Title of Invention

ANTIBODY (“11C7”) ANTI NOGO A AND ITS PHARMACEUTICAL USE

Abstract Abstract This invention relates to molecules, such as for example monoclonal antibodies or Fab fragments thereof, which are capable of binding to the human NogoA polypeptide or human NiG or human NiG- or human NogoA-323-640 with a dissociation constant < 1000nM; polynucleotides encoding such a binding molecule; an expression vector comprising such polynucleotides; the use of such a binding molecule in the treatment of nerve repair; a pharmaceutical composition comprising such a binding molecule; and to a method of treatment of diseases associated with nerve repair.
Full Text

ANTIBODY ("11C7') ANTINOGO AND 1ST PHARMACEUTICAL USE
This invention relates to NogoA binding molecules, such as for example monoclonal antibodies or Fab fragments thereof.
Neuronal regeneration following injury in the adult central nervous system (CNS) is limited due to the presence of the inhibitory myelin environment that ensheaths axons and formation of scar tissue. In the last few years important insights have been gained into the molecular understanding why the CNS is unable to spontaneously repair itself following injury. Inhibitory molecules in the myelin are the major impediment for the axonal regeneration, particularly immediately after the injury. So far NogoA, Myelin-Associated Glycoprotein (MAG) and myelin-oligodendrocyte glycoprotein (OMgp) have been characterised as potent inhibitors of neurite outgrowth. In addition, myelin also contains other inhibitory components, such as, chondroitin sulphate proteoglycans. Nogo-A is a member of the reticulon protein family and it has at least two biologically active and pharmacologically distinct domains termed Amino-Nogo and Nogo-66. While the receptor site for the former is not known so far, Nogo-66 inhibits neuronal growth in vitro and in vivo via the neuronal receptor NgR. In addition to Nogo-66, MAG and OMgp also bind to the NgR with high affinity and inhibit neurite outgrowth.
Potential new research approaches currently pursued for enhancement of nerve repair include digestion of scar tissue using an enzyme chondroitinase ABC, bridging techniques using Olfactory ensheathing cells and stem cells and protein growth factors to boost neuronal growth. Blocking actions of neurite outgrowth inhibitors by modulation of intracellular signalling mediators such as Rho , a membrane-bound guanosine trisphosphatase (GTPase), which appears to be a key link in the inhibition of axonal growth. Cyclic adenosine monophosphate (cAMP) which can overcome myelin associated inhibition in vitro and induce regeneration in vivo. Use of peptide inhibitor of the NgR receptor (NEP 1-40) to induce neuronal regrowth and functional recovery in rats following spinal injury.
In addition to the use of the approaches described above, attention has also focused upon the use of certain monoclonal antibodies to neutralize neurite growth inhibitory molecules of the central and peripheral nervous system, in particular to neutralize the neurite growth inhibitory activity of NogoA. Thus it has been shown that the monoclonal antibody IN-1 or the

IN-1 Fab fragment thereof induce neurite outgrowth in vitro and enhance sprouting and regeneration in vivo (Schwab ME et al. (1996) Physiol. Rev. 76, 319-370). Testing different domains of the NogoA for neurite growth inhibitory acitvity have delineated several inhibitory domains in the molecule (Chen et al. 2000) Nature 403, 434-439; GrandPre eta I. (2000) Nature 403, 439-444; Prinjha et ai. (2000) Nature 403, 383-384; see also detailed analysis in Example 1).
Natural immunoglobulins or antibodies comprise a generally Y- shaped multimeric molecule having an antigen-binding site at the end of each upper arm. The remainder of the structure, in particular the stem of the Y mediates effector functions associated with the immunoglobulins. Antibodies consists of a 2 heavy and 2 light chains. Both heavy and light chains comprise a variable domain and a constant part. An antigen binding site consists of the variable domain of a heavy chain associated with the variable domain of a light chain. The variable domains of the heavy and light chains have the same general structure. More particularly, the antigen binding characteristics of an antibody are essentially determined by 3 specific regions in the variable domain of the heavy and light chains which are called hypervariable regions or complementarity determining regions (CDRs). These 3 hypervariable regions alternate with 4 framework regions (FRs) whose sequences are relatively conserved and which are not directly involved in binding. The CDRs form loops and are held in close proximity by the framework regions which largely adopt a 3-sheet conformation. The CDRs of a heavy chain together with the CDRs of the associated light chain essentially constitute the antigen binding site of the antibody molecule. The determination as to what constitutes an FR or a CDR region is usually made by comparing the amino acid sequence of a number of antibodies raised in the same species. The general rules for identifying the CDR and FR regions are general knowledge of a man skilled in the art and can for example be found in the webside (http://www.bioinf.orq.uk/abs/).
It has now surprisingly been found that a novel monoclonal mouse antibody (hereinafter called "11C7") raised against a polypeptide fragment of rat NogoA (SEQ ID NO; 1) and of the lgG1 type has better properties than the NogoA antibodies of the prior art especially with regard to the binding affinity to NogoA of different species including tire homo sapiens and with regard to its higher NogoA neurite outgrowth neutralizing activity at a given antibody concentration. Moreover it is now possible to construct other NogoA binding molecules having the same hypervariable regions as the said antibody.

Accordingly, the invention provides binding molecules to a particular region or epitope of NogoA (hereinafter referred to as "the Binding Molecules of the invention" or simply "Binding Molecules"). Preferably the Binding Molecules of the invention bind to human NogoA_623-640 (orthologous fragment against which 11C7 was raised; = SEQ ID NO: 6), human Nig-D20 (orthologous to the .smallest fragment of NogoA with neurite outgrowth inhibitory activity, SEQ ID NO: 24), human NogoA (SEQ ID NO: 5) or human NiG (which is the most potent neurite outgrowth inhibitory fragment of NogoA and starts at amino acid No. 186 and ends at amino acid No. 1004 of human NogoA, = SEQ ID NO: 5) with a dissociation constant (Kd) Thus, in a further preferred embodiment the Binding Molecules (at a concentration of 1 mg/ml, more preferably at 0.1 mg/ml even more preferably at 0.01 mg/ml culture medium) enhance the number of neurites of rat cerebellar granule cells on a substrate of rat spinal cord protein extract by at least 20%, preferably 50%, most preferred 100% compared to the number of neurites of rat cerebellar granule cells which are treated with a control antibody that does not bind to the human NogoA, human NiG, human Nig-D20 or NogoA_623-640 polypeptide (i.e. that has a dissociation constant > 1000 nM).
In a further preferred embodiment the Binding Molecules of the invention comprises at least one antigen binding site, said antigen binding site comprising in sequence, the hypervariable regions CDR1 -11C7, CDR2-11C7 and CDR3-11C7; said CDR1 -11C7 having the amino acid sequence SEQ ID NO: 8, said CDR2-11C7 having the amino acid sequence SEQ ID NO: 9, and said CDR3-11C7 having the amino acid sequence SEQ ID NO: 10; and direct equivalents thereof.
In a further aspect of the invention, the Binding Molecule of the invention comprises at least one antigen binding site, said antigen binding site comprising either

a) in sequence the hypervariable regions CDR1-11C7, CDR2-11C7 and CDR3-11C7; said CDR1 -11C7 having the amino acid sequence of SEQ ID NO: 8, said CDR2-11C7 having the amino acid sequence of SEQ ID NO: 9, and said CDR3-11C7 having the amino acid sequence SEQ ID NO: 10; or
b) in sequence the hypervariable regions CDRV-11C7, CDR2'-11C7 and CDR3'-11C7, said CDR1'-11C7 having the amino acid sequence of SEQ ID NO: 11, said CDR2'-11C7 having the amino acid sequence of SEQ ID NO: 12, and said CDR3'-11C7 having the amino acid sequence of SEQ ID NO: 13; or
c) direct equivalents thereof.
In a further aspect of the invention, the Binding Molecule of the invention comprises at least
a) a first domain comprising in sequence the hypervariable regions CDR1 -11C7, CDR2-11C7 and CDR3-11C7; said CDR1 -1107 having the amino acid sequence of SEQ ID NO: 8, said CDR2-11C7 having the amino acid sequence of SEQ ID NO: 9, and said CDR3-11C7 having the amino acid sequence SEQ ID NO: 10; and
b) a second domain comprising in sequence the hypervariable regions CDR1'-11C7, CDR2I-11C7 and CDR3'-11C7, said CDR1'-1107 having the amino acid sequence of SEQ ID NO: 11, said CDR2'-11C7 having the amino acid sequence of SEQ ID NO: 12, and said CDR3'-11C7 having the amino acid sequence of SEQ ID NO: 13; or
c) direct equivalents thereof.
Moreover, the invention also provides the following Binding Molecule of the invention, which comprises at least one antigen binding site comprising
a) either the variable part of the heavy chain of 1107 (SEQ ID NO: 2); or
b) the variable part of the light chain of 1107 (SEQ ID NO: 3), or direct equivalents thereof.
When the antigen binding site comprises both the first and second domains, these may be located on the same polypeptide molecule or, preferably, each domain may be on a different chain, the first domain being part of an immunoglobulin heavy chain or fragment thereof and
the second domain being part of an immunoglobulin light chain or fragment thereof.
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Examples of Binding Molecules of the invention include antibodies as produced by B-cells or hybridomas and chimeric or humanized antibodies or any fragment thereof, e.g. F(ab')2; and Fab fragments, as well as single chain or single domain antibodies.

A single chain antibody consists of the variable domains of an antibody heavy and light chains covalently bound by a peptide linker usually consisting of from 10 to 30 amino acids, preferably from 15 to 25 amino acids. Therefore, such a structure does not include the constant part of the heavy and light chains and it is believed that the small peptide spacer should be less antigenic than a whole constant part. By "chimeric antibody" is meant an * antibody in which the constant regions of heavy or light chains or both are of human origin while the variable domains of both heavy and light chains are of non- human (e.g. murine) origin. By "humanized antibody" is meant an antibody in which the hypervariable regions (CDRs) are of non-human (e.g. murine) origin, while all or substantially all the other parts of the immunoglobulin e.g. the constant regions and the highly conserved parts of the variable domains, i.e. the framework regions, are of human origin. A .humanized antibody may however retain a few amino acids of the murine sequence in the parts of the framework regions adjacent to the hypervariable regions.
Hypervariable regions may be associated with any kind of framework regions, preferably of murine or human origin. Suitable framework regions are described in "Sequences of proteins of immunological interest", Kabat E.A. et alt US department of health and human services, Public health service, National Institute of Health. Preferably the constant part of a human heavy chain of the Binding Molecules may be of the lgG4 type, including subtypes, preferably the constant part of a human light chain may be of the K or X type, more preferably of the K type.
Monoclonal antibodies raised against a protein naturally found in all humans may be developed in a non-human system e. g. in mice. As a direct consequence of this, a xenogenic antibody as produced by a hybridoma, when administered to humans, elicits an undesirable immune response, which is predominantly mediated by the constant part of the xenogenic immunoglobulin. This clearly limits the use of such antibodies as they cannot be administered over a prolonged period of time. Therefore it is particularly preferred to use single chain, single domain, chimeric or humanized antibodies which are not likely to elicit a substantial allogenic response when administered to humans.
In view of the foregoing, a more preferred Binding Molecule of the invention is selected from a chimeric antibody, which comprises at least

a) one immunoglobulin heavy chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions CDR1 -11C7, CDR2-11C7 and CDR3-11C7 and (ii) the constant part or fragment thereof of a human heavy chain; said CDR1 -11C7 having the amino acid sequence (SEQ ID NO: 8), said CDR2-11C7 having the amino acid sequence (SEQ ID NO: 9), and said CDR3-11C7 having the amino acid sequence (SEQ ID NO: 10), and
b) one immunoglobulin light chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions CDR1'-11C7, CDR2'-11C7 and CDR3'-11C7 and (ii) the constant part or fragment thereof of a human light chain; said CDR1 '-11C7 having the amino acid sequence (SEQ ID NO: 11), said CDR2'-11C7 having the amino acid sequence (SEQ ID NO:. 12), and said CDR3'-11C7 having the amino acid sequence (SEQ ID NO: 13); or
direct equivalents thereof.
Alternatively, a Binding Molecule of the invention may be selected from a single chain binding molecule which comprises an antigen binding site comprising
a) a first domain comprising in sequence the hypervariable CDR1 -11C7, CDR2-11C7 and CDR3-11C7; said CDR1 -11C7 having the amino acid sequence (SEQ ID NO: 8), said CDR2-11C7 having the amino acid sequence (SEQ ID NO: 9), and said CDR3-11C7 having the amino acid sequence (SEQ ID NO: 10); and
b) a second domain comprising in sequence the hypervariable CDR1 '-11C7, CDR2'-11C7 and CDR3'-11C7; said CDR1 '-11C7 having the amino acid sequence (SEQ ID NO: 11), said CDR2'-11C7 having the amino acid sequence (SEQ ID NO: 12), and said CDR3'-
11C7 having the amino acid sequence (SEQ ID NO: 13); and
c) a peptide linker which is bound either to the N- terminal extremity of the first domain and
to the C-terminal extremity of the second domain or to the C-terminal extremity of the
first domain and to the N-terminal extremity of second domain;
or direct equivalents thereof.
As it is well known, minor changes in an amino acid sequence such as deletion, addition or substitution of one or several amino acids may lead"to*an allelic form of the original protein which has substantially identical properties. Thus, by the term "direct equivalents thereof" is meant either any single domain Binding Molecule of the invention (molecule X)

(i) in which each of the hypervariable regions CDR1, CDR2, and CDR3 of the Binding Molecule is at least 50 or 80% homologous, preferably at least 90% homologous, more preferably at least 95, 96, 97, 98, 99% homologous to the equivalent hypervariable regions of CDR1 -11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10), whereas CDR1 is equivalent to CDR1-11C7, . CDR2 is equivalent to CDR2-11C7, CDR3 is equivalent to CDR3-11C7; and
(ii) which is capable of binding to the human NogoA, human NiG, human NiG-D20, or
human NogoA_623-640, preferably with a dissociation constant (Kd) any binding molecule of the invention having at least two domains per binding site (molecule
X')
(iii) in which each of the hypervariable regions CDR1, CDR2, CDR3, CDR1', CDR2' and CDR3' is at least 50 or 80% homologous, preferably at least 90% homologous, more preferably at least 95, 96, 97, 98, 99% identical to the equivalent hypervariable regions of CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9), CDR3-11C7 (SEQ ID NO: 10), CDR1'-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12), and CDR3'-11C7 (SEQ ID NO: 13), whereas CDRIjs equivalent to CDR1-11C7, CDR2 is equivalent to CDR2-11C7, CDR3 is equivalent to CDR3-11C7, CDR1' is equivalent to CDR1'-11C7, CDR2' is equivalent to CDR2'-11C7, CDR3' is equivalent to CDR3'-11C7;and
(iv) which is capable of binding the human NogoA, human NiG, human NiG-D20, or
human NogoA_623-640, preferably with a dissociation constant (Kd) Thus further embodiments of the inventions are for example a Binding Molecule which is capable of binding to the human NogoA, human NiG, human NiG-D20, or human NogoA_623-640 with a dissociation constant • in sequence the hypervariable regions CDR1, CDR2, and CDR3, of which each of the hypervariable regions are at least 50%, preferably 80, 90, 95, 96, 97, 98, 99% homologous to their equivalent hypervariable regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); or
• in sequence the hypervariable regions CDR1\ CDR2\ and CDR3\ of which each of the hypervariable regions are at least 50%, preferably 80, 90, 95, 96, 97, 98, 99%

homologous to their equivalent hypervariable regions CDRV-11C7 (SEQ !D NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13).
Furthermore, a Binding Molecule which is capable of binding the human NogoA, human NiG, human NiG-D20, or human NogoA_623-640 with a dissociation constant • a first antigen binding site comprising in sequence the hypervariable regions CDR1, CDR2, and CDR3, of which each of the hypervariable regions are at least 50%, preferably 80, 90, 95, 96, 97, 98, 99% homologous to their equivalent hypervariable regions CDR1 -11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); and
• a second antigen binding site comprising in sequence the hypervariable regioos . CDR1\ CDR2', and CDR3\ of which each of the hypervariable regions are at least 50%, preferably 80, 90, 95, 96, 97, 98, 99% homologous to their equivalent hypervariable regions CDRV-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12)
. andCDR3'-HC7(SEQIDNO:13).
This dissociation constant may be conveniently tested in various assays including, for example, the biosensor affinity method described in the example 7. In addition, the binding and functional effect of the Binding Molecules may be shown in a bioassay, e.g. as described below.
The constant part of a human heavy chain may be of the y1; Y2; y3; Y4; a1; a2; 5 or £ type, preferably of the y type, more preferably of the y4; type, whereas the constant part of a human light chain may be of the K or A type (which includes the A1; A2; and A3 subtypes) but is preferably of the K type. The amino acid sequence of all these constant parts are given in Kabat et al (Supra).
Conjugates of the binding molecules of the invention, e. g. enzyme or toxin or radioisotope conjugates, are also included within the scope of the invention.
"Polypeptide", if not otherwise specified herein, includes any peptide or protein comprising amino acids joined to each other by peptide bonds, having an amino acid sequence starting at the N-terminal extremity and ending at the C-terminal extremity. Preferably the

polypeptide of the present invention is a monoclonal antibody, more preferred is a chimeric (also called V-grafted) or humanised (also called CDR-grafted) monoclonal antibody. The humanised (CDR-grafted) monoclonal antibody may or may not include further mutations introduced into the framework (FR) sequences of the acceptor antibody.
A functional derivative of a polypeptide as used herein includes a molecule having a qualitative biological activity in common with a polypeptide to the present invention, i.e. having the ability to bind to the human NogoA, human NiG, human NiG-D20, or human NogoA_623-640. A functional derivative includes fragments and peptide analogs of a poipypeptide according to the present invention. Fragments comprise regions within the sequence of a polypeptide according to the present invention, e.g. of a specified sequence. The term "derivative" is used to define amino acid sequence variants, and covaient modifications of a polypeptide according to the present invention, e.g. of a specified sequence. The functional derivatives of a polypeptide according to the present invention, e.g. of a specified sequence,, e.g. of the hypervariable region of the light and the heavy chain, preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95, 96, 97, 98, 99% overall sequence homology with the amino acid sequence of a polypeptide according to the present invention, e.g. of a specified sequence, and substantially retain the ability to bind the human NogoA, human NiG, human NiG-D20, or human NogoA_623-640.
The term "covaient modification" includes modifications of a polypeptide according to the present invention, e.g. of a specified sequence; or a fragment thereof with an organic proteinaceous or non-proteinaceous derivatizing agent, fusions to heterologous polypeptide sequences, and post-translational modifications. Covaient modified polypeptides, e.g. of a specified sequence, still have the ability bind to the human NogoA, human NiG, human NiG-D20, or human NogoA_623-640 by crosslinking. Covaient modifications are traditionally introduced by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected sides or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deaminated under mildly acidic conditions. Other post-translational

modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, tyrosine or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side.chains, see e.g. T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983). Covalent modifications e.g. include fusion proteins comprising a polypeptide according to the present invention, e.g. of a specified sequence and their amino acid sequence variants, such as immunoadhesins, and N-terminal fusions to heterologous signal sequences.
"Homology" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known.
"Amino acid(s)" refer to all naturally occurring L-ct-amino acids, e.g. and including D-amino acids. The amino acids are identified by either the well known single-letter or three-letter designations.
The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences as compared to a polypeptide according to the present invention, e.g. of a specified sequence. Amino acid sequence variants of a polypeptide according to the present invention, e.g. of a specified sequence, still have the ability to bind to human NogoA or human NiG or more preferably to NogoA_623-640. Substitutional variants are those that have at least one amino acid residue removed and a different amino acid inserted in its place at the same position in a polypeptide according to the present invention, e.g. of a specified sequence. These substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Insertional variants are those with one or more amino acids inserted immediately adjacent fo an amino acid at a particular position in a polypeptide according to the present invention, e.g. of a specified sequence. Immediately adjacent to an amino acid means connected to either the a-carboxy or a-amino functional group of the amino acid. Deletional variants are those with one or more amino acids in a polypeptide

according to the present invention, e.g. of a specified sequence, removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the molecule.
A binding molecule of the invention may be produced by recombinant DNA techniques. In view of this, one or more DNA molecules encoding the binding molecule must be constructed, placed under appropriate control sequences and transferred into a suitable host organism for expression.
In a very general manner, there are accordingly provided
(i) DNA molecules encoding a single domain Binding Molecule of the invention, a single
chain Binding Molecule of the invention, a heavy or light chain or fragments thereof of
a Binding Molecule of the invention; and (ii) the use of the DNA molecules of the invention for the production of a Binding
Molecule of the invention by recombinant means.
The present state of the art is such that the skilled man will be able to synthesize the DNA molecules of the invention given the information provided herein i.e. the amino acid sequences of the hypervariable regions and the DNA sequences coding for them. A method for constructing a variable domain gene is for example described in EP 239 400 and may be briefly summarized as follows: A gene encoding a variable domain of a monoclonal antibody of whatever specificity is cloned. The DNA segments encoding the framework and hypervariable regions are determined and the DNA segments encoding the hypervariable regions are removed so that the DNA segments encoding the framework regions are fused together with suitable restriction sites at the junctions. The restriction sites may be generated at the appropriate positions by mutagenesis of the DNA molecule by standard procedures. Double stranded synthetic CDR cassettes are prepared by DNA synthesis according to the sequences given CDR1-11C7, CDR2-HC7, CDR3-11C7, CDRV-11C7, CDR2'-11C7 and CDR3'-11C7 above. These cassettes are provided with sticky ends so that they can be ligated at the junctions to the framework by standard protocol for achieving a DNA molecule encoding an immunoglobulin variable domain.
Furthermore, it is not necessary to have access to the mRNA from a producing hybridoma cell line in order to obtain a DNA construct coding for the monoclonal antibodies of the

invention. Thus PCT application WO 90/07861 gives full instructions for the production of a monoclonal antibody by recombinant DNA techniques given only written information as to the nucleotide sequence of the gene.
The method comprises the synthesis of a number of oligonucleotides, their amplification by the PCR method, and their splicing to give the desired DNA sequence.
Expression vectors comprising a suitable promoter or genes encoding heavy and light chain constant parts are publicly available. Thus, once a DNA molecule of the invention is prepared it may be conveniently transferred in an appropriate expression vector.
DNA molecules encoding single chain antibodies may also be prepared by standard methods, for example, as described in WO 88/1649.
In a particular embodiment of the invention, the recombinant means for the production of some of the Binding Molecules of the invention includes first and second DNA constructs as described below:
The first DNA construct encodes a heavy chain or fragment thereof and comprises
a) a first part which encodes a variable domain comprising alternatively framework and
hypervariable regions, said hypervariable regions comprising in sequence DNA-CDR1-
11C7 (SEQ ID NO: 15), DNA-CDR2-11C7 (SEQ ID NO: 16) and DNA-CDR3-11C7 (SEQ ID NO: 17); this first part starting with a codon encoding the first amino acid of the variable domain and ending with a codon encoding the last amino acid of the variable domain, and
b) a second part encoding a heavy chain constant part or fragment thereof which starts with
a codon encoding the first amino acid of the constant part of the heavy chain and ends
with a codon encoding the last amino acid of the constant part or fragment thereof,
followed by a non-sense codon.
Preferably, the second part encodes the constant part of a human heavy chain, more preferably the constant part of the human y4 chain. This second part may be a DNA fragment of genomic origin (comprising introns) or a cDNA fragment (without introns).

The second DNA construct encodes a light chain or fragment thereof and comprises
a) a first part which encodes a variable domain comprising alternatively framework and hypervariable regions; said hypervariable regions comprising in sequence DNA-CDR1'-11C7 (SEQ ID NO: 17), DNA-CDR2'-11C7 (SEQ ID NO: 18) and DNA-CDR3'-11C7 (SEQ ID NO: 19), this first part starting with a codon encoding the first amino acid of the variable domain and ending with a codon encoding the last amino acid of the variable domain, and
b) a second part encoding a light chain constant part or fragment thereof which starts with a codon encoding the first amino acid of the constant part of the light chain and ends with
a codon encoding the last amino acid of the constant part or fragment thereof followed by a non-sense codon.
Preferably, the second part encodes the constant part of a human light chain, more preferably the constant part of the human K chain.
The first or second DNA construct advantageously comprises a third part which is located upstream of the first part and which encodes part of a leader peptide; this third part starting with the codon encoding the first amino acid and ending with the last amino acid of the leader peptide. This peptide is required for secretion of the chains by the host organism in which they are expressed and is subsequently removed by the host organism. Preferably, the third part of the first DNA construct encodes a leader peptide having an amino acid sequence substantially identical to the amino acid sequence of the heavy chain leader sequence as shown in SEQ ID NO: 21 (starting with the amino acid at position -19 and ending with the amino acid at position -1). Also preferably, the third part of the second DNA construct encodes a leader peptide having an amino acid sequence as shown in SEQ ID NO: 23 (light chain, starting with the amino acid at position -18 and ending with the amino acid at position -1).
Each of the DNA constructs are placed under the control of suitable control sequences, in particular under the control of a suitable promoter. Any kind of promoter may be used, provided that it is adapted to the host organism in which the DNA constructs will be transferred for expression. However, if expression is to take place in a mammalian cell, it is particularly preferred to use the promoter of an immunoglobulin gene.

The desired antibody may be produced in a cell culture or in a transgenic animal. A suitable transgenic animal may be obtained according to standard methods which include micro injecting into eggs the first and second DNA constructs placed under suitable control sequences transferring the so prepared eggs into appropriate pseudo- pregnant females and selecting a descendant expressing the desired antibody.
When the antibody chains have to be produced in a cell culture, the DNA constructs must first be inserted into either a single expression vector or into two separate but compatible expression vectors, the latter possibility being preferred.
Accordingly, the invention also provides an expression vector able to replicate in a prokaryotic or eukaryotic cell line which comprises at least one of the DNA constructs above described.
Each expression vector containing a DNA construct is then transferred into a suitable host organism. When the DNA constructs are separately inserted on two expression vectors, they may be transferred separately, i.e. one type of vector per cell, or co- transferred, this latter possibility being preferred. A suitable host organism may be a bacterium, a yeast or a mammalian cell line, this latter being preferred. More preferably, the mammalian cell line is of lymphoid origin e.g. a myeloma, hybridoma or a normal immortalized B-cell, but does not express any endogeneous antibody heavy or light chain.
It is also preferred that the host organism contains a large number of copies of the vectors per cell, if the host organism is a mammalian cell line, this desirable goal may be reached by amplifying the number of copies according to standard methods. Amplification methods usually consist of selecting for increased resistance to a drug, said resistance being encoded by the expression vector.
In another aspect of the invention, there is provided a process for producing a multi-chain binding molecule of the invention, which comprises (i) culturing an organism which is transformed with the first and second DNA constructs of the invention and (ii) recovering an active binding molecule of the invention from the culture.

Alternatively, the heavy and light chains may be separately recovered and reconstituted into an active binding molecule after in vitro refolding. Reconstitution methods are well-known in the art; Examples of methods are in particular provided in EP 120 674 or in EP 125 023. Therefore a process may also comprise (i) culturing a first organism which is transformed with a first DNA construct of the
invention and recovering said heavy chain or fragment thereof from the culture and (ii) culturing a second organism which is transformed with a second DNA construct of
the invention and recovering said light chain or fragment thereof from the culture and (iii) reconstituting in vitro an active binding molecule of the invention from the heavy
chain or fragment thereof obtained in (i) and the light chain or fragment thereof
obtained in (ii).
In a similar manner, there is also provided a process for producing a single chain or single
domain binding molecule of the invention which comprises
(i) culturing an organism which is transformed with a DNA construct respectively
encoding a single chain or single domain binding molecule of the invention and (ii) recovering said molecule from the culture.
The binding molecules of the invention exhibit very good nerve repair activity as shown, for example, in the granule cell neurite outgrowth model.
1. Granule cell neurite outgrowth assay (in vitro)
Neurite outgrowth from dissociated cerebellar granule cells are determined as described (Niederost et al. (1999) J.Neurosci. 19: 8979-8989). Briefly, cerebella are removed from decapitated postnatal day 5-7 rats and dissociated by trypsin treatment. To reduce fibroblast contamination, the cells are preplated onto bacterial dishes. 75*000 cells are then cultured per well in 4-well Greiner tissue culture (Huber & Co AG,Rheinach, Basel) dishes (well surface: 1 cm2) in medium (Neurobasal with B27 serum replacement, Invitrogen). Culture dishes are coated with poly-L-lysine (Sigma). Chaps extracted proteins from total spinal cord homogenates of adult rats (Spilimann et al. (1998) J. Biol. Chem. 273: 19283-19293) is coated at protein concentrations of 0.5 till 8 \ig per well over night at 4°C and washed. The binding molecules of the invention are then pre-incubated for 30 min on the test substrate and removed before the cells are added. Cerebellar granule cells are added and incubated for 24 hours. To stop the experiment, 2 ml of 4 % buffered formaldehyde is

slowly added to the culture dishes. Cultures are then stained by immunofluorescence for the growth-associated protein GAP-43 and with Hoechst for cell nuclei (Granule cells are stained with Hoechst in order to see if all the cells have neurites (neurite visualised with anti-GAP-43)). Three pictures are taken randomly at a defined distance of the upper, lower and lateral edge of each well with a 40x objectif on a Zeiss Axiophot Fluorescence Microscope. All the neurites in a field are counted on number-coded, randomly arranged photographs*. The response (outgrowth of the granule cell neurites) is dose-dependent in the range of about 0.1 - 10 \xg total protein per well (the specific activities of a given preparation vary within this range).
Enhancement of neurite outgrowth of cerebellar granule cell in the non-permissive environment of the above prepared spinal cord extract by preincubation with a binding molecule of the invention may be observed. E.g. a typical profile for the neutralizing effect of the mouse 11C7-lgG1 antibody in the granule cell neurite outgrowth model is given below: Assay 1:

The neutralizing activity of the molecules of the invention may also be estimated by measuring the regenerative sprouting and neurite outgrowth in the in vivo spinal cord injury model as follows:
2. Spinal cord injury model {in vivo)
Adult Lewis rats are injured microsurgical^ by transecting the dorsal half of the spinal cord
bilaterally at the level of the 8th thoracic vertebra. Laminectomy, anesthesia and surgery are

described in Schnell and Schwab 1993 (Eur.J. Neurosci. 5: 1156 - 1171). Controls or binding molecules of the invention are applied in two different ways: either by implanting 10 freshly harvested hybridoma cells into one side of the cerebral cortex (grafted animals) or, alternatively, by an implanted intraventricular canula linked to a subcutaneously implanted 2ml Alzet (Alza Corporation, Palo Alto) pump (pump animals). - Hybridoma grafted animals: Rats are immunosuppressed for 7 - KXdays with cyclosporin A and sacrificed by transcardial perfusion with 4% buffered formalin 14 days after injury. - Pump animals: Binding molecules of the invention (e.g. at 3.3 mg/ml for mouse 11C7) are filled into 2 ml pumps delivering 0.5 p.l/h into the lateral ventricle for 2 weeks. Pumps are implanted at the time of the spinal cord lesion, and rats are sacrificed 2 weeks later.
Neuroanatgmical tracing: The motor and sensory corticospinal tract is traced by injecting the anterograde tracer biotin dextran amine (BDA) into the cortex of the side opposite to the pump or the graft. BDA is transported to the spinal cord within 10 - 14 days and visualized using diaminobenzidine (DAB) as a substrate as described in Brosamle et al.f (2000 J.Neurosci. 20: 8061-8068).
Evalutation of anatomical results: Two methods of evaluation are used: a semi-quantitative and a quantitative one. Semi-quantitative estimation of intensity of sprouting and regeneration: Complete sagittal section series of number-coded, randomly mixed animals are evaluated for the presence and density of regenerating sprouts^ostral to the lesion using the following definitions: regenerative sprouts are fibers emanating from the transected CST; they are long, irregular in their course, much less branched than the normal grey matter collaterals, and they growth towards and ventrally or laterally around the lesion. Regenerative sprouts often end in a growth cone which can be small and bulbouse or large and branched. Density of sprouting is rated on a scale of 0 - 3 for each animal. - Long distance regeneration: fibers that can be followed through the lesion into the caudal spinal cord are considered long-distance regenerating fibers. Their maximal distance from the lesion site can be measured, but is often a minimal distance as some unlesioned fibers from the small ventral funiculus CST are often present; their branches mix with those of regenerating axons and make distinction dffficult.
Fiber counts (quantitative assay): A line positioned at -0.5 mm rostral to the end of the transected CST is posed on alternating sections of the grey matter, and all intersections with

CST fibers (normal collaterals or sprouts) are counted. Similar lines are positioned caudal to the lesion at a distance of +0.5, +2 and +5 mm from the lesion center. Intersecting fibers are counted and the 3 levels are added to a sum reflecting CST fibers in the caudal spinal cord. These caudal fibers are divided by the number of fibers -0.5 mm rostral to the CST end to obtain a ratio.
Two weeks after a spinal cord injury destroying about 40 % of the spinal cord segment T8, mainly in the dorsal half, including both main CSTs: tracing of the CST in control animals show a moderate degree of reactive sprouting of the tract. This phenomenon corresponds to the spontaneous sprouting in response to injury well known in the literature. Injured rats being treated with the binding molecules of the invention or with pumps delivering the binding molecules of the invention may show an enhanced sprouting at the lesion site and regeneration of damaged axons neurite outgrowth of damaged neurites.
Therefore the invention also provides
(i) the use of the binding molecules of the invention in the nerve repair of a mammalian nervous system, in particular human nervous system,
(ii) a method of repairing nerves of a mammalian nervous system, in particular human nervous system which comprises administering an effective amount of the binding molecules of the invention to a patient in need of such treatment, or
(iii) a pharmaceutical composition for nerve repair of a mammalian nervous system, in particular human nervous system which comprises the binding molecules of the invention and a pharmaceutical^ acceptable carrier or diluent.
In particular, the binding molecules of the invention are useful for axonal regeneration and improved sprouting after nerve fiber damage. Thus the molecules of the invention have a wide utility in particular for human subjects. For example the binding molecule of the invention are useful in the treatment of various diseases of the peripheral (PNS) and central (CNS) nervous system, i.e. more particularly in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Amyotrophic lateral sclerosis (ALS), Lewy like pathologies or other dementia in general, diseases following cranial, cerebral or spinal trauma, stroke or a demyeliating disease. Such demyelinating diseases include, but are not

limited to, multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, pontine myelmolysis, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease. In one example, administration of the binding molecules of the invention can be used to treat a demyelinating disease associated with NogoA protein. In another example, cells which express the binding molecules of the invention may be transplanted to a site spinal cord injury to facilitate axonal growth throughout the injured site. Such transplanted cells would provide a means for restoring spinal cord function following injury or trauma. Such cells could include olfactory ensheathing cells and stem cells of different lineages of fetal nerve or tissue grafts.
In addition, the Binding Molecules of the invention are useful for the treatment of degenerative ocular disorders which may directly or indirectly involve the degeneration of retinal or corneal cells including ischemic retinopathies in general, anterior ischemic optic neuropathy, all forms of optic neuritis, age-related macular degeneration, diabetic retinopathy, cystoid macular edema (CME), retinitis pigmentosa, Stargardt's disease, Best's vitelliform retinal degeneration, Leber's congenital amaurosis and other hereditary retinal degenerations, pathologic myopia, retinopathy of prematurity.and Leber's hereditary optic neuropathy, the after effects of corneal transplantation or of refractive corneal surgery, and herpes keratitis.
Furthermore, it was shown that NogoA plays a role in psychiatric conditions, in particular schizophrenia and depression. Hence, the binding molecules of the invention are useful for the treatment of psychiatric conditions, in particular schizophrenia and depression.
The Binding Molecules of the invention can be provided alone, or in combination, or in sequential combination with other agents. For example, the binding molecules of the invention can be administered in combination with anti-inflammatorv agents such as but not limited to corticosteroids following stroke or spinal cord injury as a means for blocking further neuronal damage and inhibition of axonal regeneration, Neurotrophic factors such as NGF, BDNF or other drugs for neurodegenerative diseases such as Exelon™ or Levodopa. As used herein, two agents are said to be administered in combination when the two agents are

administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
For the treatment of psychiatric conditions, in particular schizophrenia or depression, the Binding Molecules of the invention can be provided alone or in combination in particular with other agents selected from the group consisting of (a) anti-epileptic drugs selected from barbiturates and derivatives thereof, benzodiazepines, carboxamides, hydantoins, succinimides, valproic acid and other fatty acid derivates and other anti-epileptic drugs, (b) conventional antipsychotics, (c) atypical antipsychotics and (d) antidepressants.
The term "barbiturates and derivatives thereof" as used herein includes, but is not limited to Phenobarbital and primidon. The term "benzodiazepines" as used herein includes, but is not limited to clonazepam, diazepam and lorazepam. The term "carboxamides" as used herein includes, but is not limited to carbamazepine, oxcarbazepine and 10-hydroxy-10,11-dihydrocarbamazepine. The term "hydantoins" as used herein includes, but is not limited to phenytoin. The term "succinimides" as used herein includes, but is not limited to ethosuximide and mesuximide. The term 'Valproic acid and other fatty acid derivates" as used herein includes, but is not limited to valproic acid sodium salt, tiagabine hydrochloride monohydrate and vigrabatrine. The term "other anti-epileptic drugs" as used herein includes, but is not limited to levetiracetam, lamotrigine, gabapentin and felbamate.
The term "conventional antipsychotics" as used herein includes, but is not limited to haloperidol and fluphenazine.
The term "atypical antipsychotics" as used herein relates to Clozaril, risperidone, olanzapine, quetiapine, ziprasidone and aripiprazol.
The term "antidepressants" as used herein includes, but is not limited to selective serotonin reuptake inhibitors (SSRI's), or selective serotonin and norepinephrine reuptake inhibitors (SNRI-s). An SSRI's suitable for the present invention can be selected from fluoxetine, fuvoxamine, sertraline, paroxetine, citalopram and escitalopram. * '
The structure of the active ingredients identified by code nos., generic or trade names may be taken from the actual edition of the standard compendium "The Merck Index" or from

databases, e.g. Patents International (e.g. IMS World Publications). The corresponding content thereof is hereby incorporated by reference. Any person skilled in the art is fully enabled to identify the active ingredients and, based on these references, likewise enabled to manufacture andtest the pharmaceutical indications and properties in standard test models, both in vitro and in vivo.
For the indications mentioned above, the appropriate dosage will, of course, vary depending upon, for example, the particular molecule of the invention to be employed, the mode of administration and the nature and severity of the condition being treated. The Binding Molecules of the invention are conveniently administered by pumps or injected as therapeutics at the lesioned site, e.g. they can be administered directly into the CNS intracranial^ or into the spine intrathecally to the lesioned site.
Pharmaceutical compositions of the invention may be manufactured in conventional manner. E.g. a composition according to the invention comprising the molecules of the invention is preferably provided in lyophilized form. For immediate administration it is dissolved in a suitable aqueous carrier, for example sterile water for injection or sterile buffered physiological saline.
To aid in making up suitable compositions, the binding molecules of the invention and optionally a second drug enhancing the effect of the Binding Molecules of the invention, may be packaged separately within the same container, with instructions for mixing or concomitant administration. Optional second drug candidates are provided above.
The synergistic effect of a combination of the binding molecules of the invention and growth factors such as NGF may be demonstrated in vivo by the spinal cord injury model described above.
Brief description of the drawing
Figure 1: Sequence Comparison: Sequence comparison of the NiG from different species,
showing the immunogenic-peptide sequence for the 11C7 mAb.
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

In the following examples all temperatures are in degree Celsius (°C).
The monoclonal antibody of attention in the Examples is a Binding Molecule according to th> present invention comprising the variable part of the light chain (SEQ ID NO: 3) and the variable part of the heavy chain (SEQ ID NO: 2).


Example 1: NiG-D20 (SEQ ID NO: 24) is one of the neurite outgrowth inhibitory fragments of NogoA
Methods:
a) Rat Nogo-A deletion library: Deletion constructs are made using internal restriction sites, by Exonucleaselll/Mung Bean Nuclease treatment and by PCR with rat Nogo-A-specific primers on rat Nogo- (method as in WO00/31235): rat Nogo-A (aa 1-1163; DNA as shown hereafter related to the amino acids of rat NogoA (SEQ ID NO: 26), e.g. aa 1-1163 means that the cDNA construct encodes for polypeptide which starts at the amino acidl and ends at amino acid 1163 of the rat polypeptide sequence of NogoA), rat Nogo-B (aa 1-172 + 976-1163), rat Nogo-C (Nogo-C N-terminal 11 aa + aa 976-1163), rat Nogo-66 (aa 1019-1083), rat GST-Nogo-66 (aa 1026-1091), rat NiR-G (aa 1-979), rat NiR (1-172), rat NiR-Dl (aa 1-31), rat NiR-D2 (aa 59-172), rat NiR-D3 (aa 1-31 + 59-172), rat EST-Nogo1 (aa 762-1163), rat NiG (aa 174-979), rat ISHG-D1 (aa174-909), rat NiG-D2 (aa 174-865), rat NiG-D3 (aa 172-723), rat NiG-D4 (aa 172-646), rat NiG-D5 (aa 293-647), rat NiG-D6 (aa 763-975), rat NiG-D7 (aa 174-235 + 294-979), rat NiG-D8 (aa 218-653), rat NiG-D9 (aa 172-259 + 646-974), rat NiG-D10 (aa 293-979), rat NJG-D11 (aa 209-268), rat NiG-D12 (aa 198-233), rat NiG-D13 (aa 174-216), rat NiG-D14 (aa 174-260), rat NiG-D15 (aa 174-190 + 493-979);-rat NiG-D16 (aa 174-190 + 621-979), rat NiG-D17 (aa 174-190 + 259-979), rat NiG-D18 (aa 174-190 + 263-979), rat NJG-D19 (aa 763-865), rat NiG-D20 (aa 544-725), rat NiG-D21 (aa 812-918), rat NiG-D22 (aa 866-975), rat NiG-D23 (aa 914-975), rat NiG-D24 (aa 544-685), rat NiG-D25 (aa 614-725), rat NiG-D26 (aa 544-613), rat NiG-D27 (aa 581-648), rat NiG-D28 (aa 614-685), rat NiG-D29 (aa 648-725), rat NIG-D30 (aa 682-725), rat NiG-D31 (aa 544-580), rat NiG-D32 (aa 581-613), rat NiG-D33 (aa 614-648), rat NiG-D34 (aa 648-685), rat NiG-D35 (aa 260-556), rat NiG-D36 (aa 260-415). NiR-G and NiR-a are derived from Nogo-A-pET28 by restriction enzyme digestions. NiG is derived from NiR-G by restriction digestion and MungBean Nuclease treatment. NiG-D1, -D3, -D4, -D5, -D7, -D8, -D9, -D10 derived from NiG-pET28 by restriction enzyme digestions. NiG-D15, -D16, -D17, -D18 derived from NiG-pET28 by Exonuclease III digestion. NiR-b, NiR-D1, -D2, -D3 derived by PCR with NiR-a-pET28 as a template. NiG-D2, -D6, -D11, -D12, -D13, -D14, -D19, -D20, -D21, -D22, -D23, D24, -D25, -D26, -D27, -D28, -D29, -D30, -D31, -D32, -D33, -D34, -D35, -D36 derived by PCR using NiG-pET28 as a template. All constructs subcloned into pET28. pET28 used for all the constructs mentioned aboved. pGEX-6P used for GST-Nogo66 and pET26 for periplasmic expression of rat NiG. Human GST-Nogo-66 (aa 1055-1120 of human Nogo-A)

is cloned by PCR on human NogoA DNA (SEQ ID NO: 4) as a template. Deletion constructs are then cloned into pET28 vector (Novagen), pGEX-6P (Amersham Pharmacia Biotech) and pET26 vector (Novagen). Human GST-Nogo-66 corresponds to the GST-nogo protein published by GrandPre et al. (supra). Synthetic rat peptide 4
EELVQKYSNSALGHVNSTIKELRRL (SEQ ID NO: 27) corresponds to the human peptide 4 (Human peptide 4 has been shown to be the inhibitory region of the Nogo-66 domain (GrandPre et al., 2000)). The orthologous rat peptide has a single mismatch C->S (see peptide 4 sequence in GrandPre et al., 2000, supra). Synthetic Pro/Ser-rich peptide PSSPPPSSPPPSSPPPS (SEQ ID NO: 28) as well as rat peptide 4 have been produced and HPLC-purified by Primm SA. Human NogoA_623-640 (SEQ ID NO: 6) is synthesised and purified by Research Genetics Inc.
b) Generation of human Nogo-A expression constructs (pRK7-hNogo-A): A human cDNA library constructed in lambda gt10 (Clontech) is screened with duplicate filter sets using standard procedures. Fragments of human Nogo-A are amplified by PCR from human whole brain cDNA (Clontech) using a standard protocol and subsequently cloned into pBIuescript, digested and isolated, or used as screening probes directly. A 400bp Xhol/Smal fragment is used as 5' probe, the 3' probe is amplified with primers CA-NA-2F: 5'-AAG CAC CAT TGA ATT CTG CAG TTC C-3' (SEQ ID NO: 29) and CA-NA-3R: 5'-AAC TGC AGT ACT GAG CTC CTC CAT CTG C-3' (SEQ ID NO: 30). Positive clones are isolated, subcioned and sequence confirmed. To obtain a full length human Nogo-A cDNA, overlapping clones are assembled using an unique EcoRI restriction site in the human Nogo-A sequence and subcioned into Bluescript vector, named Pbsnogoa. To obtain pRK7-hNogo-A, the full length cDNA was inserted into the eukaryotic expression vector pRK-7 by directional cloning.
c) Generation of human NiG (hNiG) expression plasmids (pET28a-hNiG) for bacterial production: A hNiG encoding DNA fragment is subcioned into BamHI/Xhol of pET28a (Novagen), after PCR amplification of the respective coding region from Pbsnogoa, in frame with the N-terminal His- and T7-tag for bacterial expression, using primer sets: forward 5'-GTC GCG GAT CCA TGG AGA CCC TTT TTG CTC TTC-3' (SEQ ID NO: 31); reverse 5'-GTT CTC GAG TTA TGA AGT TTT ACT CAG-3' tSEQ ID NO: 32). The final plasmid is termed pET28a-hNiG. hNiG was then expressed in E.coli BL21 pRP by induction with 1 mM Isopropyl-beta-D-thiogalactopyranoside (IPGT).

d) Generation of mouse NiG-exon3 (mNiG-exon3) expression plasmid: The region encoding
mouse exon 3 is amplified from mouse genome BAC template with primers: forward 5'-GTG
CGG ATC CAT GGA TTT GAA GGA GCA GC-3' (SEQ ID NO: 33); reverse 5'-GTT TCT
CGA GTG AAG TTT TAT TCA GCT C-3' (SEQ ID NO: 34) and subcloned into the
BamHI/Xhol cloning sites of pET28a. The final plasmid construct is named pET28a-mNiG-
exon3.
Cloning of monkey NiG: PolyA RNA is isolated from frozen monkey brain tissue and cDNA are synthesised using an oligo dT primer. Two overlapping fragments covering the 5' and the 3' region of the cDNA are amplified by PCR using sequence-specific primers and a proofreading enzyme. The primers are designed using the known sequence of the human NiG cDNA. For amplification of the 5' fragment the primers are 5'-TCCACCCCGGCCGCGCCCAA-3' (SEQ ID NO: 35) and 5'-AATGATGGGCAAAGCTGTGCTG-3* (SEQ ID NO: 36), for the 3'-fragment 51-GGTACAAAGATTGCTTATGAAACA-3' (SEQ ID NO: 37) and 5'-AGCAGGGCCAAGGCAATGTAGG-3' (SEQ ID NO: 38). The two fragments are then subcloned and for each fragment at least 4 independent clones were sequenced. The full length cDNA is assembled by overlapping PCR using the primers mentioned above and the resulting product is cloned and sequenced again.
e) Production of recombinant NogoNiG proteins and the Nogo-A-deletion library as defined
above: The bacterial Nogo-A-deletion library is expressed in Escherichia coli. Proteins are
extracted either by repeated sonication in sonication buffer (20 mM Tris, 50 mM NaH2P04,
100 mM NaCI, pH 8.0) with 0.75 mg/ml Lysozyme, by solubilisation with B-Per™ (Pierce) or
with 8 M urea. NiG expressed with pelB-leader is obtained from the periplasmic space
according to the Novagen protocol for periplasmic protein purification. Supernatants of
pET28-constructs are purified using the Co2+-Talon™ Metal Affinity Resin (Clontech) in a
batch procedure. 8 M urea and B-Per™ solubilised lysates are brought to non-denaturing
conditions by increasingly substituting the buffer with sonication buffer during the resin-batch
procedure. Proteins are eluted with 250 mM imidazole in sonication buffer on a gravity
columm(BioRad). NiG proteins are further purified by gel filtration on Superdex 200 HiLoad
16/60. Supernatants of pGEX-6P constructs are purified with G-sepharose column in a batch
procedure according to manufacturer indications (Amersham Pharmacia). Cleavage of GST-
Nogo-66 is done by incubating solubilised GST-Nogo-66 with PreScission protease and

subsequent HPLC purification. Gel electrocution is performed by preparative SDS-PAGE of IMAC-purified recombinant Nogo and elution with BioRad Electro-Eluter into 50 mM Tris, pH 7.4, 100 mM NaCI, 0.2% (w/v) CHAPS for 1 hr at 250 mA and followed by 30 s of reversed electrode polarities. Protein concentrations of chromatography-purified proteins are determined using Pierce Coomassie Stain and BSA as standard protein. Protein concentrations of gel eluted proteins are estimated based on band intensity of silver-stained gels (Merril CR, Dunau ML, Goldman D (1981) A rapid sensitive silver stain for polypeptides in polyacrylamide gels. Analyt.Biochem. 110:201-207) with BSA as a standard.
f) Production of recombinant NogoA fragments in CHO cells: A 3119 bp fragment resulting from a partial Hindi digest of rat Nogo-A cDNA, NiR-G, is cloned into pSecTag2 expression vectors (Invitrogen, Groningen, The Netherlands). Transfection of pNiR-G into CHO cells results in intracellular, cytoplasmic expression of NiR-G. Stable NiR-G CHO cell lines are selected with 250 ng/ml Zeocin (Invitrogen). Recombinant NiR-G from cell lysate is purified over a Ni2+-NTA column (Qiagen AG, Basel, Switzerland). Rat NiG-D20.and Nogo-66 are cloned into pAPtag5 vector by PCR. Transfection of pNiG-D20-AP into CHO cells results in NiG-520-AP that was secreted into the culture supernatant. Stable pNiG-D20-AP and pNogo-66-AP cell lines were selected with 250 \ig/m\ Zeocin (Invitrogen). Both cell lines are adapted to serum-free medium (Gibco) conditions and grown in a cell-line chamber (Integra). Supernatants are tenfold concentrated prior to use, and the concentration of fusion protein is assessed as described elsewhere (Flanagan JG, Leder P (1990) The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 63:185-194).
g) 3T3 fibroblast and CHO spreading assays: The 3T3 spreading assays are performed as described previously (Spillmann AA, Bandtlow CE, Lottspeich F, Keller F, Schwab ME (1998) Identification and characterization of a bovine neurite growth inhibitor (bNI-220). J.Biol.Chem. 273:19283-19293). CHO spreading assays are performed essentially the same way as for 3T3 fibroblasts. Briefly, CHO cells are split 1:2. 24 hrs later they are trypsinised in PBS-EDTA for 30 s and ~8'000 CHO cells are plated onto culture dishes precoated with 5, 1, 0.5 and 0.2 fxg/well NiG or Nogo-66. After 30-45 min the cells are fixed with 4% (w/v) PFA, 5% (w/v) sucrose and then analysed as described Spillmann et al, supra). -100 cells are counted per well with light microscopy; criterion of spreaded ceils: (a) attachement to the dish AND (b) extended morphology indicative for lamellipodia; under light microscopy the cells appear darker and larger than not spreaded, round cells; non-spreaded cells are

considered those cells that are (a) not attached to the dish OR (b) attached to the dish, but small, rounded, without detectable lamellipodia protruding on the dish. The ratio between spreaded and not spreaded cells defines the degree of non-permissiveness of the substratum.
h) PC12 Neurite outgrowth assays: PC12 neurite outgrowth assays are performed as described previously (Rubin BP, Spillmann AA, Bandtlow CE, Keller F, Schwab ME (1995) Inhibition of PC-12 cell attachment and neurite outgrowth by detergent solubilized CNS myelin proteins. Europ. J. Neurosci. 7: 2524-2529). PC12 cells (a PC12 cell clone able to grow independently of laminin obtained from Moses Chao, New York) are primed for two days with 50-100 ng/ml NGF (Harlan Biopreducts, Indianapolis) to DMEM, 5% foetal calf serum, 10% horse serum, 100 U/ml Penicillin and 0.5 mg/ml Streptomycin (Pen-Strep from Gibco-BRL). PC12 cells are detachde mechanically, trypsinised for 5 minutes with 0.05% trypsin (Sigma) in HBSS (Gibco) and plated at a density of 3,000-5,000 cells/cm2 in culture medium with 100 ng/ml NGF. Assays were stopped after 24 hrs by adding 4% (w/v) PFA, 5% (w/v) sucrose in PBS, pH8. Cell culture dishes were coated for PC12 cells the same way as for 3T3 cells.
i) Retinal ganglion cell stripe assays: The retinal ganglion cell stripe assay is performed according to Vielmetter (see Vielmetter J, Stolze B, Bonhoeffer F, Stuermer CA (1990) In vitro assay to test differential substrate affinities of growing axons and migratory cells. Exp. Brain Res. 81:283-287) with modifications (see Schmalfeldt Mf Bandtlow CE, Dours-Zimmermann MT, Winterhalter KH, Zimmermann DR (2000) Brain derived versican V2 is a pottnt inhibitor of axonal growth. J. Cell Sci. 113:807-816). Explants are evaluated after fixation with 4% (w/v) PFA, 0.1% (v/v) glutaraldehyde in PBS for 10 min at RT. For immunostainings, fixed explants are blocked for 1 hr at RT with RNO-blocking solution (0.5% (w/v) BSA, 0.3% (w/v) TopBlock (Juro Supply), 0.1% (w/v) NaN3 in PBS), permeabilised for 10 min with 0.05% (v/v) Tx-100 in RNO-blocking solution, frozen for one minute at -20 °C and incubated with primary antibodies (AS Bianca for NiR, AS Laura for Nogo-A, NiR-G, NiG, NiG-D3 and NiG-D20, Novagen mAb anti-T7 for Nogo-C and beta-Gal control protein). After washing with PBS, FITC- and TRITC (FITC: Fluorescein-lsoThioCyanate: TRITC: Tetramethyl Rhodamine lsoThiocyanate)-conjugated antibodies (Jackson ImmunoResearch

Laboratories) are added (1:150) to the explants. The samples are coverslipped in 50% (v/v) glycerol, 25 mM NaHC03, 40 mM NaCI, 1% (w/v) p-Phenylendiamine (Sigma).
Results:
a) Two regions in the N-terminal part of Nogo-A are inhibitory for spreading of3T3 fibroblasts: In order to identify the regions of Nogo-A responsible for the inhibition of 3T3 fibroblast spreading, a library of 50 Nogo deletion constructs is made and recombinant proteins are expressed in bacteria (see method 1a). The apparent EC50 for inhibition of 3T3 fibroblast spreading was approximately 400-500 ng/0.1 ml Nogo-A coated overnight per cm2 of culture dish (-4 pmol/cm2). Treatment of Nogo-A or its fragments with 8 M urea results in a strong decrease of inhibitory activity, indicating that conformation is important. The analysis of Nogo fragments in the fibroblast spreading assay reveals that at least two stretches of the Nogo-A protein mediate inhibition of the spreading of freshly plated fibroblasts; namely NiR-D2 (aa 59-172) and NiG-D20 (aa 544-725). All the fragments derived from the NiG-region displaying inhibitory activity (e.g. NiG-D4 and NiG-D8) partially overlap with NiG-D20. Minor inhibitory activity at high protein concentration is seen for NiG-D19 within the NiG-D6 region. Nogo-C, Nogo-66 and rat Peptide 4 (shown to bethe inhibitory region of Nogo-66 by GrandPre et al., 2000) are not inhibitory for fibroblast spreading. These data show that the anti-spreading activity of Nogo-A on 3T3 fibroblasts resides in two defined stretches located at the N-terminus (NiR-D2) and within the Nogo-A-specific part (NiG-D20) of the protein. Non-specific physico-chemical properties (acidity of the fragments, structural effects due to proline and serine residues) are not responsible for this effect. The C-terminal RTN domain is not involved in the inhibition of fibroblast spreading.
b) NiG~D20 Region of Nogo-A is inhibitory for neurite outgrowth: To determine whether the fragments of Nogo-A that are non-permissive for cell spreading are also inhibitory for neurite outgrowth, a series of bacterially produced Nogo-A fragments as well as eukaryotically produced Nogo-AP chimeras in different neuronal assays are tested. In the stripe assay (method 1), neurites avoid laminin/Nogo-A coated stripes, growing on the laminin-only stripes, whereas stripes coated with laminin/beta-Galactosidase are not circumvented. Full-length Nogo-A is strongly non-permissive for retinal ganglion cell (RGC) neurite outgrowth, while the N-terminal part (NiR) had only marginal effects. Nogo-C activity is indistinguishable from the control protein beta-Galactosidase. The Nogo-A-specific region NIG-D20 appears to

contain the main region responsible for the non-permissive activity on RGC neurite outgrowth; the growth cones stop when encountering NiG-D20-coated stripes. The nonpermissive effect is concentration-dependent. At lower Nogo-A concentrations the number of crossing fibers increased. No obvious difference is observed between nasal and temporal RGC neurites concerning their responsiveness to Nogo-A regions. A laminin-independent, NGF-responsive clone of PC12 cells is primed with 50 ng/ml NGF for 24 hrs and then plated onto dishes coated with bacterially produced Nogo fragments at 0.1-3 pg/cm2. Neurite outgrowth is scored one day later. The Nogo-A-specific region (NiG) and its fragment NiG-520 strongly inhibited PC12 neurite outgrowth. In contrast, the N-terminal fragment NiR has only minor activity, detectable only at high protein concentration. Nogo-C and Nogo-66 are inactive.
Example 2: Presence of binding site(s) for NiR-G and NiG-D20 on 3T3 fibroblasts and rat cortical brain membranes:
Methods:
a) Radioactive labelling and binding experiments: IMAC-purified NiG-D20 is iodinated by ANAWA Trading SA (Wangen, Switzerland) (2,030 Ci/mmol) using Lactoperoxidase and purified by reverse-phase HPLC. Membranes from rat brain cortex are prepared as described (Olpe HR, Karlsson G( Pozza MF, Brugger F, Steinmann M, Van Riezen H, Fagg G, Hall RG, Froestl W, Bittiger H (1990) CGP 35348: a centrally active blocker of GABAB receptors. Eur.J.Pharmacol. 187:27-38). Binding is performed for 1 hr at RT essentially as described (Kaupmann K, Huggel K, Heid J, Flor PJ, Bischoff S, Mickel SJ, McMaster G, Angst C, Bittiger H, Froestl W, Bettler B (1997) Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors. Nature 386:239-246.) using 1.5 ml tubes preincubated for 2 hrs with 1 % (w/v) bovine serum albumin to reduce non-specific binding. Membrane homogenates in HEPES buffer pH 7.4 (125 mM NaCI, 5 mM KCI, 0.6 mM MgCI2, 1.8 mM CaCI2l 20 mM HEPES, 6 mM dextrose) containing protease inhibitors (Roche Diagnostics, Mannheim, FRG) are incubated with 1.3 nM iodinated NiG-D20 in the absence or presence of increasing concentrations of unlabelled NJG-D20.
b) Flow cytometry: Flow cytometry and cell sorting are performed on a Cytomation MoFlo high-speed cell sorter (Fort Collins, Colorado). The flow cytometsr is equipped with an argon-ion/UV Enterprise II laser tuned to 488 nm with 130 mW of power. Fluorescein (FITC)

fluorescence is collected through a 530/40 nm bandpass filter. For analysis 3T3 fibroblasts are detached with Cell Dissociation Buffer (Gibco). The pre-formed complex used to detect binding of NiR-G to 3T3 fibroblasts is prepared as follows: NiR-G and anti-Myc antibody (9E10) are incubated at a 1:1 molar ratio for 30 min at 4 9C. Next, FITC conjugated F(ab)2 Goat Anti Mouse IgG is added and incubated for additional 30 min at 4 QC . The resulting molar ratio of the trimeric complex is 1:1:0.5. The complex is added to 1x106 3T3 fibroblasts in a final volume of 0.1 ml, incubated for 2 hrs at 4 9C, washed, and analysed by flow cytometry.
Results:
Presence of binding site(s) for Nogo-A-specific active fragments on 3T3 fibroblasts and rat cortical brain membranes: Since the NiR-D2 and NiG-020 regions of Npgo-A are inhibitory for cell spreading and neurite outgrowth despite the absence of Nogo-66 and independently of NgR, the presence of a separate, Nogo-A-specific receptor has to be postulated. Thus binding studies are performed of multimerised, myc-tagged and IMAC-purified NiR-G to living 3T3 fibroblasts that are analysed by flow cytometry. Ab-complexed NiR-G is binding efficiently to 3T3 cells as seen by a fluorescence shift of over 90% of the 3T3 cells. In contrast, 3T3 cells are not labelled after incubation with the 9E10 primary mouse anti-myc mAb complexed with a FITC-conjugated secondary F(ab)2 goat anti-mouse IgG nor with the secondary Ab alone. To test binding of NiG-D20 to rat cortical membranes, [125l]-labelled NiG-D20 in a radioligand binding assay is used. At a concentration of 1.3 nM of [125l]-NiG-D20, evidence for a specific NiG-D20 binding sites on brain membranes as shown by a concentration-dependent competition of radioligand binding by unlabelled NiG-D20 is found. These results show that aminoterminal fragments of Nogo-A can bind to the surface of 3T3 cells and to rat cortical membranes, demonstrating the presence of membrane-bound, Nogo-A-specific binding sites or receptor(s).
Example 3: Generation of mouse 11C7-IgG1
Mice (C3H- and C57BI6/J-strains) are immunised subcutaneously with the synthetic peptide SYDSIKLEPENPPPYEEA (= rat NogoA_623-640; SEQ ID NO: 1), corresponding to a particular epitope in NiG-D20. This epitope is highly conserved in human, cynomologus monkey and mouse NiG-D20 Nogo-A specific region and starts at amino acid 623 and ends

at amino acid 640 of the human NogoA amino acid sequence (SEQ ID NO: 5) (See also sequence alignment: Figure 1).
ha.
mAb 11C7 has been obtained out of a fusion of rat NogoA__623-640 with the carrier protein Key hole limped hemagglutinin (KLH) immunised mice. Monoclonal antibodies have been screened by ELISA on rat NogoA_623-640-KLH, rat NogoA_623-640 free peptide and a nonrelated peptide-KLH. In a further screen, the mAbs have been tested by ELISA on NiR-G versus b-Galactosidase, both expressed as his-tagged proteins and purified by metal affinity chromatography . Subsequently, the mAbs have been tested for recognition of Nogo-A on Western blot of oligodendrocyte and brain lysates (rat origin). Antibodies are tested for recognition of the protein in immunocytochemistry of rat Nogo-A-transfected CHO or COS cells and of endogenous Nogo-A of rat oligodendrocytes (permeabilised cells). They have also been tested for surface binding to living rat oligodednrocytes. Species crossreactivity is tested on recombinant NiG of rat, mouse, human and bovine origin by ELISA and on endogenous rat, mouse, human and monkey Nogo-A by Western blot of tissue or cell extracts.
Western blot analysis: SDS-PAGE and Westernblotting are performed as described earlier (Huber AB, Weinmann O, Brosamie C, Oertie T, Schwab ME (2002) Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J. Neurosci. 22: 3553-3567), blocking is done with 3% (w/v) Top Block (Juro Supply, Lucerne, Switzerland). Antibodies are diluted as follows: Purified monoclonal 11C7 or hybridoma supernatants 1:150. Secondary antibodies are HRP-conjugate anti-mouse ((Pierce; 1:5000.)
1:50,000). Hybridisation with the 11C7 antibody is carried out over night at 4°C. For detection the ECL detection reagents from Amersham Pharmacia are used.
Results:
The 11C7 mAb identifies the 190 kD Nogo-A band on a Western blot of oligodendrocyte cell
culture homogenate. 11C7 also identifies human NiG, Cynomolgus NiG cell lysate and rat
NiG-D20 in western blots. 11C7 mAb is characterised as a lgG1 isotype (IsoStrip Kit,
Roche).
Example 4: Characterisation of the mouse 11C7 mAb

Immunocytochemistry: Optic nerve oligodendrocytes are prepared as described (Schwab, Caroni, 1988, Neuron). Three to five day-old cultures grown on poly-L-lysine coated coverslips are washed twice with PBS, fixed in 4% (w/v) paraformaldehyde (PFA), 5% (w/v) sucrose in PBS for 15 min at room temperature (RT) and non-specific binding is blocked with 10% (v/v) FCS. Cells were then incubated with mouse 11C7 (1:100). Secondary antibodies are goat-anti-mouse TRITC (Jackson ImmunoResearch Laboratories). For cell surface staining, two day-old rat optic nerve cultures are incubated with monoclonal antibody in medium for 25 min at RT. Secondary alkaline phosphatase conjugated antibodies (Milan Analytica, Lausanne) are used at 1:7,500 in 0.1 M maleic acid with 1% (w/v) blocking reagent (1 hr). The cultures are washed twice with maleic acid buffer, once with alkaline phosphatase buffer (0.1 M Tris-HCI pH 9.5, 0.1 M NaCI, 5 mM MgCI2) and the staining is developed for 3 hrs at room temperature with 0.175 mg/ml BCIP (Sigma) and 0.338 mg/ml NBT (Sigma) in alkaline phosphatase buffer.
NogoA__623-640 epitope of Nogo-A present at the cell surface of cultured oligodendrocytes: Living cultures of oligodendrocytes incubated with mouse 11C7 mAb stain the differentiated oligodendrocyte cell bodies and their radial processes. The control mouse IgG and the antibodies against the intracellular protein CNPase do not stain the living cells. Preincubation of mouse 11C7 with the corresponding immunogenic peptide (= rat NogoAJ323-640 SED ID NO: 1) reduces staining to background levels (competitive assay). Cell surface staining is present on all major and small processes and on the cell body. Thus, the Nogo-A specific part of the molecule recognised by mouse 11C7 mAb is exposed to the extracellular space on the plasma membrane of oligodendrocytes.
Production and Purification of mouse 11C7 mAb:mA 10-L glas bioreactor is used for continuous-mode cultivation of the hybridoma clone producing the mouse 11C7 mAb. The bioreactor is equipped with a marine impeller placed in a center tube for gentle agitation, a spin filter for cell retention, and coiled silicone tubing for bubble-free aeration. The hybridoma cells are cultivated in our RPMI based serum free medium. The medium is
inoculated with cells at 3.7 x 105/ml. After 28 hours continuous medium flow through the
*
bioreactor is started with a rate of 0.5 fermentor volumes /day (5 liters/day). Another 24 hours later the flow rate is increased to its final level of 1 fermentor volume/day (10 liters/day). After 1 week the culture reaches a steady state with 11 x 105cells/ml and the process is continued for another week. The titer of the mouse 11C7 mAb is determined daily

by HPLC. A total of 150 liters culture supernatant is harvested from the bioreactor, sterile filtered for removal of cells and cell debris. 150 L culture supernatant are concentrated to about 6 L using a Pellikon tangential flow device (Millipore ; 10 kDa cut-off). The concentrated supernatant is purified in 3 runs over a 220 ml bed volume column of Protein A Sepharose CI-4B (Pharmacia ; 11 cm bed height). Briefly, the culture supernatant after pH correction to 8.1 is loaded at 4 ml/min and the column washed to base-line at 8 ml/min using. 100 mM Na2HP04, pH 8.1. Bound material is finally eluted at 8 ml/min using 50 mM NaH2P04, pH 3.0, 140 mM NaCI and immediately neutralized (pH 7.0) with 5 N NaOH and sterile filtered. Absorbance is monitored at 280 nm. Portion of the purified material are eventually further concentrated by ultrafiltration and/or dialyzed against PBS. All the buffers used in the purification are filtered on a 10 kDa ULTRASETTE™ tangential flow device (Filtron Technology Corporation) in order to remove possible endotoxin contaminations. For the same reason the Protein A resin is extensively washed with 20% ethanol and all tubings/pumps treated with 0.1 M NaOH prior to use. Protein concentration is measured spectrophotometrically at 280 nm using a reference absorption of 1.35 for 1 mg/ml. Purity is routinely assessed by SDS-PAGE under reducing conditions using 4-20% Novex gradient gels. Endotoxin content is measured by the classical Limulus Amoebocyte Lysate (LAL) reaction according to the manufacturer instructions (Endotell AG, Allschwil, Switzerland).
Generation of Fab fragments: A portion of mouse 11C7 mAb is extensively dialyzed against 100 mM Na-actetate, pH 5.5, 2 mM EDTA and adjusted to a concentration of 6 mg/ml. F3b fragments are generated by papain digestion (1:200 w/w ratio) in the presence of 0.25 mM
cysteine. The reaction is allowed to proceed for 16 hours at 37 °C and then stopped by the addition of the specific papain inhibitor E64 (N-[N-(L-3-trans-carboxirane- 2-carbonyl)-L-leucyl]-agmatine) in large excess (10 //M). The digested antibody is then passed over a column of protein A Sepharose Fast Flow in order to remove intact material and Fc fragments. The Fab fraction is extensively dialysed against PBS and concentrated to about 3 mg/mi. (Papain and E64 are from Roche Molecular Biochemicals ).
HPLC, Mass Spectrometry and N-terminal amino acid sequencing of VL and VH regions: a) Reduction and Alkylation: Purified, dried 11C7 antibody are dissolved in 40 ul of 8M urea, 0.4M NH4HCO3, pH 8.3. 60 ug DTT (Calbiochem), pre-dissolved in 10 ul of the same buffer as the protein, are added. Reduction is performed at 50°C for 30 min under argon (100 fold molar excess of DTT over protein thiols). After reduction, the sample is cooled

to room temperature. 304 ug of iodoacetamide (Sigma Ultra, 1-1149) dissolved in the same buffer as the protein is added. Carboxamidomethylation is carried out at room temperature for 15 min in the dark. 1 |il (3-mercaptoethanoI is added to quench the • reaction.
b) Isolation of Heavy- and Light-Chain: Carboxamidomethylated heavy and light chains of antibody are isolated by Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) on a Hewlett Packard 1090M HPLC System with DR5 pumping system and diode-array UV detector. The conditions for chromatography are: PerSeptive Biosystems Poros 2.1x100 mm column packed with R1/H material; flow is 0.5 ml/min; solvents: (A) 0.1% TFA in water and (B) 0.09% TFA / acetonitril/water 9:1; gradient 25-70% B in 8 minutes at 80°C; detection at 218 / 280 nm.
c) LC-ESI-MS: Mass spectrometry is carried out using a Q-Tof (Micromass, Manchester, UK) quadrupole time-of-flight hybrid tandem mass spectrometer equipped with a Micromass Z-type electrospray ionization source (ESI). Acquisition mass range is typically m/z 500-2000. Data are recorded and processed using MassLynx software. Calibration of the 500-2500 m/z scale is achieved by using the multiple-charged ion peaks of horse heart myoglobin (MW 16951.5).
d) HPLC-MS of heavy and light chain: Separation of reduced and carboxamidomethylated heavy and light chain is performed on a HP1100 HPLC system (Hewlett Packard, Palo-Alto, CA, USA) employing a 1mmx150mm LC Packings column packed with Perseptive Biosystems POROS R1/H. The column is held at 60°C. Sample volumes of 10 nl are injected onto the column using a CTC PAL autosampler (CTC, Zwingen, Switzerland) fitted with a Valco model C6UW HPLC valve (Valco, Houston, TXf USA) and a 10 \i\ injection loop. HPLC was controlled by MassLynx software (Micromass, Manchester, UK). UV detection is at 214 nm. Eluent A is water containing 0.05% TFA. Eluent B is a 1:9 mixture of water: acetonitrile containing 0.045% TFA. A gradient from 20% B to 90% B is run in 20 minutes at 80 °C. The flow rate is typically 60 nl/min. The total flow from the LC system is introduced into the UV detection cell, then the ESI source without any splitting. The HPLC system is controlled and the signal from the UV detector is processed using MassLynx software (Micromass, Manchester, UK). The following 5 signals are detected:
Table 1:

Measured: Signal interpretation
A= 50959.0 Da B= 51119.5 Da H-Chain with carboxamidomethyl-cysteine (CAMCys)* Signal A+162 Da (= hexose)**


d) N-terminal amino acid sequencing of VL and VH regions: Collected H+L chains peaks form HPLC are used for sequence analysis. Amino acid sequences are determined on a Hewlett Packard G1000A N-terminal Protein Sequencing System. The system performs automated Edman chemistry on protein samples retained on miniature adsorptive biphasic columns. An optimized chemistry method (double couple 3.0) is used to enhance chemical efficiency, minimize lags and herewith extend sequence analysis to about 50 residues. Analysis of PTH-amino acids is performed on an on-line Hewlett Packard HP1090 HPLC System equipped with a ternary pumping system and a narrowbore (2.1mm x 25cm) PTH column.
Results:
From mass analysis homogeneous heavy and light chain of mouse 11C7-lgG1 are determined. The H-chain is single glycosylated and there are two forms with a difference on the C-terminal Lysine. Total mass analysis of heavy and light chain shows a single mass for both chains. HPLC chromatography of mouse 11C7-lgG1 shows a single peak. After HPLC purification followed by reduction and alkylation pure heavy and light chain are available. N-terminal sequence degradation is performed on light-chain and heavy-chain. 45 to 55 amino acids from the N-terminal sequence of L-chain and H-chain are identified by sequence degradation. Light Chain


Example 5: Cloning of the heavy and light chain genes of mouse 11C7 mAb
Total RNA is prepared from 107 hybridoma cells (clone 11C7) using TriPure reagent (Roche diagnostics, Germany, Cat.# 1667157) according to the manufacturers instructions. For cDNA synthesis, mRNA is isolated from above prepared total RNA using Oligotex Resin (Qiagen, Germany, cat. # 70022).
cDNA is generated by reverse transcription using the following conditions: 2 /vlmRNA, 2 //I 10 x reverse transcription buffer, 2 /yl (dT)2o primer (10 //M), 0.5 /J\ RNasin (Promega, 40 U/ml), 2 fj\ dNTPs (5 mM each), 1 p\ Omniscript™ reverse transcriptase (Qiagen, Cat # 205110), 10.5 //I ddH20, Reactions hr at 37°C. For PCR amplification of cDNA encoding for the VH and VL the proofreading enzyme ProofStart™ DNA polymerase is used.
PCR of light and heavy chain: Reaction mix: 2//I cDNA , 5//110 x reaction buffer, 3/yl dNTPs (5 mM each), 2/vl 5'primer (10//M) (see Table 2), 2//I 3'primer (10/yM) (see Table 2), 1 //I ProofStart (Qiagen, Cat # 202203), 36 //I ddH20. PCR conditions: 95°C/5 min, (95°C/40 sec, 53°C/1 min, 72°C 1 min) x 35, 72°C/10 min. The resulting PCR products are ligated directly into pCRbluntTOPO (Invfrrogen). The ligation mix is transfected into TOP 10 cells (Invitrogen) and several clones are picked. The nucleotide sequences of the variable part of the heavy chain of the 11C7 mAb (V-H, SEQ ID NO: 43) and of the light chain of the 11C7 mAb (V-Lf SEQ ID NO: 44) cDNas are determined on an ABI sequencer. The subsequent amino acid sequence of V-H and V-L are shown in SEQ ID NO: 2 (V-H) and SEQ ID NO: 3 (V-L). Primers used for PCR amplification of the VH and VL cDNAs; all primers are synthesized by MWG Biotech, Germany.



Example 6: Binding of 11C7 and Fab to Nogo-A domains using ELISA
Greiner 96 well PS plates (#655161) are coated with 0.4-2ug/ml Nogo protein fragments in PBS (100ul/weil) covered and incubated 4 hours at room temperature. Plates are flicked and refilled with 200ul/well blocking buffer (PBS+2% BSA), covered and incubated. 1h at RT or overnight at 4 °C, then washed 4 times with water and PBS. Different concentrations of mouse 11C7 mAb or 11C7 Fab are diluted in PBS +2% BSA (100 ul/well), and incubated 2h at RT or overnight at 4 °C. Wash step is repeated and Goat anti-mouse IgG conjugated with horse radish peroxidase (HRP) at a dilution of 1:5000 (ICN #55550) in PBS/0.1 %BSA /0.1%Nonidet 40 (100 ul/well) is added and incubated. 2h at RT or overnight at 4 °C and wash step is repeated. HRP reaction is started by adding 100 ul/well BM blue POD (Roche #1484281) and incubated in the dark at RT for 15 minutes . H2S04 50ul/well 1M is added to stop HRP substrate reaction and the optical density is determinated using a microplate reader (Packard Spectra Count) set to 450nm.
The mouse 11C7 mAb binds to human NiG, rat NiG, mouse NiG, rat NiG-D20 and peptide 472 at very low concentrations of 0.02 to 2.5 nM. Binding to human NiG, rat NiG, mouse NiG at very low concentration is confirmed by the very high affinity (Kd 0.1 - 0.44nM Biosensor affinity measurements) and is consistent with the fact that 472 peptide with the exception of 2-3 amino acids is identical in human compared to rat and mouse equivalent region. The specificity of the binding is indicated by the fact that the mouse 11C7 mAb does not show any binding at all to rat NiG-D6 and Nogo-66 fragments over the same concentration range. The Fab monovalent fragment bound to human NiG and rat NiG-D20 at concentrations 0.025 to 25nM and showed no binding to rat NiG-D6 and Nogo-66 fragments over the same concentration range. The Kd measured by Biosensor was 7.14 nM for human NiG.

Example 7: Biosensor affinity measurements for mouse 11C7-!gG1 and Fab to Nogo-A domains
The affinity of the mouse 11C7 mAb and of the 11C7 Fab are measured by surface plasmon resonance (SPR) using a BIAcore 2000 optical biosensor (Biacore, Uppsala, Sweden) according to the manufacture's instructions (see Figure 2). Recombinant human, mouse, and rat NIG are covalently attached to three separate flow cells of a CM5 sensor chip using amine-coupling chemistry. Briefly; the carboxymethlyladed dextran matrix is activated by injecting 35ul of a solution containing 0.025M NHS and 0.1 M EDC. For the immobilization on the sensor chip the recombinant mouse, human, and rat NIG are diluted in 0.01 M citrate buffer at a pH varying between 3.5 and 4.5 and injected at a flow rate of 5ul/min to achieve coupling levels allowing affinity measurements. The deactivation of the remaining NHS-ester group is performed by injection of 35ul of 1M ethanoiamine hydrochloride (pH 8.5). The surface of the sensor chip is regenerated by injecting 5ul 0.1 M HCI. For the measurement of the affinity the antibodies are injected at different concentration, ranging from 0.50nM to 100nM at a flow rate of 200 ul/min. After each injection the sensor chip surface is regenerated with the injection of 10 ul 0.1 M HCI without loss of binding activity on the .... surface. The kinetic constants, ka and kd and the affinity constants KA and KD are evaluated using the BIAevaluations 3.0 software supplied by the manufacturer.
Affinity measurement in BIAcore: The kinietc and the affinity binding constants of the mouse 11C7 mAb and the 11C7 derived monovalent Fab fragment to recombinat NogoA are measured in real time using surface plasmon resonance (SPR) technology (Biacore). For this analysis recombinant human, mouse and rat NIGs are coupled on three independent sensor chip surfaces and different concentrations of the antibodies are injected. Kinetic parameters of the binding interactions are derived from the sensorgrams by non-linear curve fitting. The affinity constants at equilibrium of mouse 11C7-igG1 are KD= 0.1 nM, KD= 0.4nM and KD= 0.19nM for human, rat, and mouse NIG respectively (table 3). For the 11C7 derived Fab fragment the affinity constant to human NIG is KD= 7.14nM. The lower affinity of the Fab fragment results from a decrease of both kinetic constants, association and dissociation {ka, kd). Lower affinity of the Fab fragment compared to the complete antibody is probably related to the avidity effect, which is lacking in the monomeric Fab.





SEQUENCE LISTING
Novartis AG
Organic Coirpound
4-32761P1/UNZ
44
PatentIn version 3.]
1
18
PRT
Rattus norvegicus


PEPTIDE
(1)..(18)
rat NogoA_623-640
1
Ser Tyr Asp Ser ILe Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu
1 10 15
Glu Ala
2
221
PRT
Mus musculus

CHAIN
(1)..(221) • •
Variable part of Heavy Chain of 11C7 with leader sequence

2
Met Asp Phe Gly Leu ILe Phe Phe ILe Val Gly Leu Leu Lys Gly Val
15 10 15
Gin Cys Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro
20 25 30
Gly Gly Ser Leu Lys Leu Ser Cys Val Val Ser Gly Phe Asp Phe Arg
35 40 45
Arg Asn Trp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp lie Gly Glu lie Asn Pro Asp Ser Ser Lys lie Asn Tyr Thr Pro
65 70 75 80
Ser Leu Lys Asp Lys Phe ILe ILe Ser Arg Asp Asn Ala Lys Asn Thr
85 90 95
Leu Tyr Leu GIn Val Ser Thr Val Arg Ser Glu Asp Thr Ala Leu Tyr
100 105 110
Tyr Cys Val Arg Pro Val Trp Met Tyr Ala Met Asp Tyr Trp Gly Gin
115 120 125
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

130 135 140
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gin Thr Asn Ser Met Val Thr
145 150 155 160
■ ******
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
165 170 175
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
180 185 . . 190
Leu Gin Ser Asp Leu Tyr Thr Leu Ser. Ser Ser Val Thr Val Pro Ser
195 200 205
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
210 215 220
3
238
PRT
Mus musculus

CHAIN

(1)..(238)
Light Chain of 11C7 with leader sequence
3
Met Ser Pro Ala GIn Phe Leu Phe Leu Leu Val Leu Trp ILe Arg Glu
1 5 10 15
Thr Ser Gly Asp Val Leu Leu Thr GLn Thr Pro Leu Thr Leu Ser ILe
20 25 30
Thr He Gly Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu
35 40 45
Leu His Ser Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gin Arg Pro
50 55 60
Gly Gin Ser Pro Lys Arg Leu ILe Tyr Leu Val Ser Lys Leu Asp Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys ILe Ser Arg Val Glu Ala Glu Asp Leu- Gly Leu Tyr Tyr Cys
100 105 110
Trp Gin Gly Thr His Phe Pro Gin Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125
Glu ILe Lys Arg Ala Asp Ala Ala Pro Thr Val Ssr ILe Fhe Pro Pro
130 135 140
Ser Ser Glu Gin Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
145 150 155 160
Asn Asn Phe Tyr Pro Lys Asp ILe Asn Val Lys Trp Lys ILe Asp Gly
165 170 175
Ser Glu Arg Gin Asn Gly Val Leu Asn Ser Trp Thr Asp Gin Asp Ser
180 185 190
Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
195 200 205
Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr Kis Lys Thr
210 215 220
Ser Thr Ser Pro ILe Val Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
4 3919 DNA

Homo sapiens

CDS
(1)..(3579)
Human NogoA
4
atg gaa gac ctg gac cag tct cct ctg gtc teg tec teg gac age cca 48
Met Glu Asp Leu Asp Gin Ser Pro Leu Val Ser Ser Ser Asp Ser Pro
15 10 15
ccc egg ccg cag ccc gcg ttc aag tac cag ttc gtg agg gag ccc gag 96
Pro Arg Pro Gin Pro Ala Phe Lys Tyr Gin Phe Val Arg Glu Pro Glu
20 25 30
gac gag gag gaa gaa gag gag gag gaa gag gag gac gag gac gaa gac 144
Asp Glu Glu Glu Glu Glu Glu Glu. Glu Glu Glu Asp Glu Asp Glu Asp
35 40 45
ctg gag gag ctg gag gtg ctg gag agg aag ccc gec gee ggg ctg tec 192
Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly Leu Ser
50 55 60
gcg gec cca gtg ccc ace gec cct gee gec ggc gcg ccc ctg atg gac 240
* Ala Ala Pro Val Pro Thr Ala Pro Ala Ala Gly Ala Pro Leu Met Asp
65 70 75 80
ttc gga aat gac ttc gtg ccg ccg gcg ccc egg gga ccc ctg ccg gee 288 Phe Gly Asn Asp Phe Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala

85 90 95
get ccc ccc gtc gec ccg gag egg cag ccg tct rgg gac ccg age ceg 336 Ala Pro Pro Val Ala Pro Glu Arg Gin Pro Ser Trp Asp Pro Ser Pro
100 105 110
gtg teg teg ace gtg ccc gcg cca tec ccg ctg ret get gee gca gtc 334
Val Ser Ser Thr Val Pro Ala Pro Ser Pro Leu Ser Ala Ala Ala Val
115 120 125
teg ccc tec aag etc cct gag gac gac gag cct ccg gec egg cct ccc 432
Ser Pro Ser Lys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro
130 135 . . 140
cct cct ccc ccg gec age gtg age ccc cag gca gag ccc gtg tgg ace 430
Pro Pro Pro Pro Ala Ser Val Ser Pro Gin Ala Glu Pro Val Trp Thr
145 150 155 160
ccg cca gec ccg get ccc gee gcg ccc ccc tec ace ccg gec gcg ccc 528 Pro Pro Ala Pro Ala Pro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro
165 170 175
aag cgc agg ggc tec teg ggc tea gtg gat gag ace ctt ttt get ctt 576 Lys Arg Arg Gly Ser Ser Gly Ser Val Asp Glu Thr Leu Phe Ala Leu
180 185 190
cct get gca tct gag cct gtg ata cgc tec tct gca gaa aat atg gac 624
Pro Ala Ala Ser Glu Pro Val lie Arg Ser Ser Ala Glu Asn Met Asp
195 200 205
ttg aag gag cag cca ggt aac act att teg get ggt caa gag gat ttc 672
Leu Lys Glu Gin Pro Gly Asn Thr lie Ser Ala Gly Gin Glu Asp Phe
210 215 220 - -
cca tct gtc ctg ctt gaa act get get tct ctt cct tct ctg tct cct 720
Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu Pro Ser Leu Ser Pro
225 230 235 240

etc tea gec get tct ttc aaa gaa cat gaa tac ctt ggt aat ttg tea 763 Leu Ser Ala Ala Ser Phe Lys Glu His Glu Tyr Leu Gly Asn Leu Ser
245 250 2.55
aca gta tta ccc act gaa gga aca ctt caa gaa aat gtc agt gaa get 816 Thr Val Leu Pro Thr Glu Gly Thr Leu Gin Glu Asn Val Ser Glu Ala
260 265 270
tct aaa gag gtc tea gag aag gca aaa act eta etc ata gat aga gat 864
Ser Lys Glu Val Ser Glu Lys Ala Lys Thr Leu Leu lie Asp Arg Asp
275 280 285
tta aca gag ttt tea gaa tta gaa tac tea gaa atg gga tea teg ttc 912
Leu Thr Glu Phe Ser Glu Leu Glu Tyr Ser Glu Met Gly Ser Ser Phe
290 295 300
agt gtc tct cca aaa gca gaa tct gec gta ata gta gca aat cct agg 960
Ser Val Ser Pro Lys Ala Glu Ser Ala Val He Val Ala Asn Pro Arg
305 "' 310 315 320
gaa gaa ata ate gtg aaa aat aaa gat gaa gaa gag aag tta gtt agt 1008 Glu Glu lie He Val Lys Asn Lys Asp Glu Glu Glu Lys Leu Val Ser
325 330 335
aat aac ate ctt cat aat caa caa gag tta cct aca get ctt act aaa 1056
Asn Asn He Leu His Asn Gin Gin Glu Leu Pro Thr Ala Leu Thr Lys
340 345 350
ttg gtt aaa gag gat gaa gtt gtg tct tea gaa aaa gca aaa gac agt 1104
Leu Val Lys Glu Asp Glu Val Val Ser Ser Glu Lys Ala Lys Asp Ser
355 360 365
ttt aat gaa aag aga gtt gca gtg gaa get cct atg agg gag gaa tat 1152
Phe Asn Glu Lys Arg Val Ala Val Glu Ala Pro Met Arg Glu Glu Tyr
370 375 380

gca gac ttc aaa cca ttt gag cga gta tgg gaa gtg aaa gat agt aag 1200
Ala Asp Phe Lys Pro Phe Glu Arg Val Trp Glu Val Lys Asp Ser Lys
385 390 395 400
gaa gat agt gat atg ttg get get gga ggt aaa ate gag age aac ttg 1248 Glu Asp Ser Asp Met Leu Ala Ala Gly Gly Lys He Glu Ser Asn Leu
405 410 415
gaa agt aaa gtg gat aaa aaa tgt ttt gca gat age ctt gag caa act 1296 Glu Ser Lys Val Asp Lys Lys Cys Phe Ala Asp Ser Leu Glu Gin Thr
420 425 430
aat cac gaa aaa gat agt gag agt agt aat gat gat act tct ttc ccc 1344
Asn His Glu Lys Asp Ser Glu Ser Ser Asn Asp Asp Thr Ser Phe Pro
435 440 445
agt acg cca gaa ggt ata aag gat cgt tea gga gca tat ate aca tgt 1392
Ser Thr Pro Glu Gly He Lys Asp Arg Ser Gly Ala Tyr He Thr Cys
450 455 460
get ccc ttt aac cca gca gca act gag age att gca aca aac att ttt 1440
Ala Pro Phe Asn Pro Ala Ala Thr Glu Ser He Ala Thr Asn He Phe
465 470 475 480
cct ttg tta gga gat cct act tea gaa aat aag ace gat gaa aaa aaa 1488 Pro Leu Leu Gly Asp Pro Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys
485 490 495
ata gaa gaa aag aag gec caa ata gta aca gag aag aat act age ace 1536
He Glu Glu Lys Lys Ala Gin He Val Thr Glu Lys Asn Thr Ser Thr
500 505 510
aaa aca tea aac cct ttt ctt gta gca gca cag gat tct gag aca gat 1584
Lys Thr Ser Asn Pro Phe Leu Val Ala Ala Gin Asp Ser Glu Thr Asp
515 520 525
tat gtc aca aca gat aat tta aca aag gtg act gag gaa gtc gtg gca 1632

Tyr Val Thr Thr Asp Asn Leu Thr Lys Val Thr Glu Glu Val Val Ala
530 535 540
aac atg cct gaa ggc ctg act cca gat tta gta cag gaa gca tgt gaa 1533
Asn Met Pro Glu Gly Leu Thr Pro Asp Leu Val Gin Glu Ala Cys Glu
545 550 555 560
agt gaa ttg aat gaa gtt act ggt aca aag att get tat gaa aca aaa 1723 Ser Glu Leu Asn Glu Val Thr Gly Thr Lys He Ala Tyr Glu Thr Lys
565 570 575
atg gac ttg gtt caa aca tea gaa gtt atg caa gag tea etc tat cct 1775 Met Asp Leu Val Gin Thr Ser Glu Val Met Gin Glu Ser Leu Tyr Pro
580 585 590
gca gca cag ctt tgc cca tea ttt gaa gag tea gaa get act cct tea 1824
Ala Ala Gin Leu Cys Pro Ser Phe Glu Glu Ser Glu Ala Thr Pro Ser
595 600 605
cca gtt ttg cct gac att gtt atg gaa gca cca ttg aat tct gca gtt 1872
Pro Val Leu Pro Asp He Val Met Glu Ala Pro Leu Asn Ser Ala Val
610 615 620
cct agt get ggt get tec gtg ata cag ccc age tea tea cca tta gaa 1920
Pro Ser Ala Gly Ala Ser Val He Gin Pro Ser Ser Ser Pro Leu Glu
625 630 635 640
get tct tea gtt aat tat gaa age ata aaa cat gag cct gaa aac ccc 1968 Ala Ser Ser Val Asn Tyr Glu Ser He Lys His Glu Pro Glu Asn Pro
645 650 655
cca cca tat gaa gag gee atg agt gta tea eta aaa aaa gta tea gga 2016
Pro Pro Tyr Glu Glu Ala Met Ser Val Ser Leu Lys Lys Val Ser Gly
660 665 670
ata aag gaa gaa att aaa gag cct gaa aat att aat gca get ctt caa 2064 He Lys Glu Glu He Lys Glu Pro Glu Asn He Asn Ala Ala Leu Gin

675 680 685
gaa aca gaa get cct tat ata tct att gca tgt gat tta att aaa gaa 2112
Glu Thr Glu Ala Pro Tyr lie Ser lie Ala Cys Asp Leu lie Lys Glu
690 595 700
aca aag ctt tct get gaa cca get ccg gat ttc tct gat tat tea gaa 2160
Thr Lys Leu Ser Ala Glu Pro Ala Pro Asp Phe Ser Asp Tyr Ser Glu
705 710 715 720
atg gca aaa gtt gaa cag cca gtg cct gat cat tct gag eta gtt gaa 2208 Met Ala Lys Val Glu Gin Pro Val Pro Asp His Ser Glu Leu Val Glu
725 . . 730 735
gat tec tea cct gat tct gaa cca gtt gac tta ttt agt gat gat tea 2256
Asp Ser Ser Pro Asp Ser Glu Pro Val Asp Leu Phe Ser Asp Asp Ser
740 745 750
ata cct gac gtt cca caa aaa caa gat gaa act gtg atg ctt gtg aaa 2304
He Pro Asp Val Pro Gin Lys Gin Asp Glu 'Thr Val Met Leu Val Lys
755 760 765
gaa agt etc act gag act tea ttt gag tea atg ata gaa tat gaa aat 2352
Glu Ser Leu Thr Glu Thr Ser Phe Glu Ser Met He Glu Tyr Glu Asn
770 775 780
aag gaa aaa etc agt get ttg cca cct gag gga gga aag cca tat ttg 2400
Lys Glu Lys Leu Ser Ala Leu Pro Pro Glu Gly Gly Lys Pro Tyr Leu
785 790 795 800
gaa tct ttt aag etc agt tta gat aac aca aaa gat ace ctg tta cct 2448 Glu Ser Phe Lys Leu Ser Leu Asp Asn Thr Lys Asp Thr Leu Leu Pro
805 810 - - 815
gat gaa gtt tea aca ttg age aaa aag gag aaa att cct ttg cag atg 2496
Asp Glu Val Ser Thr Leu Ser Lys Lys Glu Lys He Pre Leu Gin Met
820 825 330

gag gag etc agt act gca gtt tat tea aat gat gac tta ttt att tct 2544
Glu Glu Leu Ser Thr Ala Val Tyr Ser Asn Asp Asp Leu Phe He Ser
835 840 845
aag gaa gca cag ata aga gaa act gaa acg ttt tea gat tea tct cca 2592
Lys Glu Ala Gin He Arg Glu Thr Glu Thr Phe Ser Asp Ser Ser Pro
850 855 860
att gaa att ata gat gag ttc cct aca ttg ate agt tct aaa act gat 2640
He Glu He He Asp Glu Phe Pro Thr Leu He Ser Ser Lys Thr Asp
865 870 875 880
tea ttt tct aaa tta gec agg gaa tat act gac eta gaa gta tec cac 2688 Ser Phe Ser Lys Leu Ala Arg Glu Tyr Thr Asp Leu Glu Val Ser His
885 890 895
aaa agt gaa att get aat gec ccg gat gga get ggg tea ttg cct tgc 2736
Lys Ser Glu He Ala Asn Ala Pro Asp Gly Ala Gly Ser Leu Pro Cys
900 905 910
aca gaa ttg ccc cat gac ctt tct ttg aag aac ata caa ccc aaa gtt 2784
Thr Glu Leu Pro His Asp Leu Ser Leu Lys Asn He Gin Pro Lys Val
915 920 925
gaa gag aaa ate agt ttc tea gat gac ttt tct aaa aat ggg tct get 2832
Glu Glu Lys He Ser Phe Ser Asp Asp Phe Ser Lys Asn Gly Ser Ala
930 935 940
aca tea aag gtg etc tta ttg cct cca gat gtt tct get ttg gec act 2880
Thr Ser Lys Val Leu Leu Leu Pro Pro Asp Val Ser Ala Leu Ala Thr
945 950 955 960
caa gca gag ata gag age ata gtt aaa ccc aaa gtt ctt gtg aaa gaa 2928 Gin Ala Glu He Glu Ser He Val Lys Pro Lys Val Leu Val Lys Glu
965 970 975

get gag aaa aaa ctt cat tec gat aca gaa aaa gag gac aga tea cca 2976 Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp Arg Ser Pro
980 985 990
tct get ata ttt tea gca gag ctg agt aaa act tea gtt gtt gac etc 3024
Ser Ala lie Phe Ser Ala Glu Leu Ser Lys Thr Ser Val Val Asp Leu
995 1000 1005
ctg tac tgg aga gac att aag aag act gga gtg gtg ttt ggt gec 3069
Leu Tyr Trp Arg Asp lie Lys Lys Thr Gly Val Val Phe Gly Ala
1010 1015 1020
age eta ttc ctg ctg ctt tea ttg aca gta ttc age att gtg age 3114
Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser He Val Ser
1025 1030 1035
gta aca gee .tac att gee ttg gec ctg etc tct gtg ace ate age 3159
Val Thr Ala Tyr He Ala Leu Ala Leu Leu Ser Val Thr He Ser
1040 1045 1050
ttt agg ata tac aag ggt gtg ate caa get ate cag aaa tea gat 3204
Phe Arg He Tyr Lys Gly Val He Gin Ala He Gin Lys Ser Asp
1055 1060 1065
gaa ggc cac cca ttc agg gca tat ctg gaa tct gaa gtt get ata 3249
Glu Gly' His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala He
1070 1075 1080
tct gag gag ttg gtt cag aag tac agt aat tct get ctt ggt cat 3294
Ser Glu Glu Leu Val Gin Lys Tyr Ser Asn Ser Ala Leu Gly His
1085 1090 1095
gtg aac tgc acg ata aag gaa etc agg cgc etc ttc tta gtt gat 3339
Val Asn Cys Thr He Lys Glu Leu Arg Arg Leu Phe Leu Val Asp
1100 1105 1110
gat tta gtt gat tct ctg aag ttt gca gtg ttg atg tgg gta ttt 3384

Asp Leu Val Asp Ser Leu Lys Phe Ala Val Leu Met Trp Val Phe
1115 1120 1125
ace tat gtt ggt gec ttg ttt aat ggt ctg aca eta ctg att ttg 3429
Thr Tyr Val Gly Ala Leu Phe Asn Gly Leu Thr Leu Leu lie Leu
1130 1135 1140
get etc att tea etc ttc agt gtt cct gtt ate tat gaa egg cat 3474
Ala Leu lie Ser Leu Phe Ser Val Pro Val lie Tyr Glu Arg His
1145 1150 1155
cag gca cag ata gat cat tat eta gga ctt gca aat aag aat gtt 3519
Gin Ala Gin lie Asp His Tyr Leu Gly Leu Ala Asn Lys Asn Val
1160 1165 1170
aaa gat get atg get aaa ate caa gca aaa ate cct gga ttg aag 3564
Lys Asp Ala Met Ala Lys lie Gin Ala Lys lie Pro Gly Leu Lys
1175 1180 1185
cgc aaa get gaa tga aaacgcccaa aataattagt aggagttcat ctttaaaggg 3619 Arg Lys Ala Glu 1190
gatattcatt tgattatacg ggggagggtc agggaagaac gaaccttgac gttgcagtgc 3679
agtttcacag atcgttgtta gatctttatt tttagccatg cactgttgtg aggaaaaatt 3739
acctgtcttg actgecatgt gttcatcatc ttaagtattg taagctgeta tgtatggatt 3799
taaaccgtaa tcatatcttt ttcctatctg aggcactggt ggaataaaaa acctgtatat 3859
tttactttgt tgcagatagt cttgccgcat cttggcaagt tgcagagatg gtggagctag 3919
5

1192
PRT
Homo sapiens
5
Met Glu Asp Leu Asp Gin Ser Pro Leu Val Ser Ser Ser Asp Ser Pro
15 10 15
Pro Arg Pro Gin Pro Ala Phe Lys Tyr Gin Phe Val Arg Glu Pro Glu
20 25 30
Asp Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Glu Asp
35 "' 40 45
Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly Leu Ser
50 55 60
Ala Ala Pro Val Pro Thr Ala Pro Ala Ala Gly Ala Pro Leu Met Asp
65 70 75 80
Phe Gly Asn Asp Phe Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala
85 90 95
to
Ala Pro Pro Val Ala Pro Glu Arg Gin Pro Ser Trp I-JSV Pro Ser Pro
100 105 110

Val Ser Ser Thr Val Pro Ala Pro Ser Pro Leu Ser Ala Ala Ala Val
115 120 125
Ser Pro Ser Lys Leu Pro Glu Asp Asp Glu Pre Pro Ala Arg Pro Pro
130 135 140
Pro Pro Pro Pro Ala Ser Val Ser Pro Gin Ala Glu Pro Val Trp Thr
145 150 155 160
Pro Pro Ala Pro Ala Pro Ala Ala Pro Pro Ser Thr Pro Ala Ala Pro
165 170 175
Lys Arg Arg Gly Ser Ser Gly Ser Val Asp_Glu Thr Leu Phe Ala Leu
180 185 190
Pro Ala Ala Ser Glu Pro Val lie Arg Ser Ser Ala Glu Asn Met Asp
195 200 205
Leu Lys Glu Gin Pro Gly Asn Thr lie Ser Ala Gly Gin Glu Asp Phe
210 215 220
Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu Pro Ser Leu Ser Pro
225 230 235 240
Leu Ser Ala Ala Ser Phe Lys Glu His Glu Tyr Leu Gly Asn Leu Ser
245 250 255

Thr Val Leu Pro Thr Glu Gly Thr Leu Gin Glu Asn Val Ser Glu Ala
260 265 270
Ser Lys Glu Val Ser Glu Lys Ala Lys Thr Leu Leu lie Asp Arg Asp
275 280 285
Leu Thr Glu Phe Ser Glu Leu Glu Tyr Ser Glu Met Gly Ser Ser Phe
290 295 300
Ser Val Ser Pro Lys Ala Glu Ser Ala Val lie Val Ala Asn Pro Arg * •
305 310 315 320
.Glu Glu He He Val Lys Asn Lys Asp Glu Glu Glu Lys Leu Val Ser
325 330 335
Asn Asn He Leu His Asn Gin Gin Glu Leu Pro Thr Ala Leu Thr Lys
340 345 350
Leu Val Lys Glu Asp Glu Val Val Ser Ser Glu Lys Ala Lys Asp Ser
355 360 365
Phe Asn Glu Lys Arg Val Ala Val Glu Ala Pro Met Arg Glu Glu Tyr
370 375 380
Ala Asp Phe Lys Pro Phe Glu Arg Val Trp Glu Val Lys Asp Ser Lys
385 390 395 400
Glu Asp Ser Asp Met Leu Ala Ala Gly Gly Lys He Glu Ser Asn Leu

405 410 415
Glu Ser Lys Val Asp Lys Lys Cys Phe Ala Asp Ser Leu Glu Gin Thr
420 425 430
Asn His Glu Lys Asp Ser Glu Ser Ser Asn Asp Asp Thr Ser Phe Pro
435 440 445
Ser Thr Pro Glu Gly lie Lys Asp Arg Ser Gly Ala Tyr He Thr Cys
450 455 - * 460
Ala Pro Phe Asn Pro Ala Ala Thr Glu Ser He Ala Thr Asn He Phe
465 470 475 480
Pro Leu Leu Gly Asp Pro Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys
485 490 495
He Glu Glu Lys Lys Ala Gin He Val Thr Glu Lys Asn Thr Ser Thr
500 505 510
Lys Thr Ser Asn Pro Phe Leu Val Ala Ala Gin Asp Ser Glu Thr Asp
515 520 525
Tyr Val Thr Thr Asp Asn Leu Thr Lys Val Thr Glu Glu Val Val Ala
530 535 540 - -
Asn Met Pro Glu Gly Leu Thr Pro Asp Leu Val Gin Glu Ala Cys Glu
545 .550 555 560

Ser Glu Leu Asn Glu Val Thr Gly Thr Lys He Ala Tyr Glu Thr Lys
565 570 575
Met Asp Leu Val Gin Thr Ser Glu Val Met Gin Glu Ser Leu Tyr Pro
580 585 590
Ala Ala Gin Leu Cys Pro Ser Phe Glu Glu Ser Glu Ala Thr Pro Ser
595 600 605
Pro Val Leu Pro Asp lie Val Met Glu Ala Pro Leu Asn Ser Ala Val
610 615 620
Pro Ser Ala Gly Ala Ser Val He Gin Pro Ser Ser Ser Pro Leu Glu
625 " 630 635 640
Ala Ser Ser Val Asn Tyr Glu Ser He Lys His Glu Pro Glu Asn Pro
645 650 655
Pro Pro Tyr Glu Glu Ala Met Ser Val Ser Leu Lys Lys Val Ser Gly
660 665 670
He Lys Glu Glu He Lys Glu Pro Glu Asn He Asn Ala Ala Leu Gin
675 680 685
Glu Thr Glu Ala Pro Tyr He Ser He Ala Cys Asp Leu He Lys Glu
690 695 700

Thr Lys Leu Ser Ala Glu Pro Ala Pro Asp Phe Ser Asp Tyr Ser Glu
705 710 715 720
Met Ala Lys Val Glu Gin Pro Val Pro Asp His Ser Glu Leu Val Glu
725 730 735
Asp Ser Ser Pro Asp Ser Glu Pro Val Asp Leu Phe Ser Asp Asp Ser
740 745 750
He Pro Asp Val Pro Gin Lys Gin Asp Glu Thr Val Met Leu Val Lys
755 760 765
Glu Ser Leu Thr Glu Thr Ser Phe Glu Ser Met He Glu Tyr Glu Asn
770 775 780
Lys Glu Lys Leu Ser Ala Leu Pro Pro Glu Gly Gly Lys Pro Tyr Leu
785 790 795 800
Glu Ser Phe Lys Leu Ser Leu Asp Asn Thr Lys Asp Thr Leu Leu Pro
805 810 815
Asp Glu Val Ser Thr Leu Ser Lys Lys Glu Lys He Pro Leu Gin Met
820 825 830
Glu Glu Leu Ser Thr Ala Val Tyr Ser Asn Asp Asp Leu Phe He Ser
835 840 845

Lys Glu Ala Gin lie Arg Glu Thr Glu Thr Phe Ser Asp Ser Ser Pro
850 855 360
lie Glu lie lie Asp Glu Phe Pro Thr Leu lie Ser Ser Lys Thr Asp
865 870 875 880
Ser Phe Ser Lys Leu Ala Arg Glu Tyr Thr Asp Leu Glu Val Ser His
885 890 895
Lys Ser Glu lie Ala Asn Ala Pro Asp Gly Ala Gly Ser Leu Pro Cys
900 905 910
Thr Glu Leu Pro His Asp Leu Ser Leu Lys Asn lie Gin Pro Lys Val
915 920 925
Glu Glu Lys lie Ser Phe Ser Asp Asp Phe Ser Lys Asn Gly Ser Ala
930 935 940
Thr Ser Lys Val Leu Leu Leu Pro Pro Asp Val Ser Ala Leu Ala Thr
945 950 955 960
Gin Ala Glu He Glu Ser He Val Lys Pro Lys Val Leu Val Lys Glu
965 970 " 975
Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp Arg Ser Pro
980 985 S90
Ser Ala He Phe Ser Ala Glu Leu Ser Lys Thr Ser Val Val Asp Leu

995 1000 1005
Leu Tyr Trp Arg Asp He Lys Lys Thr Gly Val Val Phe Gly Ala
1010 1015 1020 ...■
Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser lie Val Ser
1025 1030 1035
Val Thr Ala Tyr He Ala Leu Ala Leu Leu Ser Val Thr He Ser
1040 - • 1045 1050
Phe Arg - He Tyr Lys Gly Val He Gin Ala He Gin Lys Ser Asp
1055 1060 1065
Glu Gly His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala He
1070 1075 1080
Ser Glu Glu Leu Val Gin Lys Tyr Ser Asn Ser Ala Leu Gly His
1085 1090 1095
Val Asn Cys Thr He Lys Glu Leu Arg Arg Leu Phe Leu Val A-sp
1100 1105 1110
Asp Leu Val Asp Ser Leu Lys Phe Ala Val Leu Met Trp Val Phe
1115 1120 1125
Thr Tyr Val Gly Ala Leu Phe Asn Gly Leu Thr' Leu Leu He Leu
1130 1135 1140

Ala Leu He Ser Leu Phe Ser Val Pro Val He Tyr Glu Arg His
1145 1150 1155
Gin Ala Gin He Asp His Tyr Leu Gly Leu Ala Asn Lys Asn Val
1160 1165 1170
Lys Asp Ala Met Ala Lys He Gin Ala Lys He Pro Gly Leu Lys
1175 1180 1185
Arg Lys Ala Glu 1190
6
18
PRT
Homo sapiens

PEPTIDE
(1). .T13)
Human NogoA_623-640

6
Asn Tyr Glu Ser lie Lys His Glu Pro Glu Asn Pro Pro Pro Tyr Glu
15 10 15
Glu Ala
7
819
PRT.
Homo sapiens

PEPTIDE
(1)..(819)
human Nig
7
Asp Glu Thr Leu Phe Ala Leu Pro Ala Ala Ser Glu Pro Val He Arg
15 10 15

Ser Ser Ala Glu Asn Met Asp Leu Lys Glu Gin Pro Gly Asn Thr lie
20 25 30
Ser Ala Gly Gin Glu Asp ?he Pro Ser Val Leu Leu Glu Thr Ala Ala
35 40 45
Ser Leu Pro Ser Leu Ser Pro Leu Ser Ala Ala Ser Phe Lys Glu His
50 55 60
Glu Tyr Leu Gly Asn Leu Ser Thr Val Leu Pro Thr Glu Gly Thr Leu
65 70 75 - 80 "
Gin Glu Asn Val Ser Glu Ala Ser Lys Glu Val Ser Glu Lys Ala Lys
85 90 95
Thr Leu Leu lie Asp Arg Asp Leu Thr Glu Phe Ser Glu Leu Glu Tyr
100 105 110
Ser Glu Met Gly Ser Ser Phe Ser Val Ser Pro Lys Ala Glu Ser Ala
115 120 125
Val lie Val Ala Asn Pro Arg Glu Glu lie lie Val Lys Asn Lys Asp
130 135 140
Glu Glu Glu Lys Leu Val Ser Asn Asn lie Leu His Asn Gin Gin Glu
145 150 155 160

Leu Pro Tbr Ala Leu Thr Lys Leu Val Lys Glu Asp Glu Val Val Ser
165 170 175
Ser Glu Lys Ala Lys Asp Ser Phe Asn Glu Lys Arg Val Ala Val Glu
180 185 190
Ala Pro Met Arg Glu Glu Tyr Ala Asp Phe Lys Pro Phe Glu Arg Val
195 200 205
Trp Glir Val Lys Asp Ser Lys Glu Asp Ser Asp Met Leu Ala Ala Gly
210 215 220
Gly Lys lie Glu Ser Asn Leu Glu Ser Lys Val Asp Lys Lys Cys Phe
225 230 235 240
Ala Asp Ser Leu Glu Gin Thr Asn His Glu Lys Asp Ser Glu Ser Ser
245 250 255
Asn Asp Asp Thr Ser Phe Pro Ser Thr Pro Glu Gly lie Lys Asp Arg
260 265 270
Ser Gly Ala Tyr lie Thr Cys Ala Pro Phe Asn Pro Ala Ala Thr Glu
275 280 285
Ser He Ala Thr Asn He Phe Pro Leu'Leu Gly Asp Pro Thr Ser Glu
290 295 300
Asn Lys Thr Asp Glu Lys Lys He Glu Glu Lys Lys Ala Gin He Val

305 310 315 320
Thr Glu Lys Asn Thr Ser Thr Lys Thr Ser Asn Pro Phe Leu Val Ala
325 330 335
Ala Gin Asp Ser Glu Thr Asp Tyr Val Thr Thr Asp Asn Leu Thr Lys
340 345 350
Val Thr Glu Glu Val Val Ala Asn Met Pro Glu Gly Leu Thr Pro Asp
355 360 365
Leu Val Gin Glu Ala Cys Glu Ser Glu Leu Asn Glu Val Thr Gly Thr
370 375 380
Lys lie Ala Tyr Glu Thr Lys Met Asp Leu Val Gin Thr Ser Glu Val
385 390 395 400
Met Gin Glu Ser Leu Tyr Pro Ala Ala Gin Leu Cys Pro Ser Phe Glu
405 410 415
Glu Ser Glu Ala Thr Pro Ser Pro Val Leu Pro Asp lie Val Met Glu
420 425 430
Ala Pro Leu Asn Ser Ala Val Pro Ser Ala Gly Ala Ser Val He Gin
- - 435 " "440 445
Pro Ser Ser Ser Pro Leu Glu Ala Ser Ser Val Asn Tyr Glu Ser He
450 455 460

Lys His Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu Ala Met Ser Val
465 470 475 480
Ser Leu Lys Lys Val Ser Gly lie Lys Glu Glu He Lys Glu Pro Glu
485 490 495
Asn He Asn Ala Ala Leu Gin Glu Thr Glu Ala Pro Tyr He Ser He
500 505 510
Ala Cys Asp Leu He Lys Glu Thr Lys Leu Ser Ala Glu Pro Ala Pro
515 520 525 -
Asp Phe Ser Asp Tyr Ser Glu Met Ala Lys Val Glu Gin Pro Val Pro
530 535 540
Asp His Ser Glu Leu Val Glu Asp Ser Ser Pro Asp Ser Glu Pro Val
545 550 555 560
Asp Leu Phe Ser Asp Asp Ser He Pro Asp Val Pro Gin Lys Gin Asp
565 570 575
Glu Thr Val Met Leu Val Lys Glu Ser Leu Thr Glu Thr Ser Phe Glu
580 585 590
Ser Met He Glu Tyr Glu Asn Lys Glu Lys Leu Ser Ala Leu Pro Pro
595 600 605

Glu Gly Gly Lys Pro Tyr Leu Glu Ser Phs Lys Leu Ser Leu Asp Asn
610 515 620
Thr Lys Asp Thr Leu Leu Pro Asp Glu Val Ser Thr Leu Ser Lys Lys
625 630 635 640
Glu Lys lie Pro Leu Gin Met Glu Glu Leu Ser Thr Ala Val Tyr Ser
645 650 655
Asn Asp Asp Leu Phe lie Ser Lys Glu Ala Gin lie Arg Glu Thr Glu
660 665 ' 670
Thr Phe Ser Asp Ser Ser Pro lie Glu lie lie Asp Glu Phe Pro Thr
675 680 635
Leu lie Ser Ser Lys Thr Asp Ser Phe Ser Lys Leu Ala Arg Glu Tyr
690 695 700
Thr Asp Leu Glu Val Ser His Lys Ser Glu lie Ala Asn Ala Pro Asp
705 710 715 720
Gly Ala Gly Ser Leu Pro Cys Thr Glu Leu Pro His Asp Leu Ser Leu
725 730 735
Lys Asn lie Gin Pro Lys Val Glu Glu Lys lie Ser ?he Ser Asp Asp
740 745 750

Phe Ser Lys Asn Gly Ser Ala Thr Ser Lys Val Leu Leu Leu Pro Pro
755 760 765
Asp Val Ser Ala Leu Ala Thr Gin Ala Glu He Glu Ser He Val Lys
770 775 780
Pro Lys Val Leu Val Lys Glu Ala Glu Lys Lys Leu Pro Ser Asp Thr
785 ' 790 795 800
- Glu Lys Glu Asp Arg Ser Pro Ser Ala He Phe Ser Ala Glu Leu Ser
805 810 815
Lys Thr Ser
8
10
PRT
Mus musculus

BINDING
(1)..(10)
hypervariable part of heavy chain cf 11C7

3
Gly Phe Asp Phe Arg Arg Asn Trp Met Ser
15 10
9
17
PRT
Mus musculus

BINDING
(1).. (17)
hypervariable part of heavy chain of 11C7
9
Glu lie Asn Pro Asp Ser Ser Lys lie Asn Tyr Thr Pro Ser Leu Lys
1 5* 10 15
Asp

10
9
PRT
Mus musculus

BINDING
(1)..(9)
hypervariable part of heavy chain of 11C7
10
Pro Val Trp Met Tyr Ala Met Asp Tyr
1 5
11
16
PRT
Mus musculus


BINDING
(1).. (16)
hypervariable part of light chain of 11C7
11
Lys Ser Ser Gin Ser Leu Leu His Ser Asp Gly Lys Thr Tyr Leu Asn
1,5 10 15
12
7
PRT
Mus musculus

BINDING
(1)..(7)
hypervariable part of light chain of 11C7

12
Leu Val Ser Lys Leu Asp Ser
1 5
13
9
PRT
Mus musculus

* * -
BINDING
(1).. (9)
hypervariable part of light chain of 11C7
13
Trp Gin Gly Thr His Phe Pro Gin Thr
1 5
^ »
14

30
DNA
Mus musculus

misc_binding
(1)..(30)
DNA-CDR1-11C7
14
ggattcgatt ttagaagaaa ttggatgagt
15
51
DNA
Mus musculus
'
misc_binding

(1)..(51)
DNA-CDR2-11C7
15
gaaattaatc cagatagcag taagataaac tatacgccat ctctaaagga t
16
27
DNA
Mus musculus

irdsc_binding
(1)..(27)
DNA-CDR3-11C7
16
ccggtctgga tgtatgctat ggactac
17

48
DNA
Mus musculus

misc_binding
(1)r.(48)
DNA-CDR'l-irC7
17
aagtcaagtc agagcctctt gcatagtgat ggaaagacat atttgaat
18
21
DNA
Mus musculus

misc_binding

(1)..(21)
DNA-CDR'2-11C7
18
ctggtgtcta aactggactc t
19
27
DNA
Mus musculus

misc_binding
(1)..(27)
DNA-CDR'3-11C7
19
tggcaaggta cacattttcc tcagacg
20

54
DNA
Mus musculus

CDS
(1)..(54)
leader sequence for heavy chain of 11C7
20
atg gat ttt ggg ctg att ttt ttt att gtt ggt ctt tta aaa ggg gtc
Met Asp Phe Gly Leu He Phe Phe He Val Gly Leu Leu Lys Gly Val
15 10 15
cag tgt Gin Cys
21
18
PRT
Mus musculus

21
Met Asp Phe Gly Leu He Phe Phe He Val Gly Leu Leu Lys Gly Val
1 5 10 15
Gin Cys
22
57
DNA
Mus musculus

CDS
(1).. (57)
leader sequence for HC7-light chain
22
atg agt cct gcc cag ttc ctg ttt ctg tta gtg etc tgg att egg gaa
Met Ser Pro Ala Gin Phe Leu Phe Leu Leu Val Leu Trp He Arg Glu
15 10 15

ace age ggt Thr Ser Gly
23
19
PRT
Mus musculus
23
Met Ser Pro Ala Gin Phe Leu Phe Leu Leu Val Leu Trp lie Arg Glu
15 10 15
Thr Ser Gly
24
181
PRT
Homo sapiens


PEPTIDE (1)..(181) human Nig-D20
24
Gly Thr Lys He Ala Tyr Glu Thr Lys Met Asp Leu Val Gin Thr Ser
15 10 15
Glu Val Met Gin Glu Ser Leu Tyr Pro Ala Ala Gin Leu Cys Pro Ser
20 25 30
Phe Glu Glu Ser Glu Ala Thr Pro Ser Pro Val Leu Pro Asp He Val
35 40 45
Met Glu Ala Pro Leu Asn Ser Ala Val Pro Ser Ala Gly Ala Ser Val
50 55 60
He Gin Pro Ser Ser Ser Pro Leu Glu Ala Ser Ser Val Asn Tyr Glu
65 70 75 80
Ser He Lys His Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu Ala Met
85 90 95
Ser Val Ser Leu Lys Lys Val Ser Gly He Lys Glu Glu He Lys Glu
100 105 110

Pro Glu Asn lie Asn Ala Ala Leu Gin Glu Thr Glu Ala Pro Tyr lie
115 120 125
Ser He Ala Cys Asp Leu He Lys Glu Thr Lys Leu Ser Ala Glu Pro
130 135 140
Ala Pro Asp Phe Ser Asp Tyr Ser Glu Met Ala Lys Val Glu Gin Pro
145 150 155 160
Val Pro Asp His Ser Glu Leu Val Glu Asp Ser Ser Pro Asp Ser Glu
165 170 175
Pro Val Asp Leu Phe 180
25
3492
DNA
Rattus norvegicus

CDS
(1)..(3492)

rat NogoA
25
atg gaa gac ata gac cag teg teg ctg grc tec teg tec acg gac age
Met Glu Asp lie Asp Gin Ser Ser Leu Val Ser Ser Ser Thr Asp Ser
15 10 15
ccg ccc egg cct ccg ccc gee ttc aag tac cag ttc gtg acg gag ccc
Pro Pro Arg Pro Pro Pro Ala Phe Lys Tyr Gin Phe Val Thr Glu Pro
20 25 30
gag gac gag gag gac gag gag gag gag gag gac gag gag gag gac gac
Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Glu Asp Asp
35. 40 45
gag gac eta gag gaa ctg gag gtg ctg gag agg aag ccc gca gec ggg
Glu Asp Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly
50 55 - 60
ctg tec gca get gcg gtg ccg ccc gee gec gec gcg ccg ctg ctg gac
Leu Ser Ala Ala Ala Val Pro Pro Ala Ala Ala Ala Pro Leu Leu Asp
65 70 75 80
ttc age age gac teg gtg ccc ccc gcg ccc cgc ggg ccg ctg ccg gee Phe Ser Ser Asp Ser Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala
85 90 95
gcg ccc cct gee get cct gag agg cag cca tec tgg gaa cgc age ccc
Ala Pro Pro Ala Ala Pro Glu Arg Gin Pro Ser Trp Glu Arg Ser Pro
100 105 110
gcg gcg ccc gcg cca tec ctg ccg ccc get gee gca gtc ctg ccc tec
Ala Ala Pro Ala Pro Ser Leu Pro Pro Ala Ala Ala Val Leu Pro Ser
115 120 125

aag etc cca gag gac gac gag cct ccg gcg agg ccc ccg cct ccg ccg
Lys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro Pro Pro Pro
130 135 140
cca gec ggc gcg age ccc ctg gcg gag ccc gec gcg ccc cct tec acg
Pro Ala Gly Ala Ser Pro Leu Ala Glu Pro Ala Ala Pro Pro Ser Thr
145 * 150 155 160
ccg gec gcg ccc aag cgc agg ggc tec ggc tea gtg gat gag ace ctt Pro Ala Ala Pro Lys Arg Arg Gly Ser Gly Ser Val Asp Glu Thr Leu
165 170 175
ttt get ctt cct get gca tct gag cct gtg ata ccc tec tct gca gaa
Phe Ala Leu Pro Ala Ala Ser Glu Pro Val lie Pro Ser Ser Ala Glu
180 185 190
aaa att atg gat ttg atg gag cag cca ggt aac act gtt teg tct ggt
Lys He Met Asp Leu Met Glu Gin Pro Gly Asn Thr Val Ser Ser Gly
195 200 205
caa gag gat ttc cca tct gtc ctg ctt gaa act get gec tct ctt cct
Gin Glu Asp Phe Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu Pro
210 215 220
tct eta tct cct etc tea act gtt tct ttt aaa gaa cat gga tac ctt
Ser Leu Ser Pro Leu Ser Thr Val Ser Phe Lys Glu His Gly Tyr Leu
225 230 235 240
ggt aac tta tea gca gtg tea tec tea gaa gga aca att gaa gaa act Gly Asn Leu Ser Ala Val Ser Ser Ser Glu Gly Thr He Glu Glu Thr
245 250 255
tta aat gaa get tct aaa gag ttg cca gag agg gca aca aat cca ttt
Leu Asn Glu Ala Ser Lys Glu Leu Pro Glu Arg Ala Thr Asn Pro Phe
260 265 270
gta aat aga gat tta gca gaa ttt tea gaa tta gaa tat tea gaa atg

Val Asn Arg Asp Leu Ala Glu Phe Ser Glu Leu Glu Tyr Ser Glu Met
275 280 285
gga cca tct ttt aaa ggc tec cca aaa gga gag tea gcc ata tta gta 912
Gly Ser Ser Phe Lys Gly Ser Pro Lys Gly Glu Ser Ala lie Leu Val
290 295 300
gaa aac act aag gaa gaa gta att gtg agg agt aaa gac aaa gag gat 960
Glu Asn Thr Lys Glu Glu Val lie Val Arg Ser Lys Asp Lys Glu Asp
305 310 315 320
tta gtt tgt agt gca gcc ctt cac agt cca caa gaa tea cct gtg ggt 1008 Leu Val Cys Ser Ala Ala Leu His Ser Pro Gin Glu Ser Pro Val Gly
325 330 335
aaa gaa gac aga gtt gtg tct cca gaa aag aca atg gac att ttt aat 1056
Lys Glu Asp Arg Val Val Ser Pro Glu Lys Thr Met Asp lie Phe Asn
340 345 350
gaa atg cag atg tea gta gta gca cct gtg agg gaa gag tat gca gac 1104
Glu Met Gin Met Ser Val Val Ala Pro Val Arg Glu Glu Tyr Ala Asp
355 360 365
ttt aag cca ttt gaa caa gca tgg gaa gtg aaa gat act tat gag gga 1152
Phe Lys Pro Phe Glu Gin Ala Trp Glu Val Lys Asp Thr Tyr Glu Gly
370 375 380
agt agg gat gtg ctg get get aga get aat gtg gaa agt aaa gtg gac 1200
Ser Arg Asp Val Leu Ala Ala Arg Ala Asn Val Glu Ser Lys Val Asp
385 390 395 400
aga aaa tgc ttg gaa gat age ctg gag caa aaa agt ctt ggg aag gat 1248 Arg Lys Cys Leu Glu Asp Ser Leu Glu Gin Lys Ser Leu Gly Lys Asp
405 410 415
agt gaa ggc aga aat gag gat get tct ttc ccc agt ace cca gaa cct 1296 Ser Glu Gly Arg Asn Glu Asp Ala Ser Phe Pro Ser Thr Pro Glu Pro

420 425 430
gtg aag gac age tec aga gca tat att ace tgt get tec ttt ace tea 1344
Val Lys Asp Ser Ser Arg Ala Tyr lie Thr Cys Ala Ser Phe Thr Ser
435 440 445
gca ace gaa age ace aca gca aac act ttc cct ttg tta gaa gat cat 1392
Ala Thr Glu Ser Thr Thr Ala Asn Thr Phe Pro Leu Leu Glu Asp His
450 455 460
act tea gaa aat aaa aca gat gaa aaa aaa ata gaa gaa agg aag gec 1440
Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys lie Glu Glu Arg Lys Ala
465 470 475 * - 480
caa att ata aca gag aag act age ccc aaa acg tea aat cct'ttc ctt 1488 Gin lie lie Thr Glu Lys Thr Ser Pro. Lys Thr Ser Asn Pro Phe Leu
485 490 495
gta gca gta cag gat tct gag gca gat tat gtt aca aca gat ace tta 1536 Val Ala Val Gin Asp Ser Glu Ala Asp Tyr Val Thr Thr Asp Thr Leu'
500 505 510
tea aag gtg act gag gca gca gtg tea aac atg cct gaa ggt ctg acg 1584
Ser Lys Val Thr Glu Ala Ala Val Ser Asn Met Pro Glu Gly Leu Thr
515 520 525
cca gat tta gtt cag gaa gca tgt gaa agt gaa ctg aat gaa gec aca 1632
Pro Asp Leu Val Gin Glu Ala Cys Glu Ser Glu Leu Asn Glu Ala Thr
530 535 540
ggt aca aag att get tat gaa aca aaa gtg gac ttg gtc caa aca tea 1680
Gly Thr Lys lie Ala Tyr Glu Thr Lys Val Asp Leu Val Gin Thr Ser
545 550 * 555 560
gaa get ata caa gaa tea ctt tac ccc aca gca cag ctt tgc cca tea 1728 Glu Ala lie Gin Glu Ser Leu Tyr Pro Thr Ala Gin Leu Cys Pro Ser
565 570 575

ttt gag gaa get gaa gca act ccg tea cca gtt ttg cct gat att gtt 1776 Phe Glu Glu Ala Glu Ala Thr Pro Ser Pro Val Leu Pro Asp lie Val
580 585 590
atg gaa gca cca tta aat tct etc ctt cca age get ggt get tct gta 1824
Met Glu Ala Pro Leu Asn Ser Leu Leu Pro Ser Ala Gly Ala Ser Val
595 600 605
gtg cag ccc agt gta tec cca ctg-gaa gca cct cct cca gtt agt tat 1872
Val Gin Pro Ser Val Ser Pro Leu Glu Ala Pro Pro Pro Val Ser Tyr
610 615 620
gac agt ata aag ctt gag cct gaa aac ccc cca cca tat gaa gaa gec 1920
Asp Ser lie Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu Ala
625 630 635 640
atg aat gta gca eta aaa get ttg gga aca aag gaa gga ata aaa gag 1968 Met Asn Val Ala Leu Lys Ala Leu Gly Thr Lys Glu Gly He Lys Glu
645 - 650 655
cct gaa agt ttt aat gca get gtt cag gaa aca gaa get cct tat ata 2016 Pro Glu Ser Phe Asn Ala Ala Val Gin Glu Thr Glu Ala Pro Tyr He
660 665 670
tec att gcg tgt gat tta att aaa gaa aca aag etc tec act gag cca 2064
Ser He Ala Cys Asp Leu He Lys Glu Thr Lys Leu Ser Thr Glu Pro
675 680 635
agt cca gat ttc tct aat tat tea gaa ata gca aaa ttc gag aag teg 2112
Ser Pro Asp Phe Ser Asn Tyr Ser Glu He Ala Lys Phe Glu Lys Ser
690 695 700
gtg ccc gaa cac get gag eta gtg gag gat tec tea cct gaa tct gaa 2160
Val Pro Glu His Ala Glu Leu Val Glu Asp Ser Ser Pro Glu Ser Glu
705 710 715 720

cca gtt gac tta ttt agt gat gat teg act cct gaa gtc cca caa aca 2208 Pro Val Asp Leu Phe Ser Asp Asp Ser lie Pro Glu Val Pro Gin Thr
725 730 735
caa gag gag get gtg atg etc atg aag gag agt etc act gaa gtg tct 2256 Gin Glu Glu Ala Val Met Leu Met Lys Glu Ser Leu Thr Glu Val Ser
740 745 750
gag aca gta gec cag cac aaa gag gag aga ctt agt gee tea cct cag 2304
. Glu Thr Val Ala Gin His Lys Glu Glu Arg Leu Ser Ala Ser Pro Gin
755 760 765
gag eta gga aag cca tat tta gag tct ttt cag ccc aat tta cat agt 2352
Glu Leu Gly Lys Pro Tyr Leu Glu Ser Phe Gin Pro Asn Leu His Ser
770 775 780
aca aaa gat get gca tct aat gac att cca aca ttg ace aaa aag gag 2400
Thr Lys Asp Ala Ala Ser Asn Asp lie Pro Thr Leu Thr Lys Lys Glu
785 790 795 800
aaa att tct ttg caa atg gaa gag ttt aat act gca att tat tea aat 2448 Lys lie Ser Leu Gin Met Glu Glu Phe Asn Thr Ala lie Tyr Ser Asn
805 810 815
gat gac tta ctt tct tct aag gaa gac aaa ata aaa gaa agt gaa aca 2496
Asp Asp Leu Leu Ser Ser Lys Glu Asp Lys lie Lys Glu Ser Glu Thr
820 825 830
ttt tea gat tea tct ccg att gag ata ata gat gaa ttt ccc acg ttt 2544
Phe Ser Asp Ser Ser Pro lie Glu lie lie Asp Glu Phe Pro Thr Phe
835 840 845
gtc agt. get aaa gat gat tct cct aaa tta gec aag gag tac act gat 2592
Val Ser Ala Lys Asp Asp Ser Pro Lys Leu Ala Lys Glu Tyr Thr Asp
850 855 860
eta gaa gta tec gac aaa agt gaa att get aat ate caa age ggg gca 2640

Leu Glu Val Ser Asp Lys Ser Glu lie Ala Asn lie Gin Ser Gly Ala
865 870 875 880
gat tea ttg cct tgc tta gaa ttg ccc tgt gac ctt tct ttc aag aat 2688 Asp Ser Leu Pro Cys Leu Glu Leu Pro Cys Asp Leu Ser ?he Lys Asn
885 890 895
ata tat cct aaa gat gaa gta cat gtt tea gat gaa ttc tec gaa aat 2736
lie Tyr Pro Lys Asp Glu Val His Val Ser Asp Glu Phe Ser Glu Asn
900 905 910
agg tec agt gta tct aag gca tec ata teg cct tea aat gtc tct get 2784
Arg Ser Ser Val Ser Lys Ala Ser lie Ser Pro Ser Asn Val Ser Ala * '
915 920 925
ttg gaa cct cag aca gaa atg ggc age ata gtt aaa tee aaa tea ctt 2832
Leu Glu Pro Gin Thr Glu Met Gly Ser lie Val Lys Ser Lys Ser Leu
930 935 940
acg aaa gaa gca gag aaa aaa ctt cct tct gac aca gag aaa gag gac 2880
Thr Lys Glu Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp
945 950 955 960
aga tec ctg tea get gta ttg tea gca gag ctg agt aaa act tea gtt 2928 Arg Ser Leu Ser Ala Val Leu Ser Ala Glu Leu Ser Lys Thr Ser Val
965 970 975
gtt gac etc etc tac tgg aga gac att aag aag act gga gtg gtg ttt 2976 Val Asp Leu Leu Tyr Trp Arg Asp lie Lys Lys Thr Gly Val Val Phe
980 985 990
ggt gcc age tta ttc ctg ctg ctg tct ctg aca gtg ttc age att gtc 3024
•Gly Ala Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser lie Val
995 1000 1005
agt gta acg gcc tac att gcc ttg gcc ctg etc teg gtg act ate 3069 Ser Val Thr Ala Tyr lie Ala Leu Ala Leu Leu Ser Val Thr lie

1010 1015 1020
age ttt agg ata tat aag ggc gtg ate cag get ate cag aaa tea 3114
Ser Phe Arg He Tyr Lys Gly Val He Gin Ala He Gin Lys Ser
1025 1030 1035
gat gaa ggc cac cca ttc agg gca tat tta gaa tet gaa gtt get 3159
Asp Glu Gly His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala
1040 1045 1050
ata tea gag gaa ttg gtt cag aaa tac agt aat tct get ctt ggt 3204
He Ser Glu Glu Leu Val Gin Lys Tyr Ser Asn Ser Ala Leu Gly
1055 1060 . . 1065
cat gtg aac age aca ata aaa gaa ctg agg egg ctt ttc tta gtt 3249
His Val Asn Ser Thr lie Lys Glu Leu Arg Arg Leu Phe Leu Val
1070 1075 1080
gat gat tta gtt gat tec ctg aag ttt gca gtg ttg atg tgg gtg 3294
Asp Asp Leu Val Asp Ser Leu Lys Phe Ala Val Leu Met Trp Val
1085 1090 1095
ttt act tat gtt ggt gee ttg ttc aat ggt ctg aca eta ctg att 3339
Phe Thr Tyr Val Gly Ala Leu Phe Asn Gly Leu Thr Leu Leu He
1100 1105 1110
tta get ctg ate tea etc ttc agt att cct gtt att tat gaa egg 3384
Leu Ala Leu He Ser Leu Phe Ser He Pro Val He Tyr Glu Arg
1115 1120 1125
cat cag gtg cag ata gat cat tat eta gga ctt gca aac aag agt 3429 His Gin Val Gin He Asp His Tyr Leu Gly Leu Ala Asn Lys Ser
1130 1135 1140 - '
gtt aag gat gee atg gee aaa ate caa gca aaa ate cct gga ttg 3474
Val Lys Asp Ala Met Ala Lys He Gin Ala Lys He Pro Gly Leu
1145 1150 1155

aag cgc aaa gca gat tga
Lys Arg Lys Ala Asp 1160
26
1163
PRT
Rattus norvegicus
26
Met Glu Asp lie Asp Gin Ser Ser Leu Val Ser Ser Ser Thr Asp Ser
1 5 - 10 15
Pro Pro Arg Pro Pro Pro Ala Phe Lys Tyr Gin Phe Val Thr Glu Pro
20 25 30
Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Glu Asp Asp
35 40 45
Glu Asp Leu Glu Glu Leu Glu Val Leu Glu Arg Lys Pro Ala Ala Gly
50 55 60
Leu Ser Ala Ala Ala Val Pro Pro Ala Ala Ala Ala Pro Leu Leu Asp
65 70 75 80

Phe Ser Ser Asp Ser Val Pro Pro Ala Pro Arg Gly Pro Leu Pro Ala
85 90 95
Ala Pro Pro Ala Ala Pro Glu Arg Gin Pro Ser Trp Glu Arg Ser Pro
100 105 110
Ala Ala Pro Ala Pro Ser Leu Pro Pro Ala Ala Ala Val Leu Pro Ser
115 120 125
Lys Leu Pro Glu Asp Asp Glu Pro Pro Ala Arg Pro Pro Pro'Pro Pro
130 135 140
Pro Ala Gly Ala Ser Pro Leu Ala Glu Pro Ala Ala Pro Pro Ser Thr
145 150 ** 155 160
Pro Ala Ala Pro Lys Arg Arg Gly Ser Gly Ser Val Asp Glu Thr Leu
165 170 175
Phe Ala Leu Pro Ala Ala Ser Glu Pro Val lie Pro Ser Ser Ala Glu
180 185 190
Lys He Met Asp Leu Met Glu Gin Pro Gly Asn Thr Val Ser Ser Gly
195 200 205
Gin Glu Asp Phe Pro Ser Val Leu Leu Glu Thr Ala Ala Ser Leu Pro
210 215 220

Ser Leu Ser Pro Leu Ser Thr Val Ser Phe Lys Glu His Gly Tyr Leu
225 230 235 240
Gly Asn Leu Ser Ala Val Ser Ser Ser Glu Gly Thr He Glu Glu Thr
245 250 255
Leu Asn Glu Ala Ser Lys Glu Leu Pro Glu Arg Ala Thr Asn Pro Phe
260 265 270
Val Asn Arg Asp Leu Ala Glu Phe Ser Glu Leu Glu Tyr Ser Glu^M^t
275 280 285
Gly Ser Ser Phe Lys Gly Ser Pro Lys Gly Glu Ser Ala He Leu Val
290 295 300
Glu Asn Thr Lys Glu Glu Val He Val Arg Ser Lys Asp Lys Glu Asp
305 310 315 320
Leu Val Cys Ser Ala Ala Leu His Ser Pro Gin Glu Ser Pro Val Gly
325 330 335
Lys Glu Asp Arg Val Val Ser Pro Glu Lys Thr Met Asp He Phe Asn
340 345 350
Glu Met Gin Met Ser Val Val Ala Pro Val Arg Glu Glu Tyr Ala Asp
355 360 365
Phe Lys Pro Phe Glu Gin Ala Trp Glu Val Lys Asp Thr Tyr Glu Gly

370 375 380
Ser Arg Asp Val Leu Ala Ala Arg Ala Asn Val Glu Ser Lys Val Asp
385 390 395 400
Arg Lys Cys Leu Glu Asp Ser Leu Glu Gin Lys Ser Leu Gly Lys Asp
405 410 415
Ser Glu Gly Arg Asn Glu Asp Ala Ser Phe Pro Ser Thr Pro Glu Pro
420 425 430
Val Lys Asp Ser Ser Arg Ala Tyr lie Thr Cys Ala Ser Phe Thr Ser
435 440 445
Ala Thr Glu Ser Thr Thr Ala Asn Thr Phe Pro Leu Leu Glu Asp His
450 455 460
Thr Ser Glu Asn Lys Thr Asp Glu Lys Lys lie Glu Glu Arg Lys Ala
465 470 475 480
Gin lie lie Thr Glu Lys Thr Ser Pro Lys Thr Ser Asn Pro Phe Leu
485 490 495
Val Ala Val Gin Asp Ser Glu Ala Asp Tyr Val Thr Thr Asp Thr Leu
500 505 510.
Ser Lys Val Thr Glu Ala Ala Val Ser Asn Met Pro Glu Gly Leu Thr
515 520 525

Pro Asp Leu Val Gin Glu Ala Cys Glu Ser Glu Leu Asn Glu Ala Thr
530 535 540
Gly Thr Lys He Ala Tyr Glu Thr Lys Val Asp Leu Val Gin Thr Ser
545 550 555 560
Glu Ala He Gin Glu Ser Leu Tyr Pro Thr Ala Gin Leu Cys Pro Ser
565 570 575
Phe Glu Glu Ala Glu Ala Thr Pro Ser Pro Val Leu Pro Asp He Val
580 585 590
Met Glu Ala Pro Leu Asn Ser Leu Leu Pro Ser Ala Gly Ala Ser Val
595 600 605
Val Gin Pro Ser Val Ser Pro Leu Glu Ala Pro Pro Pro Val Ser Tyr
610 615 620
Asp Ser He Lys Leu Glu Pro Glu Asn Pro Pro Pro Tyr Glu Glu Ala
625 630 635 640
Met Asn Val Ala Leu Lys Ala Leu Gly Thr Lys Glu Gly He Lys Glu
645 650 655
Pro Glu Ser Phe Asn Ala Ala Val Gin Glu Thr Glu Ala Pro Tyr He
660 665 670

Ser lie Ala Cys Asp Leu lie Lys Glu Thr Lys Leu Ser Thr Glu Pro
675 680 685
Ser Pro Asp Phe Ser Asn Tyr Ser Glu lie Ala Lys Phe Glu Lys Ser
690 695 700
Val Pro Glu His Ala Glu Leu Val Glu Asp Ser Ser Pro Glu Ser Glu
705 710 715 720
Pro Val Asp Leu Phe Ser Asp Asp Ser lie Pro Glu Val Pro Gin Thr
725 730 735
Gin Glu Glu Ala Val Met Leu Met Lys Glu Ser Leu Thr Glu Val Ser
740 " 745 750
Glu Thr Val Ala Gin His Lys Glu Glu Arg Leu Ser Ala Ser Pro Gin
755 760 765
Glu Leu Gly Lys Pro Tyr Leu Glu Ser Phe Gin Pro Asn Leu His Ser
770 775 780
Thr Lys Asp Ala Ala Ser Asn Asp lie Pro Thr Leu Thr Lys Lys Glu
785 790 795 800
»
Lys lie Ser Leu Gin Met Glu Glu Phe Asn Thr Ala lie Tyr Ser Asn
805 810 815

Asp Asp Leu Leu Ser Ser Lys Glu Asp Lys lie Lys Glu Ser Glu Thr
820 825 830
Phe Ser Asp Ser Ser Pro lie Glu lie lie Asp Glu Phe Pro Thr Phe
835 840 845
Val Ser Ala Lys Asp Asp Ser Pro Lys Leu Ala Lys Glu Tyr Thr Asp
850 855 860
Leu Glu Val Ser Asp Lys Ser Glu lie Ala Asn He QJn Ser Gly Ala
865 870 875 880
Asp Ser Leu Pro Cys Leu Glu Leu Pro Cys Asp Leu Ser Phe Lys Asn
885 890 895
He Tyr Pro Lys Asp Glu Val His Val Ser Asp Glu Phe Ser Glu Asn
900 905 910
Arg Ser Ser Val Ser Lys Ala Ser He Ser Pro Ser Asn Val Ser Ala
915 920 925
Leu Glu Pro Gin Thr Glu Met Gly Ser He Val Lys Ser Lys Ser Leu
930 935 940
Thr Lys Glu Ala Glu Lys Lys Leu Pro Ser Asp Thr Glu Lys Glu Asp
945 950 955 960
Arg Ser Leu Ser Ala Val Leu Ser Ala Glu Leu Ser Lys Thr Ser Val

965 970 975
Val Asp Leu Leu Tyr Trp Arg Asp lie Lys Lys Thr Gly Val Val Phe
980 985 990
Gly Ala Ser Leu Phe Leu Leu Leu Ser Leu Thr Val Phe Ser lie Val
995 1000 1005
Ser Val Thr Ala Tyr He Ala Leu Ala Leu Leu Ser Val Thr He
1010 1015 1020
Ser Phe Arg He Tyr Lys Gly Val He Gin Ala He Gin Lys Ser
1025 1030 1035
Asp Glu Gly His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala
1040 1045 1050
He Ser Glu Glu Leu Val Gin Lys Tyr Ser Asn Ser Ala Leu Gly
1055 1060 1065
His Val Asn Ser Thr He Lys Glu Leu Arg Arg Leu Phe Leu Val
1070 1075 1080
Asp Asp Leu Val Asp Ser Leu Lys Phe Ala Val Leu Met Trp Val
1085 1090 . .1095
Phe Thr Tyr Val Gly Ala Leu Phe Asn Gly Leu Thr Leu Leu He
1100 1105 1110

Leu Ala Leu He Ser Leu Phe Ser He Pro Val He Tyr Glu Arg
1115 1120 1125
His Gin* Val Gin He Asp His Tyr Leu Gly Leu Ala Asn Lys Ser
1130 1135 1140
Val Lys Asp Ala Met Ala Lys He Gin Ala Lys He Pro Gly Leu
1145 1150 1155
Lys Arg Lys Ala Asp 1160
27
25
PRT
Rattus norvegicus

PEPTIDE
(1). . (25)
rat PEP4

27
Glu Glu Leu Val Gin Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn
15 10 15
Ser Thr lie Lys Glu Leu Arg Arg Leu
20 25
28
17
PRT
Artificial Sequence

PRO/SER rich peptide

PEPTIDE
(1)..(17)
> Synthetic peptide
28

Pro Ser Ser Pro Pro Pro Ser Ser Pro Pro Pro Ser Ser Pro Pro Pro
1 5 10 15
Ser
29
25
DNA
Artificial Sequence

CA-NA-2F

primer_bind
(1)..(25)
CA-NA-2F primer
29
aagcaccatt gaattctgca gttcc

I

30
28
DNA
Artificial Sequence

CA-NA-3R
primer_bind (1J,.. (28)
30
aactgcagta ctgagctcct ccatctgc
31
33
DNA
Artificial Sequence


forward 5'

primer_bind
(1).. (33)
forward primer
31
gtcgcggatc catggagacc ctttttgctc ttc
32
27
DNA
Artificial Sequence

reverse 5'


primerjoind
(1)..(27)
reverse primer
32
gttctcgagt tatgaagttt tactcag
33
29
DNA
Artificial Sequence

forward 5'-l

primer_bind
(1).. (29)
primer * -
33

gtgcggatcc atggatttga aggagcagc
34
28
DNA
Artificial Sequence

reverse 5'-l

primer__bind
(1)..(28)
primer
34
gzttctcgag tgaagtttta ttcagctc
35
20
DNA

Artificial Sequence

5' primer

primer„bind
(1)..(20)
primer
35
tccaccccgg ccgcgcccaa
36
22
DNA
Artificial Sequence

5' primer 2


primer_bind
(1)..(22)
primer
36
aatgatgggc aaagctgtgc tg
37
24
DNA -
Artificial Sequence

3' primer

primer_bind
(1)..(24)
primer

37
ggtacaaaga ttgcttatga aaca
38
22
DNA
Artificial Sequence

3' primer 2

pr imer__bind
(1)..(22)
primer
38
agcagggcca aggcaatgta gg
39

28
DNA
Artificial Sequence

S'-VL leader

pr imer__bind
(1)..(28)
primer
39
aatatgagtc ctgcccagtt cctgtttc
40
32
DNA
Artificial Sequence


3'-Ck

primer_bind
(1)..(32)
primer
40
ttaggaattc ctaacactct cccctgttga a
41
31
DNA
Artificial Sequence

5'-VH leader

primer_bind

(1)..(31)
primer
41
aatatggatt ttgggctgat tttttttatt
42
24
DNA
Artificial Sequence

3 '-CH hinge

primer_bind
(1)..(24)
primer
42
aattgggcaa cgttgcaggt gacg

43
663
DNA
Mus musculus

irdsc_binding
(1)..(663)
DNA variable part of heavy chain 11C7
43
atggattttg ggctgatttt ttttattgtt ggtcttttaa aaggggtcca gtgtgaggtg 60
aagcttctcg agtctggagg tggcctggtg cagcctggag gatccctgaa actctcctgt 120
gtagtctcag gattcgattt tagaagaaat tggatgagtt gggtccggca ggctcctggg 180
aaagggctag aatggattgg agaaattaat ccagatagca gtaagataaa ctatacgcca 240
tctctaaagg ataaattcat catctccaga gacaatgcca agaatacgct gtacctgcaa 300
gtgagcacag tgagatctga ggacacagcc ctttattact gtgtgagacc ggtctggatg 360
tatgctatgg actactgggg tcaaggaacc tcagtcaccg tctcctcagc caaaacgaca 420
cccccatctg tctatccact ggcccctgga tctgctgccc aaac~aactc catggtgacc 480

ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 540
tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 600
agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 660
gcc 663
44
717
DNA
Mus musculus

misc_binding
(1)..(717)
variable part of light chain of 11C7
44
atgagtcctg cccagttcct gtttctgtta gtgctctgga ttcgggaaac cagcggtgat 60
gttctgttga cccagactcc tctcactttg tcgataacca ttggacaacc agcctccatc 120
tcttgcaagt caagtcagag cctcttgcat agtgatggaa agacatattt gaattggttg 180

ttacagaggc caggccagtc tccaaagcgc ctaatctatc tggtgtctaa actggactct 240
ggagtccctg acaggttcac - tggcagtgga tcagggacgg atttcacact gaaaatcagc 300
agagtggagg ctgaggattt gggactttat tattgctggc aaggtacaca ttttcctcag 360
acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660
actcacaaga catcaacttc acccattgtc aagagcttca acaggggaga gtgttag 717







Claims:
1.) A binding molecule which is capable of binding to the human NogoA polypeptide (SEQ ID NO: 5) or human NiG (SEQ ID NO: 7) or human NiG-D20 (SEQ ID NO: 24) or human NogoA_623-640 (SEQ ID NO: 6) with a dissociation constant 2.) A binding molecule which is capable of binding to the human NogoA polypeptide (SEQ ID NO: 5) or human NiG (SEQ ID NO: 7) or human NiG«D20 (SEQ ID NO: 24) or human NogoA_623-640 (SEQ ID NO: 6) with a dissociation constant • in sequence the hypervariable regions CDR1, CDR2, and CDR3, of which each of the hypervariable regions are at least 50% homologous to their equivalent hypervariable regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); or
• in sequence the hypervariable regions CDR1\ CDR2', and CDR3\ of which each of the hypervariable regions are at least 50% homologous to their equivalent hypervariable regions CDRV-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13).
3.) A binding molecule which is capable of binding to the human NogoA polypeptide (SEQ ID NO: 5) or human NiG (SEQ ID NO: 7) or human NiG-D20 (SEQ ID NO: 24) or human NogoA„623-640 (SEQ ID NO: 6) with a dissociation constant • a first antigen binding site comprising in sequence the hypervariable regions CDR1, CDR2, and CDR3, of which each of the hypervariable regions are at least 50% homologous to their equivalent hypervariable regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); and
• a second antigen binding site comprising in sequence the hypervariable regions CDR1\ CDR2\ and CDR3', of which each of the hypervariable regions are at least 50% homologous to their equivalent hypervariable regions CDRV-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13).
4.) A binding molecule which comprises at least one antigen binding site, said antigen binding site comprising either

• in sequence the hypervariable regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); or
• in sequence the hypervariable regions CDR1 '-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13); or
• direct equivalents thereof.
5.) A binding molecule comprising
• a first antigen binding site comprising in sequence the hypervariable regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (SEQ ID NO: 10); and
• a second antigen binding site comprising in sequence the hypervariable regions CDRV-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13); or
• direct equivalents thereof.
6.) The binding molecule according to claims 1 to 5 which comprises at least
• one immunoglobulin heavy chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions regions CDR1-11C7 (SEQ ID NO: 8), CDR2-11C7 (SEQ ID NO: 9) and CDR3-11C7 (S£Q ID NO: 10) and (ii) the constant part or fragment thereof of a human heavy chain; and
• one immunoglobulin light chain or fragment thereof which comprises (i) a variable domain comprising in sequence the hypervariable regions CDR1'-11C7 (SEQ ID NO: 11), CDR2'-11C7 (SEQ ID NO: 12) and CDR3'-11C7 (SEQ ID NO: 13) and (ii) the constant part or fragment thereof of a human light chain; or
• direct equivalents thereof.

7. The binding molecule according to claim 6 in which the constant part or fragment thereof of the human heavy chain is of the y4 type and the constant part or fragment thereof of the human light chain is of the K type.
8. The binding molecule according to claims 1 to 7, which is a chimeric or*iumanised monoclonal antibody.

9. A binding molecule comprising polypeptide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3.
10. A polynucleotide comprising polynucleotides encoding a binding molecule according to any of claims 1 to 9.
11. A polynucleotide comprising either

• polynucletide sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; or
• polynucletide sequences as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

12. An expression vector comprising polynucleotides according to any one of claims 10 or 11.
13. An expression system comprising a polynucleotide according to any one of claims 10 or
11, wherein said expression system or part thereof is capable of producing a polypeptide of any one of claims 1 to 9, when said expression system or part thereof is present in a compatible host cell.
14. An isolated host cell which comprises an expression system according to claim 13.
15. The use of a binding molecule according to any one of claims 1 to 9 as a pharmaceutical.
16. The use of a binding molecule according to any one of claims 1 to 9 in the treatment of nerve repair.
17. A pharmaceutical composition comprising a binding molecule according to any one of claims 1 to 9 in association with at least one pharmaceutically acceptable carrier or diluent.

Amethod of treatment of diseases associated with nerve repair comprising administering to a subject in need of such treatment an effective amount of a binding nolecule according to any one of claims 1 to 9.


Documents:

1212-chenp-2005 correspondence others-15-07-2009.pdf

1212-chenp-2005-abstract.pdf

1212-chenp-2005-claims.pdf

1212-chenp-2005-correspondnece-others.pdf

1212-chenp-2005-correspondnece-po.pdf

1212-chenp-2005-description(complete).pdf

1212-chenp-2005-form 1.pdf

1212-chenp-2005-form 18.pdf

1212-chenp-2005-form 26.pdf

1212-chenp-2005-form 3.pdf

1212-chenp-2005-form 5.pdf

1212-chenp-2005-pct.pdf

EXAMINATION REPORT REPLY.PDF


Patent Number 244384
Indian Patent Application Number 1212/CHENP/2005
PG Journal Number 50/2010
Publication Date 10-Dec-2010
Grant Date 06-Dec-2010
Date of Filing 10-Jun-2005
Name of Patentee UNIVERSITAT ZURICH
Applicant Address Prorektorat Forschung, Ramistrasse 71, CH-8006 Zurich
Inventors:
# Inventor's Name Inventor's Address
1 OERTLE, Thomas Casa al lago, CH-6576 Gerra/Gambarogno
2 SCHWAB, Martin, E. Waffenplatz Strasse 79, CH-8002 Zurich
3 BARSKE, Carmen Birkenweg 10, 79540 Loerrach
4 MIR, Anis, Khusro Rue des Vosges 6, F-68870 Bartenheim
5 SCHNELL, Lisa Schwanengasse 9, CH-8001 Zurich
6 VITALITI, Alessandra 6981 Bedigliora, CH-
7 ZURINI, Mauro Benkenstrasse 92a, CH-4102 Binningen
PCT International Classification Number C07K 16/18
PCT International Application Number PCT/EP2003/013960
PCT International Filing date 2003-12-09
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0228832.2 2002-12-10 U.K.