WO2018060480A1

WO2018060480A1 - Anti-mesothelin antibodies

Info

Publication number: WO2018060480A1
Application number: PCT/EP2017/074872
Authority: WO
Inventors: John Wijdenes; Andrew Clark
Original assignee: Mab Designs Ltd.
Priority date: 2016-09-30
Filing date: 2017-09-29
Publication date: 2018-04-05
Also published as: GB201616699D0

Abstract

The present invention relates to antibodies which bind to mesothelin. In particular, the present invention provides antibodies that bind to the membrane-bound form of mesothelin on cells and do not bind significantly to the soluble form of mesothelin. The invention also relates to immunoconjugates and compositions comprising such antibodies. The invention also provides methods of producing such antibodies. The invention further provides the use of such antibodies for therapeutic and diagnostic purposes.

Description

Anti-mesothelin antibodies

This invention relates generally to the field of antibodies, in particular antibodies (e.g. Fabs) that specifically bind to membrane bound mesothelin whilst showing a very low affinity for the soluble form of the same antigen. The antibodies (e.g. Fabs) of the invention may be useful in specific delivery of pharmacologic agents to mesothelin-positive cells as well as in eliciting an immune effector activity on mesothelin-positive cells, for example, tumour cells and precursors.

A difficulty that is commonly encountered when treating patients who have cancer with cytotoxic small molecule drugs is that the cytotoxin causes damage to normal tissues as well as cancerous tissues. One approach to obtain higher specificity for the cancer tissue is the use of antibodies that can target specific antigens, expressed on cancer cells, that are not expressed or are expressed at a lower level on normal cells. These target antigens can be exploited using antibodies to specifically kill antigen-bearing tumour cells by a variety of mechanisms including inhibiting the biological activity of the antigen, eliciting an immune effector activity by complement dependent cytotoxicity (CDC) and/or antibody dependent cellular cytotoxicity (ADCC), or by delivering immuno- or radio conjugates that, when delivered to the antigen-bearing cell, specifically kill the target cell.

In this regard, although there has been some considerable success in the treatment of cancer using antibodies, there have also been a high number of failures. Initial expectations were high, as it was believed that the target antigen could be selected to be present on the cancer cell but not on normal tissue. The selectivity of the antibody should thus ensure that only the cancer cells were treated and the tumour cells were killed in a more selective manner than could be achieved with conventional chemo- or radio-therapies. For some of the blood borne cancers or leukaemias, antibody therapy has proven to be significantly effective, but this has not been matched in the treatment of solid tumours, as, with the exception of Herceptin and Avastin, antibody therapies have not proven particularly beneficial.

The lack of efficacy in the treatment of solid tumours may be the result of a number of factors:

a) Solid tumours are dense and poorly vascularised internally. The antibody molecule is large and may simply take too long to penetrate the tumour mass. b) Tumours are heterogeneous and the antigen target may not be present on the surface of all cells within the tumour mass.

c) Tumour cells shed antigen from the cell surface, and the soluble circulating antigen is binding the therapeutic antibody.

Factors a) and b) have been addressed by attaching to the therapeutic antibody a killing agent that acts in a localised but non-discriminatory way. This is referred to as antibody conjugation. These agents include radio-isotopes of elements such as Yttrium, Iodine, Indium or Cobalt. A range of chemotoxic agents have also been targeted in this way, including conventional cytotoxic drugs, toxins such as ricin and calicheamicin and pro-drugs of several forms. The localisation to the tumour of a non-selective killing agent addresses both the issues of penetration and the heterogeneity of the tumour. However, it does not address the issue of shed antigen.

Alternative methods to address factor a) involve the administration of the antibody in very large amounts to try and reach all the cells in the interior of the tumour, but in some cases large amounts of antibody cannot be given because of undesirable side effects. In addition, immune-conjugates comprising a killing agent such as those described above cannot be given in large amounts because of the non-specific side effects of the radio-isotope, drug or toxin. When antibodies are administered at lower doses, it is believed that the shed antigen levels mentioned in factor c) can be high enough to present a significant problem and to interfere with the action of the immune-conjugate.

Studies carried out by the Pastan group have recognised the potential problems associated with shed antigen in the blood and also in the interstitial space of tumours and have made the observation that the concentration of a particular shed tumour antigen, mesothelin, within the tumour and the blood is lowered by chemotherapy. They have suggested that the reduction in the levels of shed antigen by chemotherapy should enhance the efficacy of immunoconjugate therapies (Zhang et al, PNAS, 2007, 104(43): 17099-17104; Zhang and Pastan, Clin Cancer Research 2008: 14(24): 7981 -7986). Prinssen et al (Cancer Immunol Immunotherapy, 1998, 47:39-46) also observed a problem with the circulating (shed) antigen MUC1 in ovarian cancer patients in that the administration of a therapeutic radiolabeled antibody directed against the MUC1 antigen was resulting in the formation of radiolabeled immune complexes in the circulation which were then accumulating to problematic levels in the liver. To try and overcome the problem of circulating radioactive immune complexes and their accumulation in the liver, this group came up with the strategy to administer a dose of unlabelled antibody (to mop up the circulating antigen) before they administered the

radiolabeled antibody.

The present inventors however have devised a very different way of addressing the problem of shed antigen by developing a method which enables antibodies to be generated which can distinguish between the membrane bound form and the soluble form of the same antigen. Antibodies which bind the membrane bound form of the antigen but not the soluble form would not be decoyed by soluble (shed) antigen in the circulation or in the interstitial spaces of the tumour but would instead target straight to the tumour cell membranes. Such antibodies thus provide an alternative and advantageous solution to the problem of shed antigen as well as having many other applications. Although such antibodies may well be useful therapeutically in an unconjugated form, or through the effects of CDC or ADCC mechanisms, when coupled to killing agents such as those described above and elsewhere herein, such antibodies should significantly overcome all three of the factors identified above. It is also likely that such antibodies (in either a "naked" unconjugated form or coupled to killing agents) will be effective at lower doses which should result in fewer side effects and a more cost effective treatment.

Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesised as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form and a 40 kDa membrane-bound form (Yamaguchi, et al. J. Biol. Chem. 269, 805-808, 1994). Mesothelin is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in a number of different cancers, including mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, and ovarian cancer (Hassan et al., Clin. Cancer Res. 10:3937-3942, 2004; McGuire et al., N. Engl. J. Med. 334:1 -6, 1996; Argani et al., Clin. Cancer Res. 7:3862-3868, 2001 ; Hassan et al., Appl. Immunohistochem. Mol.

Morphol. 13:243-247, 2005; Li et al., Mol. Cancer. Ther. 7:286-296, 2008; U.S. Pat. No. 7,081 ,518). In particular, it has been reported that a majority of serous carcinomas of the ovary and adenocarcinomas of the pancreas express high levels of mesothelin (Yen et al., Clin. Cancer Res. 12:827-831 , 2006). In addition, high levels of mesothelin have been detected in greater than 55% of lung cancers and greater than 70% ovarian cancers (Hassan et al., Appl. Immunohistochem. Mol. Morphol. 13:243-247, 2005; Ho et al., Clin. Cancer Res. 13(5): 571 -1575, 2007). The limited expression of mesothelin on normal cells makes it a viable target for tumour immunotherapy. Administration of antibodies against mesothelin has been proposed as a strategy for treatment of mesothelioma as well as lung, ovarian, and pancreatic cancer.

In 1992, Chang et al. described monoclonal antibodies that recognised antigens on human ovarian carcinoma cells (Chang, et al., Am. J. Surg. Pathol. 1992 16:259-68). This antibody, called K1 , was chemically conjugated to a truncated form of Pseudomonas exotoxin and found to bind mesothelin-positive cells and cancer cells. However, it was not useful as an immunotoxin conjugate due to its poor internalisation.

US. Pat. No. 6,083,502 describes mesothelin and uses for targeting and diagnosing mesothelin-positive cells using antibody K1 . Subsequent single chain antibodies were produced that bound with high-affinity and had potent antitumor activity on mesothelin-positive tumours as a conjugate. One such single chain antibody is SS1 (scFv)-PE38 which has a high binding affinity (Kd of 0.7 nM) to mesothelin. This single chain antibody is a stabilised form in which a disulfide bond connects the light and heavy chain domains. SS1 (scFv)-PE38 has been shown to have activity in killing tumour cells by internalisation of the single chain antibody- immuno-toxin complex (Hassan, et al., Clin. Cancer Res., 8: 3520-6, 2002; Hassan, et al., Proc. Am. Soc. Clin. Oncol., 21 : 29a, 2002). Other groups have also developed antibodies that can bind to mesothelin and found overexpression of this antigen to be associated with various cancers (Scholler, et al., Proc. Natl. Acad. Sci. U.S.A. 1999 Sep. 28; 96(20):1 1531 -6; Ordonez, Am. J. Surg. Pathol. 27:1418-28, 2003). US. Pat. No. 6,809,184 describes a single chain high affinity antibody that binds to mesothelin at a different epitope than the K1 antibody. This antibody fragment was found to internalise in mesothelin-positive cells as a single chain fragment linked to an immunotoxin. The antibody was named SS 1 . Attempts to develop immune-conjugated antibodies that can specifically target mesothelin have been performed with little success due to poor internalisation and/or affinity

(Hassan, et al., J Immunother. 2000 Jul-Aug; 23(4):473-9). This lack of

internalisation could be due to low affinity or antibody composition and/or epitope binding. In addition, generation of the monoclonal antibody (mAb) K1 as an immune-conjugate was attempted because the unconjugated form was not cytotoxic itself (Hassan, et al., Clin. Cancer Res., 10:3937-3942, 2004).

In the context of membrane bound or soluble antigen it is not exclusively the case that the antigens are either attached to the cell membrane or present in a soluble form in the extracellular environment. Current research suggests that there are a broad variety of non-cellular bodies consisting of membrane components and intra- and cell surface moieties present in tissues. Extracellular vesicles (EVs) are signalling organelles that are released by many cell types and are highly conserved in both prokaryotes and eukaryotes. Based on the mechanism of biogenesis, these membranous vesicles can be classified as exosomes, shedding microvesicles, and apoptotic bodies/blebs. It is becoming clearer that these EVs mediate signal transduction in both autocrine and paracrine fashion by the transfer of proteins and RNA. While the role of EVs including exosomes in pathogenesis is well established, very little is known about their function in normal physiological conditions. Recent evidences allude that EVs can mediate both protective and pathogenic effects depending on the precise state - (Proteomics. 2015 Jan;15(2-3):260-

71.doi:10.1002/pmic. 201400234. Epub 2014 Dec 18. Extracellular vesicles including exosomes are mediators of signal transduction: are they protective or pathogenic Gangoda L, Boukouris S, Liem M, Kalra H, Mathivanan S).

With regard to the discrimination between a 'membrane bound' antigen and its soluble form there are clearly a whole range of intermediate forms with the antigen of interest present on the surface of an EV. It has been clearly established that antigen presented in this way can bind to endogenous antibodies which target the tumour and inhibit their action - (C. Battke, R. Ruiss, U. Welsch et al., "Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC," Cancer Immunology, Immunotherapy, vol. 60, no. 5, pp. 639-648, 201 1 ).

It is also the case that therapeutic antibodies intended to target cellular membrane bound antigen can bind to antigen present on EVs and that this has a neutralising effect. EVs, in this instance exosomes "can also function to neutralize antibody-based drugs. Exosomes secreted by HER2-overexpressing breast carcinoma cell lines express a full-length HER2 molecule, enabling them to bind to the HER2 antibody Trastuzumab both in vitro and in vivo. The exosome-antibody interactions inhibit the overall effect of Trastuzumab on the proliferation of cancer cells by reducing antibody binding to cancer cells" - (V. Ciravolo, V. Huber, G. C. Ghedini et al., "Potential role of HER2-overexpressing exosomes in countering Trastuzumab based therapy," Journal of Cellular Physiology. 227 658-667 2012). Such antibody sequestration was also demonstrated to reduce the antibody- dependent cytotoxicity effect on tumour cells by immune effector cells. (The Roles of Tumor-Derived Exosomes in Cancer Pathology Chenjie Yang and Paul D.

Robbins Clinical and Developmental Immunology Volume 201 1 , Article ID 842849, 1 1 pages doi:10.1 155/201 1/842849). A similar effect has been reported for the anti-CD20 antibody Rituximab. "Exosomes carried the CD20 target antigen and acted as decoy targets upon rituximab exposure, allowing lymphoma cells to escape from humoral

immunotherapy. In vivo, exosomes also bound the anti-CD20 antibody rituximab in humans who had received the antibody for therapeutic purposes. Approximately half of all of the plasma rituximab was found to be fixed to exosomes 3 h after the end of the rituximab infusion." (T. Aung, B. Chapuy, D. Vogel et al., "Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3," Proceedings of the National Academy of Sciences of the United States of America , vol. 108, no. 37, pp. 15336-15341 , 201 1 ).

The same authors go on to state "our data are comparable to previous pharmacokinetic findings at initiation of rituximab therapy. However the high fraction of rituximab bound to exosomes indicates that significant proportions of rituximab in the serum are not in a soluble state and thus are not available for lymphoma cell attack."

It is clear from the evidence presented above that the amount of a soluble therapeutic antibody available to bind an antigen on a cell membrane is influenced not only by the physical availability of that antigen, but also by the level of the antigen present in either soluble form (so called 'shed' antigen) or also by the levels of antigen present on EVs of many different conformations.

Advantageously, the present invention provides for anti-mesothelin antibodies (e.g. Fabs) which recognise the membrane bound mesothelin antigen on cells whilst not significantly recognising the soluble form of the same antigen.

Surprisingly and advantageously, preferred antibodies also do not significantly recognise the EV bound form of the same antigen (mesothelin). In some

embodiments, these antibodies (e.g. Fabs) may be internalised within the target cell or may effect CDC or ADCC mechanisms.

It is clear that there are reasonable grounds to expect that an antibody which recognises the membrane bound antigen but not antigen present in either the 'shed' soluble form or antigen present on the surface of EVs might be expected, as a result of this greater selectivity for the target cell, to demonstrate an enhanced therapeutic profile. In one aspect, the present invention provides an antibody, for example an isolated antibody which binds to (or specifically recognises) mesothelin, wherein said antibody:

(i) binds to the membrane-bound form of mesothelin on cells;

(ii) does not bind significantly to the soluble form of mesothelin; and

(iii) does not bind significantly to mesothelin on extracellular vesicles.

In one embodiment, the present invention provides an antibody comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:5, or a sequence substantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, the antibody comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 or 24, or a sequence substantially homologous to any one of these sequences.

Alternatively or in addition, in an embodiment of the invention, the antibody comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 7 or 25, or a sequence substantially homologous to any one of these sequences.

Alternatively or in addition, in an embodiment of the invention, the antibody comprises a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, the antibody comprises a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, the antibody comprises a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10 or 28, or a sequence substantially homologous to any one of these sequences.

Thus, in certain embodiments, the invention provides an antibody comprising one or more heavy chain CDR domains, wherein the heavy chain CDR domain is selected from the group consisting of (or comprising):

(a) a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:5, or a sequence substantially homologous thereto;

(b) a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6 or 24, or a sequence substantially homologous thereto; and (c) a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 7 or 25, or a sequence substantially homologous thereto.

The invention also provides, in certain embodiments an antibody comprising one or more light chain CDR domains, wherein the light chain CDR domain is selected from the group consisting of (or comprising):

(a) a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:

8, or a sequence substantially homologous thereto;

(b) a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO:

9, or a sequence substantially homologous thereto; and

(c) a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10 or 28, or a sequence substantially homologous thereto.

In certain preferred embodiments, the antibody comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a sequence substantially homologous thereto and

(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a sequence substantially homologous thereto.

More preferably, a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto, and/or a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, are also present.

In certain preferred embodiments, the antibody comprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25, or a sequence substantially homologous thereto, and

(b) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28, or a sequence substantially homologous thereto.

More preferably, a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8 or a sequence substantially homologous thereto, and/or a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, are also present. In one preferred embodiment, the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially homologous thereto, and CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a sequence substantially homologous thereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a sequence substantially homologous thereto, are present individually or in combination.

In one preferred embodiment, the heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 24, or a sequence substantially homologous thereto, and CDR3 comprising the amino acid sequence of SEQ ID NO: 25, or a sequence substantially homologous thereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, and CDR3 comprising the amino acid sequence of SEQ ID NO: 28, or a sequence substantially homologous thereto, are present individually or in combination.

Viewed alternatively, in certain embodiments, the present invention provides an antibody comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 7, or a sequence substantially homologous thereto and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:8, or a sequence substantially homologous thereto. In certain embodiments, the present invention provides an antibody comprising a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:25, or a sequence substantially homologous thereto, and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

Viewed alternatively, in certain embodiments, the present invention provides an antibody comprising a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 7, or a sequence substantially homologous thereto, and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

In certain embodiments, the present invention provides an antibody comprising a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 25, or a sequence substantially homologous thereto, and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

Viewed alternatively, in certain embodiments, the present invention provides an antibody comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 7, or a sequence substantially homologous thereto, and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto.

In certain embodiments, the present invention provides an antibody comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 5, or a sequence substantially homologous thereto, and/or a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially homologous thereto.

Said antibody optionally further comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 25, or a sequence substantially homologous thereto, and/or a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, or a sequence substantially homologous thereto, and/or further comprises a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, or a sequence substantially homologous thereto, and/or a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 9, or a sequence substantially homologous thereto.

Certain preferred antibodies of the invention comprise one or more, or all, of the CDRs selected from the group consisting of (or comprising) SEQ ID NOs: 5, 6, 7, 8, 9 and 10, or a sequence substantially homologous to any one of the foregoing SEQ ID NOs.

Certain preferred antibodies of the invention comprise one or more, or all, of the CDRs selected from the group consisting of (or comprising) SEQ ID NOs: 5, 24, 25, 8, 9 and 28, or a sequence substantially homologous to any one of the foregoing SEQ ID NOs. Certain preferred antibodies comprise two or more of the light chain CDRs of SEQ ID NOs: 8, 9 and 10; or 8, 9 and 28, or sequences substantially homologous to any one of the foregoing SEQ ID NOs.

Especially preferred antibodies comprise 3 of the light chain CDRs of SEQ ID NOs: 8, 9 and 10; or 8, 9 and 28, or sequences substantially homologous to any one of the foregoing SEQ ID NOs (i.e. one of each of the aforementioned light chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto).

Other certain preferred antibodies comprise two or more of the heavy chain CDRs of SEQ ID NOs: 5, 6 and 7; or 5, 24 and 25, or sequences substantially homologous to any one of the foregoing SEQ ID NOs.

Especially preferred antibodies comprise 3 of the heavy chain CDRs of SEQ ID NOs: 5, 6 and 7; or 5, 24 and 25, or sequences substantially homologous to any one of the foregoing SEQ ID NOs (i.e. one of each of the aforementioned heavy chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the light chain

CDRs of SEQ ID NOs: 8, 9 and 10, or sequences substantially homologous to any one of these sequences (i.e. one of each of the aforementioned light chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto), and 3 of the heavy chain CDRs of SEQ ID NOs: 5, 6 and 7, or sequences substantially homologous to any one of these sequences (i.e. one of each of the aforementioned heavy chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the light chain CDRs of SEQ ID NOs: 8, 9 and 28, or sequences substantially homologous to any one of these sequences (i.e. one of each of the aforementioned light chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto), and 3 of the heavy chain CDRs of SEQ ID NOs: 5, 24 and 25, or sequences substantially homologous to any one of these sequences (i.e. one of each of the aforementioned heavy chain CDR1 and CDR2 and CDR3 or sequences substantially homologous thereto).

Certain especially preferred antibodies comprise a heavy chain CDR1 domain of SEQ ID NO: 5, a heavy chain CDR2 domain of SEQ ID NO: 6, and a heavy chain CDR3 domain of SEQ ID NO: 7, or sequences substantially

homologous to any one of the aforementioned sequences; and/or comprise a light chain CDR1 domain of SEQ ID NO: 8, a light chain CDR2 domain of SEQ ID NO: 9, and a light chain CDR 3 domain of SEQ ID NO: 10, or sequences substantially homologous to any one of the aforementioned sequences. Certain especially preferred antibodies comprise a heavy chain CDR1 domain of SEQ ID NO: 5, a heavy chain CDR2 domain of SEQ ID NO: 24, and a heavy chain CDR3 domain of SEQ ID NO: 25, or sequences substantially homologous to any one of the aforementioned sequences; and/or comprise a light chain CDR1 domain of SEQ ID NO: 8, a light chain CDR2 domain of SEQ ID NO: 9, and a light chain CDR 3 domain of SEQ ID NO: 28, or sequences substantially homologous to any one of the aforementioned sequences.

In a further embodiment, the invention provides an antibody that comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said heavy chain variable region comprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 5,

(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 6, and

(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 7.

In a preferred aspect of this embodiment, one or more of said light chain variable region CDRs are selected from the group consisting of (or comprising):

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 8,

(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 9, and

(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 10. Preferably, 2 or 3 of said light chain variable region CDRs are selected from the above group.

Antibodies comprising sequences which are substantially homologous to one or more of the aforementioned sequences are also provided in this embodiment.

(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 24, and

(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 25.

(i) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 8,

(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 9, and

(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 28. Preferably, 2 or 3 of said light chain variable region CDRs are selected from the above group.

Antibodies comprising sequences which are substantially homologous to one or more of the aforementioned sequences are also provided in this embodiment. Certain further preferred embodiments of the invention provide an antibody that comprises:

a VH domain that comprises one, two or three of the heavy chain CDRs of SEQ ID NOs: 5, 6 or 7, or sequences substantially homologous to one or more of SEQ ID NOs: 5, 6 or 7, and/or a VL domain that comprises one, two or three of the light chain CDRs of SEQ ID NOs: 8, 9 or 10, or sequences substantially homologous to one or more of SEQ ID NOs: 8, 9 or 10.

Certain further preferred embodiments of the invention provide an antibody that comprises:

a VH domain that comprises one, two or three of the heavy chain CDRs of SEQ ID NOs: 5, 24 or 25, or sequences substantially homologous to one or more of SEQ ID NOs: 5, 24 or 25, and/or a VL domain that comprises one, two or three of the light chain CDRs of SEQ ID NOs: 8, 9 or 28, or sequences substantially homologous to one or more of SEQ ID NOs: 8, 9 or 28.

Certain preferred embodiments of the invention provide an antibody comprising a VH domain that has the amino acid sequence of SEQ ID NO: 3 or 21 , or a sequence substantially homologous thereto, and/or a VL domain that has the amino acid sequence of SEQ ID NO: 4 or 22, or a sequence substantially homologous thereto.

Further preferred embodiments provide an antibody comprising a VH domain that has the amino acid sequence of SEQ ID NO: 3 or 21 and a VL domain that comprises 3 light chain CDRs. Preferably said light chain CDRs have SEQ ID NOs 8, 9 and 10; or 8, 9 and 28.

Further preferred embodiments provide an antibody comprising a VL domain that has the amino acid sequence of SEQ ID NO: 4 or 22 and a VH domain that comprises 3 heavy chain CDRs. Preferably said heavy chain CDRs have SEQ ID NOs 5, 6 and 7; or 5, 24 and 25.

Further preferred embodiments provide an antibody comprising a VH domain that has the amino acid sequence of SEQ ID NO: 3, or a sequence substantially homologous thereto, and/or a VL domain that has the amino acid sequence of SEQ ID NO: 4, or a sequence substantially homologous thereto.

Further preferred embodiments provide an antibody comprising a VH domain that has the amino acid sequence of SEQ ID NO: 21 , or a sequence substantially homologous thereto, and/or a VL domain that has the amino acid sequence of SEQ ID NO: 22, or a sequence substantially homologous thereto. Antibodies based on the 1 h07 antibody or 3C02 antibody sequences set forth in Tables 1 and 2 are preferred. Antibodies based on the 1 h07 antibody set forth in Table 1 are particularly preferred.

The invention is exemplified by monoclonal antibodies 1 h07 (also referred to herein as 1 H7) and 3C02, sequences of which are shown in Tables 1 and 2 herein. The CDR domains, VH and VL domains of the 1 h07 and 3C02 antibodies are shown in Tables 1 and 2 herein. Antibodies comprising these CDR domains or VH and VL domains (or sequences substantially homologous thereto) are preferred aspects of the invention.

Certain examples of substantially homologous sequences are sequences that have at least 65% identity to the amino acid sequences disclosed. In certain embodiments, the antibodies of the invention comprise at least one light chain variable region that includes an amino acid sequence region of at least about 65%, 70% or 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% or 95% and most preferably at least about 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4 or 22; and/or at least one heavy chain variable region that includes an amino acid sequence region of at least about 65%, 70% or 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% or 95% and most preferably at least about 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 or 21.

Other preferred examples of substantially homologous sequences are sequences containing conservative amino acid substitutions of the amino acid sequences disclosed.

Other preferred examples of substantially homologous sequences are sequences containing 1 , 2 or 3, preferably 1 or 2 (more preferably 1 ), altered amino acids in one or more of the CDR regions disclosed. Such alterations might be conserved or non-conserved amino acid substitutions, or a mixture thereof.

In such embodiments, preferred alterations are conservative amino acid substitutions.

In one preferred embodiment, the present invention provides an antibody that comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ

ID NO:8 or a sequence substantially homologous thereto, (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and

(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10 or a sequence substantially homologous thereto; and/or

wherein said heavy chain variable region comprises:

(d) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:5 or a sequence substantially homologous thereto,

(e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6 or a sequence substantially homologous thereto, and

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto;

wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence, or wherein said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence.

In a particularly preferred embodiment, the invention provides an antibody that comprises:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8, a VL CDR2 of SEQ ID NO:9, and a VL CDR3 of SEQ ID NO:10, and

a VH domain that comprises a VH CDR1 of SEQ ID NO:5, a VH CDR2 of SEQ ID NO:6, and a VH CDR3 of SEQ ID NO:7.

In a preferred embodiment, the present invention provides an antibody, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%) and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%).

In a particularly preferred embodiment, the invention provides an antibody comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:3 and a VL domain that comprises the amino acid sequence of SEQ ID NO:4.

In another preferred embodiment, the present invention provides an antibody that comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ

ID NO:8 or a sequence substantially homologous thereto,

(b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and (c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:28 or a sequence substantially homologous thereto; and/or

wherein said heavy chain variable region comprises:

(e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:24 or a sequence substantially homologous thereto, and

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto;

a VL domain that comprises a VL CDR1 of SEQ ID NO:8, a VL CDR2 of SEQ ID NO:9, and a VL CDR3 of SEQ ID NO:28, and

a VH domain that comprises a VH CDR1 of SEQ ID NO:5, a VH CDR2 of SEQ ID NO:24, and a VH CDR3 of SEQ ID NO:25.

In a preferred embodiment, the present invention provides an antibody, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:22, or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%) and/or wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO:21 , or a sequence having at least 80% sequence identity thereto (e.g. at least 85%, 90%, 95% or 98%).

In a particularly preferred embodiment, the invention provides an antibody comprising a VH domain that comprises the amino acid sequence of SEQ ID NO:21 and a VL domain that comprises the amino acid sequence of SEQ ID NO:22.

In all embodiments, antibodies containing substantially homologous sequences retain the ability to bind to the membrane-bound form of mesothelin on cells, without significantly binding to the soluble-form of mesothelin. Preferably, antibodies containing substantially homologous sequences also do not bind significantly to mesothelin on extracellular vesicles. Preferably, antibodies containing substantially homologous sequences retain all of the properties described in relation to the 1 h07 and/or 3C02 antibodies.

Further examples of substantially homologous amino acid sequences in accordance with the present invention are described elsewhere herein. The CDRs of the antibodies of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and/or effective engineered antibodies. Thus, the V_H, V_L and individual CDR sequences of the invention are preferably provided within or incorporated into an appropriate framework or scaffold to enable antigen binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1 , FR2, FR3 and/or FR4, as appropriate to form an appropriate scaffold, or may correspond to consensus framework regions, for example identified by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, e.g. T cell receptor frameworks can be used.

Appropriate sequences that can be used for framework regions are well known and documented in the art and any of these may be used. Preferred sequences for framework regions are one or more of the framework regions making up the V_H and/or V_L domains of the invention, i.e. one or more of the framework regions of the 1 h07 or 3C02 antibodies, as disclosed in Tables 1 and 2, or framework regions substantially homologous thereto, and in particular framework regions that allow the maintenance of antigen specificity, for example framework regions that result in substantially the same or the same 3D structure of the antibody. In certain preferred embodiments, all four of the variable light chain (SEQ ID NOs:15, 16, 17 and 18) and/or variable heavy chain (SEQ ID NOs:1 1 , 12, 13 and 14) framework regions (FR), as appropriate, or FR regions substantially

homologous thereto, are found in the antibodies of the invention. In other preferred embodiments, all four of the variable light chain (SEQ ID NOs:33, 34, 35 and 36) and/or variable heavy chain (SEQ ID NOs:29, 30, 31 and 32) framework regions (FR), as appropriate, or FR regions substantially homologous thereto, are found in the antibodies of the invention.

In further embodiments of the present invention, the VH CDR2 has or comprises an amino acid sequence of SEQ ID NO: 37 (I Y P G N X₆ D T). In these embodiments X₆ can be any amino acid. In a preferred embodiment X₆ is R or S. Thus, a preferred VH CDR2 has or comprises the amino acid sequence of SEQ ID NO: 38 (I Y P G N R/S D T). Preferred VH CDR2 sequences of this embodiment are SEQ ID NOs: 6 or 24.

In further embodiments of the present invention, the VH CDR3 has or comprises an amino acid sequence of SEQ ID NO: 39 (T R G X₄ X₅ G X₇ Y F D Xn). In these embodiments X₄, X_5, X₇ and Xn can be any amino acid. Preferably one or more, most preferably all, of these X residues are selected from the following group: X₄ is V or I; X₅ is I or R, X₇ is I or S; and Xn is Y or V. Thus, a preferred VH CDR3 has or comprises the amino acid sequence of SEQ ID NO: 40 (T R G V/l l/R G l/S Y F D Y/V). For example, preferred VH CDR3 sequences of this embodiment have or comprise SEQ ID NOs: 7 or 25.

In further embodiments of the present invention, the VL CDR3 has or comprises an amino acid sequence of SEQ ID NO: 41 (Q Q X₃ N E X₆ P Xs T). In these embodiments X₃, X₆ and X₈ can be any amino acid. Preferably one or more, most preferably all, of these X residues are selected from the following group: X₃ is N or S; X₆ is A or D; and X₈ is L or Y. Thus, a preferred VL CDR3 has or comprises the amino acid sequence of SEQ ID NO: 42 (Q Q N/S N E A/D P L/Y T). For example, preferred VL CDR3 sequences of this embodiment have or comprise SEQ ID NOs: 10 or 28.

In one embodiment, the present invention provides an antibody that comprises:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:41 , and/or a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence

substantially homologous thereto, a VH CDR2 of SEQ ID NO:37, and a VH CDR3 of SEQ ID NO:39.

In some such embodiments a preferred "substantially homologous" sequence is a sequence containing 1 , 2 or 3, preferably 1 or 2, altered amino acids compared with a given CDR sequence. In some such embodiments, the VH CDR3 is preferably SEQ ID NO:7. In some such embodiments, the VH CDR3 is preferably SEQ ID NO:25. In some such embodiments, the VH CDR2 is preferably SEQ ID NO:6. In some such embodiments, the VH CDR2 is preferably SEQ ID NO:24. In some such embodiments, the VL CDR3 is preferably SEQ ID NO:10. In some such embodiments, the VL CDR3 is preferably SEQ ID NO:28.

In one embodiment, the present invention provides an antibody that comprises:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:42, and/or a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence

substantially homologous thereto, a VH CDR2 of SEQ ID NO:38, and a VH CDR3 of SEQ ID NO:40. In some such embodiments a preferred "substantially homologous" sequence is a sequence containing 1 , 2 or 3, preferably 1 or 2, altered amino acids compared with a given CDR sequence. In some such embodiments, the VH CDR3 is preferably SEQ ID NO:7. In some such embodiments, the VH CDR3 is preferably SEQ ID NO:25. In some such embodiments, the VH CDR2 is preferably SEQ ID NO:6. In some such embodiments, the VH CDR2 is preferably SEQ ID NO:24. In some such embodiments, the VL CDR3 is preferably SEQ ID NO:10. In some such embodiments, the VL CDR3 is preferably SEQ ID NO:28.

In some embodiments, the present invention provides an antibody (e.g. a Fab fragment) that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43, or a sequence having at least 80% identity thereto, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 44, or a sequence having at least 80% identity thereto.

In some embodiments, the present invention provides an antibody (e.g. a Fab fragment) that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:47, or a sequence having at least 80% identity thereto, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 48, or a sequence having at least 80% identity thereto.

In embodiments of the invention where one or more of the CDR sequences contain an X_x residue, then CDRs with sequences which are substantially homologous thereto are also encompassed within the invention. In some such embodiments a preferred "substantially homologous" sequence is a sequence containing 1 , 2 or 3, preferably 1 or 2, altered amino acids or amino acid

substitutions compared with a given CDR sequence. In some such embodiments said alterations or substitutions in amino acid residues can include one or more of the X_x residues or can be at residues other than the X_x residues. In other such embodiments said alterations are in a mixture of the X_x residues and the non-X_x residues.

In other embodiments of the invention, antibodies comprise:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:41 or 42, or a sequence substantially homologous thereto, and a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence substantially homologous thereto, a VH CDR2 of SEQ ID NO:37 or 38, or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO:39 or 40, or a sequence substantially homologous thereto. In other embodiments of the invention, antibodies comprise:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:10 or a sequence substantially homologous thereto, and a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence substantially homologous thereto, a VH CDR2 of SEQ ID NO:24 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO:25 or a sequence substantially homologous thereto.

In other embodiments of the invention, antibodies comprise:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:41 or 42 or a sequence substantially homologous thereto, and a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence substantially homologous thereto, a VH CDR2 of SEQ ID NO:37 or 38, or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO:39 or 41 , or a sequence substantially homologous thereto.

In other embodiments of the invention, antibodies comprise:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:28 or a sequence substantially homologous thereto, and a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence substantially homologous thereto, a VH CDR2 of SEQ ID NO:6 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO:7 or a sequence substantially homologous thereto.

In another aspect, the present invention provides an antibody, for example an isolated antibody, which binds to (or specifically recognises) mesothelin, wherein said antibody:

(i) binds to the membrane-bound form of mesothelin on cells; and (ii) does not bind significantly to the soluble form of mesothelin.

Preferred embodiments of this aspect of the invention include antibodies comprising one or more of the antibody sequences (e.g. CDR sequences and/or VH domain and/or VL domain sequences) that are described elsewhere herein in connection with other aspects of the present invention. Thus, discussion of various features of the antibodies of other aspects of the invention and preferred

embodiments apply mutatis mutandis to this aspect of the invention. By way of example, in one preferred embodiment said isolated antibody which (i) binds to the membrane-bound form of mesothelin on cells and (ii) does not bind significantly to the soluble form of mesothelin comprises:

a VL domain that comprises a VL CDR1 of SEQ ID NO:8 or a sequence

substantially homologous thereto, a VL CDR2 of SEQ ID NO:9 or a sequence substantially homologous thereto, and a VL CDR3 of SEQ ID NO:41 (preferably SEQ ID NO:42), and/or

a VH domain that comprises a VH CDR1 of SEQ ID NO:5 or a sequence

substantially homologous thereto, a VH CDR2 of SEQ ID NO:37 (preferably SEQ ID NO:38), and a VH CDR3 of SEQ ID NO:39 (preferably SEQ ID NO:40).

In some such embodiments a preferred "substantially homologous" sequence is a sequence containing 1 , 2 or 3, preferably 1 or 2, altered amino acids compared with a given CDR sequence.

By way of another example, in one preferred embodiment, said isolated antibody which (i) binds to the membrane-bound form of mesothelin on cells and (ii) does not bind significantly to the soluble form of mesothelin comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ

ID NO:8 or a sequence substantially homologous thereto,

(b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto and

wherein said heavy chain variable region comprises:

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7 or a sequence substantially homologous thereto.

Optionally, said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence, or said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence.

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto,

(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:28 or a sequence substantially homologous thereto; and/or

wherein said heavy chain variable region comprises:

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:25 or a sequence substantially homologous thereto.

In another aspect, the present invention provides an antibody, for example an isolated antibody, which binds to (or specifically recognises) mesothelin.

embodiments apply mutatis mutandis to this aspect of the invention.

(i) binds to the membrane-bound form of mesothelin on cells; and

(ii) does not bind significantly to any other form of mesothelin.

More specifically, in preferred embodiments of this aspect of the invention, the antibody does not bind significantly to any non-cellular form of mesothelin or to any other form of mesothelin which can act as a decoy or a competitive or a sequestering entity to the binding of the antibody to the membrane-bound form of mesothelin on cells.

embodiments apply mutatis mutandis to this aspect of the invention.

In one aspect, the present invention provides an antibody which binds to mesothelin, said antibody comprising at least one heavy chain variable region and/or at least one light chain variable region as described elsewhere herein (e.g. having 3 VH CDRs and/or 3 VL CDRs of the 1 h07 antibody, or having 3 VH CDRs and/or 3 VL CDRs of the 3C02 antibody). Preferably, such antibodies have one or more or all (preferably all) of the properties described herein in relation to the 1 h07 and/or 3C02 antibodies.

As mentioned above, the present invention provides antibodies, preferably isolated antibodies, which bind to mesothelin, wherein said antibodies bind to the membrane-bound form of mesothelin on cells and do not bind significantly to the soluble form of mesothelin. Preferably, said antibodies do not bind significantly to mesothelin on extracellular vesicles.

Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesised as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form and a 40 kDa membrane-bound form (Yamaguchi, et al. J. Biol. Chem. 269, 805-808, 1994).

In accordance with the present invention, the mesothelin may be from any species, e.g. mouse or human. In a preferred embodiment the mesothelin is human mesothelin.

The term "membrane-bound form of mesothelin on cells" refers to mesothelin which is attached to, associated with, embedded in, or otherwise bound to a cell membrane on a cell, or is a component of a cell membrane on a cell. Thus, the membrane-bound form of mesothelin on cells can be referred to, for example, as a cell surface form of mesothelin or a cell surface mesothelin molecule. Such membrane bound forms will thus in many cases represent a native or natural form of mesothelin, for example the form found on cells which naturally express or overexpress mesothelin.

Unless otherwise evident, in the context of the present invention the term "cells" is used to refer to mesothelin-positive (mesothelin expressing) cells. In the context of the present invention the term "cells" is used to refer to nucleus- containing cells.

In some embodiments, the membrane-bound form of mesothelin on cells is the membrane-bound form of mesothelin on tumour cells (e.g. solid tumour cells). Put another way, in some embodiments, the membrane-bound form of mesothelin on cells is tumour-associated mesothelin. In some embodiments, the membrane- bound form of mesothelin on cells is the membrane-bound form of mesothelin on tumour cell lines (e.g. HeLa cells, or e.g. MDA-MB231 cells, HT29 cells or A549 cells). In some embodiments, the membrane-bound form of mesothelin on cells is the membrane-bound form of mesothelin on CHO cells that have been transfected with (and thus express) mesothelin, preferably human mesothelin.

A preferred membrane-bound form of mesothelin on cells is a 40kDa membrane-bound form. A 40kDa membrane-bound form of mesothelin may be derived from a 69 kDa precursor (pre-protein/precursor protein) (Yamaguchi et al., supra).

Membrane-bound mesothelin on cells may be derived from a number of different isoforms of the mesothelin precursor protein (e.g. derived from isoform 2 e.g. derived from the mesothelin precursor protein as set forth in SEQ ID NO:51 , or derived from isoform 1 , e.g. derived from the mesothelin precursor protein as set forth in SEQ ID NO:52). Antibodies of the present invention may bind to a membrane-bound form of mesothelin on cells derived from any precursor isoform.

Typically, mesothelin precursor proteins (e.g. SEQ ID NO: 51 ) are proteolytically processed, with a signal peptide (e.g. residues 1 -36 of SEQ ID NO:51 ) being cleaved-off, a region (fragment) corresponding to megakaryocyte potentiating factor (MPF) (e.g. residues 37-286 of SEQ ID NO:51 ) being cleaved-off and secreted as MPF, and a C-terminal region (fragment) (e.g. residues 607-630 of SEQ ID NO:51 ) also being cleaved-off in the mature protein. Thus, a membrane- bound form of mesothelin on cells (e.g. amino acid residues 287 to 606 of SEQ ID NO:51 ) may be considered to be a mature form of mesothelin, from which a signal peptide, MPF, and a C-terminal region has been removed. In some embodiments, the membrane-bound form of mesothelin on cells is at least 100 amino acids in length, at least 150 amino acids in length, at least 200 amino acids in length, at least 250 amino acids in length, preferably at least 300 amino acids in length (e.g. 320 amino acids). In some embodiments, the

membrane-bound form of mesothelin on cells is up to 320 amino acids in length.

A preferred membrane-bound form of mesothelin on cells comprises (or consists of) amino acid residues 287 to 606 of SEQ ID NO:51 . Thus, antibodies of the present invention preferably bind to this membrane-bound form of mesothelin on cells. Antibodies of the invention may bind to a membrane-bound form of mesothelin on cells that corresponds to this membrane-bound form of mesothelin on cells (e.g. in a different mesothelin isoform or mesothelin from a different species).

Another membrane-bound form of mesothelin on cells may comprise (or consist of) amino acid residues 296 to 606 of SEQ ID NO:51 . Thus, antibodies of the present invention may bind to this membrane-bound form of mesothelin on cells. Antibodies of the invention may bind to a membrane-bound form of mesothelin on cells that corresponds to this membrane-bound form of mesothelin on cells (e.g. in a different mesothelin isoform or mesothelin from a different species).

Antibodies of the present invention do not bind to the signal peptide of a mesothelin precursor protein (the signal peptide that is cleaved off during proteolytic processing of a mesothelin precursor protein). Antibodies of the present invention do not bind a region (fragment) of a mesothelin precursor protein corresponding to megakaryocyte potentiating factor (MPF), (MPF that is cleaved off during proteolytic processing of a mesothelin precursor protein). Antibodies of the present invention do not bind to a C-terminal region (fragment) that is cleaved off from a mesothelin precursor protein during proteolytic processing.

Antibodies of the present invention do not bind to amino acid residues 1 -36 of SEQ ID NO:51. Antibodies of the present invention do not bind to amino acid residues 37-286 of SEQ ID NO:51 . Antibodies of the present invention do not bind to amino acid residues 607-630 of SEQ ID NO:51 .

Antibodies of the present invention preferably do not bind to amino acid residues 287-290 of SEQ ID NO: 51 (or to an epitope comprising one or more of said residues). Antibodies of the present invention preferably do not bind to amino acid residues 409-416 of SEQ ID NO:51 (or to an epitope comprising one or more of said residues). Antibodies of the present invention preferably do not bind to amino acid residues 599-606 of SEQ ID NO:51 (or to an epitope comprising one or more of said residues). Antibodies of the present invention preferably do not bind to amino acid residue 309 of SEQ ID NO:51.

The membrane-bound form of mesothelin may be attached (typically C- terminally attached) to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor (GPI-anchor). Preferred antibodies of the invention do not bind to the GPI- anchor (e.g. do not bind directly to the GPI-anchor itself).

The ability of an antibody to bind to a membrane-bound form of mesothelin on cells can be readily tested for using methods that are routine in the art. For example, flow cytometry can be used. In an exemplary flow cytometry method, mesothelin expressing cells (e.g. HeLa cells or cells transfected with mesothelin) are incubated with the antibody under investigation and the antibody bound to the mesothelin on the cell is detected by fluorescence, for example the antibody is fluorescently labelled. Such labelling can for example be carried out by incubating the cell-antibody mixture with a second antibody which recognises the antibody under investigation (e.g. an anti-myc antibody if the antibody under investigation is myc tagged) and a yet further antibody (third) antibody which is fluorescently labelled (such a third antibody recognises the second antibody). Alternatively, the second antibody can also carry the fluorescent label. Accordingly, if the antibody under investigation binds to the membrane-bound form of mesothelin on the cell, the cell becomes fluorescently labelled and such cells, and thus antibodies which have the ability to bind to a membrane-bound form of mesothelin on cells, can be readily identified using a flow cytometer. An exemplary method of testing for the ability of an antibody to bind to a membrane-bound form of mesothelin on cells is described in the Example.

Another method for testing for the ability of an antibody to bind to a membrane-bound form of mesothelin is immunohistochemistry. Another method for testing for the ability of an antibody to bind to a membrane-bound form of mesothelin is microscopy (e.g. confocal microscopy) of cells that have become fluorescently labelled as a result of antibody binding to membrane-bound mesothelin.

As discussed elsewhere herein, antibodies of the present invention do not bind significantly to the soluble form of mesothelin.

The soluble form of mesothelin refers to a form of mesothelin which is present in solution or in a soluble phase. Thus, this form of mesothelin is not associated with a membrane and is not particulate and not in the form of an insoluble aggregate or precipitate. A preferred form of soluble mesothelin is (or corresponds to) mesothelin which was associated with the surface of a cell, e.g. a disease associated cell such as a tumour cell, and has been shed or lost from the cell membrane to become a soluble-form of mesothelin, for example by cleavage such as proteolytic cleavage (such a form of mesothelin can also be referred to as "shed" or the "shed" form of mesothelin). Thus, a soluble form of mesothelin can be a form which is derivable from the membrane bound form by cleavage. Such proteolytic cleavage may be cleavage by tumor necrosis factor-a converting enzyme (TACE), a member of the MMP/ADAM family. TACE has been identified as a mesothelin sheddase (Zhang Y, et al. Cancer Res. 201 1 Sep 1 ;71 (17):5915-22). The soluble antigen can also be synthesised in the cytoplasm and secreted by endocytosis and/or other mechanisms. Soluble antigen can also result from alternative splicing.

A preferred soluble form of mesothelin to which antibodies of the invention do not bind significantly is a soluble form of the membrane-bound mesothelin that has been shed from (cleaved-off from, e.g. by TACE) the cells. Thus, a soluble form of mesothelin may comprise (or consist of) the same primary amino acid sequence as (or contained in) the membrane-bound form of mesothelin on cells, or comprise (or consist of) a substantial portion (fragment) of the primary amino acid sequence of the membrane-bound mesothelin on cells. For example, a soluble form of mesothelin may comprise (or consist of) an amino acid sequence having at least 100, at least 200, at least 250, at least 275, preferably at least 300 (e.g. 308 or 313 or e.g. up to 308 or up to 313) amino acids that correspond to the amino acid sequence of the membrane-bound form of mesothelin on cells.

A preferred soluble form of mesothelin (e.g. a recombinant soluble form) comprises (or consists of) amino acid residues 291 -590 of SEQ ID NO:52. In some embodiments, a soluble form of mesothelin has a sequence corresponding thereto (e.g. in a different mesothelin isoform or mesothelin from a different species).

Another preferred soluble form of mesothelin (e.g. a recombinant soluble form) comprises (or consists of) amino acid residues 291 -598 of SEQ ID NO:51. In some embodiments, a soluble form of mesothelin has a sequence corresponding thereto (e.g. in a different mesothelin isoform or mesothelin from a different species).

Another preferred soluble form of mesothelin comprises (or consists of) amino acid residues 287-594 of SEQ ID NO:51. In some embodiments, a soluble form of mesothelin has a sequence corresponding thereto (e.g. in a different mesothelin isoform or mesothelin from a different species).

Another preferred soluble form of mesothelin comprises (or consists of) amino acid residues 287-599 of SEQ ID NO:51. In some embodiments, a soluble form of mesothelin has a sequence corresponding thereto (e.g. in a different mesothelin isoform or mesothelin from a different species).

In some embodiments, a soluble form of mesothelin does not comprise (or consist of) the entire (full-length) amino acid sequence of the corresponding membrane-bound form of mesothelin on cells because when mesothelin is shed or lost (or cleaved-off e.g. by TACE) from the cell membrane to become a soluble-form of mesothelin, a short amino acid sequence (or "stub" or fragment) may be left behind on the cell membrane (i.e. may still be attached to the cell membrane). Thus, after cleavage there may be a soluble (shed) form of mesothelin and a residual short amino acid sequence that remains membrane-bound (a "stub"). Cleavage may occur for example after residues 594 and/or 599 of SEQ ID NO:51 .

Preferred antibodies of the invention do not bind to such a "stub". Thus, the "membrane-bound form of mesothelin on cells" in accordance with the present invention is preferably not a protein consisting solely of the "stub" (i.e. it is not a protein consisting only of a portion or fragment of mesothelin that is still attached to the cell membrane after shedding or cleavage of mesothelin e.g. by TACE).

Thus, preferred antibodies of the invention do not bind to the portion (or fragment) of mesothelin that remains on (or attached to) the cell membrane after a soluble form of mesothelin has been shed (or cleaved off by a protease, e.g. by TACE). Such portions (or fragments or "stubs") may be, for example, up to 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25 or 50 amino acids in length (e.g. 7 or 12 amino acids in length).

In some embodiments, the "stub" (stub protein) has (e.g. consists of) the amino acid sequence of residues 595-606 of SEQ ID NO:51 (or corresponding residues, e.g. in a different mesothelin isoform or mesothelin from a different species). In some embodiments, the "stub" (stub protein) has (e.g. consists of) the amino acid sequence of residues 600-606 of SEQ ID NO:51 (or corresponding residues, e.g. in a different mesothelin isoform or mesothelin from a different species).

Shed (or soluble) mesothelin may be found in the blood (serum mesothelin) but also may be found in the interstitial space of tissues, for example in the interstitial space of tumors. The soluble form of mesothelin can thus exist naturally, or correspond to a naturally occurring form of soluble mesothelin.

Unless otherwise clear from the context, the 30/31 kDa NH2-terminal secreted protein (fragment) derivable by proteolytic cleavage of a mesothelin pre- protein (precursor protein) (as described in the art, for example in Yamaguchi, 1994, supra.) is not itself considered to be mesothelin (or a soluble form of mesothelin) in accordance with the present invention. The 30/31 kDa fragment is also referred to in the art as megakaryocyte potentiating factor (MPF). Megakaryocyte potentiating factor (MPF) is not considered to be mesothelin (or a soluble form of mesothelin) in accordance with the present invention. Of course, antibodies of the present invention do not bind to the 30/31 kDa NH2-terminal secreted fragment (the

30/31 kDa fragment (MPF)) (or to a corresponding NH2-terminal secreted fragment) as this has a different amino acid sequence from the membrane-bound form of mesothelin. Antibodies of the present invention do not bind to amino acid residues 37-286 of SEQ ID NO:51 , which corresponds to MPF.

The soluble mesothelin can be from any appropriate source, i.e. any sample or source in which the mesothelin is present in a soluble form.

One appropriate and preferred source is recombinant mesothelin (e.g.

recombinant human mesothelin). Recombinant human mesothelin is commercially available (e.g. from RayBiotech, e.g. Cat. No. 230-00043-10). Recombinant human mesothelin may comprise (or consist of) amino acid residues 291 -590 of SEQ ID NO:52.

In the case of mesothelin shed from a tumour (or indeed mesothelin shed from any cell into the circulation) then an appropriate source might be blood from a relevant subject (e.g. patients that are afflicted with the tumour or otherwise contain the cells in question from which the mesothelin is shed). Whole blood or serum (e.g. serum derived from a metastatic cancer patient, e.g. a metastatic colon cancer patient or a metastatic pancreatic cancer patient) could provide a source of soluble mesothelin.

Soluble mesothelin (or soluble mesothelin related peptides, SMRP, to which antibodies on the invention preferably do not bind significantly) may be mesothelin in a physiological fluid (e.g. serum), for example a physiological fluid (e.g. serum) from a cancer patient having a cancer which expresses mesothelin (e.g. a metastatic colon cancer patient or a metastatic pancreatic cancer patient). Another source of soluble mesothelin would be to carry out in vitro cultures of relevant cells which have the membrane bound form of mesothelin and to use the culture medium as a source of the shed (soluble) mesothelin. For example, the soluble form of mesothelin could be produced by utilizing recombinant methods, where cells can be engineered to express or overexpress the membrane bound form of mesothelin using appropriate recombinant techniques, for example by transfecting the cells with an expression vector designed to overexpress mesothelin. In such systems, whilst overexpression of the membrane bound form of mesothelin will occur, equally, as shedding is a natural process, relevant soluble forms of mesothelin will be shed into the culture medium which can then be used as a further source for the soluble form of mesothelin. If necessary, appropriate proteases could be used to increase the amount of shed mesothelin by cleavage.

In some embodiments, antibodies of the invention do not bind significantly to soluble mesothelin that lacks a GPI (GPI anchor).

Whether or not (or the extent/level at which) an antibody is able to bind to the soluble form of mesothelin can be readily tested for using methods that are routine in the art. For example, an ELISA (enzyme-linked immunosorbent assay) assay may be used.

In simple terms, in such an ELISA an amount of a soluble form of the antigen (mesothelin) is affixed to a surface (e.g. coated on the well of a plate), and then an antibody under investigation is washed over the surface so that it can, if it recognizes the antigen, bind to the antigen. This antibody (or a secondary antibody that binds thereto) is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antibody bound to antigen in the sample can be determined through the magnitude of the fluorescence.

Radioimmunoassays could also be used to determine whether or not (or the extent/level at which) an antibody is able to bind to the soluble form of mesothelin.

Importantly, antibodies of the present invention bind to the membrane-bound form of mesothelin on cells and do not bind significantly to the soluble form of mesothelin. This combination of properties is particularly important from the point of view of antibody-based therapies (e.g. solid tumour therapy). As described above, antibodies which bind the membrane bound form of mesothelin on cells (e.g. tumour cells) but not the soluble form would not be decoyed by soluble (shed) mesothelin in the circulation or in the interstitial spaces of the tumour but would instead target straight to the cell membranes of disease cells (e.g. tumour cells). Preferred antibodies of the invention retain the ability to bind to the membrane-bound form of mesothelin on cells and to not bind significantly to the soluble form of mesothelin in the presence of any concentration, or any physiological concentration (e.g. any concentrations observed in the human or animal body) of soluble mesothelin. For example, the ability is retained even when high concentrations of soluble mesothelin are present, for example concentrations of up to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ng/ml, or even at concentrations of up to 150, 200, 250, 300, 350, 380, 390 or 400 ng/ml, or even at concentrations of up to 0.5, 1 , 2, 3, 4 or 5 μg/ml.

Antibodies of the present invention thus discriminate between the membrane bound form of mesothelin on cells and soluble forms of mesothelin. Antibodies of the invention positively discriminate for the membrane bound form of mesothelin on cells. Antibodies of the present invention may thus be considered specific for the membrane-bound form of mesothelin on cells.

Antibodies of the present invention may thus bind to a conformational epitope on mesothelin. Such a conformational epitope of mesothelin is present on membrane-bound mesothelin on cells but not present (or is significantly diminished or altered) on the soluble form of mesothelin. Without wishing to be bound by theory, it is believed that the conformational epitope on membrane-bound mesothelin on cells to which antibodies of the present invention (e.g. 1 H7) bind is generated by molecular interactions between the mesothelin and the

glycosylphosphatidylinositol (GPI) anchor via which the membrane-bound form of mesothelin is attached to the cell membrane. However, preferred antibodies of the invention do not bind to the GPI-anchor (e.g. do not bind directly to the GPI-anchor itself).

Although corresponding (or identical) primary amino acid sequences (linear amino acid sequences) may be present in the membrane-bound form of mesothelin on cells and in the soluble form of mesothelin, preferred antibodies of the present invention are advantageously able to discriminate between the different forms, e.g. by recognising a conformational rather than a linear epitope, e.g. recognise a neo- epitope or neo-conformational epitope present in the membrane-bound form of mesothelin on cells but not in the soluble form of mesothelin. Preferably, antibodies of the invention do not bind significantly to mesothelin when present on extracellular vesicles. Mesothelin on extracellular vesicles is mesothelin which is attached to, associated with, embedded in, or otherwise bound to the membrane of an extracellular vesicle, or is a component of a membrane of an extracellular vesicle. Thus, preferably antibodies of the invention do not bind significantly to the membrane bound form of mesothelin when present on

extracellular vesicles. Mesothelin on extracellular vesicles typically has the same primary amino acid sequence as membrane-bound mesothelin on cells.

Extracellular vesicles are non-cellular bodies comprising (or consisting of) membrane components and intra- and cell surface moieties which can be present in the body, for example in tissues. Extracellular vesicles are released by many cell types and are highly conserved in prokaryotes and eukaryotes. Thus, they generally reflect the antigenic content of the cells from which they originate.

Extracellular vesicles can act as signalling organelles that are released by many cell types. Extracellular vesicles are membranous vesicles. Extracellular vesicles do not contain a nucleus. Based on the mechanism of biogenesis extracellular vesicles can be classified as exosomes, microvesicles (shedding microvesicles) or apoptotic bodies/blebs.

In a preferred embodiment, antibodies do not bind significantly to mesothelin on extracellular vesicles from tumour cells (e.g. solid tumour cells). In some embodiments, antibodies do not bind significantly to mesothelin on extracellular vesicles from a tumour cell line, e.g. HeLa.

In a preferred embodiment, antibodies do not bind significantly to mesothelin on exosomes, for example do not bind significantly to mesothelin on exosomes from tumour cells (e.g. solid tumour cells). In some embodiments, antibodies do not bind significantly to mesothelin on exosomes from a tumour cell line, e.g. HeLa.

Exosomes are nanosized membrane vesicles. Exosomes are endosome- derived, 30-1 OOnm small membrane vesicles released by most cell types including tumour cells (Yang and Robbins, supra). Exosomes can be defined as vesicles formed by "inward/reverse budding" of the limiting membrane of the multivesicular bodies (MVBs) in the late endocytic compartment and released upon the fusion of MVB with the plasma membrane. They are for example characterized by a size of 30-1 OOnm in diameter and a density of 1 .13-1.19 g/ml in a sucrose gradient and can be sedimented at 100,000 x g. Exosomes typically show a "cup-shaped" or "saucer- like" morphology when analysed by electron microscopy. Exosomes contain cytosolic and membrane proteins derived from the parental cells. Thus, exosomes (and other extracellular vesicles) derived from mesothelin positive cells will generally also have mesothelin present. Tumour-derived exosomes usually contain tumour antigens (Yang and Robbins, supra).

In some embodiments, antibodies do not bind significantly to mesothelin on isolated exosomes. Methods of isolating or purifying exosomes are known in the art (Theny et al., Current Protocols in Cell Biology (2006), 3.22.1 - 3.22.29. Exosomes can be isolated by ultracentrifugation (e.g. a series of ultracentrifugations). In some embodiments, antibodies do not bind significantly to mesothelin on exosomes present in a culture medium in which cells (e.g. tumour cells such as HeLa cells) have been grown.

In some embodiments, antibodies of the invention do not bind significantly to mesothelin on microvesicles (shedding microvesicles), for example do not bind significantly to mesothelin on microvesicles from tumour cells (e.g. solid tumour cells). In some embodiments, antibodies do not bind significantly to mesothelin on microvesicles from a tumour cell line, e.g. HeLa.

Generally, the term "microvesicles" refers to vesicles shed from the plasma membrane. Microvesicles have a relatively larger size (100-1000nm) than exosomes and can be sedimented at 10,000 x g (Yang and Robbins, supra).

Microvesicles originate directly from the plasma membrane of the cell and reflect the antigenic content of the cells from which they originate.

In some embodiments, antibodies of the invention do not bind significantly to mesothelin on apoptotic bodies/blebs. In certain embodiments, antibodies of the invention do not bind significantly to mesothelin on apoptotic bodies/blebs from tumour cells (e.g. solid tumour cells). In some embodiments, antibodies do not bind significantly to mesothelin on apoptotic bodies/blebs from a tumour cell line, e.g. HeLa.

Antibodies of the present invention may thus bind to a conformational epitope on mesothelin. The conformational epitope of mesothelin may be present on membrane-bound mesothelin on cells but not present (or significantly altered or diminished) on mesothelin on extracellular vesicles.

Although corresponding (or identical) primary amino acid sequences (linear amino acid sequences) may be present in the membrane-bound form of mesothelin on cells and in mesothelin on extracellular vesicles, preferred antibodies of the present invention are advantageously able to discriminate between the different forms, e.g. by recognising a conformational rather than a linear epitope, e.g.

recognising a neo-epitope or neo-conformational epitope present in the membrane- bound form of mesothelin on cells but not in the mesothelin on extracellular vesicles (and preferably also not on the soluble form of mesothelin, as described above).

The preferred property of not binding significantly to mesothelin on extracellular vesicles is important and advantageous from the point of view of antibody-based therapies (e.g. solid tumour therapy). As described above, antibodies which bind the membrane bound form of mesothelin on cells (e.g. tumour cells), but do not significantly bind mesothelin on extracellular vesicles would not be decoyed (or sequestered) by mesothelin on extracellular vesicles (e.g. exosomes in the blood circulation) but would instead target straight to the cell membranes of disease cells (e.g. tumour cells).

Preferred antibodies of the present invention thus discriminate between the membrane bound form of mesothelin on cells and mesothelin on extracellular vesicles (e.g. exosomes). Antibodies of the invention positively discriminate for the membrane bound form of mesothelin on cells.

A convenient way of identifying (carrying out the screening for) antibodies which can bind to the membrane-bound form of mesothelin on cells but not bind (or not significantly bind) to the soluble form of mesothelin or to mesothelin on extracellular vesicles will be the use of some kind of competition assay. Thus, an assay can be used where a sample of soluble mesothelin (e.g. recombinant human mesothelin or HeLa cell supernatant or concentrated HeLa cell supernatant) or a source of mesothelin on extracellular vesicles (e.g. isolated exosomes) is introduced in order to assess whether the soluble mesothelin or mesothelin on extracellular vesicles has the ability to compete for the binding of a candidate antibody to a source of the membrane bound form of mesothelin on cells. If the soluble mesothelin or the mesothelin on extracellular vesicles can compete to a significant extent then this is indicative that the antibody candidate is not specific for the membrane bound form of mesothelin on cells (as it also binds the soluble form or an extracellular vesicle form). If the soluble mesothelin or the mesothelin on extracellular vesicles cannot compete to a significant extent then this is indicative that the antibody candidate has the ability to discriminate between the membrane bound form of mesothelin on cells and the soluble or extracellular vesicle forms.

Thus, by way of example, in an appropriate competition assay, a candidate antibody is regarded as discriminatory if the ability of said antibody to bind to the membrane bound form of mesothelin on cells is not significantly effected/competed by the addition of said soluble form of mesothelin or said mesothelin on extracellular vesicles, e.g. the reduction in binding is less than 2 fold, 1.8 fold, 1 .6 fold, 1 .4 fold, 1 .2 fold or around 1 .0 fold, i.e. that there is no significant or no reduction in binding. In such assays, said soluble form of mesothelin or said mesothelin on extracellular vesicles is added at an appropriate concentration (or range of concentrations, for example in the form of serial dilutions) such that significant competition (e.g. a reduction in binding of at least 2-fold, 3-fold, or 4-fold) would occur if the candidate antibody had the ability to bind to both the membrane bound form of mesothelin on cells and the soluble form of mesothelin or extracellular vesicle form.

Alternatively viewed, in an appropriate competition assay (e.g. as described elsewhere herein), a candidate antibody may bind to the membrane-bound form of mesothelin on cells but not be significantly effected/competed by the addition of said soluble form of mesothelin or said mesothelin on extracellular vesicles, e.g. the reduction in binding, if observed at all, is a less than 50% reduction, a less than 40% reduction, a less than 30% reduction, a less than 20% reduction, a less than 10%, a less than 5% reduction or is around a 0% reduction, i.e. that there is no significant or no reduction in binding. In such assays, said soluble form of mesothelin or said mesothelin on extracellular vesicles is added at an appropriate concentration (or range of concentrations, for example in the form of serial dilutions) such that significant competition (e.g. a reduction in binding of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or around 100%) would occur if the candidate antibody had the ability to bind to both the membrane bound form of mesothelin on cells and the soluble form of mesothelin or extracellular vesicle form.

A convenient and preferred way to do this screening would be to carry out flow cytometry (FACS) analysis using cells which are known to be positive for the membrane bound form of mesothelin (e.g. HeLa cells). In such an assay a significantly reduced signal when the soluble form of mesothelin or mesothelin on extracellular vesicles is added indicates that the candidate antibody binds to both the membrane bound form on cells and soluble or extracellular vesicle forms, i.e. does not discriminate, whereas a largely or significantly maintained signal when the soluble form or extracellular vesicle form is added indicates that the candidate antibody does not bind the soluble form or the extracellular vesicle form but does bind the membrane bound form (or there would be no positive signal), i.e. that the antibody can distinguish between the membrane bound form of mesothelin on cells and the soluble form of mesothelin or mesothelin on extracellular vesicles. Such methods of screening are exemplified herein (see the Example).

In a preferred competition assay for identifying (carrying out the screening for) antibodies which can bind to the membrane-bound form of mesothelin on cells but not bind (or not significantly bind) to the soluble form of mesothelin or to mesothelin on extracellular vesicles, the antibody (e.g. a Fab fragment) is incubated with either a soluble form of mesothelin (e.g. recombinant human mesothelin in PBS, or a HeLa cell supernatant, e.g. a concentrated (e.g. 10x concentrated) HeLa cell supernatant) or a source of extracellular vesicles (e.g. isolated exosomes) prior to incubating the antibody with cells (e.g. HeLa cells) (in a pre-incubation step) and then detecting the antibody that is bound to mesothelin on the cells (and thus detecting the antibody-bound cells) using flow cytometry (FACS) analysis. An assay performed with a pre-incubation step without a soluble-form of mesothelin and without a source of mesothelin on extracellular vesicles (e.g. exosomes), i.e. a pre-incubation step without a form of potentially competing mesothelin (e.g. a preincubation step with PBS only or with exosome free DMEM culture medium) can be used to establish the level (amount) of binding of the antibody to the membrane- bound form of mesothelin on cells in the absence of competition. Further details of an exemplary and particularly preferred competition assay are provided in the Example.

Although flow cytometry (FACS) analysis is convenient because of the instant and readily quantifiable fluorescent readout, other forms of assay could equally be used to screen for an antibody which can bind to the membrane bound form of mesothelin on cells but not to the soluble form of mesothelin or to mesothelin on extracellular vesicles (i.e. for antibodies which can discriminate between the membrane bound form of mesothelin on cells and the soluble form of mesothelin or the extracellular vesicle form), such as ELISA or

immunofluorescence. For example, in an ELISA assay, a sample of the membrane bound form of mesothelin on cells could be coated onto an ELISA plate (e.g.

appropriate cells could be coated onto an ELISA plate) and a similar competition assay carried out using a sample of soluble mesothelin (e.g. recombinant human mesothelin) or mesothelin on extracellular vesicles (e.g. isolated exosomes bearing mesothelin) and the candidate antibodies.

The use of competition assays are of course not required as it would readily be possible to use assays such as ELISA assays to compare the binding of a candidate to a soluble form or extracellular vesicle form of mesothelin and a membrane bound form of mesothelin on cells by having the various forms of mesothelin coated on separate ELISA plates and measuring and comparing the amount of antibody which became bound.

It may also in some cases be desirable to carry out an initial round of screening using the membrane-bound form of mesothelin on cells in order to select only the candidates which bind the membrane-bound form of mesothelin on cells and thus eliminate any antibodies which do not show this binding. The positive clones can then be subject to further screening to assess whether or not they also do not have the ability to bind to soluble mesothelin or mesothelin on extracellular vesicles, i.e. have the ability to discriminate between the membrane bound form of mesothelin on cells and the soluble form or mesothelin on extracellular vesicles. Again any appropriate method, e.g. flow cytometry (FACS) or ELISA can be used.

Non-significant (insignificant) binding to the soluble form of mesothelin or to mesothelin on extracellular vesicles generally means reproducibly (i.e. consistently observed) low or negligible binding to these forms of mesothelin. In some cases, insignificant binding can be considered to be at a background level, e.g.

comparative to or not significantly different from a level observed in a negative control experiment.

Whether or not an antibody "does not bind significantly" to the soluble form of mesothelin or to mesothelin on extracellular vesicles can be tested using, for example, the types of competition assay described elsewhere herein. Thus, "insignificant" binding to the soluble form of mesothelin or to mesothelin on extracellular vesicles means that the binding of the antibody to the soluble form of mesothelin or to mesothelin on extracellular vesicles is significantly weaker or a significantly lower affinity than its binding to the membrane bound form of mesothelin on cells. For example, if the ability of an antibody to bind to the membrane-bound form of mesothelin on cells is not significantly reduced, effected or competed (or not reduced, effected or competed) by incubation of the antibodies with a soluble form of mesothelin or mesothelin on extracellular vesicles (e.g. as tested in competition assay as described above), then it can be considered that such an antibody does not bind significantly to the soluble form of mesothelin or does not bind significantly to mesothelin on extracellular vesicles. Such antibodies are discriminatory (specific) for the membrane bound form of mesothelin on cells.

As mentioned above an antibody may be regarded as discriminatory if the ability of said antibody to bind to the membrane bound form of mesothelin on cells is not significantly reduced/effected/competed by the addition of said soluble form of mesothelin or said mesothelin on extracellular vesicles, e.g. the reduction in binding is less than 2 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold or around 1.0 fold, i.e. that there is no significant or no reduction in binding.

Thus, in an exemplary embodiment, in the context of the present invention an antibody that does not bind significantly to the soluble form of mesothelin is an antibody that binds to the membrane-bound form of mesothelin on cells, wherein said binding to the membrane-bound form of mesothelin on cells is not significantly reduced/effected/competed by the addition or presence of said soluble form of mesothelin, e.g. the reduction in binding is less than 2 fold, 1.8 fold, 1 .6 fold, 1 .4 fold, 1 .2 fold or around 1.0 fold, i.e. that there is no significant reduction in binding. Put another way, said binding of an antibody to the membrane-bound form of mesothelin on cells is reduced by less than 2 fold, 1 .8 fold, 1.6 fold, 1 .4 fold, 1.2 fold or around 1.0 fold, i.e. that there is no significant reduction in binding on the addition of or in the presence of said soluble form of mesothelin.

Alternatively viewed, in an appropriate competition assay (e.g. as described elsewhere herein), a candidate antibody may bind to the membrane-bound form of mesothelin on cells but not be significantly effected/competed by the addition of said soluble form of mesothelin, e.g. the reduction in binding, if observed at all, is a less than 50% reduction, a less than 40% reduction, a less than 30% reduction, a less than 20% reduction, a less than 10%, a less than 5% reduction or is around a 0% reduction, i.e. that there is no significant or no reduction in binding. In such assays, said soluble form of mesothelin is added at an appropriate concentration (or range of concentrations, for example in the form of serial dilutions) such that significant competition (e.g. a reduction in binding of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or around 100%) would occur if the candidate antibody had the ability to bind to both the membrane bound form of mesothelin on cells and the soluble form of mesothelin.

In an exemplary embodiment, in the context of the present invention an antibody that does not bind significantly to mesothelin on extracellular vesicles is an antibody that binds to the membrane-bound form of mesothelin on cells, wherein said binding to the membrane-bound form of mesothelin on cells is not significantly reduced/effected/competed by the addition or presence of mesothelin on extracellular vesicles, e.g. the reduction in binding is less than 2 fold, 1 .8 fold, 1 .6 fold, 1 .4 fold, 1 .2 fold or around 1.0 fold, i.e. that there is no significant reduction in binding. Put another way, said binding of an antibody to the membrane-bound form of mesothelin on cells is reduced by less than 2 fold, 1 .8 fold, 1 .6 fold, 1.4 fold, 1 .2 fold or around 1.0 fold, i.e. that there is no significant reduction in binding on the addition of or in the presence of said mesothelin on extracellular vesicles.

Alternatively viewed, in an appropriate competition assay (e.g. as described elsewhere herein), a candidate antibody may bind to the membrane-bound form of mesothelin on cells but not be significantly effected/competed by the addition of said mesothelin on extracellular vesicles, e.g. the reduction in binding, if observed at all, is a less than 50% reduction, a less than 40% reduction, a less than 30% reduction, a less than 20% reduction, a less than 10%, a less than 5% reduction or is around a 0% reduction, i.e. that there is no significant or no reduction in binding. In such assays, said mesothelin on extracellular vesicles is added at an appropriate concentration (or range of concentrations, for example in the form of serial dilutions) such that significant competition (e.g. a reduction in binding of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or around 100%) would occur if the candidate antibody had the ability to bind to both the membrane-bound form of mesothelin on cells and the extracellular vesicle form.

In some embodiments, antibodies of the present invention predominantly bind to the membrane-bound form of mesothelin on cells as opposed to the soluble- form of mesothelin. In some embodiments, antibodies of the present invention predominantly bind to the membrane-bound form of mesothelin on cells as opposed to mesothelin on extracellular vesicles.

In some embodiments, antibodies of the invention do not bind (e.g. do not measurably bind) to the soluble form of mesothelin. In some embodiments, the antibodies of the invention do not bind (e.g. do not measurably bind) to mesothelin on extracellular vesicles.

Preferably, antibodies of the invention may induce antibody dependent cellular cytotoxicity (ADCC) of mesothelin expressing cells (mesothelin positive cells). ADCC may be assayed in vitro using methods well known in the art. In other embodiments, the antibodies are not capable of inducing ADCC, or may induce little or no significant ADCC.

Preferably, antibodies of the invention may induce complement-dependent cytotoxicity (CDC) of mesothelin expressing cells (mesothelin positive cells). CDC may be assayed using well-known standard methods. In other embodiments, the antibodies are not capable of inducing CDC, or may induce little or no significant CDC. Preferably, antibodies of the invention are internalized or are capable of being internalized by cells expressing or bearing mesothelin (mesothelin-positive cells). This is particularly advantageous when the antibody is used, for example, as an immunotoxin. Exemplary immunotoxins can bind to mesothelin on the surface of a cancer cell (or other target mesothelin positive cell), enter the cell, and kill it.

Preferably, antibodies of the invention are capable of blocking one or more of the biological functions of the membrane-bound form of mesothelin on cells.

In some embodiments, antibodies of the invention may be used for the generation of anti-mesothelin CARs (chimeric antigen receptors) or CAR (chimeric antigen receptor) T cells. Thus, in one embodiment the invention provides CARs or CAR T cells comprising (or based on) an antibody of the invention (e.g. 1 h07).

As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. Therefore, an "antibody", as used herein, means "at least a first antibody". The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

In addition, where the terms "comprise", "comprises", "has" or "having", or other equivalent terms are used herein, then in some more specific embodiments these terms include the term "consists of" or "consists essentially of", or other equivalent terms.

Nucleic acid molecules comprising nucleotide sequences that encode the antibodies of the present invention as defined herein or parts or fragments thereof, or nucleic acid molecules substantially homologous thereto, form yet further aspects of the invention. Preferred nucleic acid molecules are those encoding a VH region of an antibody of the present invention (e.g., those encoding SEQ ID NOs:3 or 21 , such as SEQ ID NOs:1 or 19, respectively). Other preferred nucleic acid molecules are those encoding a VL region of an antibody of the present invention (e.g., those encoding SEQ ID NOs:4 or 22, such as SEQ ID NOs:2 or 20, respectively).

The term "substantially homologous" as used herein in connection with an amino acid or nucleic acid sequence includes sequences having at least 65%, 70% or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acid or nucleic acid sequence disclosed. Substantially homologous sequences of the invention thus include single or multiple base or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level preferred substantially homologous sequences contain up to 5, e.g. only 1 , 2, 3, 4 or 5, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in one or more of the framework regions and/or one or more of the CDRs making up the sequences of the invention. Said alterations can be with conservative or non-conservative amino acids. Preferably said alterations are conservative amino acid substitutions.

In certain embodiments, if a given starting sequence is relatively short (e.g. if a given CDR sequence is three amino acid residues in length), then fewer amino acid substitutions are present in sequences substantially homologous thereto as compared with the number of amino acid substitutions that might optionally be made in a sequence substantially homologous to a longer starting sequence. For example, in certain embodiments, a sequence substantially homologous to a starting VL CDR2 sequence in accordance with the present invention, e.g. a starting VL CDR2 sequence which in some embodiments may be three amino acid residues in length, preferably has 1 or 2 (more preferably 1 ) altered amino acids in comparison with the starting sequence. Accordingly, in some embodiments the number of altered amino acids in substantially homologous sequences (e.g. in substantially homologous CDR sequences) can be tailored to the length of a given starting CDR sequence. For example, different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence such as to achieve a particular % sequence identity in the CDRs, for example a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

Routine methods in the art such as alanine scanning mutagenesis and/or analysis of crystal structure of the antigen-antibody complex can be used in order to determine which amino acid residues of the CDRs do not contribute or do not contribute significantly to antigen binding and therefore are good candidates for alteration or substitution in the embodiments of the invention involving substantially homologous sequences.

The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the invention in substantially the same way. For example, any substantially homologous antibody should retain the ability to bind to mesothelin as described above. Preferably, any substantially homologous antibody should retain one or more of the functional capabilities of the starting antibody. Preferably, any substantially homologous antibody should retain the ability to specifically bind to the same epitope of mesothelin as recognized by the antibody in question, for example, the same epitope recognized by the CDR domains of the invention or the VH and VL domains of the invention as described herein. Binding to the same epitope/antigen can be readily tested by methods well known and described in the art, e.g. using binding assays, e.g. a competition assay. Retention of other functional properties can also readily be tested by methods well known and described in the art.

Thus, a person skilled in the art will appreciate that binding assays can be used to test whether "substantially homologous" antibodies have the same binding specificities as the antibodies and antibody fragments of the invention, for example, binding assays such as competition assays or ELISA assays as described elsewhere herein. BIAcore assays could also readily be used to establish whether "substantially homologous" antibodies can bind to mesothelin. The skilled person will be aware of other suitable methods and variations.

As outlined below, a competition binding assay can be used to test whether "substantially homologous" antibodies retain the ability to specifically bind to substantially the same epitope of mesothelin as recognized by the antibodies of the invention (e.g. 1 h07 or 3C02), or have the ability to compete with one or more of the various antibodies of the invention (e.g. 1 h07 or 3C02). The method described below is only one example of a suitable competition assay. The skilled person will be aware of other suitable methods and variations.

An exemplary competition assay involves assessing the binding of various effective concentrations of an antibody of the invention to mesothelin in the presence of varying concentrations of a test antibody (e.g. a substantially homologous antibody). The amount of inhibition of binding induced by the test antibody can then be assessed. A test antibody that shows increased competition with an antibody of the invention at increasing concentrations (i.e. increasing concentrations of the test antibody result in a corresponding reduction in the amount of antibody of the invention binding to mesothelin) is evidence of binding to substantially the same epitope. Preferably, the test antibody significantly reduces the amount of antibody of the invention that binds to mesothelin. Preferably, the test antibody reduces the amount of antibody of the invention that binds to mesothelin by at least about 95%.

In some embodiments, "substantially homologous" antibodies which retain the ability to specifically bind to substantially the same (or the same) epitope of mesothelin as recognized by the antibodies of the invention (e.g. 1 h07 or 3C02) or which have the ability to compete with one or more of the various antibodies of the invention (e.g. 1 h07 or 3C02) are preferred.

The term "competing antibodies", as used herein, refers to antibodies that bind to about, substantially or essentially the same, or even the same, epitope as a "reference antibody". "Competing antibodies" include antibodies with overlapping epitope specificities. Competing antibodies are thus able to effectively compete with a reference antibody for binding to mesothelin. Preferably, the competing antibody can bind to the same epitope as the reference antibody. Alternatively viewed, the competing antibody preferably has the same epitope specificity as the reference antibody.

"Reference antibodies" as used herein are antibodies which can bind to mesothelin in accordance with the invention and which have one or more of the CDR sequences are defined herein, preferably a VH and a VL domain as defined herein, more preferably a VH of SEQ ID NO: 3 and a VL of SEQ ID NO: 4, or a VH of SEQ ID NO: 21 and a VL of SEQ ID NO: 22. Most preferred reference antibodies are selected from 1 h07 and 3C02.

The identification of one or more competing antibodies is a straightforward technical matter now that reference antibodies such as 1 h07 and 3C02 have been provided. As the identification of competing antibodies is determined in comparison to a reference antibody, it will be understood that actually determining the epitope to which either or both antibodies bind is not in any way required in order to identify a competing antibody. However, epitope mapping can be performed using standard techniques, if desired.

Substantially homologous sequences of proteins of the invention include, without limitation, conservative amino acid substitutions, or for example alterations that do not affect the VH, VL or CDR domains of the antibodies, e.g. antibodies where tag sequences, toxins or other components are added that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g. conversion from Fab to scFv or whole antibody or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g. the conversion of an antibody molecule to IgG or a subclass thereof, e.g. lgG1 or lgG3).

A "conservative amino acid substitution", as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

Homology may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W

(Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1 , so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that may be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981 ) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Other methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.

Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:1 1 -17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. Furthermore, the Dali server at the European Bioinformatics institute offers structure-based alignments of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998). By way of providing a reference point, sequences according to the present invention having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, sequence identity etc. may be determined using the ALIGN program with default parameters (for instance available on Internet at the GENESTREAM network server, IGH, Montpellier, France).

In the following descriptions of the compositions, immunoconjugates, pharmaceuticals, combinations, cocktails, kits, first and second medical uses and all methods in accordance with this invention, the terms "antibody" and

"immunoconjugate", or an antigen-binding region or fragment thereof, unless otherwise specifically stated or made clear from the scientific terminology, refer to a range of anti-mesothelin antibodies as well as to the specific 1 h07 and 3C02 antibodies.

The terms "antibody" and "immunoglobulin", as used herein, refer broadly to any immunological binding agent that comprises an antigen binding domain, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and the antibodies of the invention may be in any one of these classes. Several of these are further divided into subclasses or isotypes, such as lgG1 , lgG2, lgG3, lgG4, and the like. The heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed a, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regions are used in the invention, IgG (e.g. IgG-i) and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types: kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains and some amino acids in the framework regions of their variable domains.

As will be understood by those in the art, the immunological binding reagents encompassed by the term "antibody" extend to all mouse and human antibodies and humanized antibodies and antigen binding fragments thereof, including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies, and fragments thereof. The term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')₂, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody;

kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-lg (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. In one preferred embodiment the antibody fragment is a Fab fragment (e.g. a murine or humanized murine Fab fragment).

The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Diabodies, in particular, are further described in EP 404 097 and WO 93/1 1 161 ; whereas linear antibodies are further described in the art.

The term "heavy chain complementarity determining region" ("heavy chain CDR") as used herein refers to regions of hypervariability within the heavy chain variable region (V_H domain) of an antibody molecule. The heavy chain variable region has three CDRs termed heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. The heavy chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

The term "heavy chain variable region" (V_H domain) as used herein refers to the variable region of a heavy chain of an antibody molecule.

The term "light chain complementarity determining region" ("light chain CDR") as used herein refers to regions of hypervariability within the light chain variable region (V_L domain) of an antibody molecule. Light chain variable regions have three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

The term "light chain variable region" (V_L domain) as used herein refers to the variable region of a light chain of an antibody molecule. Antibodies can be fragmented using conventional techniques. For example, F(ab')₂ fragments can be generated by treating the antibody with pepsin. The resulting F(ab')₂ fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')₂, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized.

Techniques for producing antibody fragments are well known and described in the art.

In certain embodiments, the antibody or antibody fragment of the present invention comprises all or a portion of a heavy chain constant region, such as an lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g. an lgG1 or lgG3 heavy chain constant region, or a portion thereof. Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. All or part of such constant regions may be produced naturally or may be wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art. When a full complement of constant regions from the heavy and light chains are included in the antibodies of the invention, such antibodies are typically referred to herein as "full length" antibodies or "whole" antibodies. In some embodiments, IgGi antibodies are preferred.

The antibodies or antibody fragments can be produced naturally or can be wholly or partially synthetically produced. Thus the antibody may be from any appropriate source, for example recombinant sources and/or produced in transgenic animals or transgenic plants, or in eggs using the IgY technology. Thus, the antibody molecules can be produced in vitro or in vivo.

Preferably, the antibody or antibody fragment comprises an antibody light chain variable region (V_L) that comprises three CDR domains and an antibody heavy chain variable region (V_H) that comprises three CDR domains. Said VL and VH generally form the antigen binding site.

An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region has a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six hypervariable regions (CDRs) confer antigen-binding specificity to the antibody.

However, it is well documented in the art that the presence of three CDRs from the light chain variable domain and three CDRs from the heavy chain variable domain of an antibody is not always necessary for antigen binding. Thus, constructs smaller than the above classical antibody fragment are known to be effective.

For example, camelid antibodies have an extensive antigen binding repertoire but are devoid of light chains. Also, results with single domain antibodies comprising VH domains alone or VL domains alone show that these domains can bind to antigen with acceptably high affinities. Thus, three CDRs can effectively bind antigen.

Thus, although preferred antibodies of the invention might comprise six CDR regions (three from a light chain and three from a heavy chain), antibodies with fewer than six CDR regions (e.g. 3 CDR regions) are encompassed by the invention. Antibodies with CDRs from only the heavy chain or light chain are also contemplated.

Preferred light chain CDR regions for use in conjunction with the specified heavy chain CDR regions are described elsewhere herein. However, other light chain variable regions that comprise three CDRs for use in conjunction with the heavy chain variable regions of the invention are also contemplated. Appropriate light chain variable regions which can be used in combination with the heavy chain variable regions of the invention and which give rise to an antibody which binds mesothelin in accordance with the invention can be readily identified by a person skilled in the art.

For example, a heavy chain variable region of the invention can be combined with a single light chain variable region or a repertoire of light chain variable regions and the resulting antibodies tested for binding to mesothelin.

If desired, similar methods could be used to identify alternative heavy chain variable regions for use in combination with preferred light chain variable regions of the invention.

The exemplary 1 h07 and 3C02 antibodies of the present invention are murine Fab fragments. Thus, in one embodiment Fab fragments (e.g. mouse Fab fragments) are preferred. In certain embodiments, "humanized" versions of the antibodies of the invention (e.g. the 1 h07 and 3C02 antibodies) are preferred. "Humanized" antibodies, which are based on substantially non-human variable region domains, e.g. mouse variable region domains in the present case, are antibodies in which certain amino acids have been changed to better correspond with the amino acids typically present in human antibodies. Methods for generating humanized antibodies are well known in the art. For example, humanized antibodies can be accomplished by inserting the appropriate CDRs (e.g. murine CDRs) into a human antibody "scaffold".

A yet further aspect of the invention provides an antibody, preferably an isolated antibody, more preferably a mouse or a humanized or a human antibody, which binds to or specifically recognizes mesothelin in accordance with the invention and which has the ability to compete with (i.e. bind to the same or substantially the same epitope as) the 1 h07 antibody and/or the 3C02 antibody (i.e. an antibody comprising the VL of SEQ ID NO:4 and the VH of SEQ ID NO:3, or an antibody comprising the VL of SEQ ID NO:22 and the VH of SEQ ID NO:21 , respectively) as described herein, or the ability to compete with an antibody comprising the same CDRs as 1 h07 and/or 3C02 (i.e. an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 10 and VH CDR sequences of SEQ ID NOs: 5, 6 and 7, or an antibody comprising VL CDR sequences of SEQ ID NOs: 8, 9 and 28 and VH CDR sequences of SEQ ID NOs: 5, 24 and 25, respectively) for binding to mesothelin in accordance with the invention. Other features and properties of other aspects of the invention apply, mutatis mutandis, to this aspect of the invention.

Binding to the same epitope/antigen can be readily tested by methods well known and described in the art, e.g. using binding assays such as a competitive inhibition assay. Thus, a person skilled in the art will appreciate that binding assays can be used to identify other antibodies and antibody fragments with the same binding specificities as the antibodies and antibody fragments of the invention.

Suitable binding assays are discussed elsewhere herein.

Preferably, the above described abilities and properties are observed at a measurable or significant level and more preferably at a statistically significant level, when compared to appropriate control levels. Appropriate significance levels are discussed elsewhere herein. More preferably, one or more of the above described abilities and properties are observed at a level which is measurably better, or more preferably significantly better, when compared to the abilities observed for prior art antibodies.

In any statistical analysis referred to herein, preferably the statistically significant difference over a relevant control or other comparative entity or measurement has a probability value of < 0.1 , preferably < 0.05. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.

In other preferred embodiments, second generation antibodies are provided that have enhanced or superior properties in comparison to an original anti- mesothelin antibody of the invention, such as 1 h07 or 3C02.

Comparisons to identify effective second generation antibodies are readily conducted and quantified, e.g. using one or more of the various assays described in detail herein or in the art. Second generation antibodies that have an enhanced biological property or activity of at least about 2-fold, 5-fold, 10-fold, 20-fold, and preferably, at least about 50-fold, in comparison to the anti-mesothelin antibodies of the present invention, as exemplified by the 1 h07 or 3C02 antibodies, are encompassed by the present invention.

In some embodiments, the antibody does not comprise (or consist of) the VH and VL domains (or regions) of the 1 h07 antibody or the VL and VH domains (or regions) of the 3C02 antibody.

In some embodiments, the antibody is a synthetic or recombinant or non- native antibody.

In some embodiments, the antibody may be a modified antibody based on an antibody having amino acid sequences of the 1 h07 or 3C02 antibody. For example, the antibody may be modified in order to decrease its immunogenicity

(e.g. immunogenicity in humans) and/or to improve its expression and/or improve its stability during its manufacture and/or to improve one or more of its functional properties. Modified antibodies, include, but are not limited to, antibodies having one or more amino acid substitutions in comparison to the starting antibody (e.g. as described elsewhere herein) and/or having modifications to side groups such as modifications to reduce or remove fucosylation (reduce or remove fucose groups), e.g. fucosylation in the Fc region of an antibody. Preferably, any modification should not alter the ability of the antibody to bind to mesothelin, e.g. in comparison with the starting (unmodified) antibody.

The antibody, binding protein and nucleic acid molecules of the invention are generally "isolated" or "purified" molecules insofar as they are distinguished from any such components that may be present in situ within a human or animal body or a tissue sample derived from a human or animal body. The sequences may, however, correspond to or be substantially homologous to sequences as found in a human or animal body. Thus, the term "isolated" or "purified" as used herein in reference to nucleic acid molecules or sequences and proteins or polypeptides, e.g. antibodies, refers to such molecules when isolated from, purified from, or substantially free of their natural environment, e.g. isolated from or purified from the human or animal body (if indeed they occur naturally), or refers to such molecules when produced by a technical process, i.e. includes recombinant and synthetically produced molecules.

Thus, when used in connection with a protein or polypeptide molecule such as light chain CDRs 1 , 2 and 3, heavy chain CDRs 1 , 2 and 3, light chain variable regions, heavy chain variable regions, and binding proteins or antibodies of the invention, including full length antibodies, the term "isolated" or "purified" typically refers to a protein substantially free of cellular material or other proteins from the source from which it is derived. In some embodiments, particularly where the protein is to be administered to humans or animals, such isolated or purified proteins are substantially free of culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically

synthesized.

The term "nucleic acid sequence" or "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid molecules may be double stranded or single stranded. The nucleic acid molecules may be wholly or partially synthetic or recombinant.

The term "fragment" as used herein refers to fragments of biological relevance, e.g. fragments that contribute to antigen binding, e.g. form part of the antigen binding site, and/or contribute to the functional properties of the mesothelin antibody. Certain preferred fragments comprise a heavy chain variable region (V_H domain) and/or a light chain variable region (V_L domain) of the antibodies of the invention. A Fab fragment is a preferred type of fragment.

A person skilled in the art will appreciate that the proteins and polypeptides of the invention, such as the light and heavy CDRs, the light and heavy chain variable regions, antibodies, antibody fragments, and immunoconjugates, may be prepared in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods. Nucleic acid fragments encoding the light and heavy chain variable regions of the antibodies of the invention can be derived or produced by any appropriate method, e.g. by cloning or synthesis.

Once nucleic acid fragments encoding the light and heavy chain variable regions of the antibodies of the invention have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region fragments into full length antibody molecules with appropriate constant region domains, or into particular formats of antibody fragment discussed elsewhere herein, e.g. Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the invention are generally incorporated into one or more appropriate expression vectors in order to facilitate production of the antibodies of the invention.

Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno- associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner that allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes and are well known in the art. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. The recombinant expression vectors of the invention may also contain a selectable marker gene that facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.

The recombinant expression vectors may also contain genes that encode a fusion moiety that provides increased expression of the recombinant protein;

increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification (for example appropriate "tags" to enable purification and/or identification may be present, e.g., His tags or myc tags).

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The terms "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid {e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the invention may be expressed in yeast cells or mammalian cells. In addition, the proteins of the invention may be expressed in prokaryotic cells, such as Escherichia coli.

Given the teachings provided herein, promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.

Alternatively, the proteins of the invention may also be expressed in non- human transgenic animals such as, rats, rabbits, sheep and pigs.

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.

N-terminal or C-terminal fusion proteins comprising the antibodies and proteins of the invention conjugated to other molecules, such as proteins, may be prepared by fusing through recombinant techniques. The resultant fusion proteins contain an antibody or protein of the invention fused to the selected protein or marker protein, or tag protein as described herein. The antibodies and proteins of the invention may also be conjugated to other proteins by known techniques. For example, the proteins may be coupled using heterobifunctional thiol-containing linkers as described in WO 90/10457, N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5 thioacetate.

A yet further aspect provides an expression construct or expression vector comprising one or more of the nucleic acid fragments or segments or molecules of the invention. Preferably the expression constructs or vectors are recombinant. Preferably said constructs or vectors further comprise the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

A yet further aspect provides a host cell or virus comprising one or more expression constructs or expression vectors of the invention. Also provided are host cells or viruses comprising one or more of the nucleic acid molecules of the invention. A host cell or virus expressing an antibody of the invention forms a yet further aspect. Suitable host cells include, but are not limited to HEK293E cells.

A yet further aspect of the invention provides a method of producing (or manufacturing) an antibody of the present invention comprising a step of culturing the host cells of the invention. Preferred methods comprise the steps of (i) culturing a host cell comprising one or more of the recombinant expression vectors or one or more of the nucleic acid sequences of the invention under conditions suitable for the expression of the encoded antibody or protein; and optionally (ii) isolating or obtaining the antibody or protein from the host cell or from the growth

medium/supernatant. Such methods of production (or manufacture) may also comprise a step of purification of the antibody or protein product and/or formulating the antibody or product into a composition including at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

In embodiments when the antibody or protein of the invention is made up of more than one polypeptide chain (e.g. certain fragments such as Fab fragments or whole antibodies), then all the polypeptides are preferably expressed in the host cell, either from the same or a different expression vector, so that the complete proteins, e.g. antibody proteins of the invention, can assemble in the host cell and be isolated or purified therefrom.

In another aspect, the invention provides a method of binding mesothelin, comprising contacting a composition comprising mesothelin with an antibody of the invention, or an immunoconjugate thereof.

In yet another aspect, the invention provides a method of detecting mesothelin, comprising contacting a composition suspected of containing mesothelin with an antibody of the invention, or an immunoconjugate thereof, under conditions effective to allow the formation of mesothelin /antibody complexes and detecting the complexes so formed.

The antibodies of the invention may also be used to produce further antibodies that bind to mesothelin. Such uses involve for example the addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antibody to form a new antibody, wherein said parent antibody is one of the antibodies of the invention as defined elsewhere herein, and testing the resulting new antibody to identify antibodies that bind to mesothelin in accordance with the invention. Such methods can be used to form multiple new antibodies that can all be tested for their ability to bind mesothelin in accordance with the invention. Preferably said addition, deletion, substitution or insertion of one or more amino acids takes place in one or more of the CDR domains.

Such modification or mutation to a parent antibody can be carried out in any appropriate manner using techniques well known and documented in the art, for example by carrying out methods of random or directed mutagenesis. If directed mutagenesis is to be used then one strategy to identify appropriate residues for mutagenesis utilizes the resolution of the crystal structure of the binding protein- antigen complex, e.g., the Ab-Ag complex, to identify the key residues involved in the antigen binding. Alanine scanning mutagenesis is also a routine method which can be used to identify the key residues involved in the antigen binding.

Subsequently, those residues can be mutated to enhance the interaction.

Alternatively, one or more amino acid residues can simply be targeted for directed mutagenesis and the effect on binding to mesothelin assessed.

Random mutagenesis can be carried out in any appropriate way, e.g., by error-prone PCR, chain shuffling or mutator E. coli strains.

Thus, one or more of the V_H domains of the invention can be combined with a single V_L domain or a repertoire of V_L domains from any appropriate source and the resulting new antibodies tested to identify antibodies which bind to mesothelin in accordance with the invention. Conversely, one or more of the V_L domains of the invention can be combined with a single V_H domain or repertoire of V_H domains from any appropriate source and the resulting new antibodies tested to identify antibodies that bind to mesothelin in accordance with the invention.

Similarly, one or more, or preferably all three CDRs of the V_H and/or V_L domains of the invention can be grafted into a single V_H and/or V_L domain or a repertoire of V_H and/or V_L domains, as appropriate, and the resulting new antibodies tested to identify antibodies that bind to mesothelin in accordance with the invention. Methods of carrying out the above described manipulation of amino acids and protein domains are well known to a person skilled in the art. For example, said manipulations could conveniently be carried out by genetic engineering at the nucleic acid level wherein nucleic acid molecules encoding appropriate binding proteins and domains thereof are modified such that the amino acid sequence of the resulting expressed protein is in turn modified in the appropriate way.

The new antibodies produced by these methods will preferably have improved functional properties, e.g. a higher or enhanced affinity (or at least an equivalent affinity) for mesothelin on the cell membrane of a cell as the parent antibodies, and can be treated and used in the same way as the antibodies of the invention as described elsewhere herein {e.g., for therapy, diagnosis, in

compositions etc.). Alternatively, or additionally, the new antibodies will have one or more other improved functional properties as described elsewhere herein.

New antibodies produced, obtained or obtainable by these methods form a yet further aspect of the invention.

Testing the ability of one or more antibodies to bind to mesothelin in accordance with the invention can be carried out by any appropriate method, which are well known and described in the art. Suitable methods are also described in the Examples section. The invention also provides a range of conjugated antibodies and fragments thereof in which the anti-mesothelin antibody is operatively attached to at least one other therapeutic or diagnostic agent. The term "immunoconjugate" is broadly used to define the operative association of the antibody with another effective agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical "conjugation". Recombinant fusion proteins are particularly contemplated. So long as the delivery or targeting agent is able to bind to the target and the therapeutic or diagnostic agent is sufficiently functional upon delivery, the mode of attachment will be suitable. In preferred immunoconjugates, active ingredients such as radionuclides, toxins (for example, the diphtheria toxin), cytokines or also cytostatic agents can be bonded (conjugated) or otherwise linked to the corresponding antibodies.

For example, in some preferred embodiments, antibodies of the invention are part of an immunotoxin or are used (e.g. used therapeutically) as part of immunotoxins. Immunotoxins are formed of an antibody (e.g. an antigen binding fragment such as a Fab fragment, or a whole antibody e.g. for use in the form of an antibody drug conjugate) linked or conjugated to a toxin. Suitable toxins are well known and described in the art, for example cytotoxic proteins derived from bacteria or plants can be used. The toxin should be capable of killing target cells once it has been taken up into said cells. Thus, preferred immunoconjugates of the invention are immunotoxins comprising an antibody of the invention linked or otherwise conjugated to a toxin.

In some embodiments, antibodies of the invention are used (e.g. used therapeutically) in their "naked" unconjugated form.

Compositions comprising at least a first antibody of the invention or an immunoconjugate thereof constitute a further aspect of the present invention.

Formulations (compositions) comprising one or more antibodies of the invention in admixture with a suitable diluent, carrier or excipient constitute a preferred embodiment of the present invention. Such formulations may be for pharmaceutical use and thus compositions of the invention are preferably pharmaceutically acceptable. Suitable diluents, excipients and carriers are known to the skilled man.

The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral, intravenal, topical or rectal

administration.

The active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.

Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as

p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers, either with an aerosol propellant or provided with means for manual compression.

The pharmaceutical compositions (formulations) of the present invention are preferably administered parenterally. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the antibody in the form of a nasal or pulmonal spray. As a still further option, the antibodies of the invention can also be administered transdermally, e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.

Suitable dosage units can be determined by a person skilled in the art.

The pharmaceutical compositions may additionally comprise further active ingredients as described above in the context of co-administration regimens.

A further aspect of the present invention provides the anti-mesothelin antibodies defined herein for use in therapy, in particular for use in the treatment of cancer.

In another aspect, the present invention provides immunoconjugates of the invention for use in therapy, in particular for use in the treatment of cancer.

In accordance with the present invention antibodies may target mesothelin positive tumour cells.

In one embodiment, solid tumours are treated.

In some embodiments, a tumour or cancer (e.g. a solid tumour) that is characterized by over-expression of mesothelin is treated.

Preferred cancers to be treated in accordance with the present invention include mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer (e.g. adenocarcinomas of the pancreas), lung cancer, ovarian cancer (e.g. serous cancers of the ovary). Other preferred cancers to be treated in accordance with the present invention include breast cancer, colon cancer, non-small cell lung cancer. In some embodiments, the cancer to be treated may be metastatic (e.g. metastatic colon cancer or metastatic pancreatic cancer).

A further aspect of the present invention provides anti-mesothelin antibodies as defined herein for use in the treatment or diagnosis of a disease (or condition) that is characterized by (or associated with) mesothelin expression, for example a disease (or condition) characterized by undesired, inappropriate, aberrant, increased or excessive mesothelin expression.

The in vivo methods and uses as described herein are generally carried out in a mammal. Any mammal may be treated, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however, the mammal is a human. Thus, the term "animal" or "patient" as used herein includes any mammal, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however, the animal or patient is a human subject. Thus, subjects or patients treated in accordance with the present invention will preferably be humans.

Alternatively viewed, the present invention provides a method of treating cancer which method comprises administering to a patient in need thereof a therapeutically effective amount of an antibody of the invention as defined herein. The present invention also provides a method of treating a disease that is characterized by mesothelin expression which method comprises administering to a patient in need thereof a therapeutically effective amount of an antibody of the invention as defined herein. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to these aspects of the invention.

A therapeutically effective amount will be determined based on the clinical assessment and can be readily monitored. Preferred cancer therapies are as described elsewhere herein.

Further alternatively viewed, the present invention provides the use of an antibody of the invention as defined herein in the manufacture of a medicament for use in therapy (e.g. therapy of a disease that is characterized by mesothelin expression). Preferred therapy is cancer therapy as described elsewhere herein (e.g. therapy of solid tumours). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

Further alternatively viewed, the present invention provides the use of an antibody of the invention as defined herein for the treatment of a disease that is characterized by mesothelin expression. A preferred use is for the treatment of cancer (as described elsewhere herein).

The compositions and methods and uses of the present invention may be used in combination with other therapeutics and diagnostics. In terms of biological agents, preferably diagnostic or therapeutic agents, for use "in combination" with an anti-mesothelin antibody in accordance with the present invention, the term "in combination" is succinctly used to cover a range of embodiments. The "in combination" terminology, unless otherwise specifically stated or made clear from the scientific terminology, thus applies to various formats of combined compositions, pharmaceuticals, cocktails, kits, methods, and first and second medical uses. The "combined" embodiments of the invention thus include, for example, where an anti-mesothelin antibody of the invention is a naked antibody and is used in combination with an agent or therapeutic agent (e.g. a chemotherapeutic agent) that is not operatively attached thereto. In other "combined" embodiments of the invention, an anti-mesothelin antibody of the invention is an immunoconjugate wherein the antibody is itself operatively associated or combined with the agent or therapeutic agent (e.g. a chemotherapeutic agent). The operative attachment includes all forms of direct and indirect attachment as described herein and known in the art.

Yet further aspects are methods of diagnosis or imaging of a subject comprising the administration of an appropriate amount of an antibody or other protein of the invention as defined herein to the subject and detecting the presence and/or amount and/or the location of the antibody or other protein of the invention in the subject.

Appropriate diseases to be imaged or diagnosed in accordance with the present invention are described elsewhere herein in connection with disease treatments.

In one embodiment, the invention provides a method of diagnosing cancer in a mammal comprising the step of:

(a) contacting a test sample taken from said mammal with one or more of the antibodies of the invention.

In a further embodiment, the invention provides a method of diagnosing cancer in a mammal comprising the steps of:

(a) contacting a test sample taken from said mammal with one or more of the antibodies of the invention;

(b) measuring the presence and/or amount and/or location of antibody- antigen complex in the test sample; and, optionally

(c) comparing the presence and/or amount of antibody-antigen complex in the test sample to a control.

In the above methods, said contacting step is carried out under conditions that permit the formation of an antibody-antigen complex. Appropriate conditions can readily be determined by a person skilled in the art.

In the above methods any appropriate test sample may be used, for example biopsy cells, tissues or organs suspected of being affected by disease or histological sections. The mammal may be a mammal (e.g. a human) that is suspected of (or at risk of) having cancer.

In certain of the above methods, the presence of any amount of antibody- antigen complex in the test sample would be indicative of the presence of disease. Preferably, for a positive diagnosis to be made, the amount of antibody-antigen complex in the test sample is greater than, preferably significantly greater than, the amount found in an appropriate control sample. More preferably, the significantly greater levels are statistically significant, preferably with a probability value of <0.05. Appropriate methods of determining statistical significance are well known and documented in the art and any of these may be used.

Appropriate control samples could be readily chosen by a person skilled in the art, for example, in the case of diagnosis of a particular disease, an appropriate control would be a sample from a subject that did not have that disease.

Appropriate control "values" could also be readily determined without running a control "sample" in every test, e.g. by reference to the range for normal subjects known in the art.

For use in the diagnostic or imaging applications, the antibodies of the invention may be labeled with a detectable marker such as a radio-opaque or radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³l, ¹²⁵l, ¹³¹ l; a radioactive emitter (e.g. α, β or γ emitters); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion; or a chemical moiety such as biotin which may be detected by binding to a specific cognate detectable moiety, e.g. labelled

avidin/streptavidin. Methods of attaching a label to a binding protein, such as an antibody or antibody fragment, are known in the art. Such detectable markers allow the presence, amount or location of binding protein-antigen complexes in the test sample to be examined.

Preferred detectable markers for in vivo use include an X-ray detectable compound, such as bismuth (III), gold (III), lanthanum (III) or lead (II); a radioactive ion, such as copper⁶⁷, gallium⁶⁷, gallium⁶⁸, indium¹¹¹, indium¹¹³, iodine¹²³, iodine¹²⁵, iodine¹³¹, mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³, technetium⁹⁹"¹ or yttrium⁹⁰; a nuclear magnetic spin-resonance isotope, such as cobalt (II), copper (II), chromium (III), dysprosium (III), erbium (III), gadolinium (III), holmium (III), iron (II), iron (III), manganese (II), neodymium (III), nickel (II), samarium (III), terbium (III), vanadium (II) or ytterbium (III); or rhodamine or fluorescein.

The invention also includes diagnostic or imaging agents comprising the antibodies of the invention attached to a label that produces a detectable signal, directly or indirectly. Appropriate labels are described elsewhere herein.

In one embodiment the method of diagnosing cancer is an in vitro method.

In one embodiment the method of diagnosing cancer is an in vivo method.

In some embodiments, if the result of a diagnostic method of the invention is indicative of cancer (e.g. a positive diagnosis of cancer is made), then an additional step of treating cancer by therapy or surgery can be performed.

The invention further includes kits comprising one or more of the antibodies, immunoconjugates or compositions of the invention or one or more of the nucleic acid molecules encoding the antibodies of the invention, or one or more

recombinant expression vectors comprising the nucleic acid sequences of the invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the invention. Preferably said kits are for use in the methods and uses as described herein, e.g. the therapeutic, diagnostic or imaging methods as described herein, or are for use in the in vitro assays or methods as described herein. The antibody in such kits may preferably be an antibody conjugate as described elsewhere herein, e.g. may be conjugated to a detectable moiety or may be an immunoconjugate. Preferably said kits comprise instructions for use of the kit components. Preferably said kits are for diagnosing or treating diseases as described elsewhere herein, and optionally comprise instructions for use of the kit components to diagnose or treat such diseases.

The antibodies of the invention as defined herein may also be used as molecular tools for in vitro or in vivo applications and assays. As the antibodies have an antigen binding site, these can function as members of specific binding pairs and these molecules can be used in any assay where the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent that comprises an antibody of the invention as defined herein and the use of such antibodies as molecular tools, for example in in vitro or in vivo assays.

TABLE OF NUCLEOTIDE AND AMINO ACID SEQUENCES DISCLOSED HEREIN AND THEIR SEQUENCE IDENTIFIERS (SEQ ID NOs) All nucleotide sequences are recited herein 5' to 3' in line with convention in this technical field.

Table 1

SEQ ID Description Sequence

NO:

Clone 1 h07

1 VH domain (nt) gaggtccagctgcagcagtctgggactgtgctggcaa ggcctggggcttccgtgaagatgtcctgcaaggcttc tggctacagctttaccaattacaggatgaactgggta aaacagaggcctggacagggtctagaatggattggtg gtatttatcctggaaatagagatactacctacaacca gaagttcaaggacaaggccaaactgactgcagtcaca tccgccaacactgcctacatggagctcagcagcctga cgaatgaggactctgcggtctattactgtacaagagg ggtaatcgggatctactttgactactggggccaaggc accactctcacagtctcctca

2 VL domain (nt) gacattgtgatgacccagtctccagcttctttggctg tgtctctagggcagagggccaccatctcctgcaaggc cagccaaagtgttgattatgatggtgatagttatatg aactggtaccaacagaaaccaggacagccacccaaac tcctcatctatgctgcatccaatctagaatctgggat cccagccaggtttagtggcagtgggtctgggacagac ttcaccctcaacatccatcctgtggaggaggaggatg ctgcaacctactactgtcagcaaaataatgaggctcc gctcacgttcggtgctgggaccaagctggagctgaaa

3 VH domain (aa) EVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMNWV

KQRPGQGLEWIGGIYPGNRDTTYNQKFKDKAKLTAVT SANTAYMELSSLTNEDSAVYYCTRGVIGIYFDYWGQG TTLTVSS

4 VL domain (aa) LDIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSY

MNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGT DFTLNIHPVEEEDAATYYCQQNNEAPLTFGAGTKLEL K Table 1

SEQ ID Description Sequence

NO:

GYSFTNYR

5 Heavy CDR1

IYPGNRDT

6 Heavy CDR2

TRGVIGIYFDY

7 Heavy CDR3

8 Light CDR1 QSVDYDGDSY

9 Light CDR2 AAS

10 Light CDR3 QQNNEAPLT

1 1 Heavy FR1 EVQLQQSGTVLARPGASVKMSCKAS

12 Heavy FR2 MNWVKQRPGQGLEWIGG

TYNQKFKDKAKLTAVTSANTAYMELSSLTNEDSAVYY

13 Heavy FR3

C

14 Heavy FR4 WGQGTTLTVSS

15 Light FR1 LDIVMTQSPASLAVSLGQRATI SCKAS

MNWYQQKPGQPPKLLIY

16 Light FR2

17 Light FR3 NLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC

FGAGTKLELK

18 Light FR4

EVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMNWV

43 Heavy chain

(aa) KQRPGQGLEWIGGIYPGNRDTTYNQKFKDKAKLTAVT

SANTAYMELSSLTNEDSAVYYCTRGVIGIYFDYWGQG TTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGA

A

44 Light chain (aa) LDIVMTQSPASLAVSLGQRATI SCKASQSVDYDGDSY

MNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGT DFTLNIHPVEEEDAATYYCQQNNEAPLTFGAGTKLEL KRADAAPTVSI FPPSSEQLTSGGASVVCFLN FYPKD I VKWKI DGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHK

gaggtccagctgcagcagtctgggactgtgctggcaaggc

45 Heavy chain Table 1

SEQ ID Description Sequence

NO:

ctggggcttccgtgaagatgtcctgcaaggcttctggcta

(nt)

cagctttaccaattacaggatgaactgggtaaaacagagg cctggacagggtctagaatggattggtggtatttatcctg gaaatagagatactacctacaaccagaagttcaaggacaa ggccaaactgactgcagtcacatccgccaacactgcctac atggagctcagcagcctgacgaatgaggactctgcggtct attactgtacaagaggggtaatcgggatctactttgacta ctggggccaaggcaccactctcacagtctcctcagccaaa acgacacccccatctgtctatccactggcccctggatctg ctgcccaaactaactccatggtgaccctgggatgcctggt caagggctatttccctgagccagtgacagtgacctggaac tctggatccctgtccagcggtgtgcacaccttcccagctg tcctgcagtctgacctctacaccctgagcagctcagtgac tgtcccctccagcacctggcccagcgagaccgtcacctgc aacgttgcccacccggccagcagcaccaaaggtggacaag aaaattgtgcccagggattgtggtgcggccgca

46 Light chain (nt) gacattgtgatgacccagtctccagcttctttggctg tgtctctagggcagagggccaccatctcctgcaaggc cagccaaagtgttgattatgatggtgatagttatatg aactggtaccaacagaaaccaggacagccacccaaac tcctcatctatgctgcatccaatctagaatctgggat cccagccaggtttagtggcagtgggtctgggacagac ttcaccctcaacatccatcctgtggaggaggaggatg ctgcaacctactactgtcagcaaaataatgaggctcc gctcacgttcggtgctgggaccaagctggagctgaaa cgggctgatgctgcaccaactgtatccatcttcccac catccagtgagcagttaacatctggaggtgcctcagt cgtgtgcttcttgaacaacttctaccccaaagacatc aatgtcaagtggaagattgatggcagtgaacgacaaa atggcgtcctgaacagttggactgatcaggacagcaa agacagcacctacagcatgagcagcaccctcacgttg accaaggacgagtatgaacgacataacagctatacct gtgaggccactcacaagacatcaacttcacccattgt caagagcttcaacaggaatgagtgttaataaggcg Table 2

SEQ ID Description Sequence

NO:

Clone 3C02

19 VH domain (nt) caggtccagctgcagcagtctgggactgtgctggcaa ggcctggggcttccgtgaagatgtcctgcaaggcttc tggctacagctttaccaactaccggatgtactgggta aaacagaggcctggacagggtctagaatggattggtg ctatttatcctggaaatagtgatactacctacaagca gaagttcaagggcaaggccaaactgactgcagtcaca tccgccagcactgcctacatggagctcagcagcctga caaatgaggactctgcggtctattactgtacaagggg gatacgggggtcgtacttcgatgtctggggcgcaggg accacggtcaccgtctcctca

20 VL domain (nt) cttgacattgtgatgacacagtctccagcttctttgg ctgtgtctctagggcagagggccaccatctcctgcaa ggccagccaaagtgttgattatgatggtgatagttat atgaactggtaccaacagaaaccaggacagccaccca aactcctcatctatgctgcatccaatctagaatctgg gatcccagccaggtttagtggcagtgggtctgggaca gacttcaccctcaacatccatcctgtggaggaggagg atgctgcaacctattactgtcagcaaagtaatgagga tccgtacacgttcggaggggggaccaagctggaaata aaa

21 VH domain (aa) QVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMYWV

KQRPGQGLEWIGAIYPGNSDTTYKQKFKGKAKLTAVT SASTAYMELSSLTNEDSAVYYCTRGIRGSYFDVWGAG TTVTVSS

22 VL domain (aa) LDIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSY

MNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGT DFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEI

K

23 or 5 Heavy CDR1 GYSFTNYR

24 Heavy CDR2 IYPGNSDT

25 Heavy CDR3 TRGIRGSYFDV

26 or 8 Light CDR1 QSVDYDGDSY

27 or 9 Light CDR2 AAS

28 Light CDR3 QQSNEDPYT Table 2

SEQ ID Description Sequence

NO:

29 Heavy FR1 QVQLQQSGTVLARPGASVKMSCKAS

30 Heavy FR2 MYWVKQRPGQGLEWIGA

31 Heavy FR3 TYKQKFKGKAKLTAVTSASTAYMELSSLTNEDSAVYY

C

32 Heavy FR4 WGAGTTVTVSS

33 Light FR1 LDIVMTQSPASLAVSLGQRATISCKAS

34 Light FR2 MNWYQQKPGQPPKLLIY

35 Light FR3 NLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC

36 Light FR4 FGGGTKLEIK

47 Heavy chain QVQLQQSGTVLARPGASVKMSCKASGYSFTNYRMYWV

KQRPGQGLEWIGAIYPGNSDTTYKQKFKGKAKLTAVT

(aa) SASTAYMELSSLTNEDSAVYYCTRGIRGSYFDVWGAG

TTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSS VTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGA

A

48 Light chain (aa) LDIVMTQSPASLAVSLGQRATISCKASQSVDYDGDSY

MNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGT DFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEI KRADAAPTVSIFPPSSEQLTSGGASVVCFLN FYPKD I VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

49 Heavy chain Caggtccagctgcagcagtctgggactgtgctggcaa

(nt) ggcctggggcttccgtgaagatgtcctgcaaggcttc tggctacagctttaccaactaccggatgtactgggta aaacagaggcctggacagggtctagaatggattggtg ctatttatcctggaaatagtgatactacctacaagca gaagttcaagggcaaggccaaactgactgcagtcaca tccgccagcactgcctacatggagctcagcagcctga caaatgaggactctgcggtctattactgtacaagggg gatacgggggtcgtacttcgatgtctggggcgcaggg accacggtcaccgtctcctcagccaaaacgacacccc catctgtctatccactggcccctggatctgctgccca aactaactccatggtgaccctgggatgcctggtcaag Table 2

SEQ ID Description Sequence

NO:

ggctatttccctgagccagtgacagtgacctggaact ctggatccctgtccagcggtgtgcacaccttcccagc tgtcctgcagtctgacctctacactctgagcagctca gtgactgtcccctccagcacctggcccagcgagaccg tcacctgcaacgttgcccacccggccagcagcaccaa ggtggacaagaaaattgtgcccagggattgtggtgcg gccgc

cttgacattgtgatgacacagtctccagcttctttggctgtgtct

50 Light chain (nt) ctagggcagagggccaccatctcctgcaaggccagccaaagtgtt gattatgatggtgatagttatatgaactggtaccaacagaaacca ggacagccacccaaactcctcatctatgctgcatccaatctagaa tctgggatcccagccaggtttagtggcagtgggtctgggacagac ttcaccctcaacatccatcctgtggaggaggaggatgctgcaacc tattactgtcagcaaagtaatgaggatccgtacacgttcggaggg gggaccaagctggaaataaaacgggctgatgctgcaccaactgta tccatcttcccaccatccagtgagcagttaacatctggaggtgcc tcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaat gtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctg aacagttggactgatcaggacagcaaagacagcacctacagcatg agcagcaccctcacgttgaccaaggacgagtatgaacgacataac agctatacctgtgaggccactcacaagacatcaacttcacccatt gtcaagagcttcaacaggaatgagtgttaataaggc

SEQ 1 D NO: 23 is identical to SEQ ID NO: 5

SEQ ID NO: 26 is identical to SEQ ID NO: 8

SEQ ID NO: 27 is identical to SEQ ID NO: 9

Table 3

SEQ ID NO: Description Sequence

37 VH CDR2 I Y P G N X₆ D T

38 VH CDR2 I Y P G N R/S D T

39 VH CDR3 TRGX₄X₅GX₇YFDX₁₁

40 VH CDR3 T R G V/I I/R G I/S Y F D Y/V

41 VL CDR3 Q Q X₃ N E X₆ P X₈ T

42 VL CDR3 Q Q N/S N E A/D P L/Y T An exemplary amino acid sequence of mesothelin precursor-protein (SEQ I D NO:51 ). This sequence is from the UN IPROT database (www.uniprot.org) and has the U NI PROT accession number Q13421 (NCBI accession number NP 037536.2) MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS LSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPL DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW SVSTMDALRGLLPVLGQPI IRSI PQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKT ACPSGKKAREI DESLIFYKKWELEACVDAALLATQMDRVNAI PFTYEQLDVLKHKLDELY PQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVA TLI DRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQ LDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVL PLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGI PNGYLVLDLS MQEALSGTPCLLGPGPVLTVLALLLASTLA

Another exemplary amino acid sequence of mesothelin precursor-protein (SEQ I D NO:52). This sequence is from the NCBI database and has the NCBI accession number AAH09272)

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFP CAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRF FSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVS CPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPS WRQPERTILRPRFRREVEKTACPSGKKAPEIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLD VLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVK GRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGS EYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVR DWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

The invention will now be further described in the following non-limiting Example with reference to the following drawings:

Figure 1 shows flow cytometry analysis of HeLa cells that have been membrane stained (fluorescently labelled) via the indicated anti-mesothelin Fab clones as described in the Example. Also included in Figure 1 are flow cytometry charts showing control data for the HeLa cells alone, Isotype Rat IgG, a commercially available anti-mesothelin antibody (R&D systems), an anti-myc antibody alone and secondary antibody alone (Cappel).

Figure 2 shows flow cytometry analysis of HeLa cells that have been membrane stained (fluorescently labelled) via the indicated anti-mesothelin Fab clones, when the Fabs either have (PBS+soluble protein 1 ^g) or have not (PBS) been pre- incubated with a soluble form of mesothelin (1 .4 μg, recombinant mesothelin, Raybiotech) prior to incubation with the HeLa cells. Figure 3 contains confocal microscope images of exosomes, serum high and serum low samples, in which the mesothelin is labelled via FITC (in green), CD63 is labelled via TexasRed (in red). Merged images are also shown.

Figure 4 depicts the results of flow cytometry analysis of HeLa cells that have been membrane stained (fluorescently labelled) via the indicated anti-mesothelin Fab clones, when the Fabs either have been pre-incubated with one of the stated forms/sources of mesothelin (serum low; serum high; exosomes; the recombinant soluble-form of mesothelin (O^g); or HeLa cell supernatants which were concentrated 10 times) prior to incubation with the HeLa cells, or have not been pre- incubated with any form/source of mesothelin (PBS; or exosome free DMEM) prior to incubation with the HeLa cells.

Figure 5 depicts the results of flow cytometry analysis of HeLa cells that have been membrane stained (fluorescently labelled) via the indicated anti-mesothelin Fab clones, when the Fabs have been pre-incubated with sera (plasma CLBL or plasma ROSI) prior to incubation with the HeLa cells. (Fam 6 = clone 1 h07 (MP01 H07 (6)); Fam 7= clone 3C02 (MP03C02 (7)); Fam 10= the family 10 clone). CLBL and ROSI correspond to metastatic cancer patient samples.

Figure 6 shows FACS analysis of staining by 1 H7-hFc on different cancer cells. CHO-meso vs CHO cells were used as a specificity control for 1 H7-hFc.

Example:

Anti-mesothelin antibodies which bind to mesothelin on cells

The inventors have identified a number of antibodies (Fabs) which bind to the membrane bound form of mesothelin on cells but do not bind to the soluble form of mesothelin. Antibodies were identified by a subtractive immunization protocol followed by phage display. The subtractive immunization and phage display protocols are described in WO2014/020139.

Fabs were produced from bacterial cultures in which the bacteria were grown in 2TY medium + ampicillin +glucose and addition of IPTG (IsoPropyl β-D-l - ThioGalactopyranoside).

The Fabs were tested as periplasmic extracts (P.E.) in binding flow cytometry (FACS) for their ability to bind to CHO-Mesothelin cells (CHO cells that have been transfected with mesothelin and thus are positive for mesothelin), CHO- WT cells (wild-type CHO cells) and HeLa cells (naturally mesothelin positive cells). In these experiments, membrane staining with the Fabs (P.E. diluted 1/5) was performed using 2x10⁵ cells. Fabs were first incubated with cells and an anti-myc antibody (9E10, Diaclone) (because the Fabs are myc tagged). Next, two washing steps were performed and cells were incubated with an anti-mouse antibody conjugated to a fluorescent dye (Cappel, MP). The cells were then analyzed by flow cytometry.

Fab binding levels were analyzed by looking at the percentage of positive signal cells obtained using the FL4-A channel detector and to the mean intensity levels of FL4 expression.

In these experiments, a number of Fabs, including clones 1 h07 and 3C02, were identified which bind to CHO-Mesothelin and HeLa cells but which do not bind to CHO-WT cells.

147 Fab clones were sequenced. These could be grouped into 35 families on the basis of their CDR3 sequences. One Fab clone from each family was tested by flow cytometry (as described above) to assess the ability to bind to mesothelin on HeLa cells. The data in Figure 1 shows that exemplary clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)) bind to mesothelin on HeLa cells.

That exemplary clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)) bind to mesothelin on HeLa cells is shown in Figure 1 by the fact that in comparison to, for example, the negative controls e.g. the anti-myc control, the 1 h07 and 3C02 Fabs cause a shift in the cell population from the lower left quadrant to the lower right quadrant of the flow cytometry plots. This is due to the fact that HeLa cells have been fluorescently labelled via the binding of these Fabs to membrane bound mesothelin. Further clones were also identified which are able to bind to mesothelin on HeLa cells (data not shown).

Data is also included in Figure 1 to show that another clone, MP02E02 (8), is not able to bind to mesothelin on HeLa cells. Also included in Figure 1 are flow cytometry charts showing control data for the HeLa cells alone, Isotype Rat IgG, a commercially available anti-mesothelin antibody (R&D systems), an anti-myc antibody alone and the secondary antibody alone (Cappel). Competition assays using the soluble form of mesothelin

A flow cytometry based competition assay was performed to assess whether the binding of anti-mesothelin antibodies (Fabs) to mesothelin on cells is altered (disturbed) by the presence of the soluble form (soluble shape) of mesothelin (recombinant human mesothelin, Raybiotech). In these experiments, positive clones (i.e. Fabs identified as having the ability to bind to mesothelin on cells) from the above-described flow cytometry screening were tested in a similar experiment in the presence (1 .4 μg) and absence of soluble mesothelin receptor (recombinant human mesothelin, Raybiotech). Before incubating the Fabs with HeLa cells (i.e. before the HeLa cells were added to the Fabs), the Fabs were pre-incubated with the soluble form of mesothelin (1 .4 μg per test) in PBS 1 X with an anti-c-myc antibody. Membrane staining of HeLa cells with the Fabs and flow cytometry analysis was then performed as described above. These experiments allowed identification of Fab clones which can bind to mesothelin on cells even when the soluble form of mesothelin is present. Put another way, using these experiments, Fab clones are identified which are specific for the cell membrane bound form of mesothelin on cells and do not significantly bind to the soluble form of mesothelin. For such Fabs the soluble form of mesothelin does not compete with the membrane bound form of mesothelin for Fab binding. Thus, anti-mesothelin Fabs are identified which can discriminate between the membrane bound form of mesothelin on cells and the soluble form of mesothelin, i.e. positively discriminate for the membrane bound form of mesothelin on cells. These experiments also allowed identification of those Fab clones which have a reduced ability (or no ability) to bind to mesothelin on cells when the soluble form of mesothelin is present.

Exemplary results from this experiment are shown in Figure 2. Clones 1 h07

(MP01 H07 (6)) and 3C02 (MP03C02 (7)) are examples of Fab clones which bind to the membrane-bound form of mesothelin on HeLa cells but do not significantly bind to the soluble-form of mesothelin. The left-hand flow cytometry chart in each of the pairs of flow cytometry charts in Figure 2 shows the situation in which the Fab has not been incubated with the soluble form of mesothelin prior to being incubated with the HeLa cells (PBS). In each of the left-hand flow cytometry charts in Figure 2 there is a significant fluorescently labelled cell population in the lower right quadrant, which is a result of the Fab binding to membrane-bound mesothelin on HeLa cells. This shows that these Fabs bind to the membrane-bound form of mesothelin on HeLa cells. The right-hand flow cytometry chart in each of the pairs of flow cytometry charts in Figure 2 shows the situation in which the Fab has been pre- incubated with the soluble form of mesothelin prior to being incubated with the HeLa cells (PBS + soluble protein 1 ^g). For clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)) a comparable fluorescently labelled cell population is observed in the lower right quadrant even when these Fabs have been pre-incubated with the soluble-form of mesothelin. This demonstrates that Fab clones 1 h07 and 3C02 do not significantly bind to the soluble-form of mesothelin and that these clones positively discriminate for the membrane-bound form of mesothelin on cells. Other discriminatory Fab clones which bind to the membrane-bound form of mesothelin and which do not bind to the soluble-form of mesothelin were also identified (data not shown).

In contrast to clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)), some Fab clones were identified which bind to the membrane-bound form of mesothelin on HeLa cells and also bind to the soluble form of mesothelin, i.e. some Fab clones were identified which do not discriminate between the membrane-bound and soluble forms of mesothelin. For example, as shown in Figure 2, clone MP05D08 (31 ) binds to both the membrane-bound and soluble forms of mesothelin. In the left- hand flow cytometry chart (PBS) for clone MP05D08 (31 ) in Figure 2 there is a fluorescently labelled cell population cell population in the lower right quadrant that is a result of the Fab binding to membrane-bound mesothelin on HeLa cells. This shows that clone MP05D08 (31 ) binds to the membrane-bound form of mesothelin on HeLa cells. However, when clone MP05D08 (31 ) is pre-incubated with the recombinant soluble form of mesothelin prior to incubation with HeLa cells, this Fab clone is not able to significantly bind to the membrane-bound form of mesothelin (there is no meaningful binding). This is evident from Figure 2, in which the right- hand MP05D08 (31 ) flow cytometry chart does not contain a significant fluorescent cell population in the lower right quadrant. Thus, clone MP05D08 (31 ) is an example of a Fab clone which recognizes both the membrane bound form and the soluble form of mesothelin.

In competition assays, the antibody must not be in excess. In order to not have an excess of Fab which might give a false impression of not having

competition we dilute the periplasmic extracts to the dilution where we see drop of signal on cell staining and use last dilution before the drop can be seen. Eight serial dilutions were performed starting from undiluted periplasmic extract Fabs, each dilution being half as concentrated as the previous. It was established that the limiting dilution retained for MP01 H07 (6) is 1/4 and the limiting dilution retained for MP03C02 (7) is 1/8. The limiting dilution retained for MP03C01 (10) is 1/2. The limiting dilution retained for MP03B05 (28) is 1/2. The limiting dilution retained for MP05D08 (31 ) is 1/2. These limiting dilutions were used in the next tests.

Quantification of mesothelin

In order to study mesothelin in different forms, HeLa cell supernatants

(which do not contain exosomes, i.e. contain soluble mesothelin) were concentrated 10 times and also exosomes were isolated from HeLa cell supernatant (via a series of ultracentrifugation). The ultracentrifugation (differential ultracentrifugation) protocol for the isolation (purification) of exosomes was in accordance with Thery et al., in Current Protocols in Cell Biology (2006), 3.22.1 - 3.22.29 (see page 3.22.2).

When assessing the concentration of mesothelin in these samples, exosome free DMEM (which is a culture medium used for HeLa cells) was used as a negative control. The concentration of mesothelin in sera derived from metastatic cancer patients (CLBL and ROSI) was also determined. To quantify the mesothelin from these different sources, a mesothelin ELISA assay was performed (BioLegend, Cat No. 438607, lot B182236). Table 4 below summarizes the various mesothelin concentrations.

Table 4

Confocal microscopy images of exosomes, serum high and serum low samples were taken. Confocal images are shown in Figure 3. The mesothelin is labelled via FITC (in green), CD63 is labelled via TexasRed (in red). These images show that mesothelin in present in exosomes and serum. CD63 is used as an exosome marker. Competition assays using various forms of mesothelin

A flow cytometry based competition assay of the type described above was performed to assess whether or not the binding of anti-mesothelin Fabs to mesothelin on cells is altered (disturbed) by the presence of other forms/sources of mesothelin (serum low; serum high; exosomes; the soluble-form of mesothelin

(O^g); HeLa cell supernatants (which were concentrated 10 times). Exosome free DMEM medium and PBS (phosphate-buffered saline) were used as controls. Fabs were pre-incubated with each form/source of mesothelin prior to incubating the Fabs with HeLa cells. Membrane staining of HeLa cells with the Fabs and flow cytometry analysis was then performed as described above. Fabs were used at the limiting dilutions described above. Anti c-myc antibody was used at 1/40. For the HeLa cell supernatant and sera assays, 80μΙ of HeLa supernatant or sera with anti-myc antibody at 1/40 was used per well. For the exosomes sample, all of the exosomes obtained after ultracentrifugation (the pellet) was resuspended in PBS in order to obtain 80μΙ with anti-myc antibody at 1/40. For the recombinant soluble-form of mesothelin 0^g of mesothelin was present in a volume of 200μΙ.

These experiments allowed identification of Fab clones which can bind to mesothelin on cells even when other forms of mesothelin are present, and also allowed identification of those Fab clones which have a reduced ability (or no ability) to bind to mesothelin on cells when other forms of mesothelin are present. Put another way, using these competition experiments, Fab clones can be identified which are specific for the cell membrane bound form of mesothelin on cells and do not significantly bind to other forms of mesothelin. In particular and surprisingly, Fab clones were identified which not only did not bind to soluble forms of mesothelin, e.g. as found in patients' serum, in HeLa supernatant, or provided as a recombinant form, but which also did not bind to mesothelin when present on exosomes. Thus, anti-mesothelin Fabs are identified which can discriminate between the membrane bound form of mesothelin on cells and other forms of mesothelin, i.e. positively discriminate for the membrane bound form of mesothelin on cells.

Exemplary results from this experiment are shown in Figure 4. Clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)) are examples of Fab clones which bind to the membrane-bound form of mesothelin on HeLa cells but do not bind to any of the other forms/sources of mesothelin tested. Figure 4 contains bar charts which depict the flow cytometry results. In Figure 4 the Y-axes correspond to the intensity of the labelling. The fact that in the bar charts for clones MP01 H07 (6) and MP03C02 (7) the bars (which reflect the ability of the Fabs to bind to membrane bound mesothelin on HeLa cells) for each of the mesothelin forms/sources that were used in the pre-incubation step have values (y-axis values) which are comparable with the bars for the PBS and exosome-free DMEM controls means that these Fabs do not bind significantly to any of the mesothelin forms/sources used in the pre-incubation step. These clones are thus shown to be specific for the membrane-bound form of mesothelin on cells. These clones do not bind

significantly to serum having a low concentration of mesothelin; do not bind significantly to serum having a high concentration of mesothelin; do not bind significantly to exosomes (isolated exosomes); do not bind significantly to the soluble (recombinant)-form of mesothelin; and do not bind significantly to

mesothelin in HeLa cell supernatants which were concentrated 10 times. Amino acid sequences of these exemplary and preferred antibodies 1 h07 and 3C02 are set forth in Tables 1 and 2 herein.

In contrast to clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)), some

Fab clones were identified which bind to the membrane-bound form of mesothelin on HeLa cells but which also bind to some other forms of mesothelin, i.e. some Fab clones were identified which do not discriminate between membrane-bound mesothelin on cells and some other forms of mesothelin. For example, as shown in Figure 4, clone MP05D08 (31 ) binds to the membrane-bound form of mesothelin on cells (see e.g. the bars for the PBS and exosome-free DMEM pre-incubations). However, clone MP05D08 (31 ) also binds to the soluble form of mesothelin. This is evidenced by the fact that a pre-incubation of this Fab with the soluble form of mesothelin results in the Fab showing no significant binding to the membrane- bound-form of mesothelin on cells in the competition assay (see the third bar from the left on the clone MP05D08 (31 ) bar chart). Thus, as described above, clone MP05D08 (31 ) is an example of a Fab clone which recognizes both the membrane bound form of mesothelin on cells and the soluble form of mesothelin.

Clone MP03B05 (28) binds to the membrane-bound form of mesothelin on cells but also shows some binding to each of the other forms/sources of mesothelin (recombinant soluble form, concentrated HeLa supernatant, exosomes (isolated exosomes), serum high and serum low). This is evidenced by the fact that the bars representing the soluble (recombinant) form of mesothelin, concentrated HeLa supernatant, exosomes (isolated exosomes), serum high and serum low pre- incubations have lower values than the bars for the PBS and exosome-free DMEM controls (Figure 4). Table 5 below shows the raw data values for the flow cytometry based petition assay data depicted in Figure 4.

Table 5

Competition assay using other plasma sources

A further competition assay was performed as described above for new sera (plasma CLBL and plasma ROSI). Fabs were pre-incubated with these new sera prior to incubation with HeLa cells and membrane staining as described above. Clones 1 h07 (MP01 H07 (6)) and 3C02 (MP03C02 (7)) were tested in these experiments. A further anti-mesothelin Fab clone, a clone from "family 10" was also tested. PBS was used as a negative control. The results are shown in Figure 5 (Fam 6 = 1 h07 (MP01 H07 (6)); Fam 7= 3C02 (MP03C02 (7)); Fam 10= the family 10 clone). Pre-incubation of Clones 1 h07 and 3C02 with either plasma CLBL or plasma ROSI (containing soluble mesothelin) did not significantly alter the ability of these Fabs to bind to the membrane bound form of mesothelin on HeLa cells. This is further evidence that Clones 1 h07 and 3C02 are specific for the membrane-bound form of mesothelin on cells. Pre-incubation of the family 10 clone with either plasma CLBL or plasma ROSI significantly reduced the ability of this Fab to bind to the membrane bound form of mesothelin on HeLa cells.

1 H7-hFc specifically binds mesothelin expressing cancer cells

We sought to determine if our discriminating antibodies target mesothelin expressing cancer cells. For this purpose, we chose two Fabs, 1 H7 as a candidate discriminatory antibody and 3C1 as a non-discriminating antibody, for chimerization with human lgG1.

1 H7 and 3C1 were chimerized at RD Biotech (Besangon, France) with human lgG1 expressing mammalian vector and transfected to CHO cells. Cell culture supernatants were collected and antibody concentration was determined by RD Biotech.

Binding capacity of 1 H7-hFc was then assessed on different cancer cell lines (Figure 6).

Using FACS analysis, we observed that 1 H7-hFc bound Hela and CHO-

Meso, a cell line over-expressing human mesothelin, but not CHO cells. 1 H7-hFc strongly bound breast cancer cell line MDA-MB231 , moderately recognized colon cancer cells (HT29 cells) and showed some binding to non-small cell lung cancer cells (A549 cells). No binding could be observed for the cervix cancer cell line C33A, which is a mesothelin negative cell line (Figure 6). In order to assess if 1 H7- hFc could recognize primary cell lines, we tested three different pancreatic cancer cells BesPac-C7, BesPac-C8 and BesPac-C12, obtained in our laboratory from ascites of pancreatic cancer patients. Flow cytometry analysis showed that 1 H7-hFc bound strongly BesPac-C7 and BesPac-C12, and to some extent to BesPac-C8 cells. Mesothelin expression on these cells was confirmed by qRT-PCR and western blotting experiments (data not shown). These results demonstrated that 1 H7-hFc selectively recognizes native mesothelin on different types of cancer cells. 1 H7 does not recognize GPI anchor

Experiments presented above demonstrate that 1 H7 is selective for the membrane associated conformation of the mesothelin protein and does not recognize other conformations such as the recombinant form or that contained within the physiological fluids from mesothelin expressing cancer patients. It remained a possibility that 1 H7 is recognizing some component of the GPI anchor or a shared epitope between the GPI anchor and the protein. In order to decipher the role of the GPI-anchor in the epitope recognition of 1 H7-hFc we assessed whether 1 H7 might be a non-specific antibody to GPI. To eliminate this hypothesis PC3 and LN-CAP cells which do not express mesothelin but highly express another GPI anchored protein CD59 were stained by 1 H7-hFc and analysed via FACS analysis. The cells were highly positive for CD59 but not for 1 H7-hFc which confirmed that 1 H7 does not recognize the GPI anchor (data not shown).

We hypothesized that 1 H7-hFc might bind a shared epitope between the GPI anchor and mesothelin. This hypothesis was also eliminated by using a FITC coupled pre-aerolysin protein, FLAER, which binds specifically to GPI (van Zanten TS et al., Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18557-62.). We determined the saturation concentration of FLAER by staining Hela cells. Then, cells were incubated with 25, 50_T and 100nM of FLAER and analyzed by flow cytometry. Relative median fluorescent intensities (RMFI) were 4.8, 5 and 5.2 for each concentration, respectively. 50nM was then selected as a saturation concentration (data not shown) and Hela cells were incubated with 50nM of FLAER one hour before staining with 1 H7-hFc. Flow cytometry analysis showed that the binding properties of 1 H7 were not altered by the presence of FLAER (data not shown). These results suggest that GPI is not involved directly in the epitope recognition by 1 H7.

Claims

1 . An isolated antibody which binds to mesothelin, wherein said isolated antibody:

(i) binds to the membrane-bound form of mesothelin on cells;

(ii) does not bind significantly to the soluble form of mesothelin; and

(iii) does not bind significantly to mesothelin on extracellular vesicles.

2. The antibody of claim 1 , wherein said antibody comprises a variable heavy (VH) CDR3 that has the amino acid sequence of T R G X₄ X₅ G X₇ Y F D Xn (SEQ ID NO:39),

wherein X₄ can be any amino acid, preferably V or I;

X₅ can be any amino acid, preferably I or R;

X₇ can be any amino acid, preferably I or S; and

Xn can be any amino acid, preferably Y or V.

3. The antibody of claim 1 or claim 2, wherein said antibody comprises a variable light (VL) CDR3 that has the amino acid sequence of Q Q X₃ N E X₆ P X₈ T (SEQ ID NO:41 ),

wherein X₃ can be any amino acid, preferably N or S;

X₆ can be any amino acid, preferably A or D; and

X₈ can be any amino acid, preferably L or Y.

4. The antibody of any one of claims 1 to 3, wherein said antibody comprises a variable heavy (VH) CDR2 that has the amino acid sequence of I Y P G N X₆ D T (SEQ ID NO:37),

wherein X₆ can be any amino acid, preferably R or S.

5. The antibody of any one of claims 1 to 4, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs,

wherein said heavy chain variable region comprises:

(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:5 or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence, or wherein said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence;

(ii) a variable heavy (VH) CDR2 that has the amino acid sequence of I Y P G N X₆ D T (SEQ ID NO:37), wherein X₆ can be any amino acid, preferably R or S;

and

(iii) a variable heavy (VH) CDR3 that has the amino acid sequence of T R G X₄ X₅ G X₇ Y F D Xn (SEQ ID NO:39),

wherein X₄ can be any amino acid, preferably V or I;

X₅ can be any amino acid, preferably I or R;

X₇ can be any amino acid, preferably I or S; and

Xii can be any amino acid, preferably Y or V.

6. The antibody of any one of claims 1 to 5, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs,

wherein said light chain variable region comprises:

(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO:8 or a sequence substantially homologous thereto wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given

CDR sequence, or wherein said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence; a variable light (VL) CDR2 that has the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto wherein said substantially homologous sequence is a sequence containing 1 , 2 or 3 amino acid substitutions compared to the given CDR sequence, or wherein said substantially homologous sequence is a sequence containing conservative amino acid substitutions of the given CDR sequence; and

a variable light (VL) CDR3 that has the amino acid sequence of Q Q X₃ N E X₆ P X₈ T (SEQ ID NO:41 ),

wherein X₃ can be any amino acid, preferably N or S;

X₆ can be any amino acid, preferably A or D; and

X₈ can be any amino acid, preferably L or Y.

7. The antibody of any one of claims 1 to 6, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

wherein said heavy chain variable region comprises:

(d) a variable heavy (VH) CDR1 that has the amino acid sequence of

SEQ ID NO:5 or a sequence substantially homologous thereto,

8. The antibody of any one of claims 1 to 7, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ

ID NO:8,

(b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9, and

(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10; and wherein said heavy chain variable region comprises:

(d) a variable heavy (VH) CDR1 that has the amino acid sequence of

SEQ ID NO:5,

(e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6, and

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7.

9. The antibody of any one of claims 1 to 6, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

wherein said heavy chain variable region comprises:

10. The antibody of any one of claims 1 to 6 or claim 9, wherein said antibody comprises at least one heavy chain variable region that comprises three CDRs and at least one light chain variable region that comprises three CDRs, wherein said light chain variable region comprises:

(a) a variable light (VL) CDR1 that has the amino acid sequence of SEQ

ID NO:8,

(b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9, and

(c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:28; and wherein said heavy chain variable region comprises:

(d) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO:5,

(e) a VH CDR2 that has the amino acid sequence of SEQ ID NO:24, and

(f) a VH CDR3 that has the amino acid sequence of SEQ ID NO:25.

1 1 . The antibody of any one of claims 1 to 8, wherein said antibody has a VH domain of SEQ ID NO: 3 or a sequence having at least 80% sequence identity thereto, and/or a VL domain of SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto.

12. The antibody of claim 1 1 , wherein said antibody has a VH domain of SEQ ID NO: 3 and a VL domain of SEQ ID NO: 4.

13. The antibody of any one of claims 1 to 6 or claims 9 or 10, wherein said antibody has a VH domain of SEQ ID NO: 21 or a sequence having at least 80% sequence identity thereto, and/or a VL domain of SEQ ID NO: 22 or a sequence having at least 80% sequence identity thereto.

14. The antibody of claim 13, wherein said antibody has a VH domain of SEQ ID NO: 21 and a VL domain of SEQ ID NO: 22.

15. The antibody of any one of claims 1 to 14, wherein said antibody is an antigen binding fragment of an antibody, preferably a Fab fragment.

16. An immunoconjugate comprising the antibody of any one of claims 1 to 15 attached to a therapeutic agent, preferably a toxin.

17. A composition comprising at least a first antibody of any one of claims 1 to 15 or an immunoconjugate thereof, preferably said composition is a

pharmaceutically acceptable composition.

18. A nucleic acid molecule comprising a nucleotide sequence that encodes an antibody according to any one of claims 1 to 15, or a nucleic acid molecule comprising a nucleotide sequence that encodes a heavy chain variable region or a light chain variable region of an antibody according to any one of claims 1 to 15.

19. A method of producing an antibody according to any one of claims 1 to 15, comprising the steps of:

(i) culturing a host cell comprising one or more nucleic acid molecules as defined in claim 18 or one or more recombinant expression vectors comprising one or more of said nucleic acid molecules under conditions suitable for the expression of the encoded antibody; and

(ii) isolating or obtaining the antibody or protein from the host cell or from the growth medium/supernatant.

20. An antibody according to any one of claims 1 to 15, or an immunoconjugate thereof, for use in therapy.

21 . The antibody, or immunoconjugate thereof, of claim 20 for use in therapy of claim 20, wherein said therapy is the treatment of cancer.

22. A method of treating cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody as defined in any one of claims 1 to 15, or an immunoconjugate thereof.

23. Use of an antibody as defined in any one of claims 1 to 15, or an

immunoconjugate thereof, in the manufacture of a medicament for use in therapy, preferably cancer therapy.