WO2012066293A1

WO2012066293A1 - Therapeutic agent

Info

Publication number: WO2012066293A1
Application number: PCT/GB2011/001620
Authority: WO
Inventors: Guiseppe D'alessio; Claudia De Lorenzo; Andrew Kelly
Original assignee: Biotecnol Inc
Priority date: 2010-11-17
Filing date: 2011-11-17
Publication date: 2012-05-24
Also published as: GB201019467D0

Abstract

The invention provides a polypeptide consisting essentially of the amino acid sequence LTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT LIDTNRSRAC HPCSPMCKGS RCWGESSEDC QSLTR (SEQ ID NO:1) or a fragment thereof.

Description

Therapeutic Agent

ErbB2 (HER2/Neu) is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors. The extracellular component of ErbB2 consists of four domains (domains I-IV).

Overexpression of the ErbB2 receptor frequently occurs in breast cancer and other malignancies, and is associated with poor prognosis and with a more aggressive clinical behaviour. Herceptin (Trastuzumab), the only humanized antiErbB2 antibody in commercial use, has proved to be effective in the immunotherapy of breast carcinoma. Cho et al (Cho, H. S., and Leahy, D. J. (2002) Science (New York, N.Y 297(5585), 1330-1333. Cho, H. S., Mason, K., Ramyar, K. X., Stanley, A. M., Gabelli, S. B.,Denney, D. W., Jr., and Leahy, D. J. (2003) Nature 421(6924), 756- 760) described the crystal structure of the extracellular region of ErbB2 both free and in complex with Herceptin (Trastuzumab) and demonstrated that Herceptin binds the C-terminal end of domain IV.

Although Herceptin is effective it can engender cardiotoxicity and a high fraction of breast cancer patients are resistant to Herceptin-treatment, or acquire or develop resistance to Herceptin during treatment.

Thus, there is a requirement for a method for identifying antibodies that antagonise expression of the ErbB2 tyrosine kinase receptor and that do not exhibit the disadvantages associated with Herceptin.

The present inventors have identified an epitope on the ErbB2 receptor. Antibodies that bind to this epitope do not exhibit the cardiotoxicity exhibited by antibodies that bind to other known epitopes on the ErbB2 receptor. The first aspect of the invention relates to a polypeptide comprising the amino acid sequence LTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT

LIDTNRSRAC HPCSPMCKGS RC WGESSEDC QSLTR (SEQ ID NO:l) or a fragment thereof. The polypeptide may consist essentially of SEQ ID NO: 1 , or a fragment thereof.

This sequence represents the amino acid residues from positions 122-195 of domain I of the extracellular region of ErbB2 sequence.

A fragment of the polypeptide of the invention comprises (or may consist of) at least 7, 8, 9, 10, 11, 12 or 13 amino acids of SEQ ID NO: l . The fragment preferably binds to an antibody which is raised against SEQ ID ΝΟ.Ί .

In one embodiment, the polypeptide of the invention extends to amino acid sequences that are at least 80% homologous with the polypeptide defined in SEQ ID NO: 1. In further embodiments, such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the amino acid sequence of the polypeptide in SEQ ID NO: 1.

As is well understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Percent homology of sequences may be determined by visual inspection and mathematical calculation. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST or ALIGN. For example, protein searches can be performed with the XBLAST program to obtain amino acid sequences homologous to protein molecules of the invention. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

In one embodiment of the first aspect of the invention, the polypeptide comprises (or consists of) the amino acid sequence SRACHPCSPMCKGS [SEQ ID NO:2] or a fragment thereof comprising at least 7, 8, 9, 10, 11, 12 or 13 amino acids of SEQ ID NO:2. The amino acid sequence of SEQ ID NO:l represents an epitope in the extracellular region of ErbB2.

The second aspect of the invention relates to a method of screening for an agent that down-regulates expression of the ErbB2 receptor comprising a) contacting a polypeptide of the first aspect of the invention with a candidate agent and b) determining binding of said candidate agent to said polypeptide.

The method of the invention can further extend to selecting agents that down-regulate expression of the ErbB2 receptor and/or to determining the extent of down-regulation of the ErbB2 receptor.

The candidate agent can be an antibody or an antibody fragment; in particular an antibody or antibody fragment derived recombinantly from phage display libraries. The antibody fragment may be a single chain variable fragment (scFv) or a Fab fragment.

The antibody can be a monoclonal or polyclonal antibody derived from immunization of animals. The antibody fragment may be cleaved from the fragment crystallizable region (Fc) of a monoclonal or polyclonal antibody to form a Fab fragment.

An antibody may also be defined as an antigen binding protein.

The antibodies of the invention can comprise a heavy chain constant region and a light chain constant region of a human antibody. Human heavy chain constant regions may be from one of five classes (IgM, IgG, IgA, IgE or IgD) or their sub-classes (IgGl, IgG2, IgG2, IgG4, IgAl or IgA2). Light chain constant regions may be from the kappa or lambda classes. The antibodies of the invention can be human

IgGl /kappa antibodies, for example. Heavy chain and light chain constant regions from non-human antibodies may also be used with the variable domains of the invention, if those constant regions have been deimmunised for use in man. An antibody fragment according to the invention comprises a stretch of amino acid residues of at least at least about 5 to 7 contiguous amino acids, at least about 7 to 9 contiguous amino acids, at least about 9 to 13 contiguous amino acids, at least about 20 to 30 or more contiguous amino acids or at least about 30 to 40 or more consecutive amino acids.

The antibody can specifically recognise a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. "Specifically recognises" in the context of this text means the antibody recognises, interacts with or binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID No: 2, without recognising, interacting significantly with or binding another target which does not structurally resemble the target of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The candidate agent may be part of a library including a phage display library such as an antibody expression library.

The third aspect of the invention provides a method of preparing an antibody or a fragment thereof comprising use of a polypeptide according to the first aspect of the invention. This may involve preparing a hybridoma comprising immunising a host with the polypeptide of the first aspect of the invention, isolating splenocytes from the immunised host and fusing the splenocytes with immortalised cells to form a hybridoma.

The step of immunising a host with the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may be carried out in any manner well known in the art for stimulating the production of antibodies (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)). This includes suspending or dissolving the antigen in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant. The amount of antigen, types of buffers and amounts of adjuvant are well known to those of skill in the art and are not limiting in any way on the present invention.

Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art. In a typical immunisation protocol, a host is injected intraperitoneally with a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 on day 1 and again about a week later. This is followed by booster injections of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 after about two weeks. Other protocols may also be utilised as long as they result in the production of B cells expressing an antibody directed to the polypeptide comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO: 2.

Alternatively, lymphocytes from the unimmunised host are isolated, grown in vitro, and then exposed to the polypeptide comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO: 2 in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out. The next step in this method is the isolation of splenocytes from the immunised host and the subsequent fusion of those splenocytes with an immortalised cell in order to form an antibody-producing hybridoma. The isolation of splenocytes from a host is well-known in the art and typically involves removing the spleen from an anesthetised host, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension. The cells are washed, centrifuged and resuspended in a buffer that lyses any red blood cells. The solution is again centrifuged and remaining lymphocytes in the pellet are finally resuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes are fused to an immortal cell line, for example a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art. The resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. The hybridomas are typically grown on a feeder layer of macrophages. Fusion methods are described in (Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59- 103 (Academic Press, 1986)), the disclosure of which is herein incorporated by reference.

The antibody or fragment thereof according to the third aspect of the invention may be a phage antibody as described in McCafferty, Griffiths, Winter and Chiswell, Notre, Vol 348, 6 December 1990 (and also for example in Ridder, Schnitz, Legay and Cram, Nature Biotechnology, 13, 255-260 (1995) and Winter, Griffiths, Hawkins and Hoogenboom, Annual Review of Immunology, Vol 12, 433-455, April 1994).

The antibodies may be made by recombinant means to obtain chimeric antibodies (variable and constant regions each from a different species) or CDR grafted antibodies (The CDR from a different species). Preferably, the antibodies are at least partly of human origin such as humanized antibodies. Also included are fully humanized antibodies (e.g. described in PCT 93/12227), and fully human antibodies made by recombinant means.

This aspect of the invention further extends to a hybridoma obtainable by the method of the third aspect of the invention.

The invention also provides a method of preparing an antibody comprising preparing a hybridoma according to the third aspect of the invention producing an antibody. The cells are allowed to grow in a selection media for sufficient time for colony formation and antibody production. This is usually between 7 and 14 days. The hybridoma colonies are then assayed for the production of antibodies specific for the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include immunoprecipitation and radioimmunoassay. The wells positive for the desired antibody production are examined to determine is one or more distinct colonies are present. If more than one colony is present, the cells may be re-cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. Positive wells with a single apparent colony are typically recloned and re-assayed to ensure only one monoclonal antibody is being detected and produced.

This aspect of the invention also extends to an antibody prepared according to the method or obtainable by the method of the fourth aspect. The fifth aspect of the invention relates to an isolated antibody that binds a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO: 2.

The antibody inhibits the signalling pathway downstream of the ErbB2 receptor signalling.

The sixth aspect of the invention provides the antibody of the fifth aspect for use in treating cancer. The cancer includes acute myeloid leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain tumours, breast cancer, bronchial adenomas/carcinoids, Burkitt lymphoma, carcinoid tumour, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer,

ependymoma, esophageal cancer, extracranial germ cell tumour, extragonadal germ cell tumours, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumour, gastrointestinal stromal tumour (GIST), germ cell tumour, extracranial, germ cell tumour, extragonadal, germ cell tumour, ovarian, gestational trophoblastic tumour, glioma, adult glioma, childhood brain stem glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, adult (primary), Hodgkin lymphoma, hypopharyngeal Cancer, hypothalamic and visual pathway glioma, childhood, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney (renal cell) cancer, laryngeal cancer, leukemia, acute lymphoblastic, leukemia, acute myeloid, leukemia, chronic lymphocytic, leukemia, chronic myelogenous, leukemia, hairy cell, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell, lung cancer, small cell, lymphoma, AIDS-related, macroglobulinemia, WaldenstrSm, malignant fibrous histiocytoma of

bone/osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, myelogenous leukemia, chronic, myeloid leukemia, acute, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, oral cavity cancer, lip and oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumours, pituitary tumour, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Kaposi's sarcoma, soft tissue, sarcoma, uterine, skin cancer (nonmelanoma), small intestine cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilm's tumour.

In one embodiment, the breast cancer is resistant to treatment with Herceptin.

The antibody can be formulated as a pharmaceutical composition. Pharmaceutical compositions for use in accordance with the present invention may comprise, in addition to the active ingredient (i.e. the antibody), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation.

The formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder.

The seventh aspect of the invention provides the use of the antibody of the fifth aspect in the manufacture of a medicament for treating cancer.

The eighth aspect of the invention provides a method of treating cancer, comprising administering the antibody of the fifth aspect of the invention to a subject.

The term 'treating' is used herein to refer to any regimen that can benefit a human or non-human animal. In one embodiment, the human or non-human animal is in need of such treatment. The treatment may be in respect of an existing condition

(therapeutic treatment) or may be prophylactic (preventative treatment). Treatment may include curative, alleviation, palliative or prophylactic effects.

More specifically, treatment includes "therapeutic" and "prophylactic" and these types of treatment are to be considered in their broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition.

Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylactic" may be considered as reducing the severity of or preventing the onset of a particular condition.

"Prophylactic" also includes preventing reoccurrence of a particular condition in a patient previously diagnosed with the condition. "Therapeutic" may also reduce the severity of an existing condition.

The antibody of the invention may be administered alone but will preferably be administered as part of a pharmaceutical composition, which will generally also comprise a suitable pharmaceutical excipient, diluent or carrier which would be selected depending on the intended route of administration.

The antibodies of the invention may be administered to a patient in need of treatment or that might benefit from such treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the form of the antibody to be administered.

Route of administration may include; parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch), some further suitable routes of administration include (but are not limited to) oral (including buccal and sublingual), rectal, nasal, topical, infusion, vaginal, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example a nebuliser or inhaler, or by an implant.

In preferred embodiments, the composition is deliverable as an injectable

composition, is administered orally, or is administered to the lungs as an aerosol via oral or nasal inhalation. For administration via the oral or nasal inhalation routes, preferably the active ingredient will be in a suitable pharmaceutical formulation and may be delivered using a mechanical form including, but not restricted to an inhaler or nebuliser device. Further, where the oral or nasal inhalation routes are used, administration is by a SPAG (small particulate aerosol generator) may be used.

For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable H, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982).

The composition is preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.

For example, in one embodiment, a suitable dose may be from 2-8mg/kg/week. The ninth aspect of the invention relates to a nucleic acid sequence, for example a RNA sequence or a DNA sequence, encoding a polypeptide of the invention.

The nucleic acid sequences of the invention are useful in the production of polypeptides of the invention.

The nucleic acid sequence of the invention can additionally comprise a promoter or other regulatory sequence which controls expression of the nucleic acid. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. It may not be necessary to utilise the whole promoter or other regulatory sequence. Only the minimum essential regulatory element may be required and, in fact, such elements can be used to construct chimeric sequences or other promoters. The essential requirement is, of course, to retain the tissue and/or temporal specificity. The promoter may be any suitable known promoter, for example, the human cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV thymidinekinase, the early and late SV40 promoters or the promoters of retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) and metallothionine promoters such as the mouse metallothionine-I promoter. The promoter may comprise the minimum comprised for promoter activity (such as a TATA elements without enhancer elements) for example, the minimum sequence of the CMV promoter.

Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence.

The polymerase chain reaction (PCR) procedure may be employed to isolate and amplify a DNA sequence encoding a desired protein sequence. Oligonucleotides that define the desired termini of the DNA sequence are employed as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA sequence into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and

Applications, Innis et al., eds., Academic Press, Inc. (1990).

The tenth aspect of the invention relates to a vector comprising the nucleic acid sequence of the second aspect of the invention. The present invention also provides recombinant cloning and expression vectors containing DNA encoding the polypeptides of the invention

The eleventh aspect of the invention relates to a host cell comprising the nucleic acid sequence of the second aspect of the invention or the vector of the third aspect of the invention. Vectors and host cells comprising nucleic acid sequences of the invention may be used to prepare the polypeptides of the invention encoded by the nucleic acid sequences. Vectors of the invention may include, among others, chromosomal, episomal and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculo-viruses, papova-viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Generally, any vector suitable to maintain, propagate or express nucleic acid to express a polypeptide in a host, may be used for expression in this regard. The twelfth aspect of the invention extends to a method of producing a polypeptide of the first aspect of the invention comprising culturing the host cell of the eleventh aspect of the invention under conditions that promote expression of the polypeptide and recovering the expressed polypeptide from the culture. In a further embodiment, the method of producing a polypeptide according to the invention further includes the step of purifying the expressed polypeptide.

Expression, isolation and purification of the polypeptides of the invention may be accomplished by any suitable technique, including but not limited to the following:

Any suitable expression system may be employed. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, may be incorporated into an expression vector used to produce a polypeptide of the invention. In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed and the mRNA translated into a fusion polypeptide comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, but allows secretion of polypeptide from the cell.

Suitable host cells for expression of polypeptides of the invention include any cell that is capable of producing posttranslationally polypeptides and includes yeast, fungi, insect and higher eukaryotic cells. Mammalian cells, and particularly human embryonic kidney (HEK), Chinese hamster ovary (CHO) or baby hamster kidney

(BHK) cells, are particularly preferred for use as host cells. Appropriate cloning and expression vectors for use with mammalian and yeast hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1986) (ISBN 0444904018).

Established cell lines of mammalian origin also may be employed, for example, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650) and HEK293 (e.g., ATCC CRL 1573) cell lines. In a preferred embodiment, the HEK-293F cell line is used. Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine or

Lipofectamine 2000 lipid reagents (Gibco/BRL) or Lipofectamine-Plus lipid reagent, can be used to transfect cells (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413- 7417, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using methods known in the art, such as, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. in

Enzymology 185:487-51 1, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotics, such as G418 (Geneticin) and hygromycin B. Cells harbouring the vector can be selected on the basis of resistance to these compounds.

Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors can be used. For example, the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534).

Yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae), can also be used. Other genera of yeast, such as Pichia (Pichia pastoris) or

Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2 [mu] yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionine, 3- phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3 -phosphate

dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-glucose isomerase, and glucokinase.

The yeast [alpha] -factor leader sequence may be employed to direct secretion of the polypeptide. The [alpha] -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81 :5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene. Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al, Proc. Natl. Acad. Sci. USA 75: 1929, 1978. A polypeptide according to the invention can be prepared using animal or plant transgenic technology. For example, the polypeptides of the invention can be produced within the mammary glands of a host female mammal. This is further discussed in US2006/0166915, the contents of which is incorporated herein. Production in transgenic plants may also be employed. Expression may be generalised or directed to a particular organ, such as a tuber (see, Hiatt, Nature 344:469-479 (1990), for example).

With respect to any type of host cell, as is known to the skilled artisan, procedures for purifying a recombinant polypeptide will vary according to such factors as the type of host cells employed and whether or not the recombinant polypeptide is secreted into the culture medium.

In general, the recombinant polypeptide can be isolated from the host cells if not secreted, or from the medium or supernatant if soluble and secreted, followed by one or more concentration, salting-out, ion exchange, hydrophobic interaction, affinity purification or size exclusion chromatography steps.

As to specific ways to accomplish these steps, the culture medium first can be concentrated using a commercially available protein concentration filter, for example, a Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium.

Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. In addition, a chromatofocusing step can be employed. Alternatively, a hydrophobic interaction chromatography step can be employed. Suitable matrices can be phenyl or octyl moieties bound to resins. In addition, affinity chromatography with a matrix which selectively binds the recombinant protein can be employed. Examples of such resins employed are lectin columns, dye columns, and metal-chelating columns. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing apolar RP-HPLC media, (e.g., silica gel or polymer resin having pendant methyl, octyl, octyldecyl or other aliphatic groups) can be employed to further purify the polypeptides. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide an isolated and purified recombinant protein.

Transformed mammalian host cells are preferably employed to express polypeptides of the invention as a secreted polypeptide in order to simplify purification. Secreted recombinant polypeptide from mammalian host cell fermentation can be purified by methods which are well known to those skilled in the art.

It is also possible to utilise an affinity column comprising a polypeptide-binding protein, such as a monoclonal antibody generated against a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilised, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the invention.

Polypeptides of the invention can be substantially purified (to substantial

homogeneity), as indicated by a single protein band upon analysis by SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Purified to substantial purity means purified to more than 90% homogenous, including over 95% homogenous.

The protein band may be visualised by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. The skilled man will recognise that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is intracellular, membrane-bound or a soluble form that is secreted from the host cell.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:

Figure 1 illustrates binding curves of Erbicin (A) or Erb-hcAb (B) to SKBR3 cells obtained by ELISA assays performed in the absence (black symbols) or in the presence (empty symbols) of N 28 antibody;

Figure 2 illustrates hydrolysis of the ECD-Erb-hcAb complex with endoprotease Glu- C. A single specific protein band around 30kDa, marked with the asterisk is present in the three sample lanes and not in the controls. Lane 1-3: Beads with ECD-Erb- hcAb after 30, 60, 120 min of incubation with Glu-C endopreprotease. Lane 4: Beads with Erb-hcAb after 120 min of incubation with Glu-C endoprotease (control). Lane 5: Markers. Lane 6: Supernatant from Erb-hcAb after 120min of incubation with Glu- C endopreprotease (control). Lane 7-9: Supernatant from ECD-Erb-hcAb after 30, 60, Figure 3 illustrates limited proteolysis of ECD with endoprotease Glu-C and detection of epitope containing region by western blot assay. Panel A: western blot with Erb- hcAb antibody of fractions from limited proteolysis after 30 and 60 min; intact ECD was loaded as control. Panel B: Colloidal Coomassie staining of fractions from limited proteolysis after 30 and 60 min; intact ECD was loaded as control. Panel C:

ECD sequence; the underlined sequence was identified by MALDI-MS analysis in the protein band at 55kDa from the 30 min Coomassie lane;

Figure 4A illustrates a Ribbon diagram of a modelled structure of Erbicin. Figures 4B-C illustrate the view of the interface region in the model of the Herceptin-like (4B) or Pertuzumab-like (4C) putative complex of Erbicin (cyan) with ECD (blue). The structures of Herceptin (green) and Pertuzumab (red) have been also reported for comparison. As can be clearly seen (Fig. 4B), the side chain of Tyr52 of Erbicin is spatially too close to the backbone atoms of ErbB2 Val286. Figure 4D illustrates the overall model of the Erbicin (cyan) and ErbB2 ECD (blue) complex from

computational docking. Details for docking calculation are described in Materials and methods. These figures are drawn with Pymol: The PyMOL Molecular Graphics System on World Wide Web. http://www.pymol.org;

Figure 5 illustrates binding curves of Erb-hcAb (black circles) or Herceptin (black squares) to the ErbB2-ECD166-179 (SRASHPSSPMSKGS) peptide obtained by ELISA assays;

Figure 6 illustrates binding of Erb-hcAb and Herceptin to ErbB2-ECD in competitive ELISA peptide assay. Erb-hcAb (A) and Herceptin (B) were preincubated with peptide 166-179 (black bars), the mutant peptide (striped bars), or unrelated peptide (empty bars) and then tested for binding to immobilized ErbB2-ECD. EXPERIMENTAL PROCEDURES

Antibodies and peptides

The antibodies used were: Herceptin (Genentech, South San Francisco, CA, USA); HRP-conjugated anti-His antibody (Qiagen, Valencia, CA, USA); horseradish peroxidise conjugated goat anti-human affinity isolated IgGl (Fc-specific, Sigma, St Louis, MO, USA). Erb-hcAb was prepared as previously described in De Lorenzo, C, Tedesco, A., Terrazzano, G., Cozzolino, R., Laccetti, P., Piccoli, R., and DAlessio, G. (2004) British Journal of Cancer 91(6), 1200-1204. The anti-ErbB2 N28 antibody was a generous gift of Dr. Michael Sela (Weizman Institute of Science, Rehovot, Israel).

The synthetic peptide corresponding to the amino acid sequence 166-179 (SRASHPSSPMSKGS) of ErbB2-ECD (extracellular domain of ErbB2), the variant peptide with His 170 replaced by Glu (SRASEPSSPMSKGS) and the unrelated control peptide (RYPHCRYRGSPPSTRK) were synthesized (95% purity) by Thinkpeptides, Magdalen Centre, Oxford Science Park (United Kingdom). ErbB2- ECD was prepared as previously described in Troise, F., Cafaro, V., Giancola, C, DAlessio, G., and De Lorenzo, C. (2008) The FEBS Journal 275(20), 4967-4979.

ErbB2-ECD - Erb-hcAb complex

Aliquots of Erb-hcAb (800μg) were immobilized onto 0.4 mL of CNBr-activated Sepharose (GE Healthcare Amersham Bioscience AB, Uppsala, Sweden). The antibody was immobilised to the agarose via secondary amine chemistry according to the manufacturer's instructions. Following the blocking of the unreacted groups with 1 M ethanolamine hydrochloride (Sigma), the resin was washed with PBS (Sigma), and soluble ErbB2-ECD (400μg) in PBS was added to the agarose containing the immobilized Erb-hcAb. Binding of the antigen was performed at 4°C by gently rotating overnight. Enzymatic hydro lyses on ErbB2-ECD - Erb-hcAb complex

Aliquots of 60μ1 of agarose beads suspension containing 300 pmol of ECD complexed with Erb-hcAb were digested with Glu-C endoprotease (Roche) and trypsin (Sigma), using an E/S ratio of 1 : 10 (w/w) in a final volume of 120μ1 of 10 mM Tris-HCl buffer, pH 7.4 at 37°C. An equivalent amount of isolated antibody was digested in the same experimental conditions and used as a control.

Aliquots of 40 μΐ of sample and control were withdrawn after 30, 60 and 120 min of reaction and centrifuged for removal of liquid phase containing unbound ECD fragments. The beads were then washed in Tris-HCl buffer and the elution of antibody bound ECD fragments was performed in Laemmli buffer (lOOmM Tris-HCl pH 6.8, 4% SDS, 0.2% blue bromophenol, 20% glycerol). Samples were fractionated by a 15% SDS-PAGE gel. The supernatant fractions containing the unbound proteins were dried under vacuum, dissolved in Laemmli buffer and loaded onto the same gel as further control. The gel was stained by Colloidal Coomassie (Pierce, Rockford, 1L).

Limited proteolysis on isolated ECD

An aliquot of 2 nmol (~140μg) of ECD was digested with Glu-C endoprotease, using two different E/S ratios (1/50 and 1/10, w/w) in a final volume of 140 μΐ of lOmM Tris-HCl buffer, pH 7.4 at 37°C. Aliquots of 70 μΐ of the digestion mixture were withdrawn after 30 and 60 min and the reactions stopped by adding 23.6μ1 of concentrated Laemmli buffer and boiling for 5 minutes.

Small aliquots of 10 μg were withdrawn from each ECD sample and used for western blot assay. All samples were fractionated on the same 15% SDS PAGE; the gel was divided and the part containing the small aliquots was submitted to western blot analysis using 20μg/ml of the primary antibody (Erb-hcAb) in 1% no-fat milk in phosphate buffer (Sigma); the secondary antibody, anti-human IgGl (Fc-specific) HRP-conjugated, was used in a dilution of 1/1000 (v/v). The portion of the gel containing larger amount of sample was stained by colloidal Coomassie and employed for mass spectrometry identification following in-gel tryptic hydrolysis. In situ hydrolyses and mass spectrometry analyses

Protein bands stained with colloidal Coomassie were excised from the gel and destained by repeated washings with 50 mM NH₄HC0₃, pH 8.0, and acetonitrile. Samples were reduced and carboxyamidomethylated with 10 mM DTT (Sigma) and 55 mM iodoacetamide (Fluka) in 50 mM NH₄HC0₃ buffer, pH 8.0. Tryptic digestion of the alkylated samples was performed at 37°C overnight, using 100 ng of trypsin.

For the MALDI-MS analysis 1 μΐ of peptide mixture was mixed with an equal volume of a-cyano-4hydroxycynnamic acid as matrix (in acetonitrile/50 mM citric acid

[70:30, v/v]), applied to the metallic sample plate and air dried. The Applied

Biosystems mass spectrometer was a MALDI Voyager DE-PRO equipped with a reflectron TOF analyser and used in delayed extraction mode. Mass calibration was performed using the standard mixture provided by the manufacturer. LC-MS/MS analyses were performed on a CHIP MS Ion Trap XCT Ultra equipped with an 1100 HPLC system and a chip cube (Agilent Technologies, Palo Alto, CA, USA). After loading, the peptide mixture (10 μΐ in 0.2% formic acid) was first concentrated and washed at 4 μΐ/min in a 40 nL enrichment column (Agilent

Technologies chip), with 0.1 % formic acid as eluent. The sample was then fractionated on a Cn reverse-phase capillary column (75 μπΐ x 43 mm) onto a CHIP (Agilent Technologies chip) at a flow rate of 200 nl/min, with a linear gradient of eluent B (0.2% formic acid in 95% acetonitrile) in A (0.2% formic acid in 2% acetonitrile) from 7% to 60%o in 50 min. Peptide analysis was performed using data- dependent acquisition of one MS scan (mass range from 400 to 2000 m/z) followed by MS/MS scans of the three most abundant ions in each MS scan.

Enzyme Linked Immuno Sorbant Assays (ELISA)

For the binding assays of Erb-hcAb antibody to the ErbB2ECD166-179

(SRASHPSSPMSKGS) peptide, a 96- well plate was coated with 20 μ ταΐ of soluble peptide in PBS, kept overnight at 4°C and blocked for 1 h at 37 °C with 5% bovine serum albumin (BSA) (Sigma) in PBS. To the plate, rinsed with PBS, were added increasing concentrations of Erb-hcAb or Herceptin (25 nM-1.2 μΜ) in ELISA buffer (PBS/BSA 1%) and incubated for 2 h at room temperature with a blank control of PBS. After rinsing with PBS, an anti-human IgGl (Fc-specific) HRP-conjugated antibody was added in ELISA buffer for antibodies detection. After 1 h at room temperature, the plate was rinsed with PBS and bound antibodies were detected by using 3,3',5,5-tetramethylbenzidine (TMB) as a substrate (Sigma). The product was measured at 450 nm using a microplate reader (Multilabel Counter Victor 3, Perkin Elmer, Cologno Monzese, Italy). The reported affinity values are the means of at least three determinations (standard deviation = 5%). The binding of Erbicin, Erb-hcAb and N28 to the receptor was tested by using ErbB2- positive SKB 3 cells, as previously described in De Lorenzo, C, Tedesco, A., Terrazzano, G., Cozzolino, R., Laccetti, P., Piccoli, R., and D'Alessio, G. (2004) British Journal of Cancer 91(6), 1200-1204. For Erbicin detection the peroxidase- conjugated anti-His mAb (Qiagen) was used; peroxidase-conjugated anti -human IgG (Fc-specific) antibodies (Sigma) or peroxidase-conjugated anti-mouse IgG antibodies (Pierce) were used for detection of human Erb-hcAb and mouse N28 antibodies, respectively.

The binding ability of Erb-hcAb or Herceptin antibodies to ErbB2-ECD was measured in the presence of increasing concentrations of three different soluble peptides: ErbB2-ECD166-179 (SRASHPSSPMSKGS), mutated ErbB2-ECD166-179 (SRASEPSSPMSKGS) and an unrelated peptide (RYPHCRYRGSPPSTRK). A 96- well plate was coated with 5 g/ml of purified ErbB2-ECD in PBS and left overnight at 4°C. After blocking, as described above, Erb-hcAb or Herceptin (50 nM) was added to the wells in triplicate before or after incubation with the peptides at increasing concentrations (60 nM-1.2 μΜ) overnight at 4°C. After 2h incubation at room temperature, the plate was rinsed with PBS and bound Erb-hcAb or Herceptin was detected as mentioned above. Standard deviations were below 10%. Computational techniques

The three-dimensional structure of Erbicin was built by homology modelling using the canonical structures method for the hypervariable loops and standard homology modelling techniques for the framework regions. Briefly, the framework structure of the light and heavy chain variable domains (VL and VH) from the PDB code 1DZB (Ay, J., Keitel, T., Kuttner, G., Wessner, H, Scholz, C, Hahn, M., and Hohne, W. (2000) Journal of Molecular Biology 301(2), 239-246) was used as the scaffolding on which the six complementarity determining regions (CDR) loops are built. The CDR loops were assigned according to the definitions proposed by Chothia and co-workers (Chothia, C, and Lesk, A. M. (1987) Journal of Molecular Biology 196(4), 901 -917. Chothia, C, Lesk, A. M., Tramontano, A., Levitt, M., Smith-Gill, S. J., Air, G., Sheriff, S., Padlan, E. A., Davies, D., Tulip, W. R., and et al. (1989) Nature

342(6252), 877-883) with the exception of the H3 CDR loop that is predicted de novo. This is a short (6 residues) loop, which should have a reduced conformational accessible space and only few conformations compatible with the rest of the protein structure. The Erbicin model was validated by the Procheck program (Laskowski, R. A., MacArthur M. V., Moss, D. S., and Thornton, J. M. (1993) Journal of Applied Crystallography 26(Pt 2), 283-291).

Rigid docking (van Dijk, A. D., Boelens, R., and Bonvin, A. M. (2005) The FEBS Journal 272(2), 293-312) of the Erbicin model onto ErbB2 ECD was performed using FTDock (Gabb, H. A., Jackson, R. M., and Sternberg, M. J. (1997) Journal of

Molecular Biology 272(1), 106-120). Given two molecules, FTDock computes the three-dimensional transformations of one of the molecules with respect to the other with the goal of maximizing surface shape complementarity while minimizing the number of steric clashes. The scoring method of FTDock also includes electrostatic filters. The candidate models were then scored according to an energy function.

The solutions were visually examined, clustered and evaluated with respect to experimental and theoretical criterions. The extensive rigid-body docking and the use of structural and biochemical data to filter the results are expected to produce a reasonable model of the complex. The final complex structure was then studied to analyze the intermolecular contacts and identify specific residue level interactions between the proteins. This protocol has allowed a successful prediction of the structure of ECD-Pertuzumab and ECD-Herceptin complexes. A protein-protein interaction server was used to identify the residues at the interface in the complex and to evaluate the interface features (McDonald, I. K., and Thornton, J. M. (1994) Journal of Molecular Biology 238(5), 777-793). The presence of putative hydrogen bonds and salt bridges were calculated with

Hbplus program (McDonald, I. K., and Thornton, J. M. (1994) Journal of Molecular Biology 238(5), 777-793). Assessment of the Erbicin model using the Procheck (Laskowski, R. A., MacArthur M. V., Moss, D. S., and Thornton, J. M. (1993) Journal of Applied Crystallography 26(Pt 2), 283-291), Prosall (Sippl, M. J. (1993) Proteins 17(4), 355-362) and CCP4 programs ((1994) Acta Crystallographica 50(Pt 5), 760- 763) suggests that it has a low energy, a good stereochemical quality, and structural features of the interface (including the surface complementarity SC value (Lawrence, M. C, and Colman, P. M. (1993) Journal of Molecular Biology 234(4), 946-950) comparable with those observed in ECD-Herceptin (PDB code: 1N8Z) and ECD- Pertuzumab{2C4} (PDB code: 1 S78) complexes.

RESULTS

The epitope recognized by EDIA is close to that of the anti-ErbB2 N-28 antibody Based on previously reported results of ELISA assays (De Lorenzo, C, Troise, F., Cafaro, V., and D'Alessio, G. (2007) British Journal of Cancer 97(10), 1354-1360), all the available anti-ErbB2 monoclonal antibodies such as Herceptin, 2c4 (Pertuzumab), 7c2 and MAB74, recognize different epitopes from that of Erbicin-derived immunoagents.

The apparent binding affinity of the compact Erb-hcAb antibody for ErbB2 on SKBR3 cells, i.e. the concentration corresponding to half-maximal saturation, is about 1 nM, comparable to the value of 4 nM, previously determined for the parental scFv (Erbicin), as reported (De Lorenzo, C, Arciello, A., Cozzolino, R., Palmer, D. B., Laccetti, P., Piccoli, R., and D'Alessio, G. (2004) Cancer Research 64(14), 4870- 4874). To determine if the novel immunoagents recognize a different epitope from that targeted by N-28 antibody (Yip, Y. L., Novotny, J., Edwards, M, and Ward, R. L. (2003) International Journal of Cancer 104(3), 303-309), competition experiments were carried out by repeating the ELISA assays on SKBR3 cells in the presence of N- 28.

In these experiments the parental scFv (Erbicin), or Erb-hcAb, was added at increasing concentrations (5-40 nM) to ErbB2 -positive cells preincubated with N-28 at a saturating concentration (50 nM) for 1 h, or to untreated cells. Binding was detected with a peroxidase-conjugated anti-His or anti -human Fc mAb capable to reveal the scFv or Erb-hcAb, respectively. As shown in Fig. 1, the presence of N-28 inhibited significantly the binding of the monovalent scFv Erbicin to the cells, whereas it slightly reduced the binding of the bivalent Erb-hcAb compact antibody. The binding ability of N-28 mAb, detected with a peroxidase-conjugated anti-mouse secondary antibody (data not shown), was unaffected by the presence of either Erbicin or Erb-hcAb. These results strongly suggest that the epitope recognized by the Erbicin-derived immunoagents is close but not overlapping to that of N-28, as Erb- hcAb is still capable of binding to the cells in the presence of N-28, although with a lower affinity.

Epitope Mapping

ErbB2-ECD - Erb-hcAb complex

Two different strategies based on the integration of limited proteolysis experiments and mass spectrometric methodologies were employed for the identification of the specific epitope on the extracellular domain (ECD) of ErbB2 receptor recognised by Erb-hcAb. The first approach is based on the protection effect exerted by the antibody on the specific interacting region that prevents hydrolysis by proteolytic enzymes. The ECD-Erb-hcAb complex was submitted to enzymatic digestions in strictly controlled conditions to identify the protein region masked by the interaction. The ErbB2-ECD - Erb-hcAb protein complex was covalently bound to agarose beads and incubated with proteases in controlled conditions of time, enzyme/substrate ratio, temperature and pH in order to maintain the complex stability and to drive the hydrolysis towards the regions of the protein not involved in binding with the antibody. A sample of Erb-hcAb was also immobilised onto the beads in the absence of ECD and used as a control.

The ErbB2-ECD - Erb-hcAb complex was initially digested with Glu-C endoprotease using an enzyme to substrate ratio of 1/10 (w/w). Three aliquots of the digestion mixture were withdrawn at 30, 60 and 120 min and the beads were separated from the supernatants by centrifugation. The beads, still containing the complex between Erb- hcAb and the ECD region involved in the interaction, were extensively washed and the protein samples eluted in denaturing conditions and fractionated by SDS-PAGE. The supernatants of the three aliquots were dried under vacuum, dissolved in

Laemmli buffer and used as a further control in the SDS-PAGE analysis.

Fig. 2 shows the corresponding gel stained by colloidal Coomassie where several bands belonging either to the antibody or to ECD were detected. A single specific protein band with an electrophoretic mobility of about 30kDa could be observed in the three sample lanes, a band absent in both controls. This result suggested that the 30kDa protein band might contain the ECD epitope specifically recognised by Erb- hcAb and protected by Glu-C digestion. The band was excised from the gel from the three sample lanes, in situ digested with trypsin and the resulting peptide mixtures were analysed by nano-LC-MS-MS.

A series of peptides mapping onto the N-Terminal ECD domain were unequivocally identified suggesting that the epitope region was located within this region of the ECD structure. On the basis of the apparent molecular mass of the fragment as estimated by electrophoretic mobility, the enzyme specificity and the arrangement of disulphide bridges in the ECD sequence, the occurrence of a single proteolytic event at Glu243 resulting in the production of the fragment 1-243 was inferred. The slight difference in molecular mass as compared with the expected mass value for this fragment, 26763 Da, could be accounted for by the presence of several glycosylation sites localised in the N-terminus domain (Asn46, Asnl02, Asnl03 and Ans237).

A second experiment carried out by using trypsin as a proteolytic probe confirmed these results as mass spectrometric analyses led to the identification of the ECD region protected by the antibody in the N-terminal domain of the protein (data not shown).

Limited proteolysis on isolated ECD

A complementary approach combining limited proteolysis on isolated ECD with western blot methodologies and protein identification by mass spectrometry was further employed to confirm the above results and finely restrict the target epitope region.

Isolated ECD samples were incubated with endoprotease Glu-C using an enzyme to substrate ratio of 1/50 for 30 and 60 min respectively. A small aliquot corresponding to 10 μg of the initial protein content was withdrawn from each samples and fractionated by SDS-PAGE, together with the remaining portion of the 30 and 60 min samples. The gel was divided and the portion containing the small aliquots was used for western blot analysis by using Erb-hcAb, whereas the remaining part of the gel was used for colloidal Coomassie staining.

The western blot analysis (Fig. 3) confirmed the presence of a large amount of undigested protein with an apparent molecular mass of 90kDa (theoretical molecular weight 69349 Da) given the presence of several glycosylation moieties. Besides the intact protein, a single band at 50kDa was recognised by Erb-hcAb only in the 30 min sample. The corresponding band from the Coomassie stained gel (Fig. 3) was excised and digested in situ with trypsin and the resulting peptide mixture was analysed by MALDI mass spectrometry and LC-MS/MS methodologies. The ECD protein sequence was almost completely mapped from residue 1 1 to 347 (Fig. 3), confirming the occurrence of the epitope recognised by Erb-hcAb in the first two domains (LI and CRl) of ECD.

In order to restrict the search for the epitope region, a second experiment was carried out with endoprotease Glu-C using a higher E/S ratio (1/10) for lh. Samples were treated as described before. The western blot analysis of the fragments released by Glu-C hydrolysis showed the presence of a small amount of intact BCD, and three immuno-positive bands at 50, 30 and 24kDa, respectively. Mass mapping experiments carried out on the 50kDa protein band excised from a preparative gel confirmed the above results indicating the occurrence of the immunoresponsive epitope within the first two ECD domains, LI and CR1. Mass analyses of the peptides from the 30 kDa protein band showed an almost complete sequence coverage of the 122-195 region of the LI domain. Moreover, the absence of the N-terminal end in the mass spectra suggested that the epitope region would be limited to the C-terminal region of LI domain. The mass spectrometric analyses of the tryptic peptides from the 24kDa protein allowed for the identification of few peptides in the 122-166 ECD region, confirming that the Erbicin recognised epitope should lie within the C-terminal half of the LI domain.

Erbicin and ErbB2 ECD complex from computational docking

To reveal the molecular bases of the different binding properties of the EDIA with respect to the previously characterized antibodies and to identify which interactions are responsible for the EDIA/ErbB2 recognition, a homology

modelling/computational docking approach has been used. We first built a three- dimensional model of Erbicin, using the canonical structures method for the hypervariable loops and standard homology modelling techniques for the framework regions. The model (Fig. 4A) has a Prosa Z-score of -6.53, a value in the range of scores typically found in proteins of similar sequence length, and shows the 96.4% of residues are in most favoured or in allowed regions of the Ramachandran map. The modelled protein is characterized by a predominantly canonical structure with a short (6 residues) H3 loop. The molecular surface is rather flat with cavities in

correspondence of the CDR loops.

To identify the structural origins of the different binding properties between Erbicin and the two immunoagents of known structures, Herceptin and Pertuzumab, we have obtained two structural models of putative complexes between Erbicin and ECD, with Erbicin bound as Herceptin or Pertuzumab, respectively. In particular, Erbicin was aligned to Herceptin (C-a rmsd = 0.94 A) in the first complex (Herceptin-like), and to Pertuzumab in the second complex (Pertuzumab-like) (C-a rmsd = 1.00 A).

These models provide valuable information on the origin of the different behaviour of Erbicin with respect to Herceptin and Pertuzumab. In particular, when compared to Herceptin, Erbicin presents a deletion in correspondence of H3 loop (6 vs. 11 residues) that prevents the binding to domain IV (Fig. 4B). The origin of the differences between Pertuzumab and Erbicin seems, instead, to be related to the substitution of residues Asp31, Asn52 e Asn54 with Ser31, Tyr52 and Gly54, respectively (see for example Fig. 4C). It should be recalled that Asp31 of

Pertuzumab forms a strong hydrogen bond with the side chain of Ser288 of ECD and participate to hydrophobic interactions with the C atoms of Val286 and Thr290 of ECD. Furthermore, ND2 atom of Asn52 forms a hydrogen bond with the backbone oxygen of Val286 of ECD, whereas OD1 and ND2 atoms of Asn54 interact with backbone atoms of Cys246 and VaI286 and with side chain atoms of Thr268.

To reveal the region of ECD involved in the interaction with EDIA, a computational docking was performed by FTDock program. These calculations were based on the model of Erbicin, here reported, and, taking into account the experimental evidences that the epitope involve ECD residues 122-195, on only domain I of ECD. The solutions were visually examined and evaluated with respect to experimental and theoretical criteria. In particular, the model should have a high surface

complementarity at the interface and should bury a surface area > 600 A² per molecule. Finally, the model should have low energy and should be reproduced when docking calculations were repeated using different programs and/or input parameters. Upon clustering the 30 solutions with the lowest energy values, we identified three potential models, one of which fulfils the previous criteria (Fig. 4D). In this model, Erbicin binds ECD in the cleft between the light and the heavy chain variable domains. A total of 23 residues form the interface that is characterized by a good surface complementarity (sc 0.55). The ECD-Erbicin complex buries about 750 A² of accessible surface area per molecule over a long groove. The peptide regions of antibody participating in direct contacts with ECD include the CDR H3 loop (Argl 00, Aspl 01, Ser 102), the CDR HI loop (Thr30, Ser31, Tyr32) and residues: Tyrl81, Serl82, Gly225, Ser226, Pro227. The ErbB2 residues at the interface mainly involve the cysteine rich fragment of region 161-189. In particular, Erbicin tightly binds the ECD region i₆₆SRACHPCSPMCKGS_]79 in which cysteine 172 forms an S-S bridge with cysteine 181 and cysteine 176 forms an S-S bridge with cysteine 189. A central role in the ECD-Erbicin interaction is played by His 170 of ECD, which fills a hydrophobic cavity lined by the side chains of tyrosines 123, 163, 164 and 181 and by Ser 182, where it may be involved in stacking interactions with one of the aromatic residues and in a hydrogen bond with the OG of the Ser.

ELISA assays with specific peptides

In order to validate the ECD-Erbicin model, which could be used with confidence for further experimental and computational work, a peptide with the amino acid sequence SRASHPSSPHSKGS (ErbB2-ECD 166-179) was synthesized and used for ELISA assays with Erb-hcAb. As a control, parallel ELISA assays were carried out using Herceptin.

As shown in Fig. 5, indirect ELISA revealed that Erb-hcAb was able to bind to the peptide SRASHPSSPMSKGS although with a lower affinity than that previously measured for the ErbB2-ECD (Troise, F., Cafaro, V., Giancola, C, DAlessio, G., and De Lorenzo, C. (2008) The FEBS Journal 275(20), 4967-4979), whereas Herceptin did not show any significant binding ability. To assess the specificity of Erb-hcAb antibody binding to the sequence 166-179, competition ELISA assays were performed. In these experiments, the binding ability of Erb-hcAb or Herceptin antibodies for ErbB2-ECD was measured in the absence or in the presence of increasing concentrations of the soluble peptide, mentioned above.

As shown in Fig. 6A, the peptide SRASHPSSPMSKGS inhibits the binding of Erb- hcAb to ECD in a dose-dependent manner, whereas it does not affect the binding of Herceptin to ECD (Fig. 6B). To further test the validity of the model, a peptide containing the same sequence but with His 170 replaced by Glu (SRASEPSSPMSKGS) was synthesized and tested as described above. Furthermore, an unrelated peptide (RYPHCRYRGSPPSTRK) was also used as a control in parallel experiments. As shown in Figs. 6A & B both the mutant and irrelevant peptides did not inhibit the binding of Erb-hcAb or Herceptin to ECD. Thus, these data provide further evidence that the epitope recognized by Erb-hcAb lies within the region 122-195 of ErbB2 domain I. The specific interaction between Erb-hcAb and the peptide SRASHPSSPHSKGS was also confirmed by fluorescence studies. Emission spectra of Erb-hcAb in the presence of this peptide and of its variant SRASEPSSPHSKGS were compared to those of the free antibody (data not shown). A variation of the signal intensity was observed only when the first peptide was added to Erb-hcAb.

Claims

1. A polypeptide consisting essentially of the amino acid sequence LTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT LIDTNRSRAC HPCSPMCKGS RCWGESSEDC QSLTR (SEQ ID NO: 1 ) or a fragment thereof.

2. A polypeptide as claimed in claim 1 , wherein the fragment comprises the amino acid sequence SRACHPCSPMCKGS (SEQ ID NO: 2).

3. A method of screening for an agent that down-regulates expression of the

ErbB2 receptor comprising a) contacting the polypeptide of claim 1 or claim 2 with a candidate agent and b) determining binding of said candidate agent to said polypeptide.

4. The method of claim 3, wherein the candidate agent is an antibody or a fragment thereof.

5. A method of preparing an antibody or a fragment thereof comprising use of a polypeptide as claimed in claim 1 or claim 2.

6. The antibody or a fragment thereof, as proposed in claim 5, which is an scFv or Fab.

7. An antibody or fragment thereof that binds the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

8. The antibody of claim 7, wherein said antibody is prepared according to the method of claim 5.

9. The antibody of claim 6, claim 7 or claim 8 for use in treating cancer.

10. Use of the antibody of claim 6, claim 7 or claim 8 in the manufacture of a medicament for treating cancer.

1 1. A method of treating cancer comprising administering the antibody of claim 6, claim 7 or claim 8 to a subject.

12. A nucleic acid encoding the polypeptide of claim 1 or claim 2.

13. A vector comprising the nucleic acid of claim 12

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13.

15. A method of producing a polypeptide according to claim 1 comprising cuituring the host cell of claim 14 under conditions that promote expression of the polypeptide and recovering the expressed polypeptide from the culture.