OA18848A - Anti - TRKA antibodies and derivatives thereof. - Google Patents

Abstract

An anti-TrkA antibody is provided that comprises: a) a variable heavy chain comprising a sequence selected fromany of BXhVH1, BXhVH2, BXhVH3, BXhVH4, BXhVH5, or HuVHWOv as shown in Figure (1a); or from variants of any of said sequences and/or b) a variable light chain comprising a sequence selected from any of BXhVL1, BXhVL2, BXhVL3, BXhVL4, BXhVL5, BXhVL6, BXhVL7 or BXhVL8; as shown in Figure (1b), or from variants of any of said sequences.TrkA-binding derivatives are also provided. Antibodies or derivatives of the present invention are useful in a number of therapies, including pain therapy.

Description

BXL-P624PCT

Antibodies and dérivatives thereof

The present invention relates to antibodies and to dérivatives thereof, especially to humanised antibodies and dérivatives thereof.

Nerve growth factor (NGF) acts through two membrane receptors. One is the relatively low affinity p75 receptor. The other is a 140 KDa high affinity receptor, known as TrkA.

NGF has potential use in the treatment of a wide range of disorders, such as various neurodegenerative disorders (including Alzheimer’s disease), diabètes and leprosy.

However NGF can hâve various undesîred agonist properties. These include an increase in pain sensitîvity. The NGF-TrkA system provides a potential target for thérapies for pain.

Various anti-TrkA antibodies hâve been produced. One such antibody is a monoclonai antibody which is referred to as 5C3 in WO 97/21732 (McGill (Jniversity). However, this was found to be a TrkA agonist and is therefore not useful for reducing pain.

Specifically, when binding to TrkA this antibody does not prevent the functional activation thereof.

An anti-TrkA monoclonai antibody known as MNAC13 is disclosed in WO 00/73344 (Societa Italiana Per La Ricerca Scientifica), from which EP-B-l 18138 (Lay Line Genomics SpA) is derived. This antibody and various dérivatives thereof are said to be effective în preventing the functional activation of TrkA in a range of biological Systems. The MNAC13 monoclonai antibody was used in a standard nociceptîon test and was found to provide remarkable hypoalgesia.

A single chain Fv (ScFv) variant of this antibody is also disclosed in WO 00/73344 and is referred to thereîn as MNAC13 ScFv. This contains the variable light and heavy chain régions of the larger antibody linked together by a linker polypeptide, which joins the Cterminus of the VL région with the N-terminus of the VH région. This variant was found to bind TrkA as efficiently as MNAC13. The sequence of the light and heavy variable régions was compared with that of the corresponding régions of the antibody described in

WO 97/21732 and it was found that there was only a low level ofoverall sequence identity therewith.

WO 06/131952 (Lay Line Genomics SpA) discloses medical uses of antî-TrkA antibodies in treating chronic pain. It provides evidence of this by using models of persistent pain, in particular the Chronic Constriction Injury (CCI) model.

WO 06/137106 (Lay Line Genomics SpA) discloses using an anti-TrkA antibody capable of inhibiting the binding between NGF and TrkA in combination with at least one opioid analgésie for treating or preventing pain. It is explained that this combination therapy allows a reduced opioid dosage to provide the same level of pain relief as a much higher dosage. This can therefore be useful in reducing the level of opioid side effects in pain therapy, because dosages can be lowered.

WO 05/061540 (Lay Line Genomics SpA & Scuolo Intemazionale Superiore Di Studi Avanzati-Sissa) discloses a method of humanisation of antibodies in which structural data obtained from crystallographic studies are used to conduct the first design stages of humanisation. As examples, WO 05/061540 takes anti-TrkA antibodies, as disclosed in WO 00/73344, and anti-NGF antibodies as starting points, and then redesigns them using the disclosed method.

Whilst the humanised antibodies disclosed in WO 05/061540 are useful, there is a need to provide additional humanised antibodies so as to expand the possibilities for effective thérapies.

The present inventors hâve now provided a range of anti-TrkA antibodies and dérivatives thereof that are not disclosed in WO 05/061540. The inventors hâve also provided data indicating the utility of such antibodies. Prior to the present invention these antibodies were simply not known in the art and the data provided could not hâve been predicted.

According to one aspect of the present invention, there is provided an anti-TrkA antibody that comprises:

a) a variable heavy chain comprising a sequence selected from any of BXhVHl (SEQ ID NO l), BXhVH2 (SEQ ID NO 2), BXhVH3 (SEQ ID NO 3), BXhVH4 (SEQ ID NO 4), BXhVH5 (SEQ ID NO 5), or HuVHWOv (SEQ ID NO 6), as shown in Figure la; or from variants of any of said sequences;

and/or

b) a variable light chain comprising a sequence selected from any of BXhVLl (SEQ ID NO 7), BXhVL2 (SEQ ID NO 8), BXhVL3 (SEQ ID NO 9), BXhVL4 (SEQ ID NO 10), BXhVL5 (SEQ ID NO 11), BXhVL6 (SEQ ID NO 12), BXhVL7 (SEQ ID NO 13) or BXhVL8 (SEQ ID NO I4); as shown in Figure Ib, or from variants of any of said sequences,

A dérivative of said antibody is also provided; wherein the dérivative is capable of binding TrkA.

More preferably, the antibody comprises both a variable heavy chain as described in a) above and a variable light chain as described in part b), i.e. it comprises one ofthe following combinations of light and heavy chains:

BXhVHlVLl, BXhVHl VL2, BXhVHlVL3, BXhVHlVL4, BXhVHlVL5,

BXhVHl VL6, BXhVHlVL7, BXhVHlVL8,

BXhVH2VLI, BXhVH2VL2, BXhVH2VL3, BXhVH2VL4, BXhVH2VL5, BXhVH2VL6, BXhVH2VL7, BXhVH2VL8,

BXhVH3VLl, BXhVH3VL2, BXhVH3VL3, BXhVH3VL4, BXhVH3VL5, BXhVH3VL6, BXhVH3VL7, BXhVH3VL8,

BXhVH4VLl, BXhVH4VL2, BXhVH4VL3, BXhVH4VL4, BXhVH4VL5, BXhVH4VL6, BXhVH4VL7, BXhVH4VL8,

BXhVH5VLl, BXhVH5VL2, BXhVH5VL3, BXhVH5VL4, BXhVH5VL5,

BXhVH5VL6, BXhVH5VL7, BXhVH5VL8, or HuVHWOv/HuVLWO.

Desirably, the dérivative ofthe antibody has at least one CDR région selected from the régions underlined in Figures l a & l b for each sequence, or from variants thereof having no more than two amino acid changes (preferably no more than one amino acid change) per underlined région.

More desirably, it has a piurality of CDR régions selected from the régions underlined în Figures la & ib for each sequence, or from variants thereof having no more than two amino acid changes (preferably no more than one amino acid change) per underlined région.

It may therefore comprise one, two, three, four, five or six of such CDR régions (optionally in combination with one or more other CDR régions).

It may preferably comprise at least the third CDR région of the heavychain, more preferably at least the third CDR région of the heavy and light chains.

Most desirably, however, it has six CDR régions corresponding to the six CDR régions underlined in Figures la & Ib for each sequence or corresponding to variants thereof having no more than two amino acid changes per underlined région.

Indeed, in most cases, it is preferred that few or no changes are made to the CDR sequences. Thus one, two, three, four, five, or even ail six CDR régions may hâve the same amino acid sequences as those shown in Figures la & Ib.

Tuming now to framework régions, it is preferred that the dérivative has at least one framework région selected from the non-underlined sequences shown in Figures la & Ib or from variants thereof that hâve at least 75% amino acid sequence identity therewith (e.g. at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity therewith).

The degree of amino acid sequence identity can be determined by simple alignments of the sequences without any gaps and determining the sequence différences.

Sequences can be aligned according to Kabat's numbering scheme and sequence identifies can then be determined accordingly (See Kabat, Sequences of Proteins of Immunological Interest. National Institutes of Health, Bethesda MD, 1987 & 1991). This numbering scheme is discussed în WO 05/061540 (Reference can also be made to Chothia & Lesk, J. Mol. Biol., 196, 901 (1987) and to Chothia et al„ Nature, 342, 878 (1989) ).

Less preferably, one or more gaps may be allowed (e.g. for one or more amino acid insertions/delettons) and gap pénalités may then be assigned.

Sequence identity can be determined using sequence analysis software e. g., BLASTN or BLASTP (available at www.ncbi.nlm.nih.gov/BLAST/). The default parameters for comparing two sequences (e.g. “Blasting two sequences against each other) by BLASTN (for nucléotide sequences) are reward for match = l, penalty for mismatch = 2, open gap = 5, extension gap = 2. When using BLASTP for protein sequences, the default parameters are reward for match = 0, penalty for mismatch = 0, open gap = 11, and extension gap = l],

More preferably, a plurality of framework régions is present and these régions are selected from the non-underlined sequences shown în Figure la & Ib or from variants thereof that hâve at least 75% amino acid sequence identity therewith (e.g. at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity.

Each chain shown in Figure la & Ib has four framework régions. Thus it is preferred that at least two, at least three or four such regions/variants thereof are present.

Most preferably, ail four framework régions or variants thereof are present.

Where one or more variant framework régions are present, it is generally preferred that the these régions do not include amino acid substitutions that would resuit in a change to an amino acid that is present in a murine sequence at the corresponding position, cf

The relevant murine amino acids that can be used for comparison are shown in mVHEP and mVLEP in Figures la & Ib respectively, with the exception that, for the purposes of this discussion, the few italicised amino acids shown in mVHEP and mVLEP are considered to be non-murine. At these positions the residues considered to be murine are given in the table below, in the order in which the italicised residues appear in the Figures.

Position	Italicised residue shown in Figure	Corresponding murine residue
Heavy chain	M	V
Heavy chain	Q	G
Light Chain	D	Q
Light Chain	S	T

Thus the percentage of humanisation of one or more framework. régions may be reduced by amino acids substitutions that do not necessarily increase the percentage of murine residues present.

These may resuit from conservative non-murine amino acid substitutions and/or from non-conservative non-murine substitutions.

However conservative substitutions are most preferred.

Amino acids can be grouped as foliows:

Group I (hydrophobie latéral chains): M, A, V, L, I;

Group II (neutral hydrophilic latéral chains): C, S, T, N, Q;

Group III (acid latéral chains): D, E;

Group IV (basic latéral chains): K, R;

Group V (residues that influence the orientation of the main chain): G, P; and Group VI (aromatic latéral chains): F, Y, W.

Conservative amino acid substitutions entail substitutions between amino acid of the same group, whilst non conservative amino acid substitutions entail an exchange between members of different groups.

Whatever sequences are present in the different régions of the light and/or heavy chains, it is preferred that an antibody or dérivative of the present invention has certain functional characteristics.

In addition to binding to TrkA. it is preferred that an antibody or dérivative of the present invention is capable of blocking or reducing the binding of NGF to TrkA.

Preferably, it is capable of blocking or reducing one or more biological activities that would otherwise be induced by the binding of NGF to the TrkA receptor.

Thus it is preferred that it is an antagonist of one or more activities induced by NGF binding to TrkA (rather than an agonist). Thus the antibodies and dérivatives thereof according to the invention suitably prevent the functional activation of TrkA. Inhibition of functional activation of TrkA by antibodies and dérivatives thereof can lead to analgesia in vivo.

Various assay procedures can be used.

A standard assay is the classical PC 12 in vitro assay in which PC12 cells are incubated with NGF and candidates are assessed to see if they are effective in reducing the extension of NGF-induced neuritic growth. This model was used in WO 00/73344, for example.

In another assay, preferred antibodies produce an OD450/630 nm value of greater than 0.1 in the TrkA-IgG binding assay illustrated by Figure 2. More preferably the OD450/630 nm value is greater than 0.2. Most preferably it is greater than 0.3.

In a further assay, preferred antibodies or dérivatives thereof provide an increase in FACS Ά staining of TFl cells in the FACS based assay described in the Examples (see Table 2). This is preferably an increase of over l.O fold. More preferably it is an increase that is at least 1.5 fold, at least 2.0 fold or at least 2.5 fold. Most preferably it is at least 3.0 fold.

Additional assays include assays for pain réduction, as described later in connection with the medical uses of the présent invention (It is partîcularly désirable for medical applications that the antibodies/derivatives thereof act as antagonists rather than agonists in respect ofthe pain response).

Desired antibodies/derivatives of the présent invention are sélective in that they bind with greater affinity to TrkA than to TrkB (Compare the black and white columns in Figure 2, for example).

For example they preferably hâve a binding affinity that is at least 2 times, at least 4 times, or at least 6 times as great for TrkA than for TrkB.

High binding affinîties to TrkA relative to TrkB resuit in greater selectivity and a lower risk of undesired side effects.

Binding affinîties can be readily assayed by comparative binding studies, such as those illustrated in Figure 2.

Tuming now to highly preferred antibodies ofthe présent invention, these comprise one of the following combinations of light and heavy chains: BXhVH3VL3, BXhVH5VLl or BXhVH5VL3.

These gave the best results in the assay illustrated by Figure 3.

Preferred dérivatives are dérivatives of BXhVH3VL3, BXhVH5VLl or BXhVH5VL3.

It will be appreciated from the foregoing discussion that a wide range of antibodies and dérivatives thereof are within the scope of the présent invention.

These hâve numerous applications, including those discussed below:

Medical applications

Antibodies or dérivatives of the present invention can be used in medicine.

They can be used to treat various disorders/conditions, as set out in various categories below.

The invention thus provides a method of treatment of the below mentioned conditions which comprises adminîstering to a subject, suitably a mammalian subject especially a human subject, in need thereof a therapeutically effective amount of an antibody or derivatîve as described herein such that the condition is thereby treated.

The invention also provides use of an antibody or derivatîve as described herein in the manufacture of a médicament for the treatment of the below mentioned conditions.

The invention also provides a kit of parts comprising an antibody or derivatîve as described herein together with instructions directing the use thereof by a subject for the treatment of the below mentioned conditions.

Here the term treatment includes therapeutic treatment of an existing disorder/condition. it also includes prophylactîc treatment. it further includes the amelioration of one or more adverse symptoms, even if a patient is not cured of a given disorder/condition. For example, pain may be alleviated or reduced.

Pain

A prefened medical use is in the treatment of pain.

According to International Association for the Study of Pain (“IASP”) pain is generally defined as An unpleasant sensory and emotional expérience associated with actual or potential tissue damage, or described in terms of such damage or both. The essential element in ail forms of pain is the activation of specialized high-threshold receptors and nerve libers to wam the organism of potential tissue damage. The involvement of inflammatory cells and processes is a common element in many pain states. The term acute pain means immédiate, generally high threshold, pain brought about by injury such as a eut, crush, bum, or by Chemical stimulation. The term chronic pain, as used herein, means pain other than acute pain, both of inflammatory and neuropathie origin. It is understood that chronic pain often Is of relatively long duration, for example, months or years and can be continuous or intermittent.

Antibodies of the present invention can be used to treat chronic pain or acute pain.

The treatment of chronic pain is preferred

The use of anti-TrkA antibodies in treating pain is discussed in WO 00/73344, in WO 05/061540 and in WO 06/131952 for example.

The pain may for example be associated with any of the following: pancreatitis, kidney stones, endometriosis, IBD, Crohn’s disease, post surgical adhesions , gall bladder stones, headaches, dysmenorrhea, musculoskeletal pain, sprains, viscéral pain, ovarian cysts, prostatitîs, cystitis, interstitial cystitis, post-operative pain, migraine, trigeminal neuralgia, pain from bums and/or wounds, pain associated with trauma, neuropathie pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing spondilitis, periarticular pathologies, oncological pain, pain from bone métastasés, HIV infection.

Various models are known for assessing pain and can be used in screening antibodies/derivatives thereof.

For example, the nociception hot plate test can be used, as disclosed in WO 00/73344, for example. The experiment can be carried out according to McMahon et al., Nature Medicine, 1, 774-780 (1995), using the antibody/derivative as immunoadhesîn. The antibody/derivative is infused subcutaneously into hind paw ofan adult rat for a period of three weeks or by an osmotic mîni-pump. The nociception sensitivity is evaluated at o intervals using a hot plate test (Eddy and Leimbach, J. Phar, Exp. Then, 107, 385393(1953)), which mimics hyperalgesia situations following inflammation or partial damage to the nerve. The nociceptive stimulus induces in such a case a response (paw licking and/or jumping) which présumés an integrated coordination higher than simple reflex. According to the test the animal is put in a pen having a plate heated to the desired température as base, usually 56°C. The latency of any of two responses (paw licking and jumping) is measured în control animais (treated with non relevant antibody) and in those treated with the anti-TrkA antibody/derivative.

As an alternative to the hot plate test, the nociceptive response to formalin can be assessed. This test is disclosed by Porto and Cavazzuti in Prog. Neurobiol., 41:565-607 (1993) and was used in WO 06/137106. It involves assessing the réduction in pain response by analyzing any subséquent réduction in paw licking when a given candidate is administered prior to testing. Saline is typically used as a négative control.

The Chronic Construction Injury (CCI) model is also a well known animal model. It involves chronic constrîction of the sciatic nerve and is used for assessment of chronic pain of a neuropathie nature. This model is described by Bennett and Xie in Pain, 33, 87107(1988). It was used in WO 06/131592, for example.

Cancer

The antibodies/derivatives can also be used in the treatment of cancer.

Various cancers express TrkA. The interaction of TrkA with NGF may be involved in tumour development (e.g. of prostate and pancreatic cancers). Indeed in certain forms of cancer, an excess of NGF can facilitate the growth and infiltration of nerve fibres. By blocking the action of NGF it is possible to significantly reduce the formation of neuromas.

Furthermore, as an alternative to simply providing a blocking effect, the antibodies/derivatives can be coupled to a cytotoxic agent and can be used to target cancer cells expressing TrkA, as discussed later in further detail.

It is not however necessary to couple the antibodies/derivatives to toxins. ADCC (antibody-dependent cell-mediated cytotoxicity) arises due to an immune response in which antibodies/derivatives, by coating target cells, can make them vulnérable to attack by the system (e.g. by T cells, by complément activation, etc.)

Neuronal disorders

The antibodies/derivatives can also be used in the treatment of various neuronal disorders.

As indicated above the antibodies/derivatives can be used to reduce the formation of neuromas.

They can also be used in the treatment of neurodegenerative disorders. As discussed earlier, NGF has potential use in the treatment of Alzheimer’s disease, but has undesired agonist properties, including an increase in pain sensitivity. Antibodies/derivatives of the présent invention may be usefùl in such treatments to reduce undesired agonist effects of NGF (see also the “Combination therapy” section below).

Furthermore, the antibodies/derivatives can be used to treat neuropathie pain, as discussed above. This may be associated with a lésion or a dysfonction of the nervous system.

Inflammatory Disorders

A still further application is in the treatment of inflammatory disorders.

NGF is released by mast cells, fîbroblasts and other cell types in the peripheral sites where inflammatory processes occur. In particular, mast cells appear to play a fondamental rôle. They produce NGF and at the same time express functional TrkA receptors at their surface. The NGF/TrkA system appears to médiate mastocyte activation through an autocrine positive feedback mechanism which allows local amplification of the algogenic inflammatory signal. Examples of inflammatory disorders that may be treated include inflammatory forms ofthe urinary tract and of the pelvic région, osteoarthritis, rheumatioid arthritis, asthma.

Other disorders

As dîscussed earlier, NGF has potential use in the treatment of diabètes and ieprosy, but has undesired agonist properties, including an increase in pain sensitivity.

Antibodies/derivatives of the present invention may be useful in such treatments to reduce undesired agonist effects of NGF (see also the ‘‘Combination therapy” section below).

Combination therapy

Antibodies or dérivatives thereof of the present invention may be used together with one or more other active agents in combination therapy. They may be used for simultaneous, sequential or concerted administration in medicine.

For example, the antibody or dérivative may be combined with an analgésie opioid. WO 06/137106 explains that small amounts of molécules able to block TrkA biological activity can potentiate the analgésie effects of opioîds.

Such opioîds include one or more compounds selected from the following: morphine, codeine, dihydrocodéine diacetylmorphine, hydrocodone, hydomorphone, levorphanol, oxymorphone, alfentanil, buprénorphine, butorphanol, fentanyl, sufentanyl, meperidine, methadone, nabulfina, propoxyphene, pentazocine, and their pharmaceutically acceptable dérivatives thereof (e.g. pharmaceutically acceptable salts thereof).

Alternatively, the antibody or dérivative may be used in combination therapy with one or more non-opioid analgésie.

A further combination is that of the antibody or dérivative with NGF. As dîscussed above, the use of NGF in the treatment of various disorders, including Alzheimer's disease, diabètes mellitus, Ieprosy, etc., had been proposed, but increases in pain sensitivity had been noted arising from agonist properties towards peripheral targets. Again, by using an antibody or dérivative of the present invention, pain sensitivity can be reduced, thereby making NGF-based thérapies more attractive.

A further combination is that of one or more antibodies or dérivatives of the present invention together with one or more other antibodies. A preferred combination is with one or more other anti-TrkA or anti-NGF antibodies. Such combinations may provide increased efficacy in treating one or more of the disorders discussed herein, relative to treatment with a single antibody. For example combinations of two or more antibodies found to be amongst the most effective in assay procedures used herein may be used.

Pharmaceutical compositions, vehicles and routes of administration

The antibodies/derivatives of the present invention can be administered by any appropriate route.

This includes (but is not limited to) intraperitoneal, intramuscular, întravenous, subcutaneous, intratracheal, oral, enterai, parentéral, intranasal or dermal administration.

Thus the invention provides a pharmaceutical composition comprising an antibody or dérivative thereof together with a pharmaceutically acceptable carrier or excipient.

The antibodies/derivatives can typically be administered for local application by injection (intraperitoneal or intracranial-typically in a cérébral ventricle-or intrapericardiac or intrabursal) of liquid formulations or by ingestion of solid formulations (in the form of pi Ils, tablets, capsules) or of liquid formulations (in the form of émulsions and solutions).

Compositions for parentéral administration commonly comprise a solution of immunoglobulîn dissolved in a compatible, preferably aqueous solution. The concentration of the antibody/derivative in these formulations can vary from less than 0.005% to 15-20% w/v. It is selected mainly according to the volumes of the liquid, viscosity, etc, and according to the particular administration mode desired.

Altematively, the antibodies/derivatives can be prepared for administration in solid form. The antibodies can be combined with different inert or excipient substances, which can include ligands such as microcrystalline cellulose, gelatin or Arabie rubber; récipients such as lactose or starch; agents such as alginic acid, Primogel or corn starch; iubricants such as magnésium stéarate, colloïdal Silicon dioxide; sweeteners such as saccharose or saccharin; or flavours, such as mint and methyl salicylate. Other pharmaceutical administration Systems include hydrogel, hydroxymethylcellulose, liposomes, microcapsules, microemulsions, microspheres, etc.

Local injections directly at a site affected by a disorder /close thereto is a preferred mode of administration if a disorder is localised.

In contrast to antî-tumour based thérapies, WO 06/131952 discusses the use of various anti-TrkA antibodies in the treatment of pain.

Here it is explained that anti-TrkA antibodies are suitably administered systemically. Systemic administration can be performed by injection, e.g. continuous intravenous infusion, bolus intravenous infusion, subeutaneous or intramuscular injection. Altematively, other forms of administration (e.g. oral, mucosal, via inhalation, sublingually, etc.) may also be used.

If desired, however, delivery of the antibody/ dérivative can be performed by local administration (e.g. intra-articular injection or subeutaneous. intramuscular injection) in the vicinity of affected tissues.

The anti-TrkA antibody/derivative will suitably be formulated in a pharmaceutical composition appropriate for the intended route of administration. Solutions for injection will suitably contain the antibody/derivative dissolved or dispersed in an aqueous medium (e.g. water for injection) as appropriate containing appropriate buffers and molarity modifiers (e.g. phosphate, sait and/or dextrose).

The treatment régime (i.e. dose, timing and répétition), can be represented by single or repeated administrations (e.g. injections) of the product by the chosen administration route.

The interval of dose administration can be subject to modifications depending on the extent and duration of the clinical response, as well as the particular individual and the individual clinical history.

Suîtably the anti-TrkA antibody/derivative has a long duration of action. In particular the clinical effect of the antibody following administration may be as long as 21 days as determined from animal studies. Furthennore, anti-TrkA antibodies/derivatives may manifest clinical benefït for a longer period than that in which its presence can be detected in a relevant biological matrix such as serum or plasma following its administration.

In light ofthe intended long duration ofaction (i.e. an effect suîtably lasting at least one week, or preferably at least two weeks e.g. at least three weeks or at least four weeks), suîtably the antibody/derivative may be administered to subjects at a frequency of not more than once per week e.g. not more than once per two weeks or once per three weeks or once per four weeks.

A suitable daily dose of the anti-TrkA antibody/derivative will typically range from O.l mg/kg to 10 mg/kg body weight.

(Using humanised anti-TrkA antibodies and a CCI model it is reported in WO 06/131592 that significant analgésie properties were observed in experimental animais at a dosage of 2 mg/kg, although lower dosages may of course be preferred for humans.)

Tuming now to administration in respect of tumours, administration may be through direct and localized injection into a tumour or a tissue near the tumour site. For systemic administration, doses vary from 0.05 mg/kg per day to 500 mg/kg per day, although dosages in the lower région of the range are preferred because they are easier to administer. Dosages can be calibrated for example to guarantee a particular level in the plasma of the antibody/derivative (in the range of about 5-30 mg/ml, preferably cf between 10-15 mg/ml) and maintain this level for a given period of time until the clinical results are achieved.

Effective methods for measuring or assessing the stage of pancreatic or prostatîc tumours are based on the measurement of the prostate spécifie antigen (PSA) in blood, on the measurement of the survival time for pancréas tumours, on the measurement of the slowing or inhibition of diffusion for métastasés in the case of both tumour types.

For direct injection at the level of a tumour site, dosage dépends on different factors including the type, stage and volume of the tumour, along with many other variables.

Depending on tumour volume, typical therapeutic doses may vary from 0.01 mg/ml to 10 mg/ml injections which can be administered with the necessary frequency.

Whatever the nature of the therapy, humanised antîbodies/derivatives may be eliminated much more slowly and require lower dosages to maintain an effective level in the plasma than non-humanised antibodies. Moreover, with high affinity antîbodies/derivatives, administration may be less frequent and less sizable than with antibodies having lower affinity.

The therapeutically effective dosage of each antibody/ derivative can be determined during the treatment by a skilled medical practitioner. If necessary, dosages can be reduced (e.g. to reduce side effects) or increased (to increase activity).

Prior to administration, préparations of antîbodies/derivatives of the invention can be stored by being frozen or lyophilized. They may then be reconstituted immedîately before use in a suitable buffer. Given that lyophilisation and reconstitution can resuit in a loss in activity, antibody administration levels can be calibrated to compensate for this fact. (For conventional immunoglobulins, IgM antibodies tend to hâve a greater loss of activity than IgG antibodies). A shelf life may also be assigned so that antîbodies/derivatives are not used after a certain period of storage. û>(

Diagnostic and prognostic applications

An antibody or dérivative thereof of the present invention can be used in the diagnosîs or prognosis of any of the diseases/ conditions discussed above in relation to medical uses.

For example it may be used to facilitate détection of TrkA positive tumour markers, as a precocious marker of the insurgence of Alzheimer’s disease, etc.

It may also be used in the diagnosis of C1PA (congénital insensitivity to pain with anhydrosîs). This is a hereditary, récessive, autosomal syndrome characterised by récurrent episodic fever, anhydrosîs, the absence of reaction to nociceptive stimuli, mental retardation and a tendency to self-mutilation. It results from mutations in the TrkA gene. Indeed an antibody or dérivative of the present invention may be used in the diagnosis or prognosis of a wide range of conditions involving aberrant expression of TrkA (compared to expression of TrkA in a healthy individual or a healthy tissue sample).

The present invention therefore includes within its scope a method comprising obtaining a biological sample obtained from a patient and contacting the sample with an antibody or derîvative of the present invention.

If desired, the antibody/derivative may be immobilised. It may be provided in the form of a diagnostic kit.

The method may then include assaying the binding of the antibody / derîvative to said sample in a quantitative or qualitative manner. If desired, this may be done with reference and/or to a positive control (indicating a healthy State) or a négative control (indicating the presence/lîkelihood of a disorder).

For diagnostic purposes, the antibodies/derivatives can be both marked with a détectable marker or can be unmarked. (The term marker” is used herein to include labels or any other détectable moiety/moiety that can trigger a détectable change.)

Unmarked antibodies can be used in combination with other marked antibodies (secondary antibodies), which are reactive against humanised, or human antibodies (e. g.

spécifie antibodies for the constant régions of human immunoglobulins).

Altematively, antibodies can be marked directly. A wide variety of markers can be used, e. g. radionuclides, fluorophores, colourings, enzymes, enzymatic substrates, enzymatic factors, enzymatic inhibitors, ligands, etc.

In particular, for diagnostic or prognostic imaging applications, a détectable agent is conjugated to the antibody that is détectable or marked with a détectable radioisotope (e.g. a radioisotope such as of iodîne, indium, technetium) or in paramagnetic manner (with paramagnetic atoms or ions, such as transition éléments, actinides and rare earths, in particular, manganèse II, copper II and cobalt II).

Imaging procedures may entail the intravenous, intraperitoneal or subeutaneous injection (in lymphatic drainage régions to identify lymph node métastasés) and may use detectors of radionuclide émissions (such as scintillation β counters) in the case of immunoscintigraphy.

If a paramagnetic marking is used instead, an NMR spectrometer can be used.

Other applications

The antibodies/derivatives thereof may be used as starting points to develop further antibodies. Thus they may be used as design tools.

They may be screened by one or more bindîng/functional assays and may therefore be part ofa drug development program.

They may be used for tissue typing, for forensic studies, etc.

They may be used as research tools

For example they may be used for further research into TrkA and/or into disorders in which TrkA binding to NGF (or other TrkA binding agents) may be implicated. They may be used to study binding and/or activation

Ail of the above applications of the antibodies/derivatives are within the scope of the present invention.

Nature of antibodies and antibody dérivatives

It will be appreciated from the foregoing description that a wîde range of antibodies and dérivatives thereof can be used in the present invention.

For the avoidance of doubt the terms antibodies” and “antibody dérivatives” are dîscussed below in further detail.

Antibodies

Antibodies of the present invention can be in the form of any desired immunoglobulin structure.

IgG and IgM are however preferred. with IgG being the most preferred. Ofthe IgG isoforms, IgGl is preferred, but other forms can be used including IgG4.

The antibodies are chimeric, i.e. they include one or more régions that are normally not associated with one another in nature. More specifically, one or more murine-derived CDR régions are present in the antibodies. but other régions (especially constant régions) are preferably human or humanised.

Humanised régions hâve more residues in common with a given human immunoglobulin région than with a corresponding mouse immunoglobulin région. Preferably, they hâve at least 75%, at least 80%. at least 85%, at least 90%, at least 95% or at least 98% identical with the human région at the amino acid sequence level. More preferably, there is 100% sequence identity over one or more non-CDR régions (e.g. constant régions).

In some cases, however it may be bénéficiai to introduce certain changes.

For example, it may be désirable to introduce changes that prevent/reduce one or more of the following:

a) activation of the complément system

b) complément mediated lysis

c) activation of T cells

d) binding to an Fc receptor.

Mutations indicated to allow one more of the above to be achieved are discussed in various patents. One or more of said mutations may therefore be included in antibodies/derivatives of the present invention

For example, US Patent No 6,194,551 proposes amino acid substitutions at amino acid positions 322, 329 and/or 331 (using the Kabat numbering System) ofthe constant heavy chain région ofthe IgG molécule and suggests that they can be used to prevent/reduce undesired activation ofthe complément system by abolishing Fc binding to Clq (see also Ward and Ghetie, Therapeutic Immunology 2: 77-94 (1995)). US Patent No 6,194,551 explains that proline is conserved at position 329 in human IgG’s. This residue (which is glycosylated and may thereby be involved in activating the complément system) is preferably replaced with alanine. However, substitution with any other amino acid is contemplated, e.g., serine, threonine, asparagine, glycine or valine. US Patent No 6,194,551 explains that proline is also conserved at position 331 in human IgGl, IgG2 and IgG3, but not IgG4 (which has a serine residue at position 331). Residue 331 is preferably replaced by alanine or another amino acid, e.g. serine (for IgG régions other than IgG4), glycine or valine. A further possibility discussed is to introduce substitutions at position 322. Lysine 322 is conserved in human IgGs, and this residue is said to be preferably replaced by an alanine residue, although a substitution with any other amino acid residue is contemplated (e.g. serine, threonine, glycine or valine).

US Patent 6,491,916 discloses that mutations in the région spanning about position 230 to about position 240 of a humanised antibody can produce particular advantages. Here it is explaîned that comparisons of antibodies that bind to Fc to those that do not bind to Fc suggest that changes in this région resuit in anti-CD3 antibodies that do not activate T cells. For example, some of the preferred antibodies comprise a mutation at position 234, at position 235, or at both. Anti-CD3 antibodies comprising one, two, three, four, five, or more mutations at one or more of positions 230, 23 i, 232,233, 234, 235, 236, 237 238 239, or 240, are expected to hâve advantages. This patent also discloses that an antibody having an IgGl Fc région and mutated to hâve alanine at both positions 234 and 235 does not bmd to the Clq component of complément and start the complement-mediated cascade. Further, it is explained that the mutation Lys 320 to Gin has an affinity for Clq only slightly weaker than the wild type but is non lytic.

US Patent 5,624,821 discloses that by changing any one of residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala, it is possible to abolish Clq binding. It points out that it is not necessary to replace the ionic residues only with Ala to abolish Clq binding, but that it will also be possible to use other alkyl-substituted non-îonic residues, such as Gly, Ile, Leu, or Val, or such aromatic non-polar residues as Phe, Tyr, Trp and Pro în place of any one ofthe three residues in order to abolish Clq binding. It will also be possible to use such polar non-ionic residues as Ser, Thr, Cys, and Met in place of residues 320 and 322, but not 318, in order to abolish Clq binding activity. US Patent 5,624,821 further discloses that replacing residue 297 (Asn) with Ala results in removal of lytic activity while only slightly reducing (about three fold weaker) affinity for Clq. It explains that it is 20 thought this anses because the alteration destroys the glycosylation site and that the presence of carbohydrate is required for complément activition. It points out that any other substitution at this site will also destroy the glycosylation site. US Patent 5,624,821 also discloses that mutations on, adjacent or close sites in the hinge link région (e.g. replacing residues 234, 236 or 237 by Ala) indicate that alterations in residues 234, 235,

236 and 237 at least affect affinity for the Fc gamma RI receptor.

Of course one or more amino acid changes (typically conservative amino acid changes) may be incorporated that do not substantially affect biological properties. Possible mutations are therefore not restricted to those discussed above.

Antibodies of whatever nature can be provided in monoclonal form (i.e. in combination with identical antibodies) or polyclonal form (i.e. in combination with different antibodies). Hybridomas capable ofproducing monoclonal antibodies ofthe present invention are also within the scope of the present invention.

Antibody dérivatives

The term “antibody dérivatives” is intended to allow for a wide range of structural changes that can be made relative to an antibody, provided that desired functional properties are retained.

Thus, for example, binding affinity to TrkA is desirably retained.

Preferably, the dérivatives are also effective in one or more ofthe functional assays described herein.

For the avoidance ofdoubt it is noted that ail ofthe following are considered to be dérivatives ofan antibody ofthe present invention:

a) a fragment of said antibody

b) a multimer comprising a plurality of fragments of said antibody (referred to herein as a “fragment multimer”)

c) a fusion product of said antibody, fragment or fragment multimer and another moiety 20 d) a variant of said antibody, fragment, fragment multimer, or fusion product, having at least 75% sequence identity therewith.

Thus the term “derivatîve” is interpreted broadly.

Turning now to fragments of the present invention, these are preferably at least seven amino acids long (Thus they are at least as long as the shortest CDR région shown in Figures la & lb for the heavy and light chains ofthe present invention). More preferably, they are at least ten, at least fifteen, or at least twenty amino acids long.

They can be produced, by means of proteolytic digestion starting from intact antibodies or by inserting stop codons in the desired positions in vectors bearing the coding DNA sequences for the variable régions of the heavy and light chain. This can be done after the CHj région to produce Fab fragments or after the hinge région to produce (Fab')₂ o<

fragments.

Denvatives in the form of ScFv chains can be obtained by joîning the variable régions of the heavy chain and ofthe light chain by means ofa linker (Huston et al, PNAS, 85, 5879 (l 988); Bird et al, Science, 242,423 (1988)). Fv or Fab fragments can be expressed in E. coli (Buchner and Rudolph, Bio/Technology, 9, 157 (1991); Skerra et al.

Bio/Technology, 9, 273 (1991)) or also in eukaiyotic cells, preferably mammal derived.

Indeed a very range of fragment forms is possible, including those discussed by Holliger & Hudson in Nature Biotechnology, Vol 23, No 9, 1126-1136 (2005).

These are ali within the scope of the présent invention. They can include fragments consisting of individual VH or VL chains (sometimes known as “domain antibodies” or “dAbs”) or even fragments of said chains (e.g. individual CDR régions). Multimeric forms are also included, such as minibodies, bis(or higher)-ScFv, diabodies, triabodies, tetrabodies, Fab multimers, etc. (referred to herein as “fragment multimers”).

Furthermore, various other moieties can be covalently linked with antibodies/fragments of the présent invention so as to provide bénéficiai properties. Such “fusion products” are within the scope of dérivatives ofthe présent invention. The moiety may for example be a diagnostic agenL a therapeutic agent, a marking agent, an agent that increases the half life ofthe product, or an agent that reduces immunogenicity (preferably in a human host).

For example, fusion products in the form of PEGylated antibodies/fragments may be provided. PEG has been predominantly used to reduce the immunogenicity and increase the circulatmg half-lives of antibodies. It may also hâve a bénéficiai effect on the use of antibodies in certain clinical settings such as tumour targeting.

The parts of a fusion product can be linked together chemically. For example this may be donc by cross-binding using heterobifunctional agents (e.g. SPDP, carbodiimide, glutaraldehyde, etc.).

In the case of fusion proteins, these are preferably made using genetic engineering techniques. Thus appropriate coding sequences based on the genetic code can be provided encoding the desired fiision protein and can then be cioned into a host cell using a suitable expression vector. Expression may be under the control of a constitutive or inducible promoter. The expressed fusion protein can be purified using standard techniques (e.g. by using immunofïinity procedures). Cell-based or cell-free expression Systems may be used.

Fusion proteins may for example comprise antibodies/fragments ofthe présent invention fùsed to cytotoxins. Résultant fusion proteins may then be used to target cells that express TrkA receptors, e.g. TrkA expressing tumour cells.

The production of various cytotoxîc immunotoxins is reported by Thorpe et al, Monoclonal Antibodies in Clinical Medicîne, Academie Press, 168 (1982). Indeed a large number of cytotoxîc agents are suitable for use in immunotoxins. Such agents include radionuclides such as iodine I3l or other isotopes of iodine, yttrium 90, rhénium 188 and bismuth 212 or other isotopes that émit alpha particles, a great number of chemotherapeutic drugs such as vindesin, methotrexate, adriamycin and cisplatin; cytotoxîc proteins, such as proteins that inhibit ribosomes (e.g. pokeweed antiviral protem, Pseudomonas exotoxin A, diphtheria toxin, ricin A and clavin of vegetable origin), or agents active at the cell surface level (e.g. phospholipase enzymes such as Phospholipase C).

Sometimes the cytotoxîc région of the immunotoxin can be immunogenic and consequently lîmit the clinical usefulness of the fusion protein in case of chronic or long term therapy.

An alternative to avoid the problem of the immunogenicity is to express in fusion with the binding domain ofthe antibody/derîvative a protein able to interact with DNA and bind to this fusion protein the vector (e.g. plasmid) that contains the toxin expression cassette. The numerous positive charges of protamin, a human protein that binds DNA, can interact in stable fashion with the négative charges ofthe DNA, generating a fusion partner for the neutral charge antibody/derivative. This is much more stable and less immunogenic than the toxin itself. After internaiization ofthe antibody-vector complex via receptor mediated endocytosis, the expression ofthe toxin causes the death ofthe cell. /

Moreover, if desired, inducible or cell-specific promoters can be provided in the toxin expression cassette. This approach is aimed at maximizing the sélective élimination of tumour cells while minimizing toxicity side effects (Chen et al, Gene Ther., 2, 116 (1995)).

Fusion proteins may also include fusions with other antibodies/derivatives. For example fusions of dAbs to spécifie antigens with other dAbs capable of binding long lasting sérum proteins (e.g. sérum albumin) hâve been used to increase sérum half life.

Variable heavy and light chain sequences of the present invention may form part of multivalent antibodies having specificity for one or more antigens, one of which is TrkA, or one or more epitopes within TrkA.

Multivalent antibodies with specificity for one or more antigens, one of which is TrkA

Expression Systems

Many expression Systems can be used to provide antibodies/derivatives ofthe present invention.

For example, prokaryotic Systems can be used and are well characterized.

E. coli is one ofthe prokaryotic hosts that is particularly useful for cloning the DNA sequences ofthe present invention. Moreover, a great number of well characterized promoters is available, e. g. from the lac or trp operon or β-lactamase or λ phage. Typically, these promoters control expression and bear binding site for the ribosome, for the correct start and finish of transcription and translation. It is possible to increase the half-hfe ofthe humanised immunoglobulins ofthe invention produced in prokaryotic Systems by conjugation with polyethylene glycol (PEG).

Other singie-cell organisms, such as yeasts, can be used for expression. The host of choice is Saccharomyces, using suitable carriers provided with expression control, réplication termination and origin sequences.

Phage-display libraries bearing sequences ofthe variable régions of immunogiobulins hâve been well reported and can be used in binding studies [Cesareni, FEBS Letts, 307, 66 ( i 992); Swimmer et al. PNAS, 89, 3756 (1992); Gram et al. PNAS, 89, 3576 (1992); Clackson étal. Nature, 352, 624 (1991); Scott & Smith, Science, 249, 386 (1990); Garrard et al. BiofTechniques, 9,1373 (1991)].

Insect cell cultures can also be used, typically utilising cells of S2 Drosophila transfected in stable fashion or cells of Spodopterafrugiperda with the expression system based on the Baculovirus (Putlitz et al. Bio/Tcchnology, 8, 651 (1990)).

Plants and cultures of vegetable cells can even be used (Larrick & Fry, Hum. Antibodies Hybndomas,2, 172 (1991); Benvenuto et al. Plant Mol. Biol, 17 865 (1991); Durin et al. Plant Mol. Biol,; 15,281 (1990); Hiatt et al. Nature, 342,76 (1989))

It is also possible to use tissue cultures ofmammal cells to express the polypeptides ofthe present invention. This can be advantageous in obtaining human glycosylation patterns. Different isotypes can be expressed. IgG 1 has proven to be the most effective isotype in the induction ofthe immune response (Winnacker, From Genes to Clones, VCH Pubhshers, NY, (1987)) whilst IgG4 is often used for diagnostic applications (Riechmann et al., Nature, 332,323 (1988)).

Mutated forms abolishing/reducing activation of complément may also be provided, as discussed earlier with reference to US Patent No 6,194,551„ US Patent 5,624,821 and/or US Patent 6,491,916.

In particular, mammalian cells are preferred. A great number of host cell lines hâve been developed for the sécrétion of intact immunogiobulins, among them are CHO cell lines, several COS cell Unes, the HeLa cells, myeloma cell lines (NSO, SP/2, YB/0 e P3X63.Ag8.653), transformed B cells or hybridomas. Expression vectors for these cells can include expression control sequences, such as a réplication origin, a promoter, an enhancer (Queen et al, PNAS, 86:10029 (1989)), and the sequences required for ribosome binding, RNA splicîng and polyadenylation, and sequences for transcription termination.

The expression control sequences of choice are promoters derived from immunoglobulin genes and from viruses, such as SV40, Adenovirus, Bovine Papilloma Virus, Cytomégalovirus and the like. Generally, the expression vector includes a selectable marker, such as the résistance to neomycin.

For the expression of humanised antibodies, it is préférable to cultivate the mammal cell hnes with a serum-free medium. For example, the HUDREG-55 cell line can easily be grown in Serum-Free and Protein-Free Hybridoma Medium Cat. No. S-2897 from Sigma (St. Louis, Mo.).

Nucleic Acids, Vectors, Transgenic animais

Nucleic acid sequences encoding the antibodies/derivatives/antibody chains of the present invention can be produced by standard techniques, given that the amino acid sequences for the key variable régions are provided herein and that corresponding coding sequences can be provided using the genetic code. These sequences can be incorporated into expression vectors and/or cloned into cells.

Indeed techniques for producing and cloning “reshaped antibodies” with rodent CDR régions and humanised framework régions are now well known. They are discussed for example in Jones, Dear, Foote, Neuberger and Winter, Nature, 321, 522-4 (1986); in Riechmann, Clark, Waldman and Winter, Nature, 332, 323-327 (1988) and in Verhoeyen, Milstein and Winter, Science, 239, 1534-1536 (1988).

Such nucleic acids can be incorporated into expression vectors, including plasmids, phage, etc., as is well known in the art and is discussed above.

Nucleic acids of the present invention can also be used to design probes or primers. These can be used for example to isolate or amplify nucleic acids of the present invention.

Probes or primers are therefore within the scope of the present invention. Typically they are at least 10, at least 15 or at least 20 bases long. Preferably they hybridise under stringent conditions to nucleic acid strands that encode antibodies/derivatives of the présent invention or to complementary strands thereof. One example of stringent hybridisation conditions involves using a pre-washing solution of 5 X SSC, 0.5% SDS l.O mM EDTA (pH 8.0) and attempting hybridisation ovemight at 55°C using 5 X SSC.

However, there are many other possibilities. Some of these are listed in Table l of

WO98/45435, for example (See especially the conditions set out under A-F of that table and, less preferably, those listed under G to L or M to R).

In a further aspect of the present invention, the nucleic acids can advantageously be used to provide transgenes for use in producing non-human transgenic animais, preferably mice. Here the antibody/derivative may be expressed in an inducible way, or under the control of constitutive promoters.

Such animais can be advantageously used to study and test drugs for human pathologies wherein the NGF/TrkA interaction is inhibited and, particularly, neurodegenerative pathologies.

The antibody/derivative can be advantageously expressed in a retrievable body fluid such as milk or sérum, from which it can be retrieved and purified using standard techniques.

Transgenes used to produce the transgenic animais may comprise the relevant coding 20 sequence(s) operatively bound to a promoter, usually in combination with an enhancer sequence, such as that ofthe rodent immunoglobulîn or the promoter/enhancer ofthe casein gene (Buhler et al., Biotechnology; 8,140 (1990); Meade et al., Bio/Technology, 8,443(1990)).

The transgenes can be transferred into the cells or embryos by means of homologous recombination. A wide range of non-human transgenic animais can be produced, including mice, rats, sheep, cows, goats, etc. (see WO 91/08216).

It will be appreciated from the foregoing description that the present invention provides a 30 range of new antibodies, dérivatives, nucleic acids, etc.

Ifdesîred, these can be provided in substantially purified form. For the purposes ofthe present invention this means that they are the majority ofthe dry weight ofa particular c ‘ 18848 composition. For example they may represent at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% of said dry weight.

They may be provided in isolated form. This means that they are removed from one or more other components with which they may be normaily associated in nature. (For example a nucleic acid may be provided in a form that is isolated from a cell).

They may be provided in a variety of other forms. For example they may be fused to heterologous moieties and/or they may be immobilised.

I0

Ail ofthe above forms are within the scope of the present invention.

The present invention will now be described by way of example only, with reference to the accompanying drawings, wherein:

Figures la & 1b show amino acid sequence alignments for various heavy and light chains.

Figure 2 shows the results for antigen binding specificity towards TrkA-IgG in respect of supematants from various clones resulting from an experiment involving transient expression of the humanised MNAC13 variants in COS-7 cells.

Figure 3 the results ofan experiment in which cellular binding ofthe antibodies to TrkA expressed on TF-1 cells was analysed by cytofluorimetric analysis.

Figure 4 shows the results of a further analysis in which the best binders identified from

Figure 3 (BXhVH3VL3, BXhVH5VLl, BXhVH5VL3, and HuMNACWOv) were compared to HuMNACWO.

Figure 5 shows the results of an assay in which different humanised candidates were assayed in parallel with murine MNAC13 antibody (muMNACEP), chimMNAC13, HuMNACWO, and Human IgG 1 as standard control.

Figure 6 shows the heavy and light chains for BXhVH5VLl, including the constant régions (the first amino acid of the constant région is underlined.)

Figure 7 shows the heavy chain for BXhVH5VLl N297A, including the constant région (the first amino acid of the constant région is underlined and the 297A position is bold ^c and underlined).

Figure 8 shows BXhVH5VLl and BXhVH5VLl N297A binding to cell lines expressing huTrkA.

Figure 9 shows the effect of various antibodies on NGF-induced MIP-1 β production in human mast cell line HMC-1.

Figure 10 shows binding of BXhVH5VLl to cell bound Fc receptors on THP1 cell line compared to BXhVH5VLl N297A.

Figure 11 shows an experiment demonstrating the analgésie effect of local intradermic injection of BXhVH5VLl N297A or control hlgG when co-injected with rhNGF

Figure 12 shows an experiment demonstrating the analgésie effect of local mtradermtc injection of muMNACEP or control mlgG when co-inj ected with rhNGF

Figure 13 shows an experiment demonstrating the analgésie effect of systemic administration of BXhVH5VLl N297A when compared to control hlgG in an animal model of NGF-induced pain.

Figure 14 shows an experiment demonstrating the analgésie effect of systemic administration of muMNACEP when compared to control hlgG in an animal model of

NGF-induced pain.

Examples

Before discussing the examples in detail, some of the nomenclature used therein is set oui below:

muMNACEP

This term is used to indicate the murine antibody MNAC13, as disclosed in EPI 181318

The heavy chain variable région of this antibody is referred to herein as mVHEP (SEQ ID NO, ]5\ The light chain variable région is referred to herein as mVLEP (SEQ ID NO.

16).

HuMNACWO

This term is used to indicate the humanised antibody MNAC13 disclosed in WO

05/061540

The heavy chain variable région of this antibody is referred to herein as HuVHWO (SEQ 30 ID NO. 17). The light chain variable région is referred to herein as HuVLWO (SEQ ID

NO. 18)

HuMNACWOv

This ternis is used to indicate a variant of the antibody disclosed in HuMNACWO (see above) in which the heavy chain CDR3 région has been replaced with a CDR3 région corresponding to that present in muMNACEP. The variant is novel and is within the scope of the present invention.

The heavy chain variable région of this antibody is referred to herein as HuVHWOv[SEQ ID NO. 6). The light chain variable région can be referred to herein as HuVLWOv. However, in order to avoid duplication, it is not shown in Figure Ib, because it is the same as HuVLWO (SEQ ID NO. 18).

ChimMNAC13

This corresponds to muMNACEP, but has human constant régions instead of mouse constant régions.

The heavy chain variable région of this antibody is referred to herein as mVHEP [SEQ ID

NO. 15)

The light chain is referred to herein as mVLEP (SEQ IDNO. 16)

3-23*01 (SEQIDNO. 19). JH4(SEQ1DNO. 20), L6*01 (SEP ID NO. 21) and JK1

SEP ID NO. 22

These are coding sequences derived from human germline genes.

They are used for assessing degrees of humanisation in Table 1. Thus if there are no changes relative to a human germline sequence it is considered that there is 100% humanisation.

[The percentage humanisation = number of changes------------- X 100] total number of residues compared

The table below shows the percentage humanisation for the different variants:

Table 1

Sequence variant	Number of murine AA in the FW/number of total AA in theFW	% humanisation 'related to FW sequence)	Number of murine AA including CDR AA/number of total AA in the variable région	% humanisation (related to complété variable sequence)
BXhVHl	0/87	100	36/123	70.7
BXhVH2	3/87	96.6	39/123	68.3
BXhVH3	3/87	96.6	39/123	68.3
BXhVH4	3/87	96.6	39/123	68.3
BXhVH5	5/87	94.2	41/123	66.7
BXhVHWO	12/87	86.2	48/123	61.0
BXhVLl	0/80	100	26/106	75.5
BXhVL2	4/80	95	30/106	71.7
BXhVL3	6/80	92.5	32/106	69.8
BXhVL4	6/80	92.5	32/106	69.8
BXhVL5	6/80	92.5	32/106	69.8
BXhVL6	8/80	90	34/106	67.9
BXhVL7	8/80	90	34/106	67.9
BXhVL8	11/80	86.2	37/106	65.1
BXhVLWO	9/80	88.8	35/106	67

It can be seen that ail of the variant variable chains hâve a degree of humanisation over the framework régions of over 85%.

“BX” sequences

The sequences labelled with a code beginning with BX” are novel sequences ofthe present invention. The letters following “BX” are either VH or VL to indicate a heavy or light variable chain respectively. The sequences are then simply numbered consecutively in the order in which they are shown in Figures la & 1b for a given chain.

There are five heavy chain sequences. Thus they are numbered:

BXhVHl (SEP IDNP. D

BXhVH2 (SEP ID NO. 2)

BXhVH3 (SEP ID NP. 3)

BXHVH4 (SEP ID NP· 4)

BXhVH5 (SEP ID NP. 5)

There are eîght light chain sequences. Thus they are numbered:

BXhVLl (SEP ID NP. 7) BXhVL2 (SEP ID NP. 8) BXhVL3 (SEP ID NP. 9) BXhVL4 (SEP IDNP. 10)

BXhVL5 (SEP ID NP. ID BXhVL6 (SEP ID NP. 12) BXhVL7 (SEP IDNP. 13) BXhVL8 (SEP IDNP. 14)

The chains can be combined in antibodies or dérivatives thereof.

The forty possible combinations hâve ail been produced and are:

BXhVHIVLl, BXhVHlVL2, BXhVHlVL3, BXhVHlVL4, BXhVHlVL5, BXhVHIVLô, BXhVHlVL7, BXhVHlVL8,

BXhVH2VLl, BXhVH2VL2, BXhVH2VL3, BXhVH2VL4_f BXhVH2VL5,

BXhVH2VL6, BXhVH2VL7, BXhVH2VL8,

BXhVH3VLl, BXhVH3VL2, BXhVH3VL3, BXhVH3VL4, BXhVH3VL5,

BXhVH3VL6, BXhVH3VL7, BXhVH3VL8,

BXhVH4VLl, BXhVH4VL2, BXhVH4VL3, BXhVH4VL4, BXhVH4VL5,

BXhVH4VL6, BXhVH4VL7, BXhVH4VL8,

BXhVH5VLl, BXhVH5VL2, BXhVH5VL3, BXhVH5VL4, BXhVH5VL5, BXhVH5VL6, BXhVH5VL7, BXhVH5VL8.

^1;N297A”

The désignation “Ν297Λ” after the name of an antibody indicated that position 297 of the heavy chain constant région is mutated from N to A.

The sequence of BXhVH5VLl N297A is provided as SEQ ID No. 23.

Expression vectors

The appropriate coding sequences were ftised to a sequence coding for a secretory signal 5' and a splice donor sequence 3' to the cDNA for cloning into an antibody expression system.

The DNA fragments were cloned into IgGl expression vectors.

These expression vectors were based on genomic sequences encoding the human constant domains and cloning cassettes for the insertion of the selected cDNA fragments of the hVH and hVL sequences.

Transient expression of the humanised MNAC13 variants in COS-7 cells and détermination of antibody titers

Each combination of Heavy and Light chain was transiently transfected in COS-7 cells and antibody tîter was determined.

The expression vectors coding for the light chain and for the heavy chain were transiently cotransfected into COS-7 cells by lipofection using Lipofectamin according to the manufacturer’s instructions (Invitrogen, Germany) in a 24-well format.

After transfection the medium was replaced by DMEM containing !0%FCS and 2%

L-glutamine and the supematants of the COS-7 cells were collected 4 days after transfection.

The antibody titer of the humanised antibodies secreted into the supematants of transfected COS-7 cells was analyzed by a sandwich ELISA.

Briefly, a mouse anti-human kappa chain recognizing antibody (BD) was immobilized on a 96 well plate, blocked and incubated with diluted supematant of transfected COS-7 cells. The presence of antibodies was detected by a POD conjugated rabbit anti-human

IgG (H+L) antibody (Dianova, Germany). A chimeric control antibody was used as a standard in concentrations from l to 10 ng/ml. The determined antibody concentrations were further adjusted by an internai standard sample having a standardized antibody concentration.

Example 1

Comparison of humanised antibody binding towards TrkA-igG in ELISA

Based on the determined antibody concentration, supematants of ail sampies were adjusted to the same antibody concentration.

The binding actîvitïes of ail humanised antibody variants were analyzed by a TrkA-igG antigen ELISA. They were compared to the binding activities of the ChimMNAC13 and HuMNACWOv.

Antibodies and antigens were thawed, aliquoted and stored at - 20°C. Aliquots of the antibodies in use were stored at 4 °C for a maximum of two weeks.

Antigen ELISA was performed as follows: Maxisorb plates (Nunc, Germany) were coated with 0.125, 0.25, 0.5, and 1 gg/ml TrkA-igG. To check the specificity of antibodyantigen binding TrkB-lgG (1 gg/ml) as a négative control was used.

Transiently expressed antibody variants were used at 1, 10, and 100 ng/ml.

Detailed procedure as follows:

Coating

Plates: Nunc MaxîSorp 96well lOOgl/well of TrkA-igG at 2 gg/ml în Carbonate Buffer 0.1M pH 9.6 (TrkB-lgG used as négative control)

Seal plate and încubate ovemight at +4°C

Wash 3 times with 200 gl of wash buffer

Blocking

Block plates by adding 200gl of SuperBiock Blocking Buffer in PBS. (Pierce Prod # 37515) to each well.

Immediately empty the plate by inversion.

Repeat two additional times.

Incubate at 37°C for 2 hours.

Primary Antibody

Discard supematant and add ΙΟΟμΙ of purified mAb appropriately diluted in TEST Buffer (standard curve range: 50-5000 pg/ml).

Seal plate and incubate at 37°C for 2 hours.

(In order to increase sensitivity incubate ovemight at +4°C)

Wash 4 times with wash buffer.

Secondary A ntibody

Add ΙΟΟμΙ of HRP-conjugated Goat anti mouse IgG (Pierce cat. 31430) diluted 1:10000 in TEST Buffer.

Incubate at 37°C for 1 hour.

Wash 4 times with wash buffer.

Development

Add ΙΟΟμΙ of Substrate solution to each well. Incubate at room température.

Stop the reaction with ΙΟΟμΙ of H2SO4 2M.

Détermine the optical density of each well using a microtiter reader at 450 nm.

Results

The results for supematants from the various clones evaluated for antîgen binding specificity by using the ELISA assay are shown in Figure 2.

Briefly, the spécifie antigen TrkA-IgG (black bars) and the négative control TRKB-IgG (white bars) were coated at 1 pg/ml concentration on different 96-well plates.

Antibodies supematants were quantified, appropriately diluted, and tested at 5 ng/ml concentration. After washing, binding was detected with the appropriated HRP-labeled secondary antibody, revealed by a chromogenic reaction and quantified by OD450/630 nm measure.

The majority of the humanised antibodies show a comparable sélective affinity for high density TrkA antigen.

In addition, their binding specificity is not significantly different from parental murine anti-human TrkA antibody and its chimeric isoform, indicating that antigen selectivity has been fully preserved along the humanisation procedure.

Example 2

Cellular binding assay of new candidates by cytofluorimetric analysis of TrkA surface expression on TF-1 cells

Procedure

Harvest cells from culture, preparîng a single cell suspension.

(In order to obtain maximum antigen expression split cells 1:3 the day before).

Distribute 0.3-0.4 x 10⁶cells/sample and wash IX with cold FACS buffer (PBS pH 7.4 +

0.1% NaN3 + 0.1% BSA).

Centrifuge at 350 x g for 5 min.

Discard supernatant and Keep tubes on ice.

Fc Receptors Blocking

Add 50 μΐ/sample of Human IgG [300 pg/ml] in FACS buffer and mix by gently vortexing.

Incubate at 4°C for 15 min.

Primary Antibody

Add 100 μΐ/sample of Primary Antibody muMNAC13 [4 μg/ml] in FACS buffer and mix by gently vortexing.

As négative control use purified mouse IgG 1 isotype control at the same concentration.

Incubate at 4°C for 30 min.

Wash 2X with 1ml of FACS buffer, spin 5 min at 350 x g, and discard the supernatant.

Secondary Antibody

Add 100 μΐ/sample of Donkey anti Mouse IgG (H+L) R-Phycoerythrîn conjugated

Jackson ImmunoResearch cat.# 715-116-151 în FACS buffer and mîx by gently vortexing.

Incubate at 4°C for 30 min.

Wash 2X with Iml of FACS buffer, spin 5 min at 350 x g, and discard the supematant.

Re-suspend in 0.5 ml of FACS Buffer.

Acquire sample data on flow cytometer.

Results

TF-1 cells were stained with supematants from ail the clones as well as HuMNACWO and HuMNACWOv antibodies as Controls (4 gg/ml) for 30 minutes at 4°C.

Staining was revealed by an appropriate PE-labeled secondary antibody and quantified by cytofluorîmetric analysis to evaluate the fluorescence intensity of the binding.

The results are shown in Figure 3, which is based upon the table below.

Table 2

		Geo Mean Fluorescence		Fold Increase
N°.	Variants	Mean	±	S.D.		Mean		S.D.
1	mVHEP/mVLEP	11.0	±	2.2		3.2	±	0.7
2	hVHWOv/hVLWO	8.9	±	1.9		2.6	±	0.6
3	hVHl/hVLl	5.7	±	0.4		1.7	±	0.1
4	hVHl/hVL2	4.6	±	0.4		1.3	±	0.1
5	hVHl/hVL3	6.1	±	0.6		1.8	±	0.2
6	hVHl/hVL4	5.1	±	0.5		1.5	±	0.2
7	hVHl/hVL5	4.5	±	0.3		1.3	±	0.1
8	hVHl/hVL6	4.9	i	0.4		1.4	±	0.1
9	hVHl/hVL7	5.1	±	0.4		1.5		0.1
10	hVHi/hVL8	5.2	i	0.1		1.5	±	0.0
11	hVH2/hVLl	9.2	±	1.3		2.6	±	0.4
12	hVH2/hVL2	6.4	±	0.7		1.8	i	0.2
13	hVH2/hVL3	10.8	±	1.3		3.1	±	0.4

14	hVH2/hVL4	6.1	±	0.3	1.8	±	0.1
15	hVH2/hVL5	6.4	±	0.2	1.8	±	0.1
16	hVH2/hVL6	6.4	±	0.7	1.8	±	0.2
17	hVH2/hVL7	6.5	±	0.8	1.9	±	0.3
18	hVH2/hVL8	6.5	±	1.0	1.9	±	0.3
19	hVH3/hVLl	8.6	±	1.5	2.5	±	0.5
20	hVH3/hVL2	7.1	±	2.1	2.0	±	0.6
21	hVH3/hVL3	12.6	±	0.6	3.6	±	0.2
22	hVH3/hVL4	7.1	±	0.1	2.0	±	0.0
23	hVH3/hVL5	6.9	±	0.5	2.0	±	0.2
24	hVH3/hVL6	6.4	±	0.5	1.8	±	0.2
25	hVH3/hVL7	7.1	±	1.0	2.0	±	0.3
26	hVH3/hVL8	6.5	±	1.2	1.9		0.3
27	hVH4/hVLl	10.4	±	2.4	3.0	±	0.7
28	hVH4/hVL2	8.3		2.5	2.4	±	0.7
29	hVH4/hVL3	10.9	±	3.0	3.1	±	0.9
30	hVH4/hVL4 Ί	8.0	i	2.2	2.3	±	0.6
31	hVH4/hVL5	8.6	±	1.7	2.5		0.5
32	hVH4/hVL6	8.0	±	1.4	2.3		0.4
33	hVH4/hVL7	8.7		2.5	2.5	±	0.7
34	hVH4/hVL8	8.3	±	1.9	2.4	±	0.6
35	hVH5/hVLl	11.3	±	2.7	3.2	±	0.8
36	hVH5/hVL2	8.6	db	2.3	2.5	±	0.7
37	hVH5/hVL3	13.7	±	2.1	3.9	±	0.6
38	hVH5/hVL4	9.1	db	2.2	2.6	±	0.7
39	hVH5/hVL5	8.3	±	2.0	2.4	±	0.6
40	hVH5/hVL6	9.1	i	1.5	2.6	±	0.5
41	hVH5/hVL7	8.6	±	2.0	2.5	±	0.6
42	hVH5/hVL8	8.3	±	1.6	2.4	±	0.5
43	huIgG	3.5		0.0	1.0		0.0

The results showed that ail the clones tested as well as HuMNACWOv positively detected the membrane-associated TrkA receptors on TFl cells though to a different extent. HuMNACWO is not able to stain TF 1 cells, which hâve a low density of surface TrkA receptors.

To further confirm, out of 40 clones tested, the best binders were selected to be further analyzed.

As evaluated in two separate experiments (Figure 4) BXHVH3VL3, BXhVH5VLl, BXhVH5VL3, and HuMNACWOv were compared with HuMNACWO.

The selected leads were confirmed good binders and slightly better performers when compared to HuMNACWOv.

The humanized antibody isoforms BXhVH5VLl N297A and BXhVH5VLl together with the reference antibodies muMNACEP and HuMNACWO were also assayed for binding capability on TF-1, HMC-1 and PC12-hTrkA cell Unes which express different levels of surface receptor hTrkA.

As shown in Figure 8, BXhVH5VLl N297A and BXhVH5VLl antibodies comparably bind ail the tested cell lines, independently of the receptor density on the cellular surface. Both antibodies appear to bind more efficiently when compared to the parental muMNACEP. HuMNACWO only binds high surface receptor density cell line PC12hTrkA.

Example 3

Comparison of humanised antibody biological activity in vitro with a prolifération assay on TF1 cells

To measure the ability of anti-human TrkA monoclonal antibodies to block cell surface TrkA- β-NGF mediated biological activity, a cell prolifération assay using a factordependent human erythroleukemic cell line, TF-1 (Kitamura, T. et al., 1989, J. Cell Physiol. 140:323-334) was used.

TF-1 cells were incubated with various concentrations of the antibodies for 0.5 hour at 37° C in a 96 fiat well culture plate.

Following this pre-incubation period, recombinant human β-NGF (rec-hu-p-NGF, R&D Systems) was added to the cell-antibody mixture.

The assay mixture in a total volume of 200 pL, containing antibody at different concentrations indicated, human β-NGF at 5.0 ng/mL and TF-l cells at 5 x 103 cells/well, was incubated at 37°C for 5 days in a humidified CCh incubator.

After that period the plates were centrifuged and after removal ofthe supematant, frozen at - 80°C in order to lyse the cells.

CyQUANT Cell Prolifération Assay Kit (Molecular Probes) was used for measuring cell prolifération according to the manufacturer’s instructions.

This experiment was performed twice.

Results

Different humanised candidates were assayed in parallel with murine MNAC13 antibody (muMNACEP), chimMNAC13, HuMNACWO and Human IgG l as standard control (Figure 5).

The IC50 were calculated for each curve and the results are given in the table overleaf.

It was found that antibody BXhVH5VLl was the best performer among the candidates.

The heavy and light chains for this antibody are therefore shown in Figure 6.

The average IC50 for murine MNAC13 over a sériés of experiments was 0.54 ± 0.47 pg/ml.

Table 3

Prolifération assay on TFl cells 1C50 (pg/ml)

Mean SD muMNACEP 0.54 0.47

EXP-1 EXP-2

ChimMNAC13	0.06	0.58
BXhVH5VLl	0.17	1.84
BXhVH3VL3	0.41	2.38
BXhVH5VL3	1.40	1.21
HuMNACWO	-	-
HuIgGstd	-	-

Example 4 Surface plasmon résonance analysis

Surface plasmon résonance analysis was used to measure the association and dissociation rate constants for binding kînetics of the different antibodies (murine, chimeric, 5 humanised variants) towards TrkA-IgG using BIACORE 2000 (Biacore AB, Uppsala, Sweden). TrkA-IgG was immobilised on aCM-5 sensorchip according to manufacturer’s conditions, in a way to achieve an immobilization density of 1100 RU. Each antibody sample was analyzed in antibody concentration ranges of 20-0.63 pg/ml. Calculations from the sensograms were performed by using the BIA évaluation version 3 (1999) software.

Analysis ofthe individual sets ofsensograms was performed with the BIA évaluation version 3 (1999) software. Among the different models tested to fit the kinetics data, the best fitting was obtained with the “separated 1:1” algorithm. In this model, only a defined range ofthe early association and dissociation curves was used for the calculation. It is assumed that during these early phases of the curve, the overlay effects like mass transfer, rebinding or others do not affect the calculations.

Results

The dissociation constant (K_D) was determined for various antibodies and is set out in the table overleaf in the order in order of Încreasing value.

The K_D value has molar units (M), which correspond to the concentration of ligand at which a binding site on a particular protein is half occupîed. The smaller the value, the more tightly bound the ligand, or the higher the affinity between ligand and protein (here between antigen and antibody).

Table 4

Antibody	Kon (1/Ms)	Kon (1/s)	KD [M]
ChimMNACl3	2.68x10’	3.53x10*	1.51x10*°
MNAC13	8.5ÛX105	1.67x10*	2.50x10*'°
BXhVH5VLl	7.68x10’	4.70x1 θ'4	6.15x10'*°
BXhVH5VL3	1.00x10°	6.38x10^	6.62x10'°
BXhVH3VL3	3.25X105	4.42x10*	1.45x10'°
HuMNACWOv	1.62x106	3.86x10J	2.48x10'9
HuMNACWO (separate experiment)	7.39x10’	3.09x10'2	4.18xl0K

It can be seen from the above table the calculated Kd for the murine and the chimeric isoforms are very comparable with one another.

They are slightly lower but of the same order of magnitude as the humanised variants BXhVH5VLl and BXhVH5VL3.

On the contrary, the humanised variants HuMNACWOv and BXhVH3VL3 display a one order of magnitude higher Kd than that observed for the murine and the chimeric variants.

However, the Kd values here are still lower than for the prior art humanised antibody

HuMNACWO.

Indeed, preferred K_D values for antibodies/variants of the present invention using this model are below 4.18 x IO^-8 (Thus they are lower than for the value for the humanised prior art antibody HuMNACWO).

More preferably they are below 2.48 x IO'⁹ (thus they are lower than the HuMNACWOv, which is a variant of HuMNACWO with the same framework régions, but with changes in the third CDR of the heavy chain).

Most preferably the Kd values are below l x IO'⁹ (thus they are of the same general order of magnitude as for the murine and the chimeric isoforms).

Consistently, the rankîng given in the above table, which is based on the calculated data using the “separated” algorithm, reflected very well the ranking obtained by visual inspections of the sensograms of ail investigated variants in overlay plots.

Example 5

Comparison of humanised antibody biological activity in vitro with a chemokine sécrétion assav on HMC-1 mast cell line

NGF acts as an important intermediate in inflammatory pain, contributing to both peripheral and central sensitization. The sensitization of peripheral nociceptors can be very rapid and can involve non-neural cells such as mast cells.

To measure the ability of anti-human TrkA monoclonal antibodies to inhibit β-NGFinduced MlPla sécrétion, a biological assay using the mast cell line, HMC-1 (Ahamed, J. et al., J Immunol. 2004 Jun 1 ; 172(11):6961-8.) was used.

HMC-1 cells (0.1 x 10⁶/well) were plated in triplicate in complété growth medium in a 96 fiat well culture plate and incubated with various concentrations of monoclonal antibody for 0.5 hour at 37° C.

Following this pre-incubation period, recombinant human β-NGF (rec-hu-fi-NGF, R&D Systems) was added to the cell-antibody mixture to a final concentration of 50 ng/ml and incubation at 37° C was extended for 6 hours in a humidified CO2 incubator.

Supematants were then harvested and levels of ΜΙΡ-Ιβ were quantified by sandwich ELISA using a DuoSet® Elisa Kit for Human CCL4/MIPI-p from R&D System (Cat. Nr. DY271).

Data obtained were expressed as % of response and analyzed with GraphPad Prism 5 software using a nonlinear régression analysis, log(inhibitor) vs. normalized response variable slope équation.

Results

BXhVH5VLl N297A antibody was assayed in parallel with murine muMNACEP, HuMNACWO, and Human IgG l as standard control. The 1C50 values were calculated for each curve and the results are shown in Figure 9. The inhibitory activity of BXhVH5VLl N297A was significantly higher than that of the humanised antibody HuMNACWO.

Example 6

BXhVH5VLl N297A and BXhVH5VLl in vitro characterization. Evaluation of binding to cellular FcRs on THP-1 cells

Human acute monocytic leukemia cell line THP1 (ATCC) was cultured in RPMI1640/GLUTAMAX (Invitrogen) + 10% Foetal Bovine Sérum (Invitrogen) + Pen/Strep. and maintaîned between 2-9x100,000 cells/ml.

Cells were harvested from culture and prepared as a single cell suspension. 03-0.4 x 10⁶celIs/sample were then distributed in 96-well round-bottom tissue culture plates (Costar, Cambridge, MA) and washed IX with cold FACS buffer.

After centrifugation at 350 x g for 5 min., supernatant is discarded and plates put on ice. Binding of IgG to FcyRs on THP-1 cells was performed by incubating monomeric IgGs in FACS Buffer starting from 30 pg/ml to 0.02 pg/ml (dilutions 1:3) in a total volume of lOOpl at 4 °C for 30 min.

Cells were then washed three times with 200pl of FACS buffer, and IgG binding détection is acheived by adding 100 pl of Donkey anti Human IgG (H+L) R18848

Phycoerythrin conjugated (Jackson Immuno Research cat# 709-1I6-149) l : 100 in FACS buffer. After gentle vortexing, cells were incubated at 4°C for 30 min.

Plates were washed 2X with 200μ! of FACS buffer, cells are finally resuspended and transferred in 0.5 ml of FACS Buffer and acquired by using a flow cytometer.

Results

Figure 10 clearly shows that as expected based on prior art disclosures (see US Patent 5,624,821 Winter) the mutated isoform BXhVH5VLl N297A is devoid of significant binding capability to cellular Fc receptors.

Example 7

In vivo Experiments

In vivo experiments that were performed to further assay the antibodies/derivatives ofthe présent invention are set out below;

NGF-mediated pain models

Nerve growth factor (NGF) and its receptor TrkA are crucial mediators ofthe pain sensations characteristic of inflammatory pain.

Classically, NGF is known as a developmental survival factor for sensory and sympathetic neurons but it continues to be synthesized in adult animais in the periphery, where it is transported retrogradely to the cell bodies of sensory neurons (Hendry et al.,1974, Otten et al 1980).

Inflammation and nerve injury cause the release of NGF which stimulâtes primary afferent fibres and induces behavioural sensitisation. Subcutaneous chronic treatment with NGF in rats causes hyperalgesia and alters local cutaneous sensation (Lewin et al., 1993; Andreev et al., 1995).

Intradermal injection of rhNGF into human forearm and masseter muscle in humans causes hyperalgesia, allodynia and alters local cutaneous sensation that began 3hrs following injection and peaked 1-7 days post injection and recovered by day 21 (Dyck et al., 1997; Svensson et al., 2003).

Thus, injections of rhNGF into the rat hindpaw were used here as a model of behavioural sensitisation that was specifically generated by NGF.

The présent experiments involved two different protocols;

1. We first examined whether intradermal injection of recombinant human (rh) NGF alone in rat paw could cause behavioural sensitisation as measured by standard nociceptive tests for hyperalgesia (Hargreave’s plantar test). We then established whether intradermal co-injection of the murine IgG. muMNACEP, human IgG, and BXhVH5VLl N297A_antibodies at a dose of lOOpg could affect the rhNGF-induced sensitisation. Murine IgGi and human IgG antibodies ware used as a négative Controls at the appropriated dosages.

2. We then established whether systemic pre-treatment of muMNACEP antibody (at doses of 8 and 1 mg/Kg, i.p.) and BXhVH5VLl N297A (doses of 8, 3, and 1 mg/Kg, i.p.) could affect the peripherally induced rhNGF sensitisation.

In the first protocol, whereby treatments where administered locally, male Lewis rats (Charles River, 5-6weeks 200g) were used with 8-9 animais per group and 4 experimental groups, injected according to the set method. Injections were carried out blind. A summary of the treatments is outlîned in the table below.

	muMNACEP /BXhVH5VLl N297A	mlgGi/hlgG
Intradermal treatments	100pg + 500ng rhNGF, n=9	100gg + 500ng rhNGF, n=9

In the second protoco, whereby treatments were administered systemically (i.p.) 24hrs prior to rhNGF paw injection, maie Lewis rats (Charles River, 5-6weeks 200 g) were used 20 with 10-12 animais per group and 10 experimental groups. Injections were carried out blind. A summary of the treatments is outlîned in the table below.

muMNACEP

mlgG]

BXhVHôVLl

hlgG

			N297A
Systemic i.p. 24hr pretreatments	Img/Kg n=10 8mg/Kg n= 10	Img/Kg n=12 8mg/Kg n= 8	Img/Kg n=10 3mg/Kg n=10 8mg/Kg n= 12	Img/Kg n=10 3mg/Kg n=10 8mg/Kg n= 12
rhNGF intradermai	500ng	500ng	500ng	500ng

Assay

Ail animais were numbered and then habituated to behaviour- testing procedures 24-48hr prior to commencement of the experiment. Behaviour readouts were the paw withdrawal 5 latency to the plantar test as a measure of hyperalgesia.

Baseline recordings were taken to establish paw withdrawal latencies. Nociceptive sensîtivity was induced by intradermal rhNGF injected at Time point 0 and behavioural nociceptive sensîtivity was monitored 30 minutes, l hour, 2 hours, 24 hours and 48 hours following rhNGF injection. Treatments were administered blind as follows:

Protocol 1: Treatment administration by intradermal injection at Time 0,

Protocol 2: Treatment administration by systemic single injection IP 24h before rhNGF paw injection.

Baseline plantar and von Frey tests were performed before drug treatments were administered.

Hyperalgesia measurements were taken 30min, Ihr, 2hrs, 24hrs and 48hrs after rhNGF injection. Three to four recordings were taken for each hindpaw ipsilateral (right paw, rhNGF-injected) and contralatéral (left paw, un-injected) to rhNGF injection.

Data from animais from individual treatment groups were collated, and means and standard déviations were calculated for the controlatéral and ipsilateral paw responses.

The presence of hyperalgesia was indicated by a significant réduction in the paw withdrawai latency (recorded in seconds) in the rhNGF-injected ipsilateral hindpaw when compared with the control contralatéral paw (by paired t-test) and when compared with the pre-injection/pre-treatment baseline (by one-way ANOVA).

Intradermal anti-hyperalgesic efficacy

We compared the anti-hyperalgesic efficacy of BXhVH5VLl N297A (Figure 11) and muMNACEP (Figure 12) (plus mlgGi and hlgG as relative Controls) by intradermal injection in this model ofNGF-induced hyperalgesia.

When BXhVH5VLl N297A and muMNACEP were co-injected with rhNGF, there was no significant development of hyperalgesia, as indicated by no significant différence between ipsilateral and contralatéral paw withdrawai responses (Figures 11 and 12).

Hyperalgesia in ipsilateral paw responses were always present following co-injection with the négative Controls (mlgGi. hlgG).

Data are represented as means ± 95% CI, before (baseline) and following intradermal injection of 500ng rhNGF with respective treatments (at arrow). Significant réduction in paw withdrawai latency ipsilateral is indicated by (p<0.05, p<0.0l, paired t-test) when compared to the contralatéral paw withdrawai.

Systemic anti-hyperalgesic efficacy

We compared the anti-hyperalgesic efficacy of BXhVH5VLl N297A, muMNACEP mlgGi, and hlgG by systemic injection in thîs model ofNGF-induced hyperalgesia.

Three different doses of BXhVH5VLl N297A and control hlgG (l, 3, and 8 mg/Kg) were tested (Figure 13). Similarly, two different doses of muMNACEP and mlgGi were tested, lmg/Kg and 8mg/Kg (Figure 14). Ail treatments were administered i.p. 24hours prior to intradermal injection with rhNGF.

Systemic pre-treatment of 8 and 3 mg/kg BXhVH5VLl N297A significantly prevented the development of hyperalgesia following rhNGF injection, as indicated by no significant différence between ipsilateral and contralatéral paw withdrawai responses (Figure 13).

Systemic pre-treatment with the murine parental antibody mMNACEP (8 mg/Kg) also prevented the development of rhNGF-induced hyperalgesia (Figure 14). However, the overall analgésie response of BXhVH5VLl N297A appeared to be better as compared with mMNACEP antibody.

At the same dose, hyperalgesia in ipsilateral paw responses was always présent following co-injection with the négative control mlgGi and hlgG.

Data are represented as means ± 95% CI, before (baseline) and following intradermal injection of 500ng rhNGF with respective treatments (at arrow). Significant réduction in paw withdrawal latency ipsilateral is indicated by ‘**’ (p<0.05, p<0.01, paired t-test) when compared to the contralatéral paw withdrawal.

Example 8

Further in vivo experiments that may be performed to further assay the antibodies/derivatives of the présent invention are set out below:

Formalin test

Mice are pretreated with the antibody/derivative intraperitoneally and 18 hours later are injected in the right dorsal footpad with 5% Formalin. Lickîng time (time spent licking the injured paw) is measured for up to 1 hour.

Chronic constriction iniurv test

Mice are subject to surgical constriction of sciatîc nerve, in order to induce a neuropathie allodynia. Animais are then treated with antibody/derivative and withdrawal response to a mechanical stimulus localized to the injured limb versus the contralatéral lîmb is measured.

Arthritis model

Rats are injected with complété Freund’s adjuvant at the tail base intradermally. Approximately three weeks late they develop a systemic poly arthritis characterized by joint pain. Animais are treated with the antibody/derivative and the analgésie effect is evaluated by the vocalization assay consisting of measurement of intensity of vocalization upon gentle manipulation of the joints.

Monkev carrageenan induced pain model

Rhésus macaques are pretreated intravenously with the antibody/derivative. The following day, animais are injected subcutaneously with carrageenan in the tail, Withdrawal time from a heat stimulus is measured.

General Points

Unless the context indicates otherwise, the following general points apply;

1) Ail references discussed herein are deemed to be incorporated by reference.

2) The term “comprises is non-limiting in that it covers “including” as well as “consisting of’. Thus the word ‘comprises’ and variations such as ‘comprise’ and ‘comprising’ will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

3) Equivalents of aspects of the invention discussed herein are considered to be within the scope of the invention, even ïf the équivalents are not specifically discussed.

Claims (62)

1. Claims

   1. An anti-TrkA antibody that comprises:

   a) a variable heavy chain comprising a sequence selected from any of BXhVHl, BXhVH2, BXhVH3, BXhVH4, BXhVH5 or HuVHWOv as shown in Figure la;

   and/or

   b) a variable light chain comprising a sequence selected from any of BXhVLl, BXhVL2, BXhVL3, BXhVL4, BXhVL5, BXhVL6, BXhVL7 or BXhVL8 as shown in Figure Ib or a dérivative of said antibody wherein the dérivative is capable of binding TrkA wherein the antibody or dérivative has at least 85% humanisation when measured over the total framework régions of the variable région of at least one of its chains (excluding the associated CDR régions).
2. 2. An antibody or dérivative according to claim 1 wherein the antibody comprises one of the following combinations of light and heavy chains:

   BXhVHl VL 1, BXhVHl VL2, BXhVHlVL3, BXhVHl VL4, BXhVHlVL5.

   BXhVHl VL6, BXhVHl VL7, BXhVHl VL8.

   BXhVH2VLl, BXhVH2VL2, BXhVH2VL3, BXhVH2VL4, BXhVH2VL5, BXhVH2VL6, BXhVH2VL7, BXhVH2VL8,

   BXhVH3VLl, BXhVH3VL2, BXhVH3VL3, BXhVH3VL4, BXhVH3VL5, BXhVH3VL6, BXhVH3VL7, BXhVH3VL8,

   BXhVH4VLl, BXhVH4VL2, BXhVH4VL3, BXhVH4VL4, BXhVH4VL5,

   BXhVH4VL6, BXhVH4VL7, BXhVH4VL8,

   BXhVH5VLl, BXhVH5VL2, BXhVH5VL3, BXhVH5VL4, BXhVH5VL5, BXhVH5VL6. BXhVH5VL7, BXhVH5VL8.

   or HuVHWOv/HuVLWO or a dérivative of said antibody; wherein the dérivative is capable of binding TrkA.
3. 3. An antibody or dérivative according to claim 1 or claim 2 wherein the antibody comprises one of the following combinations of light and heavy chains: BXhVH3VL3, BXhVHSVLl, or BXhVH5VL3.
4. 4. An antibody or dérivative according to any preceding claim that comprises one or more of CDR régions selected from the régions underlined in Figures la & 1b or from variants sqthereof having up to two amino acid changes per underlined région.
5. 5. An antibody or dérivative according to any preceding claim that comprises six CDR régions.
6. 6. An antibody or dérivative according to any preceding claim that has a plurality of framework régions selected from the non-underlined sequences shown in Figures la and/or Ib or from variants thereof that hâve at least 75% sequence identity with the nonunderlined régions.
7. 7. An antibody or dérivative according to claim 6 wherein said sequence identity is at least 85%.
8. 8. An antibody or dérivative according to claim 6 wherein said sequence identity is at least 95%.
9. 9. An antibody or dérivative according to claim 6 wherein the variants hâve no greater a percentage of murine residues present in the framework régions than are present in the corresponding non-underlined régions shown in Figures la and/or Ib.
10. 10. An antibody or dérivative according to any preceding claim that binds with greater affinity to TrkA than to Trk B.
11. 11. An antibody or dérivative according to any preceding claim that is capable of blocking or reducing the binding of NGF to the TrkA receptor.
12. 12. An antibody or dérivative according to any preceding claim that is capable of blocking or reducing one or more biological activities that would otherwise be induced by the binding of NGF to the TrkA receptor.
13. 13. An antibody or dérivative according to any preceding claim comprising a non-rodent derived constant région.
14. 14. An antibody or dérivative according to any preceding claim comprising a human constant région or a constant région with at least 75% sequence identity with a human constant région.
15. 15. An antibody or dérivative according to any preceding claim comprising a human IgG constant région or a constant région with at least 75% sequence identity therewith.
16. 16. An antibody or dérivative thereof comprising a constant région that comprises:

    a) the sequence shown in Figure 6 for the heavy chain that begins with the underlined A and terminâtes with the final K or a sequence with at least 75% sequence identity therewith;

    and /or

    b) the sequence shown in Figure 6 for the light chain that begins with the underlined R and terminâtes with the final C, or a sequence with at least 75% sequence identity therewith,
17. 17. An antibody or dérivative according to claim 16 wherein position 297 of the heavy chain constant région is mutated from N to A (N297A).
18. 18. A dérivative according to any preceding claim comprising a constant région that has one or more amino acid changes relative to a human immunoglobulin constant région that prevent/reduce one or more of the following,

    a) activation of complément

    b) complement-mediated lysis

    c) activation of T cells

    d) binding to an Fc receptor.
19. 19. An antibody or dérivative according to any preceding claim that provides an OD450/630 nm value of greater than 0.1 in the TrkA-IgG binding assay illustrated by Figure 2.
20. 20. An antibody or dérivative according to any preceding that provides an increase in FACS staining of TF1 cells in the FACS based assay described in the examples that is an increase is an increase of over 1.0 fold.
21. 21. Am antibody or dérivative according to any preceding claim that has a K_D value of less than 4.18 x 10'⁸ M in respect of TrkA-IgG binding in an assay as described in the Examples.
22. 22. An antibody or dérivative according to any preceding claim that has at least 90% humanisation when measured over the total framework régions of the variable région of at least one chain (excluding the associated CDR régions).
23. 23. An antibody or dérivative according to any preceding claim that has at least 95% humanisation when measured over the total framework régions of the variable région of at least one chain (excluding the associated CDR régions).
24. 24. An antibody or dérivative according to claim l or any of daims 22 to 23 that has at least the percentage humanisation given in any of said daims in respect of the framework régions for both the light and heavy chains.
25. 25. A dérivative of an antibody according to any preceding claim wherein the dérivative is

    a) a fragment of said antibody

    b) a fragment multimer

    c) a fusion product of said antibody fragment or fragment multimer and another moiety

    d) a variant of said antibody, fragment, fragment multimer, or fusion product, having at least 75% sequence identity therewith.
26. 26. A dérivative according to claim 25 wherein the dérivative is a fusion product and another moiety is an agent selected from the following: a diagnostic agent, a therapeutic agent, a marking agent, an agent that increases the half life and/or that reduces the îmmunogenicity of said dérivative in a human host.
27. 27. A dérivative thereof according to claim 26 wherein the therapeutic agent is a cytotoxin.
28. 28. An antibody or dérivative thereof according to any preceding claim that is PEGylated.
29. 29. An antibody or dérivative according to any preceding claim in immobilised form.
30. 30. An antibody or dérivative according to any of daims 1 to 29; for use in medicine
31. 31. An antibody or dérivative according to any of daims 1 to 29; for use in the treatment of pain.
32. 32. An antibody or dérivative according to any of daims 1 to 29; for use in the treatment of chronic pain.
33. 33. An antibody or dérivative according to any of daims 1 to 29; for use in the treatment of acute pain.
34. 34. An antibody or dérivative according to any of daims 1 to 29; for use in the treatment of pain associated with one or more of the following: pancreatitis, kidney stones, endometriosis, IBD, Crohn’s disease, post surgical adhesions , gall bladder stones,

    S-ίheadaches, dysmenorrhea, muscuioskeletal pain, sprains, viscéral pain, ovarian cysts, prostatitis, cystitîs, interstitial cystitis, post-operative pain, migraine, trigeminal neuralgia, pain from bums and/or wounds, pain associated with trauma. neuropathie pain, pain associated with muscuioskeletal diseases, rheumatoid arthritis, osteoarthritis. ankylosing spondilitîs, periarticular pathologies, oncological pain, pain from bone métastasés. HIV infection.
35. 35. An antibody or dérivative according to any of claims l to 29; for use in the treatment of cancer, a neuronal disorder (e.g, a neurodegenerative disorder), Alzheimer’s disease, diabètes mellitus, a viral disorder, an HIV mediated disorder, leprosy, or an inflammatory disorder.
36. 36. An antibody or dérivative according to any of claims 31 to 35; whereas the treatment involves ADCC.
37. 37. A combination of an antibody or dérivative according to any of claims l to 29 and an analgésie, for simultaneous. sequential or concerted administration in medicine.
38. 38. A combination of an antibody or dérivative according to any of claims l to 29 and NGF, for simultaneous, sequential or concerted administration in medicine.
39. 39. A combination of an antibody or dérivative according to any of claims l to 29 and a further anti-TrkA antibody or dérivative thereof, for simultaneous, sequential or concerted administration in medicine.
40. 40. A pharmaceutical composition comprising an antibody or dérivative thereof according to any of claims l to 29 and a pharmaceutically acceptable carrier or excipient.
41. 41. A pharmaceutical composition comprising an antibody according to any of claims l to 29 and another pharmaceutically active agent.
42. 42. A pharmaceutical composition according to claim 41; wherein said another agent is one or more of:

    a) an analgésie agent

    b) another anti-TrkA antibody or dérivative thereof

    c) NGF

    d) an anti-cancer agent.
43. 43. An antibody or dérivative thereof according to any of claims l to 29 for use în

    SS diagnosis or prognosis.
44. 44. An antibody or dérivative thereof for use in the diagnosis or prognosis of a condition involving aberrant expression of TrkA or an aberrant activity involving TrkA.
45. 45. An antibody or dérivative thereof for use in the diagnosis or prognosis of any of the diseases or disorders specified in ciaims 31 to 35.
46. 46. A method comprising obtaining a biological sample and administering an antibody or dérivative according to any of ciaims l to 29 thereto.
47. 47. A method according to claim 46 further comprising comparing the results for binding of said antibody or dérivative to said sample with a positive or négative control sample.
48. 48. A polypeptide that consists of or comprises a variable light chain of an antibody or dérivative thereof according to any of ciaims l to 29.
49. 49. A polypeptide that consists of or comprises a variable heavy chain of an antibody or dérivative thereof as described in any of ciaims l to 29.
50. 50. A polypeptide according to claim 48 or claim 49 that further comprises one or more human constant régions.
51. 51. A nucleic acid encoding an antibody or dérivative thereof according to any of ciaims l to 28 or encoding a polypeptide according to any of ciaims 48 to 50.
52. 52. A vector comprising a nucleic acid according to claim 51.
53. 53. An expression system that expresses an antibody or dérivative thereof according to any of ciaims l to 29, or a polypeptide according to any of ciaims 48 to 50 or that can be induced to provide such expression.
54. 54. A non-human transgenic mammal that expresses an antibody or dérivative thereof according to any of ciaims l to 29, or a polypeptide according to any of ciaims 48 to 50 or that can be induced to provide such expression.
55. 55. A kit of parts comprising an antibody or dérivative according to any one of ciaims I to 29 together with instructions directing the use thereof by a subject as an analgésie.

    S'3
56. 56. Use of an antibody or dérivative according to claims l to 29 in the manufacture of a médicament for use in medicine, the use comprising administering the antibody or dérivative to a subject in need thereof.
57. 57. Use of an antibody or dérivative according to claims 1 to 29 in the manufacture of a médicament for use in the treatment of pain, the use comprising administering the antibody or dérivative to a subject in need thereof.
58. 58. Use of an antibody or dérivative according to claims 1 to 29 in the manufacture of a médicament for use in the treatment of chronic pain, the use comprising administering the antibody or dérivative to a subject in need thereof.
59. 59. Use of an antibody or dérivative according to claims 1 to 29 in the manufacture of a médicament for use in the treatment of acute pain, the use comprising administering the antibody or dérivative to a subject in need thereof.
60. 60. Use of an antibody or dérivative according to claims 1 to 29 in the manufacture of a médicament for use in the treatment of pain associated with one or more of the following: pancreatitis, kidney stones, endometriosis, IBD, Crohn’s disease, post surgical adhesions, gall bladder stones. headaches, dysmenorrhea, musculoskeletal pain, sprains. viscéral pain, ovarian cysts, prostatitis, cystitis, interstitial cystitis. post-operative pain, migraine, trigeminal neuralgia, pain from bums and/or wounds, pain associated with trauma, neuropathie pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, osteoarthritis, ankylosing spondilitis, periarticular pathologies, oncological pain, pain from bone métastasés, HIV infection, the use comprising administering the antibody or dérivative to a subject in need thereof.
61. 61. Use of an antibody or dérivative according to claims 1 to 29 in the manufacture of a médicament for use in the treatment of cancer, a neuronal disorder (e.g. a neurodegenerative disorder), Alzheimer's disease, diabètes mellitus. a viral disorder, an

    18848 ίο

    HIV mediated disorder. leprosy, or an inflammatory disorder, the use comprising administering the antibody or derivative to a subject in need thereof.
62. 62. Use of an antibody or derivative according to any of claims 56 to 61, whereas the use invoives ADCC.