WO1991012272A1

WO1991012272A1 - Protein factor

Info

Publication number: WO1991012272A1
Application number: PCT/GB1991/000245
Authority: WO
Inventors: Michael Stoker; Ermanno Gherardi
Original assignee: Imperial Cancer Research Technology Limited
Priority date: 1990-02-16
Filing date: 1991-02-15
Publication date: 1991-08-22
Also published as: GB9003621D0

Abstract

A polypeptide or protein comprising at least five contiguous amino acid residues in a sequence corresponding to formula (I): VVNGIPTQTTVGX1MVSLX2YY wherein the internationally recognised 1-letter code is used to represent amino acid residues and X1 is any naturally occuring L-amino acid residue and X2 is any amino acid residue, but is preferably an L-leucine residue (L).

Description

PROTEIN FACTOR

The present invention relates to proteins, polypeptides, nucleic acid fragments, antibodies and related products and their use in diagnosis and therapy.

Scatter factor is a fibroblast-derived protein which causes separation of contiguous epithelial cells and increased local mobility of unanchored cells [Stoker and Perry an, J.Cell Sci.. 72:209-223 (1985)]. This activity may be of use in enhancing repair of wounds, such as severe burns and chronic skin ulcers, and inhibition of the activity of scatter factor or related proteins, for example using neutralising antibodies, may be of use in inhibiting metastasis of tumours.

The present inventors have elucidated the N-terminal seguence of one chain of scatter factor, thereby enabling the construction of nucleotide probes to identify RNA and DNA sequences encoding scatter factor, the isolation and cloning of mRNA and genomic or complementary DNA and thus large scale production by expression in host cells of scatter factor. This may itself be used therapeutically and diagnostically and it may also be used to generate diagnostically and therapeutically useful antibodies. Polypeptides and Proteins

Polypeptides and proteins of the invention incorporating a part or the whole of the sequence of formula (I) below are useful in generating antibodies for therapeutic and diagnostic uses and can be used as immunogens for treatment or prophylaxis of cancer. They may also be used, provided that they contain the functional portion(s) of scatter factor, to initiate or expedite wound healing. In one aspect of the invention there is therefore provided a polypeptide or protein comprising at least five contiguous amino acids in a sequence corresponding to formula (I)

WNGIPTQTTVGX¹MVSLX²YY (I) wherein the internationally recognised 1-letter code is used to represent amino acid residues and

X¹ is any naturally occurring L-amino acid residue and X² is any amino acid residue, but is preferably a L- leucine residue (L) . Statistically a sequence of 5 amino acid residues is very likely to be unique such that the proteins and polypeptides of the invention will be new.

Preferably the polypeptides and proteins of the invention comprise at least 6 contiguous amino acid residues in a seguence corresponding to formula (I) . Where polypeptides of the invention are 6 or more amino acid residues in length they must contain a portion of at least 5 amino acid residues identical to formula (I) but the remainder of the sequence may be unrelated to formula (I) , related to but not identical (ie. homologous) or identical to formula (I) . Sequences differing from formula (I) may vary by way of substitution, deletion and/or insertion of one or more amine acid residues at one or more loci. Preferably the polypeptides are longer than 20 amino acid residues and comprise a sequence of 20 amino acid residues homologous with or more preferably, identical to the sequence of formula (I) . Proteins according to the invention may comprise one polypeptide chain or more than one polypeptide chain linked covalently by disulphide bonds or otherwise or by non-covalent interactions but the invention does not extend to naturally occurring scatter factor or the 57 kDa and 30 kDa subunits thereof as produced by reductive cleavage of naturally occurring scatter factor.

Where the polypeptides of the invention comprise one or more additional sequences they may include other parts of scatter factor, and/or carrier proteins such as keyhole limpet haemocyanin, albumen or thyroglobulin or polio virus capsid protein.

The polypeptides of the invention may additionally bear linkage sugars, such as N-acetyl galactosamine on serine and/or threonine residues, or oligosaccharide moieties linked to the polypeptide via linkage sugars.

Polypeptides and proteins according to the invention may be produced by modification of naturally occurring scatter factor, by de novo synthesis or by expression of appropriate nucleic acid coding sequences or a combination of such techniques which are described below.

Polypeptides and proteins according to the invention may be produced by de novo synthesis by conventional techniques. Scatter factor may be obtained from cell culture [see Gherardi et al, Proc.Natl.Acad.Sci. , j$ : 5844- 5848 (1989)] and modified by conventional techniques [see Gherardi ibid] . Preferably the polypeptides and proteins of the invention are produced by expression of DNA encoding the polypeptide or protein, in a suitable prokaryotic or eukaryotic expression system (see Molecular Cloning: A

Laboratory Manual, 2nd Edn, Sambrook, J. , Fritsch, E.F. and Maniatis, T. ; Cold Spring Harbour Laboratory, N.Y. 1989 (hereafter "Maniatis"].

The invention further provides a process for producing a polypeptide or protein as hereinbefore defined by linking amino acid residues or oligopeptides in an appropriate sequence, or by expressing the polypeptide in a suitable expression system as hereinbefore described and recovering the polypeptide from the cell debris or culture medium. The polypeptides and proteins of the invention may be used in therapy and diagnosis or to generate antibodies for use in therapy or diagnosis.

For therapeutic use the polypeptides or proteins will be administered in effective, non-toxic amounts in order to promote invasion of wounds by epithelial cells so as to encourage wound healing. Suitably the polypeptide or protein is administered as a pharmaceutical formulation comprising a pharmaceutically acceptable carrier or diluent. Preferred carriers and diluents will be selected according to the route of administration. For oral administration, the carriers and diluents may be liquids or solids and the polypeptide or protein may be formulated as a solution, suspension or mixture. For parenteral administration the carrier or diluent will generally be sterile, pyrogen-free liguid, preferably water for injection and the polypeptide or protein will be present in solution or suspension. The carrier or diluent may optionally include accessory ingredients such as preservatives, anti-oxidants, flavouring agents, colouring agents, suspending agents such as detergents or emulgents, buffers and isotonic agents.

For topical administration the carrier or diluent will be a suitable solvent, cream or lotion base and the formulation may further comprise optional accessory ingredients such as preservatives, antioxidants , suspending agents, including detergents and emulgents, and buffers.

Routes of administration will be local, oral or parenteral, for instance subcutaneous or intramuscular injection.

Amounts to be administered will be selected according to the age, weight or body area, sex and state of health of the patient and the size of the wound(s) , ulcer(s) or area to be treated. As a guide a single dose for a normal human would generally be in the range of from 1 to 1000 ng, for instance 100 to 300 ng.

The polypeptides and proteins may be used in conventional diagnostic tests or assays, for instance to detect antibodies against scatter factor in samples of a patient's body fluid or to competitively inhibit binding of scatter factor to anti-scatter factor antibodies in competitive tests or assays for scatter factor. For diagnostic use the polypeptides and proteins of the invention may be labelled, for instance with radionuclides, fluorescent labels or enzyme labels or bound to particles or solid supports. Such labelling is achieved by conventional methods well-known in the art. Nucleic acid fragments

The present invention further provides nucleic acid fragments which encode polypeptides and proteins as hereinbefore defined or which are related hereto.

A first class of nucleic acid fragments according to the invention are fragments which encode or are complementary to fragments which encode a polypeptide or protein as defined above. Such fragments may be single- or double- stranded and they may be DNA or RNA fragments.

Single stranded fragments may be coding strands i.e. template-strand DNA or message strand RNA) or complementary strands (i.e. non-template strand DNA or anti-sense RNA) . Coding strands will contain an open reading frame comprising 15 contiguous nucleotides in a sequence of codons encoding 5 contiguous amino acid residues in a sequence corresponding to formula (I) . Complementary strands will have a sequence of 15 contiguous nucleotides in a sequence exactly complementary to a coding strand.

Double stranded nucleic acid fragments may be RNA or, preferably, DNA and comprise a coding strand and an exactly complementary strand, the coding strand comprising 15 contiguous nucleotides in an open reading frame sequence of codons encoding 5 contiguous amino acid residues in a sequence corresponding to formula (I) .

A second class of nucleic acid fragments according to the invention are fragments hybridisable with fragments of the first class. Fragments of the second class may be single or double stranded DNA or RNA but need not encode an amino acid sequence nor be complementary to a coding sequence as it is not essential that they have an exactly complementary sequence for hybridisation to occur. However, such fragments must be hybridisable to fragments of the first class at least under low stringency conditions and preferably under high stringency conditions. Such fragments differ from fragments of the first class by substitution, deletion or insertion at one or more loci. Preferably class two fragments have at least 75% homology, over the relevant region, with class one fragments, more preferably at least 90% and most preferably 95% or more homology. Thus for a fragment hybridisable with a class one fragment of only 15 bases or base pairs, the class two fragment will differ from the class one fragment at only one or two bases. Larger fragments also preferably only differ at one or two bases from class one fragments. Differences may be substitutions, deletions or insertions or any combination thereof.

Low stringency as defined above corresponds with about 75% homology. High stringency as defined above corresponds with about 95% homology.

Fragments of the invention (whether of the first or second class) are preferably at least 17, more preferably at least 18 nucleotides or nucleotide base pairs in length, more preferably at least 21 bases or base pairs and yet more preferably at least 30 nucleotide bases or base pairs in length.

Fragments longer than 15 nucleotide bases or base pairs must contain coding, complementary or hybridisable sequences of at least 15 nucleotide bases or base pairs in length as described above. The remainder of such fragments may be unrelated to the sequence of formula (I) , may encode, or be complementary to; a sequence encoding a polypeptide related to or identical to that of formula (I) or may hybridise to a sequence which encodes or is complementary to a sequence encoding; a polypeptide related to or identical to that of formula (I) .

The nucleic acid fragments of the invention may be obtained from natural sources such as mRNA or genomic DNA of cells actively expressing or capable of expressing scatter factor (in which case they must be in pure form or in a form substantially free from mRNA or DNA fragments not encoding scatter factor) . Alternatively the fragments may be produced by mutagenesis of such naturally occurring mRNA or DNA, by transcription of genomic DNA or reverse transcription from naturally occurring mRNA or transcription of genomic DNA, by partial or total de novo synthesis or by a combination of such techniques as will be described below.

Nucleic acid fragments according to the invention may be produced de novo by conventional techniques [see Maniatis] or recovered from cells, such as foetal fibroblasts, or by conventional methods and may be cloned in suitable cloning systems [Maniatis] such as described below.

Fragments according to the invention may also be produced by replication of DNA or RNA, by transcription from DNA to form RNA fragments or reverse transcription from RNA fragments to form DNA fragments. Such transcription may be in a cell free system or may be effected in cells for instance by cloning. Cell free systems include an appropriate replicase, transcriptase or reverse transcriptase, suitable nucleotide precursors and a r- cleic acid template of appropriate sequence, together with buffers and any necessary or desirable cofactors [see Maniatis] . Cloning may be conducted by conventional techniques well known in the art of recombinant DNA technology [see Maniatis] .

The invention therefore provides a process for producing nucleic acid fragments as hereinbefore defined by linking nucleotides or oligonucleotides in an appropriate sequence and recovering the fragments from the reaction medium or cell debris.

In order to produce quantities of the nucleic acids fragments of the invention, the fragments are preferably cloned by inclusion in a suitable cloning vector containing appropriate restriction endonuclease recognition and cutting sites for excision of the cloned fragments from the vector. Such constructs are cloned in suitable cloning systems such as transfected bacterial or yeast cells (e.g. Escherichia coli or Saccharomvces cerevisiae) . Suitable cloning vectors include plasmids, cosmids and viral genomic nucleic acids. Construction of the vectors and cloning thereof may be by conventional methods such as described in Maniatis.

The nucleic acid fragments of the invention may all be used as hybridisation probes and polymerase chain reaction primers for identifying DNA or RNA encoding at least a portion of scatter factor. Coding strands and double stranded fragments including a coding strand, (ie. fragments of the first class described above) may also be used in the expression of, or to construct sequences which will enable expression of, polypeptides and proteins of the invention including the expression of scatter factor.

Fragments according to the invention have a number of uses associated with the diagnosis and therapy of cancers and wound healing, for instance:

(i) Fragments of the invention may be used as primers to initiate the polvmerase chain reaction (PCR) .

This is important in, for instance, the identification of tumour cells abnormally expressing scatter factor and/or expressing scatter factor at abnormally high-level or expressing abnormal scatter factor and thus liable to induce metastasis.

The use of the PCR to detect such cells is particularly important in seeking to establish whether metastasis is likely to occur. It is also useful in detecting allele loss or translocation events (whereby the whole or a segment of the scatter factor gene is deleted or moved to an inappropriate site in the genome leading to aberrant expression of scatter factor or that part of the scatter factor molecule encoded by the translocated segment) . Such allele loss or translocation events may be indicative of cancerous or pre-cancerous abnormalities. For use as PCR primers the fragments of the present invention are preferably at least 17 bases in length. They are preferably used in conventional manner.

(ii) Fragments of the invention may be used as hybridization probes.

These hybridization probes may be used for detecting nucleic acids of complementary sequence in samples, for instance for detecting tumour cells actively aberrantly expressing scatter factor, and in the detection of allele loss and translocation events as described under (i) above. For use as probes, the fragments of the present invention are preferably at least 17 bases or base pairs in length.

The probes of the invention may be labelled with conventional detectable labels such as radionuclides, enzyme labels, fluorescent labels or other directly or indirectly detectable conventional labels. For some applications the fragmerts may be bound to a solid support. The labelling of probes and the binding of probes to solid supports are well-known technologies and may be achieved by methods known to those skilled in the art. Hybridization using these probes is conducted in conventional manner for instance as described by Maniatis. (iii) Class one fragments of the invention may be included in expression vectors and thus direct the production of polypeptides Such polypeptides have a sequence corresponding to at least a portion of scatter factor and thus have diagnostic and therapeutic uses and may be used to generate therapeutically or diagnostically useful antibodies as discussed below.

For use in the production of polypeptides and proteins, class one fragments according to the present invention will be incorporated in functional form into suitable expression systems using expression vectors and appropriate hoot cells, for instance mammalian cells. Such expression vectors include plasmids and viral genomic nucleic acids. The fragments of the invention form the whole or a part of a coding sequence accompanied by any necessary or desirable regulatory and promoter sequences, transcriptional and translational initiation and termination signals, splicing and ligation sites and marker sequences in appropriate order, reading frame register and spacing and optionally further including restriction endonuclease recognition and cutting sites.

Coding sequences including fragments according to the invention may encode fusion proteins, for instance comprising a polypeptide encoded by the fragment of the invention linked to a carrier protein, such as keyhole limpet haemocyanin, albumen or thyroglobulin. Such fusion proteins are particularly useful when the polypeptide is to be used as an immunogen. In other fusion proteins the polypeptide is linked to other parts of scatter factor. In a particular aspect of the invention the vector is viral genomic DNA and the fragment of the invention is inserted in a functional gene expressed by virus - infected cells; an example of this is when the fragment is incorporated into the gene for the capsid protein of polio virus and the vector is polio virus genomic DNA (see, for instance Burke et al. , Nature (London) 332: 81-82 (1989)).

The vector will be selected according to the host cell to be used to express the polypeptides encoded by fragments of the invention and may be constructed by conventional recombinant DNA techniques (see, for instance, Maniatis) The present invention further provides cloning and expression vectors as hereinbefore described containing fragments according to the invention and processes for their production by insertion of the fragments into the vector. Also encompassed within the invention are cells transformed or transfected with such cloning or expression vectors and processes for their production by transformation and transfection of the cells with the vector. The invention also extends to processes for cloning fragments according to the invention in cells transformed or transfected with such cloning vectors by culturing the cells, excising the fragments of the invention from the cloned vectors and recovering the fragments. The invention further extends to processes for expressing polypeptides encoded by class one fragments of the invention by culturing cells transformed or transfected with expression vectors as described above and recovering the polypeptides. The construction of vectors, transformation or transfection of cells and culturing of cells and recovery of nucleic acid fragments or polypeptides may all be accomplished by conventional methods, such as described in Maniatis. Antibodies

For use in generating antibodies, the polypeptides and proteins of the invention will be administered to a host according to a conventional immunisation schedule.

Immunisation schedules are well-known to those skilled in the art and generally comprise administration of several separate doses at intervals of from a few days to weeks or even months or years and may include, booster doses at intervals of several years. Preferably the polypeptides and proteins of the invention are administered parenterally in solution or suspension in a suitable diluent or carrier and together with a suitable adjuvant. Antibodies or antibody-producing cells are then recovered from the tissue, for instance the spleen or body fluids of the host. Antibodies are preferably recovered from the blood or serum. Antibody-producing cells are preferably recovered from the spleen or from peripheral blood samples and are then used to produce antibodies following immortalisation. Alternatively DNA from the antibody-producing cells is used in other cells to produce antibodies. These techniques will be described further below.

In a further aspect the present invention provides antibodies against a polypeptide or protein as hereinbefore defined. Such antibodies may be obtained by conventional methods [Williams, Tibtech. 6_.:36, (1988) and are useful in diagnostic and therapeutic applications, such as passive immunisation.

As used herein the term "antibody" is intended to include polyclonal and monoclonal antibodies and fragments of antibodies bearing antigen binding sites such as the F(ab')₂ fragments as well as such antibodies or fragments thereof which have been modified chemically or genetically in order to vary the amino acid residue sequence of one or more polypeptide chains, to change the species specific and/or isotype specific regions and/or to combine polypeptide chains from different sources. Especially in therapeutic applications it may be appropriate to modify the antibody by grafting either the V regions or the CDR loops of the non-human antibody into the human framework or C region sequences, or in order to vary the isotype of the antibody (see EP-A-0,239,400) . The term "antibodies" used herein is further intended to encompass antibody molecules or fragments thereof as defined above produced by recombinant DNA techniques as well as so-called "single domain antibodies" or "dAbs" such as are defined by Ward, E.S. et al. , Nature. 341:544-546 (1989) which are produced in recombinant microorganisms, such as Escherichia coli. harboring expressible DNA sequences derived from the DNA encoding the variable domain of an immunoglobulin heavy chain. By random mutation introduced for instance, during polymerase chain reaction amplification of the original DNA, dAbs may be produced with higher affinities and obtained by screening a library of such randomly mutated DNA sequences and selecting those which enable expression of polypeptides capable of specifically binding the polypeptides or proteins of the invention or scatter factor.

Antibodies according to the present invention react specifically with scatter factor.

Such antibodies may be obtained by conventional methods by immunisation of a host and recovery from a tissue or body fluid thereof or by culturing an antibody-producing cell line as described below.

Antibodies according to the invention are useful in therapy and diagnosis of cancers particularly in inhibiting or assessing the likelihood of metastasis. The antibodies may be used for other purposes including screening cell cultures for the polypeptide or protein expression product of class one nucleotide fragments of the invention, particularly the nascent expression products.

For therapeutic use the antibodies may be administered conventionally to afford passive immunity in the recipient to mutant scatter factor expressed by cancer cells.

Preferably the antibodies are administered parenterally in the form of pharmaceutical formulations comprising a pharmaceutically acceptable carrier or diluent and optionally further comprising preservatives, antioxidants, suspending agents such as detergents and emulgents, buffers and isotonic agents. Dosages will be selected according to the age, weight or body area, sex and state of health of the patient and the size and severity of the tumour to be treated. Preferably the antibodies are administered at from 1 to 20 mg, for instance 2 to 10 mg and most preferably at about 5 mg.

For diagnostic use the antibodies may be linked to or used in conjunction with a diagnostically effective ligand, such as a solid support or a label such as an enzyme label, radiolabel or fluorescent label or other directly or indirectly detectable label. The diagnostically effective ligand may be linked to the antibody or used separately. For in vivo diagnostic applications, a label is targeted to cells expressing scatter factor by the antibody linked thereto or administered separately (simultaneously or sequentially in either order) therewith, the targeted label may be used in tumour detection, localisation and imaging. For passive immunisation and targeting therapeutically or diagnostically effective ligands the antibodies will generally be administered intravenously by injection or infusion, preferably as a pharmaceutical formulation comprising a pharmaceutically acceptable diluent or carrier and optional accessory ingredients as described above. Amounts to be administered in each case will be determined according to the efficacy and serum half-life of the antibody and the patient's tolerance of the agents.

For .in vitro diagnostic use, the ligand may be a solid support or a label such as an enzyme label, chromophore, fluorophore or a radioisotope or other conventional directly or indirectly detectable label.

The use of antibodies linked to or in association with therapeutically or diagnostically effective ligands is well-known.

When administered to a patient or used in a diagnostic test or assay procedure any therapeutically or diagnostically effective ligand not linked to the antibody must be capable of specifically binding with the antibody or must be linked to a moiety itself capable of specifically binding to the antibody. The antibodies may also be used in conventional diagnostic tests and assay methods to detect cells expressing scatter factor, circulating scatter factor or degradation products thereof. For such purposes the antibodies may be linked to labels such as radionuclides, fluorescent labels and enzyme labels or to particles or solid supports. Such labelling is achieved by conventional methods well-known in the art. The assays or tests may be conducted by any known method such as radio-immuno assay or enzyme-linked immunoadsorbant assay using any conventional sandwich technique or by competitive test or assay methods. The present invention also provides an antibody as hereinbefore defined linked to a therapeutically or diagnostically effective ligand and processes for their production comprising linking a suitable ligand to the antibody optionally via a linking group. The invention further provides a diagnostic test or assay process comprising contacting a sample suspected to contain a polypeptide or protein as hereinbefore defined or scatter factor with an antibody of the invention. Preferably the antibody is incubated with the sample to permit binding of the antibody to the polypeptide, protein or scatter factor and the presence of bound or unbound antibody is then detected. The antibody may be linked to a diagnostically effective ligand or such a ligand (provided that it is capable of specifically binding with the antibody or is provided with a moiety capable of specifically binding with the antibody) is used separately. The present invention further provides a diagnostic test or assay kit comprising an antibody of the invention and, optionally, diagnostically effective ligands such as solid supports and directly or indirectly detectable labels, reagents for linking the antibody to the ligand, reagents for detecting the presence of the labels, buffers, standard solutions, positive and negative controls and samples of the antigen to be detected, for standardising, calibrating or validating the test or assay procedure Antibody Production

Antibodies according to the present invention may be produced by conventional methods. Polyclonal antibodies are generated by inoculation of a host animal. For instance a mammal such as a human or conventional laboratory primate or rodent, eg. a rat or mouse, using a polypeptide or protein as hereinbefore described. Inoculation may be by any conventional route, such as by subcutaneous, intramuscular, intravenous or intraperitoneal injection or infusion and according to any conventional immunisation schedule depending upon the nature of antibody required and the properties of the polypeptide. Polyclonal antibodies may be recovered from body fluids such as blood, plasma, or serum obtained from the inoculated animal.

The invention therefore provides a process for producing an antibody according to the invention which comprises immunising a host animal with a polypeptide or protein as hereinbefore described.

Alternatively, the invention provides a process for producing an antibody according to the invention which comprises recovering the antibody from a body fluid of a host animal inoculated with a polypeptide or protein as hereinbefore defined.

Monoclonal antibodies are produced by culturing immortalised cells which secrete antibodies as hereinbefore defined. Cells which secret such antibodies (for instance spleen cells or peripheral blood lymphocytes) are obtained from host animals inoculated as above. Immortalisation may be by any conventional technique, for instance by the use of Epstein-Barr virus or hybridization with an immortal cell line, such as a myeloma of the same or a different species to the host animal, to form a hybridoma.

The present invention therefore provides a cell capable of secreting an antibody as hereinbefore defined.

The invention further provides a process for producing such a cell comprising inoculating a host animal with a polypeptide or protein as hereinbefore defined and recovering antibody producing cells from the host animal.

The invention further provides an immortal cell line capable of secreting an antibody as hereinbefore defined or an immortal cell of such a cell line. The invention further provides a process for producing an immortal cell or cell line capable of secreting antibody according to the present invention comprising recovering antibody-secreting cells from a host animal inoculated with a polypeptide or protein as hereinbefore defined and immortalising the anti-body secreting cells. Optionally the process further comprises selecting cells capable of secreting antibodies as hereinbefore defined prior to an /or after immortalisation.

The invention further provides a process for producing a monoclonal antibody as hereinbefore described comprising culturing an immortalised cell as described above and recovering antibody from the culture medium and/or cell debris. Conventional culture conditions may be employed for n vitro or in vivo (for instance in the peritoneum) growth of the cells. Antibody recovery from body fluids (such as ascites) and culture media or cell debris is by conventional techniques, for instance, involving separation steps and/or affinity chromatography.

Antibodies according to the present invention may also be produced by expression of DNA and RNA encoding the antibodies which DNA or RNA has been recovered from antibody-secreting cells, such as cells obtained from the spleen or peripheral blood of inoculated host animals or immortalised cells as described above. Such expression may be effected using conventional expression systems employing expression vectors such as cosmids, plasmids or viral genomic nucleic acids and transforming or transfecting appropriate host cells with such expression vectors then culturing the transformed or transfected cells and recovering antibody from the culture medium or cell debris. The invention therefore further provides

(a) DNA or RNA encoding an antibody as hereinbefore described;

(b) an expression vector containing nucleic acid according to (a) above in expressible form; (c) a host cell transformed or transfected with an expression vector according to (b) above in expressible form, and (d) a process for producing an antibody as hereinbefore defined comprising culturing a cell according to (c) above under conditions suitable for expression of the nucleic acid and recovering the antibody from the culture medium or cell debris. Therapy and Diagnosis

The present invention also provides a method for therapy or diagnosis comprising administering an effective non- toxic amount of any one of the following agents:

(a) a nucleic acid fragment as hereinbefore defined;

(b) an expression vector containing a nucleic acid of (a) above; (c) a cell transformed or transfected with an expression vector of (b) above; (d) an antibody as hereinbefore defined; or

(e) a cell capable of secreting an antibody of (d) above; or

(f) a polypeptide or protein as hereinbefore defined; to a human or animal in need thereof. The administration may be by any conventional route and dosage regime and the agent may be administered in the form of a pharmaceutical composition as hereinbefore described.

The invention further provides any one of the following agents:

(a) a nucleic acid fragment as hereinbefore defined;

(b) an expression vector containing a nucleic acid of (a) above;

(c) a cell transformed or transfected with an expression vector of (b) above;

(d) an antibody as hereinbefore defined; or

(e) a cell capable of secreting an antibody of (d) above; or

(f) a polypeptide or protein as hereinbefore defined; for use in a method of treatment of the human or animal body or in a method of diagnosis practiced on the human or animal body.

The invention further provides the use of any one of the following agents: (a) a nucleic acid fragment as hereinbefore defined; (b) an expression vector containing a nucleic acid of (a) above;

(c) a cell transformed or transfected with an expression vector of (b) above;.

(d) an antibody as hereinbefore defined; or (e) a cell capable of secreting an antibody of (d) above; or (f) a polypeptide or protein as hereinbefore defined in the production of a medicament for use in a method of treatment of the human or animal body or a method of diagnosis practiced on the human and animal body.

The invention will now be illustrated by the following Examples which are not intended to limit the scope of invention in any way.

Example 1 Isolation and sequence analysis of the scatter factor 3OK protein

Scatter factor was partially purified from medium conditioned by the ras-transformed D4 clone of mouse 3T3 fibroblasts by chromatography on the cationic exchanger Mono S (1) . Fractions active in the MDCK assay (2) were pooled, dialyzed against Mono S buffer A (1) and re-applied to the Mono S column. The scatter factor peak from the second Mono S column was analysed by SDS gel electrophoresis (3) and found to contain a major band with a K = 62K and several minor contaminants. On reduction the 62K band produced bands at 9OK, 57K and 3OK (1) .

Aliquots of this material were separated on 10 x 10 x 0.3 cm Laemmli's minigels containing a 10%-20% gradient of

TITUTE SHEET polyacrylamide and transferred onto PVDF membrane (Immobilon Transfer, 0.45 urn pore size, Millipore) according to the procedure of Matsudaira (4) except that the transfer buffer had a pH of 12 and contained no methanol. The blot was stained with PAGE Blue 83 and the 3OK band excised and stored at -20°C until used for sequence analysis. This was carried out on the Applied Biosystem 470A gas-phase protein sequencer of the Medical Research Council Laboratory of Molecular Biology (Cambridge) . The sequence obtained was as follows (the first residue being the N terminal) . Val-Val-Asn-Gly-lle-Pro-Thr-Gln-Thr-Thr-Val-Gly- ? -Met-Val-Ser-Leu-(Leu)-Tyr-Tyr

Residue 13 is unassigned. Residue in position 18 is probably Leu.

References

1. Gherardi E. , Gray J. , Stoker M. , Perryman M. , & Furlong R (1989) Proc, Natl. Acad. Sci. 86: 5844-5848.

2. Stoker M, & Perryman M. (1985) J. Cell. Sci. 77: 209-223.

3. Laemmli U.K. (1970) Nature 227: 680-685.

4. Matsudaira P. (1987) 262: 10035-10038.

Claims

1. A polypeptide or protein comprising at least five contiguous amino acid residues in a sequence corresponding to formula (I) WNGIPTQTTVGX¹MVSLX²YY (I) wherein the internationally recognised 1-letter code is used to represent amino acid residues and

X¹ is any naturally occurring L-amino acid residue and X² is any amino acid residue, but is preferably a L-leucine residue (L) .

2. A pharmaceutical formulation comprising a polypeptide or protein according to claim 1 and a pharmaceutically acceptable carrier or diluent therefore.

3. A method for the treatment of the human or animal body comprising administering an effective, non-toxic amount of a polypeptide or protein according to claim 2 to a human or animal in need thereof so as to promote wound or ulcer healing in a human or animal having a wound or ulcer.

4. A polypeptide or protein according to claim l bearing a fluorescent label, enzyme label, radionuclide or bound to a particle or solid support.

5. A nucleic acid fragment encoding, or complementary to a fragment encoding a polypeptide or protein according to claim 1.

6. A nucleic acid fragment hybridisable with a fragment according to claim 5.

7. A hybridisation probe comprising a nucleic acid fragment according to claim 5 or 6 bearing a fluorescent label, enzyme label, radionuclide or bound to a solid support.

8. A cloning or expression vector comprising as an insert a fragment according to claim 5 or a cloning vector comprising as insert a fragment according to claim 6.

9. An antibody against a polypeptide or protein according to claim 1.

10. A method for treating the human or animal body comprising administering an antibody according to claim 9 in a therapeutically effective non-toxic amount to a human or animal so as to inhibit the metastatic or other deleterious effects of scatter factor.

11. A diagnostic test method comprising contacting a sample with a polypeptide or a protein according to claim 1, a nucleic acid fragment according to claim 5 or claim 6 or an antibody according to claim 9.

12. A diagnostic test kit comprising a polypeptide or protein according to claim 1, a nucleic acid fragment according to claim 5 or claim 6 or an antibody according to claim 9 and optionally further comprising any one or more accessory components selected from solid supports, directly or indirectly detectable labels, reagents for detecting the presence of the labels, buffers, standard solutions and positive and negative control materials.

13. A cell-transformed with a cloning or expression vector according to claim 8 or capable of secreting an antibody according to claim 9.

14. A polypeptide or protein according to claim 1, nucleic acid fragment according to claim 5 or claim 6 or an antibody according to claim 9 for use in the method of treatment of the human or animal body or in a method of diagnosis practised on the human or animal body.

15. Use of a polypeptide or protein according to claim 1, a nucleic acid fragment according to claim 5 or claim 6 or an antibody according to claim 9 in the production of a medicament for use in a method of treatment of the human or animal body or a method of diagnosis practised on the human or animal body.