.
In addition to the antigen, the compositions ordinarily comprise further excipients, especially adjuvants normally employed for immunization, e.g. Freund's adjuvant. In particular, complete Freund's adjuvant is used for the first immunization, whereas all further immunizations are carried out with incomplete Freund's adjuvant. The immunizing cocktail is produced by adding the antigen (immunogen), preferably as component mixture described above, to the excipient(s). The antigen is usually emulsified in this case.
Suitable as host are in particular rodents or else rabbits. These or other suitable hosts are injected with the immunizing cocktail, preferably subcutaneously. The antibody titers can be determined using an immunoassay, for example competitively using a sheep antiserum directed against host IgG, and labeled RGM protein. It is thus possible to decide towards the end of the immunization whether a particular host is suitable for obtaining antibodies. If, for example, four immunizations are carried out, the antibody titer can be determined after the third immunization, and then antibodies can be obtained from animals showing a sufficient antibody titer.
Blood is preferably taken from the hosts over several weeks or months in order to obtain the antibodies formed. It is possible finally to exsanguinate the host. Serum which comprises the desired antibodies can be obtained in a manner known per se from the blood obtained in this way. The whole serum obtained in this way can if necessary be further purified in a manner known to the skilled worker, in order to concentrate the antibody fraction present therein and, in particular, the RGM protein-recognizing antibodies.
In a particular embodiment of this method, at least one antibody of the serum which specifically recognizes the RGM protein or a derivative/equivalent thereof used as immunogen is selected. Specificity means in this connection a higher binding affinity of the antibody for the immunogen than for other, in particular immunogenically related proteins.
5.2.2 Production of Monoclonal Antibodies
Immunoglobulins useful according to the invention can be obtained using methods known per se. Thus, hybridoma technology allows monospecific antibodies for an antigen of interest to be produced. In addition, recombinant antibody techniques, such as the in vitro screening of antibody libraries, have been developed and can likewise be used to produce such specific antibodies.
Thus, for example, an animal can be immunized with the antigen of interest. This in vivo approach may further comprise establishing a series of hybridomas from the lymphocytes or spleen cells of an animal, and selecting a hybridoma which secretes an antibody which specifically binds the antigen. The animal to be immunized may be for example a mouse, rat, rabbit, chicken, camelid or sheep, or a transgenic version of one of the aforementioned animals, for example a transgenic mouse with human immunoglobulin genes which makes human antibodies after an antigen stimulus. Further types of animals which can be immunized include mice with severe combined immunodeficiency (SCID) which have been reconstituted with human peripheral mononuclear blood cells (chimeric hu-PBMC-SCID mice) or with lymphoid cells or precursors thereof, as well as mice which have been treated with lethal whole-body irradiation, subsequently protected from radiation with bone-marrow cells from a mouse with severe combined immunodeficiency (SCID) and subsequently transplanted with functional human lymphocytes (the so-called trimera system). A further type of animal to be immunized is an animal (e.g. a mouse) in whose genome an endogenous gene which encodes the antigen of interest has been switched off (“knocked out”), e.g. by homologous recombination, so that this animal recognizes the antigen as foreign after immunization with the antigen. It is clear to the skilled worker that the polyclonal or monoclonal antibodies produced by these methods are characterized and selected by using known screening methods, which include ELISA techniques, but without being restricted thereto.
According to a further embodiment, a recombinant antibody library is screened with the antigen. The recombinant antibody library can be expressed for example on the surface of bacteriophages or on the surface of yeast cells or on the surface of bacterial cells. The recombinant antibody library can be for example an scFv library or an Fab library. In a further embodiment, antibody libraries can be expressed as RNA-protein fusions.
A further approach to the production of antibodies of the invention comprises a combination of in vivo and in vitro approaches. For example, the antigen can be allowed to act on the antibody repertoire by immunizing an animal with the antigen in vivo and subsequently using the antigen for in vitro screening of a recombinant antibody library produced from lymphoid cells of the animal, or a single-domain antibody library (e.g. with heavy and/or light chains). According to a further approach, the antigen is allowed to act on the antibody repertoire by immunizing an animal with the antigen in vivo and subsequently subjecting a recombinant antibody library produced from lymphoid cells of the animal or a single-domain library to an affinity maturation. According to a further approach, the antigen is allowed to act on the antibody repertoire by immunizing an animal with the antigen in vivo, subsequently selecting single antibody-producing cells which secrete an antibody of interest, and obtaining from these selected cells cDNAs for the variable region of the heavy and light chain (e.g. by PCR) and expressing the variable regions of the heavy and light chain in vitro in mammalian host cells (which is referred to as the lymphocyte-antibody selection method or SLAM for selected lymphocyte antibody method), allowing the selected antibody gene sequences to be selected and manipulated further. Monoclonal antibodies can additionally be selected by expression cloning by expressing the antibody genes for the heavy and light chain in mammalian cells, and selecting the mammalian cells which secrete an antibody having the desired binding affinity.
The present invention makes defined antigens available in the form of RGM binding domains or polypeptides for screening and counterscreening. It is thus possible according to the invention to select those polyclonal and monoclonal antibodies which show a profile of properties which is desired according to the invention as defined above.
The method of the invention for producing antibodies can be used to produce various types of antibodies. These include substantially human antibodies, chimeric antibodies, humanized antibodies and CDR graft antibodies, and antigen-binding parts thereof.
Methods for producing antibodies of the invention are described below. A distinction is made in this connection between in vivo approaches, in vitro approaches or a combination of the two.
In vivo approaches:
Starting from cells which produce the antibody generated in vivo it is possible to produce monoclonal antibodies by standardized techniques, such as the hybridoma technique originally described by Köhler and Milstein (1975, Nature 256:495-497) (see also Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75). The technology for producing monoclonal antibody hybridomas is sufficiently well known (see in general R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36). An immortalized cell line (typically a myeloma) is for this purpose fused to lymphocytes (typically splenocytes or lymph node cells or peripheral blood lymphocytes) of a mammal immunized with the RGM protein of the invention or derivative/equivalent thereof, and the culture supernatants of the resulting hybridoma cells are screened in order to identify a hybridoma which produces a monoclonal antibody having specificity for RGM protein of the invention or for a derivative/equivalent thereof. It is possible to use for this purpose any of the many sufficiently well known protocols for fusing lymphocytes and immortalized cell lines (see also G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). The skilled worker is additionally aware of many different variations of such methods, which can likewise be used. Typically, the immortalized cell lines (e.g. a myeloma cell line) were derived from the same mammalian species as the lymphocytes. It is possible for example to establish murine hybridomas by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the invention with an immortalized mouse cell line. Preferred immortalized cell lines are mouse myeloma cell lines which are sensitive to culture medium comprising hypoxanthine, aminopterin and thymidine (HAT medium). Any one of many myeloma cell lines can be used in standard fashion as fusion partner, e.g. the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma line. These myeloma cell lines are obtainable from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (PEG). The hybridoma cells resulting from the fusion are then selected using HAT medium whereby non-fused and non-productively fused myeloma cells are killed (non-fused splenocytes die after several days because they are not transformed). Monoclonal antibody-producing hybridoma cells which specifically recognize an RGM protein of the invention or a derivative/equivalent thereof are identified by screening the hybridoma culture supernants of such antibodies e.g. by using a standard ELISA assay in order to select those antibodies which are able to bind specifically the RGM protein of the invention or a derivative/equivalent thereof.
Depending on the nature of the desired antibody, various host animals can be used for the in vivo immunization. A host which itself expresses an endogenous version of the antigen of interest can be used. Alternatively, a host which has been made deficient for an endogenous version of the antigen of interest can be used. It has been shown for example that mice which have been made deficient of a particular endogenous protein by homologous recombination on the corresponding endogenous gene (i.e. knockout mice) generate a humoral response to the protein with which they have been immunized and can therefore be used to produce high-affinity monoclonal antibodies against the protein (see, for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237; Lunn, M. P. et al. (2000) J. Neurochem. 75:404-412).
Many non-human mammals are suitable as hosts for antibody production for producing non-human antibodies against RGM protein of the invention or a derivative/equivalent thereof. These include mice, rats, chickens, camelids, rabbits and goats (and knockout versions thereof), although mice are preferred for hybridoma production. It is further possible to use a non-human host animal which expresses a human antibody repertoire for producing substantially human antibodies against a human antigen having dual specificity. Such non-human animals include transgenic animals (e.g. mice) which harbor human immunoglobulin transgenes (chimeric hu-PBMC-SCID mice) and human/mouse radiation chimeras, which are described in detail below.
According to one embodiment, the animal which is immunized with an RGM protein of the invention or derivative/equivalent thereof is a non-human mammal, preferably a mouse which is transgenic owing to human immunoglobulin genes, so that the non-human mammal makes human antibodies after an antigenic stimulus. Typically, immunoglobulin transgenes for heavy and light chain with human germline configuration are introduced into such animals, the animals having been modified so that their endogenous loci for heavy and light chain are inactive. Stimulation of such animals with antigen (e.g. with a human antigen) leads to production of antibodies which are derived from the human immunoglobulin sequences (i.e. human antibodies). Human monoclonal antibodies can be made from the lymphocytes of such animals by means of standardized hybridoma technology. For further description of transgenic mice with human immunoglobulins and their use in the production of human antibodies, see, for example, U.S. Pat. No. 5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049 (Abgenix Inc.), and U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625, 126, 5,633,425, 5,661,016, 5,770,429, 5,814,318, 5,877,397 and WO 99/45962 (Genpharm Inc.); see likewise MacQuitty, J. J. and Kay, R. M. (1992) Science 257:1188; Taylor, L. D. et al. (1992) Nucleic Acids Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature 368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol. 13:65-93; Harding, F. A. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. M. et al. (1996) Nature Biotechnology 14:845-851; Mendez, M. J. et al. (1997) Nature Genetics 15:146-156; Green, L. L. and Jakobovits, A. (1998) J. Exp. Med. 188:483-495; Green, L. L. (1999) J. Immunol. Methods 231:11-23; Yang, X. D. et al. (1999) J. Leukoc. Biol. 66:401-410; Gallo, M. L. et al. (2000) Eur. J. Immunol. 30:534-540.
In a further embodiment, the animal which is immunized with RGM protein of the invention or a derivative/equivalent thereof can be a mouse with severe combined immunodeficiency (SCID) which has been reconstituted with human peripheral mononuclear blood cells or lymphoid cells or precursors thereof. Such mice, which are referred to as chimeric hu-PBMC-SCID mice, have been demonstrated to produce human immunoglobulin responses after an antigenic stimulus. For further description of these mice and their use for generating antibodies, see, for example, Leader, K. A. et al. (1992) Immunology 76:229-234; Bombil, F. et al. (1996) Immunobiol. 195:360-375; Murphy, W. J. et al. (1996) Semin. Immunol. 8:233-241; Herz, U. et al. (1997) Int. Arch. Allergy Immunol. 113:150-152; Albert, S. E. et al. (1997) J. Immunol. 159:1393-1403; Nguyen, H. et al. (1997) Microbiol. Immunol. 41:901-907; Arai, K. et al. (1998) J. Immunol. Methods 217:79-85; Yoshinari, K. and Arai, K. (1998) Hybridoma 17:41-45; Hutchins, W. A. et al. (1999) Hybridoma 18:121-129; Murphy, W. J. et al. (1999) Clin. Immunol. 90:22-27; Smithson, S. L. et al. (1999) Mol. Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect. Diseases 180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.
In a further embodiment, the animal which is immunized with RGM protein of the invention or a derivative/equivalent thereof is a mouse which has been treated with a lethal whole-body irradiation, subsequently protected against radiation with bone-marrow cells from mice with severe combined immunodeficiency (SCID), and subsequently transplanted with functional human lymphocytes. This type of chimera, referred to as the trimera system, is used to produce human monoclonal antibodies by immunizing the mice with the antigen of interest, and subsequently producing monoclonal antibodies using standardized hybridoma technology. For further description of these mice and their use for generating antibodies, see, for example, Eren, R. et al. (1998) Immunology 93:154-161; Reisner, Y. and Dagan, S. (1998) Trends Biotechnol. 16:242-246; Ilan, E. et al. (1999) Hepatology 29:553-562; and Bocher, W. O. et al. (1999) Immunology 96:634-641.
In vitro approaches:
As alternative to the production of antibodies of the invention by immunization and selection, it is possible to identify and isolate antibodies of the invention by screening a recombinant combinatorial immunoglobulin library with an RGM protein of the invention or derivative/equivalent thereof in order thus to isolate members of the immunoglobulin library which bind specifically to the RGM protein or derivative/equivalent thereof. Kits for generating and screening display libraries are commercially available (e.g. the Recombinant Phage Antibody System from Pharmacia, catalog No. 27-9400-01; and the SurfZAP® Phage Display Kit from Stratagene, catalog No. 240612). In many embodiments, the display library is an scFv library or an Fab library. The phage display technique for screening recombinant antibody libraries has been sufficiently well described. Examples of methods and compounds which can be used particularly advantageously in the generation and screening of antibody display libraries can be found for example in McCafferty et al. WO 92/01047, U.S. Pat. No. 5,969,108 and EP 589 877 (describes in particular the display of scFv), Ladner et al. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 5,837,500 and EP 436 597 (describes for example the pill fusion); Dower et al. WO 91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527 839 (describes in particular the display of Fab); Winter et al. International Publication WO 92/20791 and EP 368,684 (describes in particular the cloning of sequences for variable immunoglobulin domains); Griffiths et al. U.S. Pat. No. 5,885,793 and EP 589 877 (describes in particular the isolation of human antibodies against human antigens using recombinant libraries); Garrard et al. WO 92/09690 (describes in particular phage expression techniques); Knappik et al. WO 97/08320 (describes the human recombinant antibody library HuCal); Salfeld et al. WO 97/29131 (describes the production of a recombinant human antibody against a human antigen (human tumor necrosis factor alpha), and in vitro affinity maturation of the recombinant antibody) and Salfeld et al. U.S. Provisional Application No. 60/126,603 and the patent applications based thereon (likewise describes the production of recombinant human antibodies against human antigen (human interleukin-12), and the in vitro affinity maturation of the recombinant antibody).
Further descriptions of screenings of recombinant antibody libraries are to be found in scientific publications such as 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; Griffiths et al., (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson 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; Barbas et al. (1991) PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; and Knappik et al. (2000) J. Mol. Biol. 296:57-86.
As alternative to the use of bacteriophage display systems it is possible to express recombinant antibody libraries on the surface of yeast cells or bacterial cells. Methods for producing and screening libraries expressed on the surface of yeast cells are described in WO 99/36569. Methods for producing and screening libraries expressed on the surface of bacterial cells are described in detail in WO 98/49286.
As soon as an antibody of interest has been identified from a combinatorial library, the DNAs which encode the light and heavy chains of the antibody are isolated by standardized techniques of molecular biology, for example by PCR amplification of DNA from the display package (e.g. the phage) which has been isolated during the screening of the library. Nucleotide sequences of genes for light and heavy antibody chains which can be used to produce PCR primers are known to the skilled worker. Many such sequences are described for example in 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 and the database for sequences of the human germline VBASE.
An antibody or antibody part of the invention can be produced by recombinant expression of the genes for light and heavy immunoglobulin chains in a host cell. For recombinant expression of an antibody, a host cell is transfected with one or more recombinant expression vectors which harbor DNA fragments which encode the light and heavy immunoglobulin chains of the antibody, so that the light and heavy chains are expressed in the host cell and preferably secreted into the medium in which the host cells are cultivated. The antibodies can be obtained from this medium. Standardized recombinant DNA methods are used to obtain genes for heavy and light antibody chains, to insert these genes into recombinant expression vectors and to introduce the vectors into host cells. Methods of this type are described for example in Sambrook, Fritsch and Maniatis (editors), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (editors) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 of Boss et al.
As soon as DNA fragments which encode the VH and VL segments of the antibody of interest have been obtained, these DNA fragments can be further manipulated using standardized recombinant DNA techniques, for example in order to convert the genes for variable regions into genes for full-length antibody chains, into genes for Fab fragments or into an scFv gene. These manipulations lead to operative linkage of a VL- or VH-encoding DNA fragment to a further DNA fragment which encodes a further protein, e.g. a constant antibody region or a flexible linker. The term “operative linkage” is intended here to mean that the two DNA fragments are connected together in such a way that the amino acid sequences encoded by the two DNA fragments remain in the reading frame (in-frame).
The isolated DNA encoding the VH region can be, converted into a gene for a full-length heavy chain for operative linkage of the DNA encoding the VH region to a further DNA molecule encoding constant regions of the heavy chain (CH1, CH2 and CH3). The sequences of genes for constant regions of human heavy chains are sufficiently well known (see, for example, 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), and DNA fragments which span these regions can be obtained by standardized PCR amplification. The constant region of the heavy chain may be a constant region from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, with preference for a constant region from IgG1 or IgG4. A gene for an Fab fragment of the heavy chain can be obtained by operative linkage of the VH-encoding DNA with a further DNA molecule which encodes only the constant region CH1 of the heavy chain.
The isolated DNA encoding the VL region can be converted into a gene for a full-length light chain (and a gene for an Fab light chain) by operative linkage of the VL-encoding DNA with a further DNA molecule which encodes the constant region CL of the light chain. The sequences of genes of the constant region of human light chains are sufficiently well known (see 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), and DNA fragments which span these regions can be obtained by standardized PCR amplification. The constant region of the light chain may be a constant kappa or lambda region, with preference for a constant kappa region.
An scFv gene can be generated by operative linkage of the VH- and VL-encoding DNA fragments to a further fragment encoding a flexible linker, e.g. the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences are expressed as continuous single-chain protein, with the VL and VH regions being connected together by the flexible linker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).
VH and VL single domains with specificity for RGM protein of the invention or a derivative/equivalent thereof can be isolated from single-domain libraries using the methods described above. Two VH single-domain chains (with or without CH1) or two VL chains or a pair of a VH chain and a VL chain having the desired specificity can be used to bind RGM proteins of the invention or derivatives/equivalents thereof.
The recombinant antibodies or antibody parts of the invention can be expressed by inserting the DNAs which encode the partial or full-length light and heavy chains into expression vectors to result in operative linkage of the genes to transcriptional and translational control sequences. The term “operative linkage” in this connection is intended to mean that an antibody gene is ligated in a vector in such a way that transcriptional and translational control sequences within the vector fulfill their intended function of regulating the transcription and translation of the antibody gene.
The expression vector and the expression control sequences are chosen so that they are compatible with the host cell used for expression. The gene for the light antibody chain and the gene for the heavy antibody chain can be inserted into separate vectors, or both genes are inserted into the same expression vector, which is usually the case. The antibody genes are inserted into the expression vector by standardized methods (e.g. ligation of complementary restriction cleavage sites on the antibody gene fragment and vector, or ligation of blunt ends, if no restriction cleavage sites are present). The expression vector may already harbor sequences for constant antibody regions before the insertion of the sequences for the light and heavy chain. One approach for example is to convert the VH and VL sequences into full-length antibody genes by inserting them into expression vectors which already encode constant regions of heavy and light chains respectively, so that there is operative linkage of the VH segment to the CH segment(s) within the vector, and also operative linkage of the VL segment to the CL segment within the vector. An additional or alternative possibility is for the recombinant expression vector to encode a signal peptide which facilitates secretion of the antibody chain from the host cell. The gene for the antibody chain can be cloned into the vector in such a way that the signal peptide is linked in reading frame with the N terminus of the gene for the antibody chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein). In addition to the genes for the antibody chain, the expression vectors of the invention may have regulatory sequences which control expression of the genes for the antibody chain in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and further expression control elements (e.g. polyadenylation signals) which control the transcription or translation of the genes for the antibody chain. Such regulatory sequences are described for example in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). The skilled worker is aware that the design of the expression vector, which includes the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the desired strength of protein expression etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements which lead to strong protein expression in mammalian cells, such as promoters and/or enhancers, which are derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (e.g. the late adenovirus major promoter (AdMLP for Adenovirus Major Late Promoter) and polyoma. For further description of viral regulatory elements and sequences thereof, see, for example, U.S. Pat. No. 5,168,062 of Stinski, U.S. Pat. No. 4,510,245 of Bell et al. and U.S. Pat. No. 4,968,615 of Schaffner et al.
In addition to the genes for the antibody chain and the regulatory sequences, the recombinant expression vectors of the invention may have additional sequences such as sequences which regulate the replication of the vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker genes facilitate the selection of host cells in which the vector has been introduced (see, for example, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all of Axel et al.). For example, it is usual for the selectable marker gene to make a host cell into which the vector has been inserted resistant to active substances such as G418, hygromycin or methotrexate. Preferred selectable marker genes include the gene for dihydrofolate reductase (DHFR) (for use in dhfr− host cells with methotrexate selection/amplification) and the neogene (for G418 selection).
For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is or are transfected into a host cell using standardized techniques. The various forms of the term “transfection” are intended to encompass a large number of techniques which are normally used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, e.g. electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention either in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells and in particular in mammalian host cells is preferred, because the probability that a correctly folded and immunologically active antibody is assembled and secreted is higher in such eukaryotic cells and especially mammalian cells than in prokaryotic cells. It has been reported that prokaryotic expression of antibody genes is inefficient for the production of large yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
Mammalian host cells preferred for the expression of recombinant antibodies of the invention include CHO cells (including dhfr CHO cells, which are described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and are used with a DHFR selectable marker as described for example in R. J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. If recombinant expression vectors which encode the antibody genes are introduced into mammalian host cells, the antibodies are produced by cultivating the host cells until the antibody is expressed in the host cells or, preferably, the antibody is secreted into the culture medium in which the host cells grow. The antibodies can be obtained from the culture medium by using standardized methods for purifying proteins.
It is likewise possible to use host cells to produce parts of intact antibodies, such as Fab fragments or scFv molecules. The invention of course includes variations of the procedure described above. For example, it may be desirable to transfect a host cell with DNA which encodes either the light chain or the heavy chain (but not both) of an antibody of the invention. If light or heavy chains which are unnecessary for the binding of the antigen of interest are present, the DNA which encodes either one such light or one such heavy chain or both is partly or completely deleted by means of recombinant DNA technology. Molecules expressed by such truncated DNA molecules likewise belong to the antibodies of the invention. It is additionally possible to produce bifunctional antibodies in which one heavy and one light chain are an antibody of the invention, and the other heavy and light chain have specificity for an antigen other than that of interest, by crosslinking an antibody of the invention with a second antibody by standardized chemical methods.
In a preferred system for recombinant expression of an antibody of the invention or antigen-binding part thereof, a recombinant expression vector which encodes both the heavy antibody chain and the light antibody chain is introduced by calcium phosphate-mediated transfection into dhfr− CHO cells. There is operative linkage within the recombinant expression vector of the genes for the heavy and light antibody chain in each case to regulatory CMV enhancer/AdMLP promoter elements in order to bring about strong transcription of the genes. The recombinant expression vector also harbors a DHFR gene which can be used to select CHO cells transfected with the vector by using methotrexate selection/amplification. The selected transformed host cells are cultivated so that the heavy and light antibody chains are expressed, and intact antibody is obtained from the culture medium. Standardized techniques of molecular biology are used in order to produce the recombinant expression vector, to transfect the host cells, to select the transformants, to cultivate the host cells and to obtain the antibody from the culture medium. Thus, the invention relates to a method for synthesizing a recombinant antibody of the invention by cultivating a host cell of the invention in a suitable culture medium until a recombinant antibody of the invention is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.
As alternative to the screening of recombinant antibody libraries by phage display, it is possible to employ further methods known to the skilled worker for screening large combinatorial libraries in order to identify the antibodies of the invention. In one type of alternative expression system, the recombinant antibody library is expressed in the form of RNA-protein fusions as described in WO 98/31700 of Szostak and Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent fusion is generated by in vitro translation of synthetic mRNAs which carry at their 3′ end puromycin, a peptidyl acceptor antibiotic, between an mRNA and the peptide or protein which it encodes. It is thus possible to enrich a specific mRNA from a complex mixture of mRNAs (e.g. a combinatorial library) on the basis of the properties of the encoded peptide or protein (e.g. of the antibody or a part thereof), such as binding of the antibody or part thereof to RGM protein of the invention or a derivative/equivalent thereof. Nucleic acid sequences which encode antibodies or parts thereof and which are obtained from the screening of such libraries can be expressed by recombinant means in the manner described above (e.g. in mammalian host cells) and additionally be subjected to a further affinity maturation by either screening mRNA-peptide fusions in further rounds, in which case mutations are introduced into the originally selected sequence(s), or by using other methods for in vitro affinity maturation of recombinant antibodies in the manner described above.
Combinations of in vivo and in vitro approaches:
The antibodies of the invention can likewise be produced by applying a combination of in vivo and in vitro approaches, such as methods in which initially RGM protein of the invention or a derivative/equivalent thereof is allowed to act on an antibody repertoire in vivo in a host animal in order to stimulate the production of RGM protein- or derivative/equivalent-binding antibodies, and subsequently further antibody selection and/or antibody maturation (i.e. optimization) is accomplished with the aid of one or more in vitro techniques. According to one embodiment, such a combined method may comprise initially immunizing a non-human animal (e.g. a mouse, rat, rabbit, chicken, camelids, goat or a transgenic version thereof or a chimeric mouse) with the RGM protein of the invention or derivative/equivalent thereof in order to stimulate an antibody response against the antigen, and subsequently producing and screening a phage display antibody library using immunoglobulin sequences from lymphocytes which have been stimulated in vivo through the action of the RGM protein or derivative/equivalent in vivo. The first step of this combined procedure can be carried out in the manner described above in connection with in vivo approaches, whereas the second step of this procedure can be carried out in the manner described above in connection with in vitro approaches. Preferred methods for the hyperimmunization of non-human animals with subsequent in vitro screening of phage display libraries produced from the stimulated lymphocytes include those described by BioSite Inc., see, for example WO 98/47343, WO 91/17271, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.
According to a further embodiment, a combined method comprises initially immunizing a non-human animal (e.g. a mouse, rat, rabbit, chicken, camelids, goat or a knockout and/or transgenic version thereof, or a chimeric mouse) with an RGM protein of the invention or derivative/equivalent thereof in order to stimulate an antibody response against the RGM protein or derivative/equivalent thereof, and selecting the lymphocytes which produce the antibodies having the desired specificity by screening hybridomas (produced for example from the immunized animals). The genes for the antibodies or single-domain antibodies are isolated from the selected clones (by standardized cloning methods such as reverse transcriptase-polymerase chain reaction) and subjected to an in vitro affinity maturation in order thus to improve the binding properties of the selected antibody or of the selected antibodies. The first step of this procedure can be completed in the manner described above in connection with the in vivo approaches, whereas the second step of this procedure can be completed in the manner described above in connection with the in vitro approaches, especially by using methods of in vitro affinity maturation such as those described in WO 97/29131 and WO 00/56772.
In a further combined method, the recombinant antibodies are generated from single isolated lymphocytes by using a procedure which is known to the skilled worker as lymphocyte antibody selection method (SLAM) and is described in U.S. Pat. No. 5,627,052, WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, a non-human animal (e.g. a mouse, rat, rabbit, chicken, camelids, goat or a transgenic version thereof, or a chimeric mouse) is initially immunized in vivo with RGM protein of the invention or a derivative/equivalent thereof in order to stimulate an immune response against the RGM protein or derivative/equivalent, and then single cells secreting antibodies of interest are selected by using an antigen-specific hemolytic plaque assay. For this purpose, the RGM protein or derivative/equivalent thereof, or structurally related molecules of interest, can be coupled to sheep erythrocytes, using a linker such as biotin, whereby individual cells which secrete antibodies of suitable specificity can be identified by using the hemolytic plaque assay. Following the identification of cells which secrete antibodies of interest, cDNAs for the variable regions of the light and heavy chains are obtained from the cells by reverse transcriptase-PCR, and these variable regions can then be expressed in conjunction with suitable constant immunoglobulin regions (e.g. human constant regions) in mammalian host cells such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences derived from lymphocytes selected in vivo can then be subjected to a further in vitro analysis and selection by, for example, expanding the transfected cells in order to isolate cells which express antibodies having the desired specificity. The amplified immunoglobulin sequences can moreover be manipulated in vitro.
6. Pharmaceutical Compositions
6.1 General
The present invention also relates to pharmaceutical compositions which comprise as active substance a protein of the invention (RGM protein; RGM protein-binding ligands such as anti-RGM protein antibodies) or a coding RGM protein nucleic acid sequence and, if appropriate, a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention may additionally comprise at least one additional therapeutic agent, e.g. one or more additional therapeutic agents for the treatment of one of the disorders described herein.
The pharmaceutically acceptable carriers include all solvents, dispersion media, coatings, antimicrobial agents, tonicity agents and agents delaying absorption, and the like, as long as they are physiologically compatible.
Pharmaceutically acceptable carriers include for example water, saline solution, phosphate-buffered saline solution, lactose, dextrose, sucrose, sorbitol, manitol, starch, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup and methylcellulose. The formulations may additionally comprise pharmaceutically acceptable carriers or conventional excipients such as lubricants, for example talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; antioxidants; antiirritants; chelating agents; coating aids; emulsion stabilizers film formers; gel formers; odor-masking agents, masking flavors, resins; hydrocolloids; solvents; solubilizers; neutralizers; permeation promoters; pigments; quaternary ammonium compounds; refatting and superfatting agents; ointment, cream or oil bases; silicone derivatives; spreading aids; stabilizers; sterilizers; suppository bases; tablet excipients such as binders, fillers, lubricants, disintegrants or coatings; propellants; desiccants; opacifiers; thickeners; waxes; plasticizers; white oils. An arrangement concerning this is based on expert knowledge as set forth for example in Fiedler, H. P., Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzende Gebiete, 4th edition, Aulendorf: ECV-Editio-Cantor-Verlag, 1996. Compare also Hager's Handbuch der Pharmazeutischen Praxis, Springer Verlag, Heidelberg.
The pharmaceutical compositions may be suitable for example for parenteral administration. For this purpose, the active substance such as, for example, the antibody is preferably prepared as injectable solutions with an active substance content of 0.1-250 mg/ml. The injectable solutions can be prepared in liquid or lyophilized form in a flintglass or vial, an ampoule or a filled syringe as dosage form.
The buffer may comprise L-histidine (1-50 mM, preferably 5-10 mM) and have a pH of 5.0-7.0, preferably of 6.0. Further suitable buffers include, without being restricted thereto, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate buffers.
Sodium chloride can be used to adjust the tonicity of the solution to a concentration of 0-300 mM (preferably 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, such as, for example, sucrose (e.g. 0-10%, preferably 0.5-1.0% (w/w)). Further suitable cryoprotectants include trehalose and lactose. Fillers can be included for a lyophilized dosage form e.g. mannitol (e.g. 1-10%, preferably 2-4% (w/w)). Stabilizers can be used both in liquid and lyophilized dosage forms, e.g. L-methionine (e.g. 1-50 mM, preferably 5-10 mM). Further suitable fillers include glycine and arginine. It is likewise possible to use surfactants, for example polysorbate 80 (e.g. 0-0.05%, preferably 0.005-0.01% (w/w)). Further surfactants include polysorbate 20 and BRIJ surfactants.
The compositions of the invention may assume a large number of forms. These include liquid, semisolid and solid dosage forms such as liquid solutions (e.g. injectable and infusible solutions, lotions, eye drops and ear drops), liposomes, dispersions or suspensions and solid forms such as oral powders, dusting powders, granules, tablets, pastilles, sachets, cachets, coated tablets, capsules such as hard and soft gelatin capsules, suppositories or vaginal pharmaceutical forms, semisolid pharmaceutical forms such as ointments, creams, hydrogels, pastes or patches. Implanted delivery devices can also be used to administer active substances of the invention. The preferred form depends on the intended mode of administration and therapeutic use. Typically, compositions in the form of injectable or infusible solutions are preferred. A suitable route of administration is, for example, parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the active substance is administered by intravenous infusion or injection. According to a further preferred embodiment, the active substance is administered by intramuscular or subcutaneous injection.
Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solution, microemulsion, dispersion, liposomal or a further ordered structure which is suitable for high active substance concentrations. Sterile injectable solutions can be produced by introducing the active compound (such as, for example, the antibody) in the required amount into a suitable solvent, if appropriate with one or a combination of the aforementioned ingredients, as required, and subsequently sterilizing by filtration. Dispersions are ordinarily prepared by introducing the active compound into a sterile vehicle which comprises a basic dispersion medium and, if appropriate, further required ingredients. In the case of a sterilized lyophilized powder for preparing sterile injectable solutions, the preferred methods of manufacture are vacuum drying and spray drying, resulting in a powder of the active ingredient and, if appropriate, further desired ingredients from a solution which has previously been sterilized by filtration. The correct flowability of a solution can be maintained by for example using a coating such as lecithin, in the case of dispersions maintaining the required particle size, or using surfactants. Prolonged absorption of injectable compositions can be achieved by incorporating an agent which delays absorption, for example monostearate salts and gelatin, into the composition.
The active substances of the invention can be administered with a large number of methods which are known to the skilled worker, although subcutaneous injection, intravenous injection or infusion represents the preferred mode of administration for many therapeutic applications. The skilled worker is aware that the route and/or mode of administration depend on the desired result. According to certain embodiments, the active compound can be prepared with a carrier which protects the compound from rapid release, for example a formulation with controlled release, including implants, transdermal patches and microencapsulated delivery systems. It is possible to use biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. The methods for preparing such formulations are generally known to the skilled worker, see, for example, Sustained und Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
According to certain embodiments, an active substance of the invention can be administered orally, for example in an inert diluent or an assimilable edible carrier. The active substance (and further ingredients if desired) can also be enclosed in a hard or soft gelatin capsule, compressed to tablets or added directly to food. For oral therapeutic administration, the active substances can be mixed with excipients and used in the form of swallowable tablets, buccal tablets, capsules, elixirs, suspensions, syrups and the like. If an active ingredients of the invention are to be administered by a route other than parenteral, it may be necessary to choose a coating of a material which prevents its inactivation.
The active substances of the invention can be administered together with one or more additional therapeutic agents which can be used in the treatment of the disorders described above.
The pharmaceutical compositions of the present invention ordinarily comprise a therapeutically effective amount or a prophylactically effective amount of at least one active substance of the invention. Dosage regimens can be chosen and adapted depending on the desired treatment, whether for example a therapeutic or prophylactic treatment is desired. For example a single dose, a plurality of separate doses distributed over time or a rising or decreasing dosage depending on the requirements of the therapeutic situation can be administered. It is particularly advantageous to formulate parenteral compositions in unit dosage form in order to facilitate administration and ensure a uniformity of dosage.
The treating physician is able to determine without difficulty the dosage form, mode of administration and dosage most suitable for the particular therapy and the particular active substance.
A therapeutically or prophylactically effective amount of an active substance of the invention may be for example in the range of 0.1-20 mg/kg and preferably 1-10 mg/kg, without being restricted thereto. It is, of course, possible for these amounts to vary depending on the nature and severity of the condition to be alleviated.
6.2 Vaccines
The RGM proteins of the invention and derivatives/equivalents thereof can be used as immunogen for vaccination of a patient to be treated.
Vaccines which can be used for this purpose generally represent a pharmaceutical composition which comprises at least one RGM protein of the invention and/or at least one derivative/equivalent of the invention thereof. The composition may additionally comprise a physiologically tolerated carrier and, if appropriate, further excipients, for example immunostimulants.
Whereas suitable carriers can in principle be chosen as desired, the nature of the carrier is generally governed by the route of administration. Thus, the vaccines of the invention can be formulated in particular in a form suitable for parenteral, for example intravenous, intramuscular and subcutaneous, administration. In these cases, the carrier preferably comprises water, saline solution, alcohol, a fat, a wax and/or a buffer.
It is possible to use any one of a large number of immunostimulants in the vaccines of the invention. For example, an adjuvant can be included. Most adjuvants comprise a substance which is intended to protect the antigen from rapid degradation, such as aluminum hydroxide or a mineral oil, and a protein derived from lipid A, Bordetella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are usually commercially available, for example complete or incomplete Freund's adjuvant; AS-2; aluminum salts such as aluminum hydroxide (if appropriate as gel) or aluminum phosphate; calcium, iron or zinc salts; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl-lipid A. Cytokines such as GM-CSF or interleukin-2, -7 or -12 can likewise be used as adjuvants.
7. Therapeutic Methods
7.1. Treatment of Neuronal Disorders
It has been disclosed in the prior art that an accumulation of RGM protein is to be observed at the site of the lesion in cases of injuries to the central nervous system (cf. Schwab et al. loc. cit.). At the same time, renewed outgrowth of the injured nerve fibers is prevented thereby. This harmful effect on nerve fiber growth is mediated by binding of RGM to the receptor molecule neogenin. Modulation, in particular inhibition, of the interaction between RGM and the receptor molecule neogenin would therefore be suitable to suppress the inhibitory activity of RGM on nerve fiber growth.
7.2. Treatment of Neoplastic Diseases
There have long been indications suggesting that neogenin might be causally connected with the development and/or progression of neoplastic diseases. Thus, for example, Meyerhardt et al. reported in Oncogene (1997) 14, 1129-1136, that neogenin was detectable in more than 50 investigated cancer cell lines, including glioblastoma, medulloblastoma, neuroblastoma cell lines, and cell lines from colorectal, breast, pancreatic and cervical carcinomas. Overexpression of neogenin was additionally observed in esophageal cancer cell lines (Hue et al., Clinical Cancer Research (2001) 7, 2213-2221). A systematic investigation of the expression profiles of 3588 genes in 211 lung adenocarcinoma patients has recently provided a further indication of the involvement of neogenin in the development and progression of the neoplastic disease (Berrar et al., J. Comput. Biol. (2005) 12 (5), 534-544).
Since it is additionally known that RGM exhibits a potential tumor-promoting effect in that it can prevent cell death through binding to the tumor cell-associated neogenin receptor (Matsunaga et al. Nature Cell Biol. 6, 749-755, 2004), a new therapeutic approach to the treatment of neoplastic diseases might be created by modulating the RGM-neogenin interaction, in particular by interrupting this interaction with the aid of specific anti-RGM antibodies.
7.3. Treatment of Iron Metabolism Disorders
RGM C, also called hemojuvelin, is of essential importance for iron metabolism in the human and animal body. Juvenile hemochromatosis is a hereditary, relatively rare iron metabolism disorder which is manifested by an iron overload in the body. This disorder is caused by mutations in the hemojuvelin molecule (cf. Huang et al., The Journal of Clinical Investigation (2005), 115, 2087-2091). The administration of functional RGM proteins of the invention or the active domains thereof therefore represents a useful therapeutic approach to alleviating such iron metabolism disorders.
7.4. Promotion of Bone Tissue Formation
There are indications in the prior art that one member of the RGM family of proteins, specifically RGM B, also known under the designation DRAGON, is involved in bone morphogenesis. Thus, for example, Samad et al. describe in JBC Papers in Press, edition of Jan. 25, 2005, the interaction between DRAGON and the type I and type II receptors of bone morphogenetic protein (BMP). A bone growth-promoting effect and thus a new therapeutic approach to the treatment of disorders with impaired bone growth or of bone injuries is therefore conceivable by administering RGM polypeptides of the invention.
8. Diagnostic Methods
Diagnostic agents which should be mentioned according to the invention are in particular RGM protein and derivatives/equivalents as defined above, and antibodies directed against them.
The present invention therefore makes it possible in particular to determine, with qualitative or quantitative improvement, the pathological states defined above by detecting disease-typical antigens or antibodies.
The determination preferably takes place using immunological methods. This is possible in principle with any analytical or diagnostic test method in which antibodies are employed. These include agglutinations and precipitation techniques, immunoassays, immunohistochemical methods and immunoblotting techniques, e.g. Western blotting or dot-blot methods. Also included are in vivo methods, for example imaging methods.
Use in immunoassays is advantageous. Those suitable are both competitive immunoassays, i.e. antigen and labeled antigen (tracer) compete for the antibody binding, and sandwich immunoassays, i.e. the binding of specific antibodies to the antigen is detected using a second, usually labeled antibody. These assays may be either homogeneous, i.e. without separation into solid and liquid phase, or heterogeneous, i.e. bound labels are separated from unbound ones, for example by solid phase-bound antibodies. The various heterogeneous and homogeneous immunoassay formats can be assigned to particular classes depending on the labeling and method of measurement, for example RIAs (radioimmunoassays), ELISA (enzyme linked immunosorbent assay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay), TRFIA (time-resolved FIA), IMAC (immunactivation), EMIT (enzyme multiplied immune test), TIA (turbidimetric immunoassay), I-PCR (immuno-PCR).
Competitive immunoassays are preferred for the antigen determination of the invention. In this case, labeled antigen (tracer) competes with the antigen of the sample to be quantified for binding to the antibody used. The amount of antigen, that is the amount of antigen, in the sample can be determined from the amount of displaced tracer with the aid of a standard curve.
Of the labels available for these purposes, enzymes have proved to be advantageous. For example, it is possible to use systems based on peroxidases, especially horseradish peroxidase, alkaline phosphatase and β-D-galactosidase. Specific substrates are available for these enzymes, and their conversion can be followed for example by photometry. Suitable substrate systems are based on p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NPT), fast red/naphthol-AS-TS phosphate for alkaline phosphatase; 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 3-dimethylaminobenzoic acid (DMAB) and 3-methyl-2-benzothiazolinone hydrazone (MBTH) for peroxidases; o-nitrophenyl β-D-galactoside (o-NPG), p-nitrophenyl β-D-galactoside and 4-methylumbellifer β-D-galactoside (MUG) for p-D-galactosidase. These substrate systems are in many cases commercially available in a form ready for use, for example in the form of tablets, which may also comprise further reagents such as expedient buffers and the like.
The coupling of labels to peptides or antibodies to prepare tracers can take place in a manner known per se. In addition, a number of labels expediently modified for conjugation to proteins are available, for example biotin-, avidin-, extravidin- or streptavidin-conjugated enzymes, maleimide-activated enzymes and the like. These labels can be reacted directly with the molecule to be used according to the invention.
If a heterogeneous immunoassay format is chosen, the antigen-antibody complex can, for the purpose of separation, be bound to the support for example via an anti-idiotype antibody, e.g. an antibody directed against rabbit IgG, coupled to the support. Supports, especially microtiter plates, which are coated with appropriate antibodies are known and commercially available.
The present invention further relates to immunoassay sets with at least one antibody described above and further components. This comprises a collection, usually as pack unit, of means for carrying out a determination of the invention. To maximize the simplicity of use, these means are preferably provided substantially ready for use. An advantageous arrangement is provided by the immunoassay in kit form. A kit usually comprises a plurality of containers for separate arrangement of components. All the components can be provided in dilution ready for use, as concentrate for dilution or as dry substance or lyophilizate for dissolving or suspending; single components or all the components may be frozen or be stored at ambient temperature until used. Sera are preferably shock-frozen, for example at −20° C., so that in these cases an immunoassay must be kept preferably at freezing temperatures before use.
Further components added to the immunoassay may be: standard protein, tracer; control serum, microtiter plates, preferably coated with antibody, buffer, for example for testing, for washing or for reacting the substrate, and the enzyme substrate itself.
General principles of immunoassays and the generation and use of antibodies as laboratory and clinical aids are to be found for example in Antibodies, A Laboratory Manual (Harlow, E., and Lane, D., Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).
9. Screening Methods
The invention also relates to methods for detecting effectors of the RGM receptor neogenin, where a sample in which an effector is suspected is incubated with an RGM protein or polypeptide, and the mixture is investigated for the formation of an effector-RGM protein complex.
Such effectors may have an agonistic, partial agonistic, antagonistic or inverse agonistic effect. They may be for example synthetic low molecular weight substances, synthetic peptides, natural or synthetic antibody molecules or natural substances.
Such methods of the invention are usually carried out as in vitro screening methods with which it is possible to select from a large number of different substances those which appear to be most promising in relation to future use.
For example, extensive substance libraries comprising a large number of potential active substances can be set up by means of combinatorial chemistry. Screening of combinatorial substance libraries for substances having a desired activity can be automated. Robot screening devices are used for efficient evaluation of the individual assays arranged preferably on microtiter plates. Thus, the present invention also relates to screening methods, i.e. both primary and secondary screening methods, in which preferably at least one of the methods described below is used. If a plurality of methods is used, this is possible sequentially or simultaneously on one and the same sample or on different samples of a substance to be investigated.
An efficient technique for carrying out such methods is the Scintillation Proximity Assay, abbreviated to SPA, which is known in the area of active substance screening. Kits and components for carrying out this assay can be purchased commercially, for example from Amersham Pharmacia Biotech. In principle, solubilized or membrane-bound receptors are immobilized on small fluoromicrospheres comprising scintillant. If, for example, a radioligand binds to the immobilized receptors, the scintillant is excited to emit light because the scintillant and radioligand are in spatial proximity.
A further efficient technique for carrying out such methods is the FlashPlateR technique known in the area of active substance screening. Kits and components for carrying out this assay can be purchased commercially, for example from NEN Life Science Products. This principle is likewise based on microtiter plates (96- or 384-well) which are coated with scintillant.
The present invention likewise relates to the substances or parts of substance mixtures which can be identified by these methods.
The invention is now explained in more detail with reference to the following nonlimiting preparation and use examples
Experimental Section
1. General Information
Assay Method 1: Demonstration of the Effect of RGM Peptides in Neuronal Outgrowth Tests with Rat Cortical Neurons
The effect of RGM peptides in vitro was investigated by carrying out neuronal outgrowth tests. For this purpose, rat cortical neurons were prepared using the following media:
Stock medium per liter: 90 ml GB5, 100 ml Minimum Essential Medium-Eagle (10×; Gibco, order No.: 21430.020), ad 1000 ml with Millipore water. pH 7.0, 280-310 mosm.
GB5 per liter: 26.6 g NaHCO3, 44.4 g glucose, sterilized by filtration.
Plating medium (PM) per liter: 0.8 mM glutamine, 100 ml of heat-inactivated fetal calf serum (FCS), 100 ml of heat-inactivated horse serum, ad 1000 ml with stock medium
Maintenance medium (MM) per liter: 0.8 mM glutamine, 100 ml of heat-inactivated horse serum, ad 1000 ml with stock medium
Trypsin solution: 0.1% trypsin, 0.04% EDTA in PBS without calcium/magnesium, sterilized by filtration.
Cortical neurons were obtained by sacrificing pregnant rats by cervical dislocation, opening the abdominal cavity thereof, removing the uterus with the embryos (embryonic day 18 (E18)) and washing in PBS. Microdissection scissors were used to cut open the uterus longitudinally, and the embryos were removed and exposed. The embryos were sacrificed by throat-cutting, the brain was removed and the forebrain cortex was dissected. The cortices are incubated each in 1 ml of a 0.1% strength trypsin solution at 37° C. for 5 minutes, the reaction is stopped with maintenance medium (MM) and, after incubation in a total of 10 ml of MM for 5 minutes, triturated with fire-polished Pasteur pipettes with decreasing orifice diameter. The cell suspension was centrifuged at 1200 rpm for 10 minutes, and the supernatant was aspirated off and discarded. The cell pellet was taken up in 8 ml of MM and cautiously resuspended. The cells were counted in a Neubauer counting chamber, and the number of cells was adjusted to about 400 000 cells/0.2 ml in MM and preaggregated. For this purpose, 200 μl of the cell suspension per well were put on glass chamber slides (LabTek, order No. 177402) and incubated at 37° C. for 24 h. Aggregates of neurons form during this. 10-80 μl of these aggregates were plated out in each well of a poly-D-lysine-coated 96-well plate (e.g. Becton Dickinson Biocoat 96-well black plate #354640) and made up where appropriate with MM to 70-90 μl. After 1 h, the RGM peptides were added in various concentrations in a volume of 10 μl and made up to 100 μl where appropriate with MM. 24-48 h later, the axonal outgrowths were initially assessed by examination under a light microscope, and the cultures were then fixed by adding 100 μl of 4% paraformaldehyde solution and stored at 4° C. for at least 12 hours. All the steps for preparing the tubulin cytoskeleton by immunofluorescent staining, with the exception of the incubation with the primary antibody, took place at room temperature. The wells were washed once with 100-300 μl of PBS for 5-15 minutes and then the cells were permeabilized by incubating in 100 μl of 0.1% Triton X-100 for 10-20 min. The wells were washed twice with 300 μl of PBS for 5-15 minutes and then incubated with 100 μl of 1% bovine serum albumin solution in PBS for 60 minutes. The primary antibody (e.g. Sigma, monoclonal anti-β-tubulin isotype III clone SDL 3D10, #T8660; Abcam, TuJI #ab14545) was diluted 1:1000 in 1% bovine serum albumin solution in PBS and incubated 50 μl per well at 4° C. overnight. The wells were washed three times with 100-300 μl of PBS for 5-15 minutes. The fluorescence-labeled secondary antibody (Jackson ImmunoResearch, Cy3 conjugated Affinity Pure Donkey Anti Mouse #715-165-151) was diluted 1:500 in 1% bovine serum albumin solution in PBS, which additionally comprised 0.5 μg/ml bisbenzimide (H33258) to visualize the cell nuclei, and 50 μl of this dilution were incubated per well at room temperature for 1-2 h or at 4° C. overnight. The wells were washed twice with 100-300 μl of PBS for 5-15 minutes, the PBS was aspirated off, and one drop (approx. 50 μl) of Fluoromount G (Southern Biotechnology Associates Inc#010001) were added to each well. Images were recorded in an inverse fluorescence microscope (Zeiss, Axiovert 200 M), detecting in each case the fluorescence of the labeled secondary antibody and the bisbenzimide fluorescence. The area covered by the two fluorescences was ascertained with the aid of an image analysis program (Media Cybernetics, Image-Pro Plus). To ascertain the axon growth index (AG-I), the difference in area between secondary antibody stain (staining of the outgrown axons and of the cell aggregates) and the bisbenzimide stain (staining of the cell aggregates) was formed and divided by the area of the bisbenzimide stain. This index therefore represents a measure of the area covered by axons in relation to the size of the cell aggregates.
Assay Method 2: Demonstration of the Effect of RGM Peptides in Neuronal Outgrowth Tests with Cortical Human Neurons
The human pluripotent carcinoma cell line Ntera (DSMZ ACC527) is an established cell culture model. In this case, axons grow out of cell aggregates and form a corona of axons around the respective aggregate.
For this purpose, 2.5×106 Ntera cells were seeded in a 175 cm2 bottle and differentiated in 10 μM retinoic acid (Sigma) (medium: D-MEM (Gibco/Invitrogen 31966-021), 10% fetal calf serum, 100 u/ml penicillin, 100 μg/ml streptomycin (both Gibco/Invitrogen)) for 3 weeks. Differentiated cells were then divided 1:6 in new bottles and cultured without retinoic acid for a further 2 days. Neuronal cells adhering to the resulting cell lawn were detached by capping and aggregated by slow shaking in Neurobasal medium (Neurobasal Medium (Gibco/Invitrogen 21103-049), 2 mM L-glutamine (Gibco/Invitrogen 25030-024), 100 u/ml penicillin, 100 μg/ml streptomycin (both Gibco/Invitrogen) in a shaken flask overnight (in an incubator).
The next day, Ntera aggregates were seeded in poly-D-lysine and laminin (Sigma) (10 μg/ml) 96-well plates (Biocoat Poly-D-Lysine Cellware 96-Well Black/Clear Plate (Becton Dickinson #35 6640). The inhibitory effect of RGM peptides and fragments was analyzed by adding various concentrations of the substances to be tested.
Assay Method 3: RGM A—Neogenin Binding Assay:
a) Materials:


    
    
        Immuno plate: Cert. Maxi Sorp F96 (NUNC, 439454)
        Recombinant human RGM A, R&D Systems; Prod.#2495-RM (260 μg/ml)
        Recombinant human neogenin Fc, Abbott; Ludwigshafen (ALU 1514/122; 425 μg/ml)
        Peroxidase-conjugated, affinity-purified mouse anti-human IgG Fc fragment Ab (Jackson Immuno Research, Code: 209-035-098 (0.8 mg/ml))
        Developer substrates: immuno Pure TMB Substrate Kit (Pierce, #34021)
        Sulfuric acid (Merck, #4.80354.1000)

b) Method:

1. RGM A binding to immuno plate:

        2.5 μg/ml RGM A (R&D) in 50 mM Na2CO3 (50 μl/well)
        Incubation at 37° C. for 1 h

2. Washing step:

        Wash 3× with PBS/0.02% Tween 20 (100 μl/well)

3. Blocking of nonspecific binding sites

        Blocking with 3% BSA in PBS/0.02% Tween (200 μl/well)
        Incubation at 37° C. for 1 h

4. Neogenin binding:

        Addition of neogenin in dilutions (initial conc. 1 μg/ml) in 1% BSA PBS/0.02% Tween
        Incubation at 37° C. for 1 h

5. Washing step:

        Wash 3× with PBS/0.02% Tween 20 (100 μl/well)

6. Antibody detection of the bound neogenin:

        Addition of HRP-coupled mouse anti-human IgG Fc fragment Ab (diluted 1:2500 in PBS/1% BSA) (50 μl/well)
        Incubation at 37° C. for 1 h

7. Washing step:

        Wash 3× with PBS/0.02% Tween 20 (100 μl/well)

8. Development

        Addition of 50 μl/well of the developing substrate (Immuno Pure TMB substrate, Pierce)
        Incubation, room temperature, 1-30 min
        Stop reaction with 50 μl of 2.5M H2SO4/well
    
    


2. Preparation Examples
Preparation Example 1
Preparation of RGM a Protein Fragments in Mammalian Cells
To characterize the active RGM A domain in axon growth and neogenin binding assay methods, RGM A (AA 168-422) and RGM A fragments 70-80 AA in size (Frag. 1: 169-238; Frag. 2: 218-284; Frag. 3: 266-335; Frag. 4: 316-386; Frag. 5: 369-238; and Frag. 6: (168-422)) was expressed in mammalian cells (HEK293) as AP fusion proteins.
For this purpose, the DNA coding for the respective fragment was cloned into the vector pDEST AP/ccdb/Myc/His (Invitrogen, Gateway Vektor System) (AP—alkaline phosphatase; PPC—PreScisson protease). The DNA coding for the respective fragment region was for this purpose amplified by PCR from the RZPD clone (clone AL136826 (DKFZp434D0727); published RZPD sequence: BC015886, AL136826)). The following oligonucleotides were employed to do this:








Fragment 169-238:









(SEQ ID NO: 11)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGGCCCCCACA



CCTCAGGACTTTCACCGAC








(SEQ ID NO: 12)









GGGGACCACTTTGTACAAGAAAGCTGGGTGCTCGTCCATCTCAGCCTGGTACACC



 


Fragment 218-284:








(SEQ ID NO: 13)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGGCCCATCATC



TTCAAGAACTTCCAGGAGTGTG








(SEQ ID NO: 14)









GGGGACCACTTTGTACAAGAAAGCTGGGTGGCGCACCACGATGGTGGTG



 


Fragment 316-386








(SEQ ID NO: 15)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGG



CCCTCAGGCCAGCACGTGGAGATCC








(SEQ ID NO: 16)









GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCAGCATTGGTGTGGAAGGCC



 


Fragment 266-335








(SEQ ID NO: 17)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGGCCCCTGCG



GGGCTGCCCCCTC








(SEQ ID NO: 18)









GGGGACCACTTTGTACAAGAAAGCTGGGTCGCCCGTGGTGAGGAGGTCG



 


Fragment 368-422








(SEQ ID NO: 19)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGGCCC



CTGCCGGTGGAGGACCTGTAC








(SEQ ID NO: 20)









GGGGACCACTTTGTACAAGAAAGCTGGGTTGCCTGGCAGGTCCCGAGTC



 


Fragment 169-422








(SEQ ID NO: 21)









GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTGGAAGTTCTGTTCCAGGGGCCC



CCACACCTCAGGACTTTCACCGAC








(SEQ ID NO: 22)









GGGGACCACTTTGTACAAGAAAGCTGGGTTGCCTGGCAGGTCCCGAGTC







The resulting PCR products was cloned by specific recombination (attL×attR) into the vector pDEST AP/ccdb/Myc/His, and the correct sequence was checked by sequencing.
HEK293 cells (ATCC CRL 1573) were transfected with the plasmids. To do this, cells were seeded at a density of 80% in 15 cm plates on the day before the transfection. The next day, DNA was mixed with Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's protocol, and the cells were incubated therewith for 12 h. After culturing under selective culturing conditions (D-MEM (Gibco/Invitrogen 31966-021), 10% fetal calf serum, 100 u/ml penicillin, 100 μg/ml streptomycin (both Gibco/Invitrogen), 150 μg/ml zeocin/invitrogen) for a further four weeks, stably expressing clones were selected by detection of alkaline phosphatase in the growth medium.
For protein production, stably expressing cells were amplified in growth medium and, when a confluence of 70% was reached, transferred into production medium (Pro293 medium (BioWhittaker/Cambrex, 12-764Q), 2 mM glutamine (Invitrogen)). After culturing for a further 4 days, the production medium was harvested, separated from cell debris and concentrated by membrane concentrators (Vivaspin30).
The expressed AP-RGM fusion proteins were then purified from the supernatants by Ni-chelate affinity chromatography. For this purpose, Ni-NTA-agarose (Qiagen) was equilibrated in buffer A (50 mM HEPES-KOH, 100 mM NaCl, 10% glycerol, 10 mM imidazole). The concentrated supernatant was then incubated with the Ni-NTA-agarose with rotation at 4° C. for 1 h. Washing three times of the Ni-NTA-agarose which was removed by centrifugation was followed by elution of bound protein with elution buffer (buffer A with 250 mM imidazole).
Analysis of the proteins took place with the aid of dot-blot, SDS-PAGE and Western blot analyses. The concentration was determined by Bradford protein determination and ELISA measurement of the AP concentration.
3. Exemplary Embodiments
Exemplary Embodiment 1
Fragment Analysis with Neogenin-Expressing HEK293 Cells
a) General:
It was previously known that the active, growth-inhibiting activity of the RGM A protein from chickens is located in the region between amino acid position 150 and 350.
Fragments of the human RGM A protein which cover this range were therefore prepared. HEK293 cells which carry the RGM receptor endogenously on their surface were then transfected with these fragments in order to prepare clonal cell cultures which produce the individual protein fragments and release them into the culture medium. All the fragments were produced as fusion proteins with attached alkaline phosphatase enzyme. The presence of the active domain in these cells should make itself noticeable by: less cellular proliferation, increased cell death and/or altered cell-substrate adhesion.
Increased cell death and less proliferation have, of course, a direct effect on the produced amount of the active fragment. The amount of the produced fragments was assessed with the aid of a semiquantitative dot-blot assay.
b) Procedure:
The RGM A fragments prepared as in preparation example 1 were introduced into HEK293 cells. Stably transfected cells were cultured to confluence in culture medium (D-MEM (Gibco/Invitrogen 31966-021), 10% fetal calf serum, 100 u/ml penicillin, 100 μg/ml streptomycin (both Gibco/Invitrogen), 150 μg/ml zeocin (Invitrogen). Subsequently, 1 ml portions of the culture supernatant were filtered through a nitrocellulose membrane (Sartorius) in a slot-blot chamber. AP-RGM fusion proteins immobilized on the membrane were detected by detecting the alkaline phosphatase activity by incubating the nitrocellulose membrane with NBT/BCIP substrate (Roche). The produced amount of the active fragment was determined with the aid of a semiquantitative dot-blot assay.
On the basis of this assay, the stable cell clones were divided into the following categories:

    
    
        strongly producing cell clones (S producer)
        moderately strongly producing clones
        weakly producing clones
        non-producing clones (0 producer).
    
    


In addition, the ratio of strongly to non-producing clones was determined.
c) Result:
The following values were found for the individual RGM A fragments assayed:












Fragment
Ratio
Proportion of


(amino acid
“S producer” versus
strongly producing


positions)
“0 producer”
cell clones [%]

















1 (169-238)
2.3
40


2 (218-284)
0.5
17


3 (266-335)
0.16
6.2


4 (316-386)
0.5
16


5 (369-422)
0.63
16


6 (168-422)
0.16
9









Fragments 3 and 6 in particular showed a pronounced activity in this assay.
Exemplary Embodiment 2
Investigation of Synthetic RGM a Peptides in the Nerve Fiber Growth Assay
For this purpose, the following RGM A peptides were synthesized and assayed in various concentrations in the nerve fiber growth assay described above (assay method 1 and assay method 2):
Peptide 1: AA 267-285
Peptide 2: AA 308-325
Peptide 3: AA 358-377
Peptide 4: AA 378-400
(Numbering based on SEQ ID NO: 2)
Of the peptides assayed to date, peptide 1 reliably and reproducibly showed inhibitory activity. It can therefore be assumed that peptide 1 (AA 267-285) forms part of the active domain of the RGM protein.
The test results are depicted in FIGS. 3 A, B, C and 4 A, B.
FIG. 3A shows that, in contrast to peptide 4, peptide 1 significantly inhibits nerve fiber growth in a concentration of 10 μg/ml in tests with rat neuronal cells (assay method 1). FIG. 3B illustrates the effect of different concentrations (0 to 30 μg/ml) of peptide 1 on nerve fiber growth of cortical neurons. A significant inhibitory activity is detectable at a concentration of more than 3 μg/ml and above. In contrast thereto, peptide 4 shows no activity under the same conditions (cf. FIG. 3C) (AG-I=axon growth index, corresponds to the total area of the neuronal aggregate with the relevant axons minus the area of the aggregate.
FIG. 4A shows that peptide 1, but not peptide 4, significantly inhibits nerve fiber growth of human Ntera cells in a concentration of 30 μg/ml (assay method 2). FIG. 4B illustrates the statistical significance of the observed inhibitory effect of peptide 1.
Exemplary Embodiment 3
Investigation of Synthetic RGM a Fragments in the Nerve Fiber Growth Assay
The six different RGM A fragments prepared as in preparation example 1 and listed again in the following were assayed for inhibitory activity in the axon growth assay (cf. above assay method 2) with human Ntera nerve cells.
Fragment 1: amino acids 169-238
Fragment 2: amino acids 218-284
Fragment 3: amino acids 266-335
Fragment 4: amino acids 316-386
Fragment 5: amino acids 369-422
Fragment 6: amino acids 168-422
(Numbering in each case based on SEQ ID NO: 2)
The results are summarized in appended FIGS. 5A, B and C.
In total, 6 different RGM A fragments were assayed in the NTera outgrowth assay. Fragments 2, 3 and 6 were active, while fragments 1, 4 and 5 were inactive.
Fragment 6 corresponds to the active, completely processed RGM A protein as has also been described in vivo. It follows from these data that an important nerve fiber growth-inhibitory domain of the human RGM A protein is located in the region of fragments 2 and 3, i.e. between amino acids 218 and 335. In order to characterize this inhibitory domain more accurately, therefore, short RGM A peptides from the regions of fragments 2 and 3 are assayed in a next step.
Exemplary Embodiment 4
Investigation of Synthetic RGM a Peptides in the Nerve Fiber Growth Assay
The peptides shown in the following table were assayed in the axon growth assay (cf. above assay method 2).








TABLE





Neighboring RGM A peptides from peptide 1 were



synthesized and employed in the axon growth assay

















Peptide 1 (267-285)
GQHVEIQAKYIGTTIVVRQ (SEQ ID NO: 8)



 


Peptide Down-1 (260-275)
KITEKVSGQHVEIQAK (SEQ ID NO: 26)


 


Peptide Up-1 (276-291)
YIGTTIVVRQVGRYLT (SEQ ID NO: 27)


 


Peptide 5-Ak (242-259)
AFVDGSKNGGDKHGANSL (SEQ ID



NO: 30)


 


Peptide 6-Ak (300-316)
VVNAVEDWDSQGLYLC (SEQ ID NO: 28)


 


Peptide 7-Ak (217-234)
TIIFKNFQECVDQKVYQA (SEQ ID



NO: 29)





Peptide 1 has already been assayed in exemplary embodiment 2.






The test results are depicted in FIGS. 6A and B.
Three of the assayed RGM A peptides led to a large reduction in nerve fiber growth in the NTera growth assay (FIG. 6A). These active RGM A peptides were the following peptides: peptide 1, peptide down-1 and peptide up-1. Inactive or only weakly active were the following peptides: peptide 5-Ak, peptide 6-Ak and peptide 7-Ak (FIG. 6B). This suggests that one of the inhibitory domains of RGM A is to be located in the region which is fixed by the 3 active RGM A peptides, i.e. the region of the human RGM A protein which extends approximately from 250 to 300, in particular from about 260-291, and thus includes the core region from position 267 to 285 (cf. SEQ ID NO: 7, 8). On the basis of the activity of the two peptides 6-Ak and 7-Ak, which showed a tendency to inhibit growth at the higher peptide concentration (FIG. 6B), it is perfectly conceivable that RGM A has further inhibitory domains and therefore the invention likewise relates to the domains in the region of about 210-260 and about 290-350.
Exemplary Embodiment 5
Neutralization of the Inhibitory Domain of RGM a with Antibodies
The intention in this experiment was to check whether polyclonal antibodies raised against active peptides of the inhibitory domain of RGM A are able to block the interaction of RGM A with its receptor neogenin and to neutralize in vitro the nerve fiber growth inhibition of the active and most potent RGM A fragment 2 (cf. exemplary embodiment 2).
Peptide down-1 was therefore used by way of example, because it forms part of the active RGM A fragment 2. Coupled to a carrier protein (LPH), this peptide was used to immunize two rabbits according to the following scheme



















Amount










of

First


Peptide
Peptide
Animal
immunization
Boost
Boost
Boost
Bleeding
Titer







Down-1
2.4 mg
8640
Day 0
Day 7
Day 14
Day 28
Day 35
1:150000


800790

8641





1:30000
























Last boost
Exsanguination
Purification









Day 56
20 ml
24.9 mg in 22.5 ml




20 ml
24.1 mg in 22 ml










After several immunizations, the raised down-1-specific antibodies were purified and employed in various assay systems.
a) RGM A—neogenin binding assay:
The procedure took place as in assay method 3; the test results are depicted in FIG. 7A.
b) Neutralization of the active RGM A fragment in the axon growth assay:
The polyclonal antibodies raised against the peptide down-1 very efficiently blocked the interaction of the RGM A protein with its receptor neogenin. In the next assay, therefore, it was investigated whether the down-1-specific antibody is able to neutralize the very potent RGM A fragment 2 (cf. exemplary embodiment 3).
The procedure took place as in assay method 2; the test results are depicted in FIG. 7B.
It is to be stated in summary that the polyclonal antibodies directed against the peptide down-1 prevent the interaction of neogenin and RGM A in the RGM Aneogenin binding assay, and almost completely neutralize the inhibitory activity of the RGM A fragment in the axon growth assay. The region between the amino-terminal amino acids 250 and 300, especially 260 and 291, of the RGM A protein is therefore particularly important for its inhibitory activity and is hereby described as novel functionally relevant domain.

## Claims

1. An isolated neogenin receptor-binding domain derived from a human RGM A as shown in SEQ ID NO:2, comprising an amino acid sequence from amino acid position 200 to 350 as shown in SEQ ID NO: 2, or from amino acid position 200 to 325 as shown in SEQ ID NO: 2, or from amino acid position 200 to 300 as shown in SEQ ID NO: 2, or from amino acid position 200 to 285 as shown in SEQ ID NO: 2, or from amino acid position 250 to 285 as shown in SEQ ID NO: 2, or from amino acid position 260 to 285 as shown in SEQ ID NO: 2, or from amino acid position 215 to 340 as shown in SEQ ID NO: 2, or from amino acid position 260 to 340 as shown in SEQ ID NO: 2, or from amino acid position 250 to 300 as shown in SEQ ID NO: 2, or from amino acid position 260 to 291 as shown in SEQ ID NO: 2, or from amino acid position 210 to 260 as shown in SEQ ID NO: 2, or from amino acid position 290 to 350 as shown in SEQ ID NO: 2, or from amino acid position 218 to 284 as shown in SEQ ID NO: 2, or from amino acid position 266 to 335 as shown in SEQ ID NO: 2, or from amino acid position 168 to 422 as shown in SEQ ID NO: 2, or SEQ ID NO: 8, or SEQ ID NO: 26, or SEQ ID NO: 27, or a functional, neogenin receptor-binding fragment thereof.

2. An antigenic polypeptide fragment of the neogenin receptor-binding domain as claimed in claim 1.

3. The antigenic polypeptide fragment as claimed in claim 2, which comprises at least 10 consecutive amino acid residues of a peptide having one of the sequences as shown in SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27. amino acids 218-284 of SEQ ID NO: 2, amino acids 266-335 of SEQ ID NO: 2, or amino acids 168-422 of SEQ ID NO: 2.

4. A composition comprising a neogenin receptor-binding domain as claimed in claim 1 or an antigenic polypeptide fragment as claimed in claim 2, and a pharmaceutically acceptable carrier.

5. The composition as claimed in claim 4, formulated for intrathecal, intravenous, subcutaneous, parenteral, nasal or inhalational administration.

6. An isolated neogenin receptor-binding domain or binding fragment of SEQ ID NO: 2, which comprises at least 10 consecutive amino acid residues from the sequence region from position 260 to 291 or 267 to 285 as shown in SEQ ID NO: 2.