WO2011139375A1

WO2011139375A1 - Antibodies directed against carbonic anhydrase ix and methods and uses thereof

Info

Publication number: WO2011139375A1
Application number: PCT/US2011/000796
Authority: WO
Inventors: Christoph Renner; Stefan Bauer; Margarita Plesko; Alzbeta Hulikova; Pawel Swietach
Original assignee: Ludwig Institute For Cancer Research Ltd; Chancellor Masters And Scholars Of The University Of Oxford; University Of Zurich
Priority date: 2010-05-06
Filing date: 2011-05-06
Publication date: 2011-11-10

Abstract

Specific binding members, particularly antibodies and fragments thereof, which bind to Carbonic Anhydrase IX (CAIX) are provided, particularly including recognizing both human and mouse CAIX and blocking or inhibiting CAIX activity. These antibodies are useful in the diagnosis and treatment of cancer and in conditions associated with hypoxia and/or elevated CAIX activity. The anti-CAIX antibodies, variable regions or CDR domain sequences thereof, and fragments thereof may also be used in therapy in combination with chemotherapeutics, immune modulators, or anti¬ cancer agents and/or with other antibodies or fragments thereof. Antibodies of this type are exemplified by the novel antibodies MSC 1 through MSC 12 whose sequences are provided herein.

Description

ANTIBODIES DIRECTED AGAINST CARBONIC ANHYDRASE IX AND

METHODS AND USES THEREOF

FIELD OF THE INVENTION

[0001 J The present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to Carbonic Anhydrase IX (CAIX) are provided, particularly including recognizing both human and mouse CAIX and blocking or inhibiting CAIX activity. These antibodies are useful in the diagnosis and treatment of cancer and in conditions associated with hypoxia and/or elevated CAIX activity. In addition, they can be used for the diagnosis and treatment of hypoxic tumors and/or of CAIX positive tumors such as renal cancer. The antibodies, variable regions or CDR domain sequences thereof, and fragments thereof of the present invention may also be used in therapy in combination with chemotherapeutics, immune modulators, or anti-cancer agents and/or with other antibodies or fragments thereof.

BACKGROUND OF THE INVENTION

[0002] Solid tumours need to find strategies to overcome oxygen- and nutrients- deficiencies due to high metabolic rate and excessive growth. Solid tumors which have regions of very low oxygen concentrations are said to be hypoxic. Hypoxia is a natural phenomenon of solid tumors and results from an insufficient vascular network. Production of angiogenic growth factors triggers neoangiogenesis, which restores the supply of tumour tissue with oxygen and nutrients. Moreover, anaerobic respiration is achieved by glycolysis. Tumor hypoxia results in resistance to ionizing radiation, resistance to chemotherapy and the magnification of mutated p53. In addition, tissue hypoxia has been regarded as a key factor for tumour aggressiveness and metastasis by activation of signal transduction pathways and gene regulatory mechanisms. Hypoxia in solid tumors promotes a strong oncogenic phenotype, has been strongly associated with tumor propagation, malignant progression and resistance to chemo- and radiotherapy (21 ), and is a phenomenon that occurs in all solid tumors. |0003] Hypoxia regulates the expression of several genes, including Hypoxia-Inducible Factor 1 (HIF- 1). The hypoxia inducible factor HIF- 1 is a transcription factor regulating gene expression due to induction by hypoxia. HIF- 1 leads to expression of target genes containing a hypoxia-responsive element (HRE), as e.g. genes involved in Glucose transport or in neoangiogenesis ( 1). HIF- 1 can activate a large number of genes including many of those responsible for cell proliferation and apoptosis, glucose metabolism, pH regulation, erythropoiesis, iron metabolism, extracellular matrix metabolism, inflammation, angiogenesis and control of vascular tone (22).

[0004] HIF- 1 is a heterodimer consisting of an inducible subunit HIF- l a and a constitutively expressed subunit HIF- 1 β (2), (3). Regulation is achieved by HIF- la which is instable under normoxia. Hydroxylation of proline residues in the oxygen-dependent degradation domain of HIF- l a leads to ubiquitination of HIF- la by the von Hippel-Lindau tumour suppressor protein (pVHL) followed by degradation in the 26S proteosome ( 1 ). Hypoxia inhibits binding of pVHL to HIF- l a, resulting in accumulation of HIF-l a and dimerization with the constitutively expressed subunit HIF- 1 β. Some cancer cell lines, for instance and particularly renal cell carcinomas, contain a mutation in pVHL leading to constitutive expression of HIF- 1 due to missing ubiquitionation and degradation of the HIF-la subunit (3).

[0005] One target of HIF-1 is the gene encoding Carbonic anhydrase IX (CAIX, CA9). CAIX is one of the most inducible and most uniformly HIF- 1 induced genes (23). Moreover because of its longer half-life (around 38 hours compared to minutes to HIF-1 (24)) and membrane location, it has become a reliable histological marker of hypoxia (25-27). The transmembrane protein is upregulated under hypoxia by HIF- 1 and, therefore, expressed by various solid cancers. The expression of CAIX, as evaluated by immunohistochemical techniques in several human cancers, has revealed a prognostic significance in renal (28), breast (29), bladder (30), head and neck (3 1 ,32), cervix carcinomas (33), soft tissue sarcoma (34) and in non-small cell lung carcinoma (35,36). In the gastrointestinal tract, its expression has been demonstrated in esophageal, gastric, colorectal, biliary, and pancreatic adenocarcinomas (range, 34%-80%) (37-39). High CAIX expression has been observed to correlate with poor prognosis in both esophageal and gastric adenocarcinoma (40). Despite elevated expression in malignancies, CAIX is generally absent from the normal tissues from which these tumors develop. Expression of CAIX in normal human tissues is restricted to the gastrointestinal tract, particularly epithelial cells, namely those lining the stomach, small intestine and gallbladder (41). It is thought to maintain gastric mucosa integrity (4).

[0006] In some cancer cells, e.g. renal cell carcinomas, an inactivating mutation in the gene encoding pVHL leads to constitutive expression of Carbonic anhydrase IX due to VHL deficiency, whereas in non-VHL tumours expression is linked to hypoxia (4), (5), (6), (7). In vitro induction of HlF- 1 can be achieved in a hypoxic chamber containing reduced oxygen levels as well as by chemical inductors mimicking hypoxic conditions e.g. cobalt chloride and dimethyloxalylglycine (DMOG) (8), (9), (10). Carbonic anhydrase IX belongs to a family of 16 isoenzymes. They are Zn-containing metalloenzymes catalyzing the reversible hydration of. carbonate to hydrogen carbonate and protons. Although other carbonic anhydrases contain the same catalytic activity, carbonic anhydrase IX shows especially high acidification rates. Carbonic anhydrases play an important role in acid-base regulation, respiration, electrolyte secretion and biosynthetic reactions with bicarbonate as a substrate ( 1), ( 1 1 ). Carbonic anhydrase IX was originally detected in HeLa cells as a cell density regulated membrane antigen named MN (12). Carbonic anhydrase IX is a transmembrane protein consisting of four domains. A short C- terminal intracellular tail is followed by the transmembrane domain. The extracellular part contains a catalytic Carbonic anhydrase domain (CA) and the N-terminal proteoglycan-like domain (PG) that is similar to the keratin sulphate attachment domain of a large proteoglycan aggrecan ( 15). The PG is absent in other Carbonic anhydrase family members known at present, making it a unique marker of Carbonic anhydrase IX (18). In tumours, the stroma is acidified due to Carbonic anhydrase IX activity (17). This acidification could be involved in tumour progression and development of metastases. New studies show that not only expression levels of Carbonic anhydrase IX but also its activity is regulated by HIF- 1 ( 14). Prevalent expression in tumour tissue as well as its catalytic activity make Carbonic anhydrase IX a promising target for tumour targeting and therapy.

[0007] Currently, there exist mainly two interesting antibodies against human carbonic anhydrase IX. The murine anti-G250 antibody recognizes the renal cell carcinoma-associated antigen G250 shown to be homologous to Carbonic anhydrase IX and MN (13). The murine antibody M75 recognizes a linear epitope in the PG domain of Carbonic anhydrase IX ( 12), ( 16), ( 18). One major drawback of the anti-G250 antibody is its inability to recognize denatured Carbonic anhydrase IX, limiting its use in immunohistochemistry. Moreover, none of the currently existing antibodies is cross-reactive to the murine isoform of Carbonic anhydrase IX or inhibits the catalytic activity of Carbonic anhydrase IX. Therefore, generation of new antibodies recognizing both isoforms, the human and the murine one, and/or which inhibit the catalytic activity of CAIX, would be highly desirable. Cross-reactivity greatly enlarges the application field of selected antibodies. They can be tested in vivo in mouse models, providing important information about binding behaviour, tumour accumulation and cytotoxicity of selected antibodies. The availability of antibodies that inhibit CAIX activity provides the opportunity for enhanced and useful therapeutic effects and clinical applications.

[0008] Thus, while the extant evidence of activity of CAIX antibodies is encouraging, the observed limitations on efficacy and anti-tumor activity remain. Accordingly, it would be desirable to develop CAIX antibodies, particularly antibodies which can be utilized in mouse animal models and which demonstrate increased efficacy and applicabi lity in diagnosis and therapy, and it is toward the achievement of that objective that the present invention is directed.

[0009] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[00010] The invention provides antibodies directed against Carbonic anhydrase IX (CAIX) for diagnostic and therapeutic purposes. In particular, antibodies specific for CAIX are provided, wherein said antibodies recognize and are capable of binding human and mouse CAIX. In particular, antibodies specific for CAIX are provided, wherein said antibodies are capable of blocking or inhibiting CAIX activity, particularly CAIX catalytic activity. Fab antibodies are particularly provided herein. The antibodies of the present invention have diagnostic and therapeutic use in cancer and in conditions associated with hypoxia and/or elevated CA IX activity. In a particular aspect the antibodies of the invention are applicable in cancers, including renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, biliary and pancreatic adenocarcinoma.

[0001 1 1 In a general aspect, the present invention provides CAIX antibodies, including antibodies directed against both human and mouse CAIX and antibodies capable of inhibiting or reducing CAIX activity. In a broad aspect, the present invention provides an isolated specific binding member, particularly an antibody or fragment thereof, including a Fab fragment and a single chain or domain antibody, which recognizes human CAIX. In a further aspect, the present invention provides an antibody or fragment thereof, which recognizes human CAIX and is selected from antibodies MSC l , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSCl 1 and MSCl 2. In a further aspect, the present invention provides an antibody or fragment thereof, which recognizes human CAIX and is selected from antibodies MSCl , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC10, MSC 1 1 and MSC12 and comprises the amino acid sequence of any of MSCl through MSC 12 as set out in FIGURES 5 through 16. In one such aspect, the invention provides an anti-CAIX antibody comprising the variable region heavy and light chain CDR sequences set out in FIGURES 5 through 16, and in TABLE 3 and TABLE 4. In an aspect, the invention provides an anti-CAIX antibody comprising the variable region heavy and light chain CDR sequences of any antibody of MSC l , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC10, MSCl 1 and MSC12, as set out in FIGURES 5 through 16.

[00012] In a particular aspect, the antibody or fragment of the invention is reactive with, capable of binding human and mouse CAIX. In a particular aspect, the antibody or fragment of the invention is reactive with, capable of binding, and specific for human CAIX. In a particular aspect, the antibody or fragment of the invention is reactive with, capable of binding human CAIX and directly affects the catalytic activity of CAIX. In an additional aspect, the antibody or fragment blocks or inhibits the catalytic activity of human CAIX and therefore reduces the activity of CAIX in a cell, particularly in a cancer or tumor cell.

[00013] The present inventors have discovered novel CAIX antibodies which are reactive to human CAIX. The novel CAIX antibodies may be selected from MSCl , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC10, MSC1 1 and MSC12. The inventors have discovered novel CAIX antibodies which are reactive to human and mouse CAIX. The novel human and mouse CAIX reactive antibody may be MSC3. The novel human and mouse CAIX reactive antibody may be selected from MSC l , MSC 7 and MSC9. The present inventors have discovered novel CAIX antibodies which are reactive to human CAIX and inhibit the activity of CAIX, particularly including inhibit the catalytic activity of CAIX. The novel CAIX inhibit antibody may be selected from MSC8. The antibodies exemplified herein include Fab antibodies and recombinant antibodies based thereon. Exemplary antibodies provided include MSCl , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC10, MSCl 1 and MSC 12 and comprise the variable region sequences as set out herein and in FIGURES 5 through 16. The antibodies have the heavy and light chain variable region sequences and comprise CDR domain region sequences as set out herein and in FIGURES 5 through 16.

[00014] The unique specificity and affinity of the antibodies and fragments of the invention provides diagnostic and therapeutic uses to identify, characterize and target conditions associated with cancer, solid tumors and/or hypoxia, particularly oxygen and/or nutrient deficient solid tumors, particularly without the problems associated with normal tissue uptake or with effects on inherent carbonic anhydrase activity, particularly CAIX in normal cells or tissues. Cancers, particularly solid tumors, which are oxygen or nutrient deficient and hypoxic and thereby trigger CAIX expression and activity are particularly susceptible to and targeted by the antibodies of the present invention. Such cancers include renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, biliary and pancreatic adenocarcinoma.

[00015] In a preferred aspect, the antibody is one which has the characteristics of the antibodies which the inventors have identified and characterized. In a particular aspect, the antibody recognizes human CAIX. In a particular such aspect the antibody is selected from MSC 1 , SC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC 1 1 and MSC 12, or active fragments thereof. In a particular preferred aspect, the antibody recognizes both human and mouse CAIX. In a particularly preferred aspect the antibody is MSC3, or active fragments thereof. In a particular preferred aspect, the antibody recognizes human CAIX and inhibits or blocke the activity of CAIX. In a particularly preferred aspect the antibody is MSC8, or active fragments thereof. In a further preferred aspect the antibody of the present invention comprises the VH and VL amino acid sequences depicted in any of FIGURES 5 through 16. In a particular aspect, the antibody of the invention comprises the heavy chain and light chain CDR sequences depicted in any of FIGURES 5 through 16. In a particular aspect of the invention the antibody is MSC3 and comprises the variable region sequences set out in FIGURE 7. In a particular aspect of the invention the antibody is MSC8 and comprises the CDR region sequences set out in FIGURE 12. In a particular aspect of the invention the antibody recognizes human CAIX and comprises the CDR region sequences selected from those set out in TABLE 3 and TABLE 4, including variants thereof.

[00016] The binding of an antibody to its target antigen is mediated through the complementarity-determining regions (CDRs) of its heavy and light chains. Accordingly, specific binding members based on the CDR regions of the heavy or light chain, and preferably both, of the antibodies of the invention, particularly of any of MSC 1 , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC1 1 and MSC 12, will be useful specific binding members for therapy and/or diagnostics. The sequences and CDRs of the antibodies are depicted in FIGURES 5 through 16. Light and heavy chain variable region sequences for CAIX antibodies are provided in TABLES 3 and 4. Antibody MSC3 comprises heavy chain CDR sequences GFTFSSYA, ISGSGGST and AKGGGTGTTVIFDY, and light chain CDR sequences VSNLGAGYE, GNS and QSYDRSLTEWV, as set out in FIGURE 7. Antibody MSC8 comprises heavy chain CDR sequences GGSFSGYY, INHSGST and ARGSGANYYDSSREPRAFDI and light chain CDR sequences SGINVDTYM, YKSESNQ and MIWHSNTWV, as set out in FIGURE 12.

[00017] Accordingly, specific binding proteins such as antibodies which are based on the CDRs of the antibody(ies) identified herein will be useful for targeting CAIX, particularly CAIX expressing cells in diseases or in cancers.. As the CAIX target of the antibodies of the invention is not significantly expressed in normal cells or cells which are not hypoxic the antibodies of the invention do no significantly bind to normal somatic^" cells, it is anticipated that there will not be significant uptake in normal tissue and there will be suitable and specific affinity for the CAIX target.

[00018] In a further aspect, the present invention provides an isolated antibody or fragment thereof capable of binding an antigen, wherein said antibody or fragment thereof comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out herein and in any of FIGURES 5 through 16.

[00019] In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member. In one such aspect, a nucleic acid encoding antibody variable region sequence having the amino acid sequences as set out in any of FIGURES 5 through 16 is provided or an antibody having CDR domain sequences as set out in any of FIGURES 5 through 16 is provided. In one aspect, a nucleic acid of any of FIGURES 5 through 16 is provided. The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes an antibody of the present invention; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the antibody VH and VL, particularly the CDR region sequences, which has a sequence or is capable of encoding a sequence as shown in any of FIGURES 5 through 16.

100020] The antibodies, fragments thereof and recombinant antibodies comprising the CDR domains according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a tumor in a human patient which comprises administering to said patient an effective amount of the antibodies, fragments thereof and recombinant antibodies of the invention.

[00021 ] The present invention also includes polypeptides or antibodies having the activities noted herein, and that display the amino acid sequences set forth and described above and in any of FIGURES 5 through 16 or are antibodies having a heavy chain and a light chain wherein the complementarity determining regions (CDRs) of the heavy and light chain comprise the amino acid sequences depicted in each or any of FIGURES 5 through 16.

[00022] The diagnostic utility of the present invention extends to the use of the antibodies of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays. In an immunoassay, a control quantity of the antibodies, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

[00023] Specific binding members of the invention may carry a detectable or functional label. The specific binding members may carry a radioactive label, such as the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶C1, ⁵¹Cr, "Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²¹I, ^{, 24}I, ¹²⁵I, ^{, 31}I, ^mIn, ^{1 17}Lu, ^{2 l l}At, ^{l 98}Au, ⁶⁷Cu, ²²⁵Ac, l³Bi, ⁹ Tc and ^{l 86}Re. When radioactive labels are used, known currently available counting procedures may be utilized to identify and quantitate the specific binding members. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art. Thus, the antibodies of the invention may be utilized in in voitro or in vivo diagnostic methods to identify and characterize cancer cells or tumors, including those which are hypoxic and/or thaose which express CAIX.

[00024] The radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques. In a further aspect of the invention, radiolabelled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy. In a still further aspect, the radiolabelled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, hypoxic cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.

[00025] Immunoconjugates or antibody fusion proteins of the present invention, wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

[00026] The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of or the activity of, for instance, CAIX. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the antibody, and one or more additional immunochemical reagents, at least one of which is a free or immobilized components to be determined or their binding partner(s).

[00027] In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the binding member, antibody, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention or treatment of cancer, including but not limited to renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, biliary and pancreatic adenocarcinoma.

[00028] The binding members and antibodies of the present invention, and in a particular embodiment the antibody whose sequences are presented in any of FIGURES 5 through 16 herein, or active fragments thereof, and single chain, recombinant or synthetic antibodies derived therefrom, particularly comprising the CDR region sequences depicted in any of FIGURES 5 through 16, can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, for administration in instances wherein therapy is appropriate, such as to treat cancer.

Such pharmaceutical compositions may also include methods of modulating the half-life of the binding members, antibodies or fragments by methods known in the art such as pegylation. Such pharmaceutical compositions may further comprise additional antibodies or therapeutic agents.

[00029] A composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, antimitotic agents, apoptotic agents or antibodies, or immune modulators. More generally these anticancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post- translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), inhibitors or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. In addition, the composition may be administered with immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines or hormones such as dexamethasone which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-CAIX antibodies or other anti-tunor antigen antibodies.

[00030] The present invention also includes antibodies and fragments thereof, which are covalently attached to or otherwise associated with other molecules or agents. These other molecules or agents include, but are not limited to, molecules (including antibodies or antibody fragments) with distinct recognition characteristics, toxins, ligands, and chemotherapeutic agents. In an additional aspect the antibodies or fragments of the invention may be used to target or direct therapeutic molecules or other agents, for example to target molecules or agents to CAIX expressing cells or to hypoxic cells, for example to cancer cells or tumor cells or oxygen and/or nutrient deficient cancer cells, tumor sites, inflammatory areas or cancerous lesions.

[00031] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS [00032] FIGURE 1: Coomassie staining of His-purified anti-Carbonic anhydrase IX Fabs. Lanes 1 - 12: Fabs MSC 1 -MSC 12. Lane 13: Fab ESC 1 1 recognizing the Fibroblast activated protein FAP was used as a negative control for all experiments.

[00033] FIGURE 2A and 2B: Binding specificity of the purified constructs. Flow cytometric analysis of Fab MSC 3 on human (A) or murine (B) cell lines induced for 48 h in 0.2% 0₂ (grey) or normoxia (white).

[00034] FIGURE 3: Affinity of Fab MSC 8 as monitored by surface plasmon resonance. Serial dilutions of purified Fab were injected on a rhCAIX-coated CM5 sensor chip. Values were corrected for binding to the reference flow cell. The solid lines represent the theoretical curves for each Fab concentration calculated according to the 1 : 1 binding model. K_A, ¾ and KD values calculated by the 1 : 1 binding model are shown in the table.

[00035] FIGURE 4A and 4B: Competition assay. A. Competition assay with M75. Grey: MSC 8 - M75, black: irrelevant Fab - M75, white: PBS - M75. B. Competition assay of MSC 8 compared to G250. Grey MSC 8 - mG250, white: PBS - mG250, black: chG250 - mG250.

[00036] FIGURE 5 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSCl . The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSCl are provided respectively in SEQ ID NOS: 1-4.

[00037] FIGURE 6 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC2. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC2 are provided respectively in SEQ ID NOS: 5-8.

[00038] FIGURE 7 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC3. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC3 are provided respectively in SEQ ID NOS: 9- 12.

[00039] FIGURE 8 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC4. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC4 are provided respectively in SEQ ID NOS: 13-16.

[00040] FIGURE 9 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC5. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC5 are provided respectively in SEQ ID NOS: 17-20.

[00041 ] FIGURE 10 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody SC6. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC6 are provided respectively in SEQ ID NOS: 21 -24.

[00042] FIGURE 11 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC7. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC7 are provided respectively in SEQ ID NOS: 25-28.

[00043] FIGURE 12 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC8. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC8 are provided respectively in SEQ ID NOS: 29-32.

|00044] FIGURE 13 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC9. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC9 are provided respectively in SEQ ID NOS: 33-36.

[00045] FIGURE 14 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSCI O. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC I O are provided respectively in SEQ ID NOS: 37-40.

[00046] FIGURE 15 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC 1 1. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC1 1 are provided respectively in SEQ ID NOS: 41 -44.

[00047] FIGURE 16 depicts the DNA sequence and amino acid sequence of the light chain variable region and the heavy chain variable region of antibody MSC 12. The CDRs are noted in blue/bold. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC 12 are provided respectively in SEQ ID NOS: 45-48.

[00048] FIGURE 17A through 17C shows selection of CAIX-specific antibodies.

Antibody binding to (A) wild-type (dashed) or CAIX-transfected (shaded) SKRC 17 cells, (B) HeLa cells under normoxic (dashed) or hypoxic (shaded) conditions and (C) murine CT26 cells under normoxic (dashed) or hypoxic (shaded) conditions was studied by flow cytometry. Sample analyses are shown for SC3 and MSC8 Fab antibody fragments. Fab ESC 1 1 recognizing human fibroblast activation protein and antibodies against either human (M75) or murine CAIX (M- 100) served as controls.

[00049] FIGURE 18 provides confirmation of selectivity of the selected antibodies.

Antibody binding to carbonic anhydrase isoforms II, IV, IX, XII and XIV was analyzed in ELISA on recombinant proteins. ESC1 1 IgG recognizing human fibroblast activation protein served as negative control. Coating was confirmed by staining with a Penta-His antibody.

Absorbance at 450 nm (A450) is shown for one representative experiment using MSC 8 IgG. Samples were measured in triplicate. Significance was assessed using a paired two-tailed T-test. **: P < 0.01 .

100050] FIGURE 19A through 19D depicts in vitro carbonic anhydrase activity assay on HCTl 16 membrane fragments. (A) Time course for C0₂ hydration with membrane-free buffer (dotted), HCT l 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC 8 Fab (dashed). (B) Carbonic anhydrase activity, normalized to a scale where activity measured in HCTl 16-CAIX and HCTl 16-EV membrane fragments are one and zero, respectively. Asterisks denote significant reduction in activity (MSC5, MSC 10„MSC12 and MSC8 Fab antibodies). (C) Time course for CO2 hydration with HCTl 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane- fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC3 IgG (dashed black), MSC8 IgG (dotted black) or acetazolamide (dashed grey). (D) Dose-response curves for MSC 8 Fab (grey) and MSC 8 IgG (black) influence on CAIX-activity (using the same normalization algorithm as in B).

[00051] FIGURE 20A through 20C provides the protocol for determining extracellular carbonic anhydrase activity in intact cells. (A) Experimental protocol indicating the composition and pH of superfusates. (B) A sample time-course of surface extracellular pH measured in HCTl 16-CAIX cells during rapid superfusate changes from NT to AmmNT and back, in the presence and absence of acetazolamide. Surface pH_e was measured using DHPE-conjugated fluorescein. (C) Cartoon showing the reactions taking place at cell surface during the addition (left) or removal (right) of extracellular ammonium chloride. [00052] FIGURE 21A through 21D depicts the determination of carbonic anhydrase activity in intact cells. Measuring extracellular pH (pH_e) transients at the surface of (A) HCTl 16- EV cells, (B) HCT l 16-CAIX cells and (C) HCTl 16-CAIX cells pre-incubated with 20 μ^ηιΐ (i) MSC3 IgG, (ii) MSC8 IgG, (iii) MSC8 Fab or (iv) continuously superfused with 1 ^πιΐ MSC8 Fab. (D) Carbonic anhydrase activity as determined by the area under measured surface pH_c transients. Higher carbonic anhydrase activity produces smaller surface pH_e transients.

[00053] FIGURE 22A and 22B depicts physiological effects of MSC8 antibody on acid- efflux from multi-cellular tissue-growths (spheroids). Spheroid pHj was measured in nine regions of interest (ROI; ROJ l =periphery, ROI9=core) during a protocol in which bulk superfusate C0₂ partial pressure was dropped rapidly from 5% to zero, whilst solution pH was maintained constant at 7.4. Data are presented as the change of pHj, relative to the average measured over the preceding 10 s. Dashed lines indicate the pHj slope measured over the initial 10 s period following superfusate C0₂-removal. (A) Core and periphery alkalinisation in (i) control spheroids (radius 123.3 ± 7.1 μιη), (ii) spheroids acutely exposed to 1 μg/ml MSC8 Fab by inclusion in the superfusate for 15 min, and spheroids incubated with 20 μg/ml (iii) MSC8 Fab for 5 h or (iv) MSC8 IgG for 48-72 h. (B) Data for initial pHj slopes are summarised for (i) Fab or (ii) IgG incubated spheroids compared to control spheroids. Asterisks indicate significant differences (P<0.05).

[00054] FIGURE 23A through 23C provides Supplementary Table 1. Summary of binding analyses performed by flow cytometry for selected Fab antibodies. Monoclonal Fab antibodies, selected by phage display, were analysed on different human and murine cell lines. (A) Flow cytometry on renal carcinoma cells not expressing CAIX (SKRC 1 ) and cells expressing CAIX constitutively due to pVHL deficiency (SKRC 52 and SKRC 59). (B) Flow cytometry on SKRC 17 MW1 cl 4 cells (stably transfected with CAIX). (C) Flow cytometry on human (HeLa) or murine (CT26) cells expressing CAIX after induction with

dimethyloxalylglycine or by incubation under 0.2% 0₂. Fab antibodies ESC 1 1 and ESC 14 recognizing human fibroblast activation protein were used as negative controls. Key: (++) GMean > l Ox background; (+) GMean > 3x background; (-) GMean < 3x background.

[00055] FIGURE 24 depicts Supplementary Fig. SI. In vitro carbonic anhydrase activity assay on CAII extracted fro lysed human red blood cells. Time course for C0₂ hydration catalysed by blood-derived CAII under control conditions (black), in the presence of 20 μg/ml MSC 8 IgG (black dashed) and 100 μΜ acetazolamide (grey dashed).

[00056] FIGURE 25 depicts Supplementary Fig. S2. Importance of buffering kinetics in determining surface extracellular pH (pH J transients. Surface pH_e transients measured in the presence of C0₂/HC0₃ ^~ (black) or Hepes (grey) buffering system.

[000571 FIGURE 26 depicts immunofluorescence using MSC 3 IgG and MSC 1 1 IgG. Immunofluorescence of sectios of C51 tumours was performed using MSC 3 IgG or MSC 1 1 IgG (red) in combination with the perfusion marker pimonidazole (green). Nuclei were stained using DAPI (blue).

DETAILED DESCRIPTION

[00058] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. ( 1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. ( 1986)]; "Immobilized Cells And Enzymes" [IRL Press, ( 1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[00059] Therefore, if appearing herein, the following terms shall have the definitions set out below.

A. TERMINOLOGY

[00060] The term "specific binding member"describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.

[00061] The term "antibody" describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CD grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567. The term "antibody(ies)" includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG l , IgG2, IgG3, IgG4, IgA l , and IgA2). Also included within the meaning of the term "antibody" are any "antibody fragment".

[00062] An "antibody fragment" means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CH I) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CH I domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S. et al., Nature 341 , 544-546 ( 1989)); (vi) a camelid antibody; (vii) an isolated complementarity determining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH 1 -VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); and (xii) other non-full length portions of heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[00063] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Patent Nos. 4,816,397 and 4,816,567.

[00064] An "antibody combining site" is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.

[00065] The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

[00066] Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v), which portions are preferred for use in the therapeutic methods described herein.

[00067] Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-cancer or anti-tumor specific antibody. The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S. S.N. 60/355,838-filed February 13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor. Thus, the anti-FAP antibodies of the invention may be utilized to direct or target agents, labels, other molecules or compounds or antibodies to stromal sites, particular areas of wound healing, inflammation, cancer or tumors.

[00068] The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

[00069] The term "antigen binding domain" describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[00070] Immunoconjugates or antibody fusion proteins of the present invention, wherein the antibodies, antibody molecules, or fragments thereof, of use in the present invention are conjugated or attached to other molecules or agents further include, but are not limited to such antibodies, molecules, or fragments conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent, antimicrobial agent or peptide, cell wall and/or cell membrane disrupter, or drug.

[00071 ] The term "specific" may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a- number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

100072] The term "comprise"generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[00073] The term "consisting essentially of refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.

[00074] The term "isolated" refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared {e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

[00075] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or '^g" mean microgram, "mg" means milligram, "ul" or "μΐ" mean microliter, "ml" means milliliter, "1" means liter.

[00076] The terms "antibody", "anti CAIX antibody", "anti-CA9 antibody", "CAIX antibody", "CA9 antibody", "human CAIX antibody", "antibody MSC 1 ", "antibody MSC2", "antibody MSC3", "antibody MSC4", "antibody MSC5", "antibody MSC6", "antibody MSC7", "antibody MSC8", "antibody MSC9", "antibody MSC10", "antibody MSC1 1 ", "antibody MSC 12", and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in any of FIGURES 5 through 16, TABLES 3 and 4, and the profile of activities set forth herein and in the Claims. The antibodies may comprise antibody nucleic acid and protein sequences, including particularly CDR region sequences CDR1 , CDR2 and CDR3 for each of the heavy and light chain variable regions, as set out in FIGURES 5 through 16 and in SEQ ID NOS: 1 -48. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "antibody", "anti CAIX antibody", "anti-CA9 antibody", "CAIX antibody", "CA9 antibody", "human CAIX antibody", "antibody MSC 1 ", "antibody MSC2", "antibody MSC3", "antibody MSC4", "antibody MSC5", "antibody MSC6", "antibody MSC7", "antibody MSC8", "antibody MSC9", "antibody MSC 10", "antibody MSC H ", "antibody MSC12" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

[00077] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL AMINO ACID

Letter 3 -Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

[00078] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

[00079] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

[00080] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

[00081] A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[00082] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

[00083 J A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[00084| Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

[00085] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[00086] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[00087] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

100088] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

100089] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

[00090] The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

[00091] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

[00092] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[00093] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by .comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

[00094] It should be appreciated that also within the scope of the present invention are DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having the same amino acid sequence as provided in any of FIGURES 5 through 16, or comprising the CDR domain region sequences set out herein or in any of FIGURES 5 through 16 but which are degenerate thereto. By "degenerate to" is meant that a different three-letter codon is used to specify ¾ particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC

Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

Isoleucine (He or I) AUU or AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU or GUC of GUA or GUG

Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC

Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG

Alanine (Ala or A) GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC

Glutamine (Gin or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic Acid (Asp or D) GAU or GAC

Glutamic Acid (Glu or E) GAA or GAG

Cysteine (Cys or C) UGU or UGC

Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG

Glycine (Gly or G) GGU or GGC or GGA or GGG

Tryptophan (Trp or W) UGG

Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

[00095] It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

[00096] Mutations can be made in the sequences encoding the amino acids, antibody fragments, heavy and light chain variable region sequences, CDR region sequences, set out in any of FIGURES 5 through 16, or in the sequences of TABLES 3 and 4, or in the sequences denoted in SEQ ID NOS: 1 -48, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing substitutions and/or conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein. In an aspect hereof, the heavy or light chain variable region sequences of an antibody of the present invention may consist of the CDR region sequences provided herein, particularly each of the CDRl , CDR2 and CDR3 region sequences, with substitutions, including conservative and nonconservative changes to the variable regions sequence outside of the CDR domains. In an aspect hereof a small number of changes even in the CDR domain region sequences may be accommodated, providing that the CAIX binding capability and/or inhibitory activity of the antibody is maintained. In one such aspect, the CDR region sequences of the antibodies hereof are distinct from the CDR region sequences of the non- MSC antibodies depicted in Table 5 and Table 6.

[00097) The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

Amino acids with uncharged polar R groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino acids with charged polar R groups (negatively charged at Ph 6.0)

Aspartic acid, Glutamic acid

Basic amino acids (positively charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

[00098] Another grouping may be those amino acids with phenyl groups:

Phenylalanine, Tryptophan, Tyrosine

[00099) Another grouping ; may be according to molecular weight {i.e., size of R groups)

Glycine 75 Alanine 89

Serine 105 Proline 1 15

Valine 1 17 Threonine 1 19

Cysteine 121 Leucine 131

Isoleucine 131 Asparagine 132

Aspartic acid 133 Glutamine 146

Lysine 146 Glutamic acid 147

Methionine 149 Histidine (at pH 6.0) 155

Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

[000100] Particularly preferred substitutions are:

- Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained;

- Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NH₂ can be maintained.

[000101] Exemplary and preferred conservative amino acid substitutions include any of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine ( ) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine ( ) for arginine (R) and vice versa.

[000102] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces, β-turns in the protein's structure.

[000103] Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions. The CDR regions of two antibodies are substantially homologous when one or more amino acids are substituted with a similar or conservative amino acid substitution, and wherein the antibody/antibodies have the profile of binding and activities of one or more of MSC 1 through MSC 12 disclosed herein.

[000104] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.

Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

1000105 J A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

[000106] The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65°C for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-R A, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T_m with washes of higher stringency, if desired.

[000107] The term 'agent' means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.

[000108] The term 'agonist' refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.

[000109] The term 'assay' means any process used to measure a specific property of a compound. A 'screening assay' means a process used to characterize or select compounds based upon their activity from a collection of compounds.

[000110] The term 'preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[000111 ] The term 'prophylaxis' is related to and encompassed in the term 'prevention', and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

1000112] 'Therapeutically effective amount' means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to gram-positive bacterial infections and growth of gram-positive bacteria, the term "effective amount" is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of infection of gram- positive bacteria, including having a bacteriocidal and/or bacteriostatic effect. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of infectious bacteria, or other feature of pathology such as for example, elevated fever or white cell count as may attend its presence and activity.

1000113] The term 'treating' or 'treatment' of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment 'treating' or 'treatment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, 'treating' or 'treatment' refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter); or both. In a further embodiment, 'treating' or 'treatment' relates to slowing the progression of a disease or reducing an infection. [000114] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

[000115] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or g" mean microgram, "mg" means milligram, "ul" or "μΐ" mean microliter, "ml" means milliliter, "1" means liter.

B. DETAILED DISCLOSURE.

[000116] The invention provides antibodies directed against Carbonic anhydrase IX (CAIX) for diagnostic and therapeutic purposes. In particular, antibodies specific for CAIX are provided, wherein said antibodies recognize and are capable of binding human CAIX. In particular, antibodies specific for CAIX are provided, wherein said antibodies recognize and are capable of binding human and mouse CAIX. In particular, antibodies specific for CAIX are provided, wherein said antibodies recognize and are capable of binding human CAIX and of inhibiting or blocking gthe activity of CAIX. Fab antibodies are particularly provided herein. The antibodies of the present invention have diagnostic and therapeutic use in conditions associated with hypoxia, including oxygen and nutrient deficient cells, particularly cancer, particularly solid tumors. In a particular aspect the antibodies of the invention are applicable in cancers, including renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, biliary and pancreatic adenocarcinoma. In a particular aspect the antibodies of the invention are applicable in renal cancer.

[000117] In a general aspect, the present invention provides CAIX antibodies, including antibodies directed against both human and mouse CAIX and antibodies capable of inhibiting or reducing CAIX activity. In a broad aspect, the present invention provides an isolated specific binding member, particularly an antibody or fragment thereof, including a Fab fragment and a single chain or domain antibody, which recognizes human CAIX. In a further aspect, the present invention provides an antibody or fragment thereof, which recognizes human CAIX and is selected from antibodies MSC 1 , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC 1 1 and SC 12. In a further aspect, the present invention provides an antibody or fragment thereof, which recognizes human CAIX and is selected from antibodies MSC1 , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, SC 1 1 and MSC 12 and comprises the amino acid sequence of any of MSC1 through MSC 12 as set out in FIGURES 5 through 16. In one such aspect, the invention provides an anti-CAIX antibody comprising the variable region heavy and light chain CDR sequences set out in FIGURES 5 through 16, and in TABLE 3 and TABLE 4. In an aspect, the invention provides an anti-CAIX antibody comprising the variable region heavy and light chain CDR sequences of any antibody of MSC1 , SC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC 1 1 and MSC 12, as set out in FIGURES 5 through 16.

[000118] In a particular aspect, the antibody or fragment of the invention is reactive with, capable of binding human CAIX. In a particular aspect, the antibody or fragment of the invention is reactive with, capable of binding human and mouse CAIX. In a particular such aspect, the antibody is MSC3 or an active fragment thereof. In a further aspect the antibody or fragment is reactive with human CAIX and inhibits CAIX activity, particularly CAIX catalytic activity. In a particular such aspect the antibody is MSC8 or an active fragment thereof. Recombinant antibodies derived from any of the antibody variable region sequences hereof are also provided.

[000119] Panels of monoclonal antibodies recognizing human CAIX can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are antibodies that mimic the activity of exemplary antibody MSC3, and have affinity for human and mouse CAIX. Of particular interest are antibodies that mimic the activity of exemplary antibody MSC8, and have affinity for human CAIX and block or inhibit CAIX activity, particularly CAIX catalytic activity. Such antibodies can be readily identified and/or screened in specific binding member activity assays.

[000120] In general, the CDR regions, comprising amino acid sequences substantially as set out as the CDR regions of any of FIGURES 5 through 16 will be carried in a structure which allows for binding of the CDR regions to CAIX, and particularly to human CAIX. CDR regions, comprising amino acid sequences substantially as set out as the CDR regions of FIGURE 7 will be carried in a structure which allows for binding of the CDR regions to CAIX, and particularly to human and mouse CAIX. CDR regions, comprising amino acid sequences substantially as set out as the CDR regions of FIGURE 12 will be carried in a structure which allows for binding of the CDR regions to CAIX, and particularly to human CAIX, and having the capability of inhibiting or reducing CAIX catalytic activity. [0001211 By "substantially as set out" it is meant that that variable region sequences, and/or particularly the CDR sequences, of the invention will be either identical or highly homologous to the specified regions of any of FIGURES 5 through 16 or in TABLES 3 and 4. By "highly homologous" it is contemplated that only a few substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3, or 1 or 2 substitutions may be made in the variable region sequence and/or in the CDR sequences. The term substantially set out as includes particularly conservative amino acid substitutions which do not materially or significantly affect the specificity and/or activity of the instant antibodies. Conservative amino acid substitutions are exemplified herein for the CDR region sequences.

1000122] Substitutions may be made in the variable region sequence outside of the CDRs so as to retain the CDR sequences. Thus, changes in the variable region sequence or alternative non-homologous or veneered variable region sequences may be introduced or utilized, such that the CDR sequences are maintained and the remainder of the variable region sesuence may be substituted.

[000123] Alternatively, substitutions may be made particularly in the CDRs. CDR sequences for the antibodies of the present invention are set out and described herein including as set out in any of FIGURES 5 through 16. Accordingly, antibodies or fragments thereof having substitutions based on the CDR regions of the heavy or light chain, and preferably both, of the antibodies of the invention, particularly of any of MSC1 , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC 1 1 and MSC 12, will be useful. Antibody MSC3 comprises heavy chain CDR sequences GFTFSSYA, ISGSGGST and AKGGGTGTTVIFDY, and light chain CDR sequences VSNLGAGYE, GNS and QSYDRSLTEWV, as set out in FIGURE 7. Antibody MSC8 comprises heavy chain CDR sequences GGSFSGYY, 1NHSGST and ARGSGANYYDSSREPRAFDI and light chain CDR sequences SGINVDTYM, YKSESNQ and MIWHSNTWV, as set out in FIGURE 12.

[000124] Antibodies of the invention having substitutions as above described and contemplated are selected to maintain the activities and specifity commensurate with the exemplary antibodies, including antibodies MSC3 and MSC8, or any of antibodies MSC 1 , SC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC I O, MSC1 1 and MSC 12 and having the characteristics as set out herein and in the claims.

[000125] The structure for carrying the CDRs of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR regions are located at locations corresponding to the CDR region of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to abat, E.A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu)).

[000126] The variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. The CDR-derived sequences of the invention, as defined in the preceding paragraph, may be introduced into a repertoire of variable domains lacking CDR regions, using recombinant DNA technology.

[000127] For example, Marks et al (Bio/Technology, 1992, 10:779-783) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR/CDRs. Marks et al further describe how this repertoire may be combined with a CDR of a particular antibody. The repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 so that suitable specific binding members may be selected. A repertoire may consist of from anything from 10⁴ individual members upwards, for example from 10⁶ to 10⁸ or I O¹⁰ members. Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature, 1994, 370:389-391 ), who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.

[000128] A further alternative is to generate novel VH or VL regions carrying the CDR- derived sequences of the invention using random mutagenesis of, for example, the Ab VH or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al ( 1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR. Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91 :3809- 3813) and Schier et al ( 1996, J. Mol. Biol. 263:551 -567). [0001291 All the above described techniques are known as such in the art and in themselves do not form part of the present invention. The skilled person will be able to use such techniques to provide specific binding members of the invention using routine methodology in the art.

[0001301 A substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as provided herein and/or known to those of skill in the art.

[000131) Although in a preferred aspect of the invention specific binding members comprising a pair of binding domains based on sequences substantially set out in any of FIGURES 5 through 16 are preferred, single binding domains based on either of these sequences form further aspects of the invention. In the case of the binding domains based on the sequence substantially set out in any of FIGURES 5 through 16, such binding domains may be used as targeting agents for CAIX, particularly in solid tumors or cancer cells, since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.

[000132] This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in U.S. Patent 5,969, 108 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al, ibid. Phage library and phage display selection systems and techniques are also provided herein.

[000133] Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, specific binding members based on the sequences of any of FIGU RES 5 through 1 6 may be attached at their C-terminal end to antibody light chain constant domains including human CK or CX chains, preferably CX chains. Simi larly, specific binding members based on the sequences of any of FIGURES 5 through 1 6, including FIG URE 7 or FIG U RE 1 2, may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes, particularly IgG 1 , IgG2b, and IgG4. IgG l is preferred.

[000134 ] The antibodies, or any fragments thereof, may be conj ugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin. or diphtheria toxin. The part of the toxin used can be the whole toxin, or any particular domain of the toxin. Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct 24; 1 333(2):C l -6; reitman et al., N Engl J Med. 2001 Jul 26;345(4):241 -7; Schnell et al., Leukemia. 2000 Jan; 14( 1 ): 129-35; Ghetie et al., Mol Biotechnol . 2001 Jul; 1 8(3):251 -68.

[0001351 Bi- and tri-specific multimers can be formed by association of di fferent scFv molecules and have been designed as cross-linking reagents for T-cel l recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb 1 ;248( l -2):47- 66; Tomlinson et al., Methods Enzymol. 2000;326:461 -79; McCall et al ., J I mmunol. 2001 May 1 5 ; 166( 1 0):6 1 12-7.

[0001361 Fully human antibodies can be prepared by immunizing transgenic mice carrying large portions of the human immunoglobulin heavy and light chains. These mice, examples of such, mice are the Xenomouse™ (Abgenix, Inc.) (US Patent Nos. 6,075, 1 81 and 6, 1 50,584), the HuMAb-Mouse™ ( edarex, Inc./GenPharm) (US patent 5545806 and 5569825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), are well known within the art. Antibodies can then be prepared by, e.g. standard hybridoma technique or by phage display. These antibodies will then contain only fully human amino acid sequences. Fully human antibodies can also be generated using phage display from human libraries. Phage display may be performed using methods well known to the ski lled artisan, and as provided herein as in Hoogenboom et al and Marks et al (Hoogenboom H R and Winter G. ( 1 992) J Mol Biol. 227(2):38 1 -8; Marks JD et al ( 1991 ) J Mol Biol. 222(3):58 1 -97; and also U.S. Patents 5885793 and 5969108). [000137] Antibodies of the invention may be labelled with a detectable or functional label. Detectable labels include, but are not limited to, radiolabels such as the isotopes ³H, ^{l 4}C, ³²P, ³⁵S, ³⁶CI, ^{5 l}Cr, "Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ^{, 2 I}I, ^{l 24}I, ^{l 25}I, ^{, 3 I} I, " 'in, ^{n 7}Lu, ^{2 l l}At, ^{, 98}Au, ⁶⁷Cu, ²²⁵Ac, ^{2 l 3}Bi, ⁹⁹Tc and ^{l 8} Re, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels (for example fluorescein, rhodamine, Texas Red) and labels used conventionally in the art for MR1- CT imaging. They also include enzyme labels such as horseradish peroxidase, β-glucoronidase, β-galactosidase, urease. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.

[000138] Also, antibodies including fragments thereof, and drugs that modulate the production or activity of the specific binding members, antibodies and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as cancer, precancerous lesions, conditions related to or resulting from hyperproliferative cell growth or the like. For example, the specific binding members, antibodies or their subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity(ies) of the specific binding members of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.

[000139] The radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy. In the instance of in vivo imaging, the specific binding members of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and ferbium. In a further aspect of the invention, radiolabeled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabeled antibodies for cancer therapy. In a still further aspect, the radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery, to remove such cells.

[000140] Immunoconjugates or antibody fusion proteins of the present invention, wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

[000141] Radioimmunotherapy (RAIT) has entered the clinic and demonstrated efficacy using various antibody immunoconjugates. ¹³ Ί labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN- 14 has been evaluated in colorectal cancer (Behr TM et al (2002) Cancer 94(4Suppl): 1373-81 ) and the same antibody with ⁹⁰Y label has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94( 1):51 -61 ). Radioimmunotherapy using monoclonal antibodies has also been assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit Rev Oncol Hematol 39(1 -2): 195-201 ; Gold DV et al (2001 ) Crit Rev Oncol Hematol 39 (1 -2) 147-54). Radioimmunotherapy methods with particular antibodies are also described in U.S. Patent 6,306,393 and 6,331 , 175. Radioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated efficacy and usefulness, including using anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim JC et al (2002) Int J Cancer 97(4):542-7; Schneebaum S et al (2001 ) World J Surg 25(12): 1495-8; Avital S et al (2000) Cancer 89(8): 1692-8; Mcintosh DG et al ( 1997) Cancer Biother Radiopharm 12 (4):287-94).

[000142] In vivo animal models of cancer or animal xenograft studies may be utilized by the skilled artisan to further or additionally screen, assess, and/or verify the specific binding members and antibodies or fragments thereof of the present invention, including further assessing CAIX, CAIX modulation and blocking, inhibiting or targeting tumor or cancer cells, particularly hypoxic cells in vivo and inhibiting tumor proliferation, progression and/or infiltration. Such animal models include, but are not limited to models of conditions associated with cancer, particularly solid tumors, including renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, bi liary and pancreatic adenocarcinoma, particularly without the problems associated with normal tissue uptake. Models of cancers, particularly solid tumor cancers, which become hypoxic and induce CAIX, are particularly susceptible to and targeted by the antibodies of the present invention. For example but without limitation, renal cancer and renal cancer cells express CAIX. This may be uti lized in xenograft experiments or in models for direct tumor targeting and/or to assess anti-tumor and anti-cancer effects of the anti-CAIX antibodies.

1000143 J Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, including by injection intramuscularly, into the bloodstream or CSF, or directly into the site of the tumor. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and the nature of the detectable or functional label attached to the antibody. Where a radionuclide is used for therapy, a suitable maximum single dose may be about 45 mCi/m², to a maximum of about 250 mCi/m². Preferable dosage is in the range of 1 5 to 40 mCi, with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi. Such therapy may require bone marrow or stem cell replacement. A typical antibody dose for either tumor imaging or tumor treatment wil l be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody in F(ab')2 form. Naked antibodies are preferably administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats, in proportion for example to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

Pharmaceutical and Therapeutic Compositions

1000144] Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site.

|000145| Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[000146] For intravenous, injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

[000147] A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, hormones, anti-mitotic agents, anti- apoptotic agents, antibodies, or immune modulators. More generally these anti-cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG 1478 and ZD 1839, STI571 , OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and/or other chemotherapeutic agents. Thus, these agents may be specific anti-cancer agents, or immune cell response modulators or may be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. The agent(s) may be specific or effective against hypoxic cells, such as azetazolamide, other sulfonamides, dithiothreitol, threitol, or beta-mercaptoethanol. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-tumor antigen antibodies.

[000148] The CAIX antibody compositions may thus include other antibodies, including more than one CAIX antibody, or may be administered in combination with other antibody compositions. As an example, the CAIX antibody(ies) may be administered with antibody(ies) or inhibitors directed against HIF- 1 and/or with inhibitors against other cancer associated antigens, particularly hypoxic cancers, such as CAXIl. Studies of CAIX and CAXIl expression in human tumors and normal adult tissues have shown co-expression of CAIX and CAXIl in regions of hypoxia in tumor tissues, but no significant co-localization in normal tissue (Ivanov S et al (2001 ) Am J Pathol 158:905-919; Liao S-Y et al (2009) BMC Develop Biology 9:22(doi : 10.1 186/1471 -213x-9-22)). Chiche et al have shown that CAIX and CAXIl proteins promote tumor cell survival and growth and are anticancer therapeutic targets (Chiche J et al (2009) Cancer Res 69:358-369). In this study, CAIX blockage by siRNA led to partial compensation by upregulation of CAXIl, and invalidation of both CAIX and CAXIl resulted in a profound decrease in tumor size and slower cell proliferation.

1000149] In addition, the present invention contemplates and includes therapeutic compositions for the use of the binding member in combination with conventional radiotherapy.

[000150] The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises an antigen capable of modulating the specific binding of the present binding member/antibody with a target cell.

[000151 ] The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

[000152] A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[000153] The therapeutic antibody- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0001541 The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of hypoxia, CAIX expression, or tumor antigen binding capacity desired.

Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and follow on administration are also variable, and may include an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain appropriate and sufficient concentrations in the blood or at the site of desired therapy are contemplated.

[000155] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

1000156] For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Diagnostic Assays

[000157] The present invention also relates to a variety of diagnostic applications, including methods for detecting the expression of or elevated presence of CAIX, CAIX-mediated mediated cancer, including renal, breast, bladder, head and neck, cervical, soft tissue sarcomas, non-small cell lung cancer, and gastrointestinal cancer, including esophageal, gastric, colorectal, biliary and pancreatic adenocarcinomaby reference to their ability to be recognized by the present specific binding member(s). Peptide complexes can be identified, targeted, labeled, and/or quantitated on hypoxic cells, cancer cells and/or tumor cells.

[000158] Diagnostic applications of the specific binding members of the present invention, particularly antibodies and fragments thereof, include in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Diagnostic assays and kits for in vitro assessment and evaluation of tumor and cancer status, may be utilized to diagnose, evaluate and monitor patient samples including those known to have or suspected of having cancer, a precancerous condition, a condition related to hyperproliferative cell growth or from a tumor sample. The assessment and evaluation of cancer, tumor and metastatic disease status is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or specific binding member, particularly an antibody, of the present invention, including combinations thereof, versus a different agent or binding member. This type of diagnostic monitoring and assessment is already in practice utilizing antibodies against the HE 2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate patients for antibody therapy using Herceptin. In vivo applications include imaging of tumors or assessing cancer status of individuals, including radioimaging.

1000159] Preferably, the antibody used in the diagnostic methods of this invention is human antibody. More preferably, the antibody is a single chain chain antibody or domain antibody. In addition, the antibody molecules used herein can be in the form of Fab, Fab', F(ab')₂ or F(v) portions of whole antibody molecules, particularly Fab.

[000160] As described in detail above, antibody(ies) to CAIX can be produced and isolated by standard methods including the phage display techniques and mutagenesis and recombinant techniques.

[000161 ] The presence of CAIX in cells can be ascertained by the usual in vitro or in vivo immunological procedures applicable to such determinations. A number of useful procedures are known. The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The "competitive" procedure is described in U.S. Patent Nos. 3,654,090 and 3,850,752. The "sandwich" procedure, is described in U.S. Patent Nos. RE 31 ,006 and 4,016,043. Still other procedures are known such as the "double antibody," or "DASP" procedure.

[000162] In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of aberrant expression of including but not limited to amplified and/or an mutation, in suspected target cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive," "sandwich," "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

[000163] Accordingly, a test kit may be prepared for the demonstration of the presence of or elevated levels of CAIX, comprising: (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present specific binding member or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

1000164] A test kit may be prepared for the demonstration of the presence of epithelial cancer, stromal cell mediated cancer, particularly selected from breast, lung, colorectal, ovarian cancer comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present specific binding member or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

[000165] In accordance with the above, an assay system for screening potential drugs effective to modulate the presence or activity of CAIX and/or the activity or binding of the antibody of the present invention may be prepared. The antigen peptide or the binding member or antibody may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the activity of the cells, binding of the antibody, or amount and extent of CAIX due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent(s).

Nucleic Acids

]000166] The present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out in any of FIGURES 5 through 16 or capable of encoding the CDR regions thereof.

[000167] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. A nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

[000168] Specific binding members and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.

[000169] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, cancer cells, ovarian cancer cells and many others. A common, preferred bacterial host is E.coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E.coli is well established in the art.

[000170] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

[000171 ] Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE- Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.

[0001721 Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non- chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCRl , pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2u plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[000173] Any of a wide variety of expression control sequences— sequences that control the expression of a DNA sequence operatively linked to it— may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. [000174] A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces , fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, BSC 1 , BSC40, and Β ΤΊ 0), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

1000175] It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

[000176] As mentioned above, a DNA sequence encoding a specific binding member can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the specific binding member amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981 ); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol. Chem. , 259:63 1 1 ( 1984). Synthetic DNA sequences allow convenient construction of genes which will express specific binding member analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native specific binding member genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

[000177] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1

Carbonic Anhydrase IX Specific Antibodies

[000178] Here, we describe the generation of novel anti-CAIX antibodies. Among the antibodies isolated and described are particularly useful and applicable CAIX antibodies, including exemplary antibody having cross reactivity for murine and human CAIX, providing applicability in biologically relevant test and animal model systems, and exemplary antibody capable of inhibiting or reducing CAIX catalytic activity.

[000179] Eukaryotically expressed human CAIX protein (rhCAIX, R&D Systems) was purchased and immobilised on beads using a mouse anti-human CAIX specific antibody (M75) (kindly provided by Egbert Oosterwijik; available from Bayer Diagnostics). These CAIX presenting beads were then used to select for specific antibody binders by phage display. The selection has been finished and-led to the isolation of twelve distinct clones (MSC 1 - MSC 12) specific for Carbonic anhydrase IX, one of them (MSC3) highly cross-reactive to murine Carbonic anhydrase IX (FIGURES 1 and 2). The binding specificity of MSC3 antibody is particularly depicted in FIGURE 2. MSC3 specifically recognizes Carbonic anhydrase IX in human or murine cell lines. Other Fabs were assessed and lead to similar results in human cell lines, however, only weak or even no signal was observed in murine cell lines (TABLE 2).

[000180] Binding specificity of the selected Fab fragments was analyzed by flow cytometry on human and murine cell lines (TABLE 1 ). Specificity seen by flow cytometry was confirmed by ELISA using recombinant proteins (TABLE 2).

TABLE 1

Cell Lines Used for Specificity Measurements in Flow Cytometry vHL deficient Transfected with CAIX Induced by Hypoxia

Human Cell Lines SKRC 1 , SKRC SKRC 17 HeLa

52 MW 1 c l4

and SKRC 59 (parental cell line: SKRC

17

Mouse CT26

[000181 ] For ELISA analysis, signals exceeding 3x the negative control were considered positive (+). Those lOx above control were judged to be highly positive (++). In flow cytometry, differences of mean fluorescence intensity of CAIX expressing cells to not-expressing cells were calculated. Differences 3x above the negative controls were considered positive (+), those l Ox above controls highly positive (++).

1000182]

TABLE 2

Summary of Binding Patterns for Antibodies MSC1-MSC12

ELISA FACS

rhCAIX rmCAIX SKRC 17 HeLa + HeLa - CT26 CT26 - MW1 cl4 DMOG Hypoxia +DMOG Hypoxia

MSC 1 ++ + ++ ++ ++ + +

SC 2 ++ - ++ ++ ++

MSC 3 ++ ++ ++ ++ ++ ++ +

MSC 4 ++ - ++ ++ ++

MSC 5 ++ + ++ ++ ++ -

MSC 6 ++ ++ ++ ++

MSC 7 ++ + ++ ++ ++ -

MSC 8 + - ++ ++ ++ - -

MSC 9 ++ + ++ ++ ++ - -

MSC 10 ++ ++ ++ ++

MSC 1 1 ++ - ++ ++ ++

MSC 12 ++ - ++ ++ ++ - -

1000183] The affinity of the different Fab fragments to human and murine Carbonic anhydrase IX was assessed by Biacore measurement and revealed medium to good affinities ranging between 2 and 60 nM for Fab fragments. The data for MSC8 are depicted in FIGURE 3. [000184] Epitope mapping by blocking assays revealed that none of the twelve new candidates are binding to the epitope recognised by G250 or M75, respectively (FIGURE 4). However, epitope overlap was found for all SC antibodies except MSC8, which seems to be distinct (data not shown).

[000185] MSC3 is highly cross-reactive to the murine Carbonic anhydrase IX, MSCl weakly cross-reactive, and MSC 1 1 not cross-reactive at all. MSC3 IgG specifically stains murine CAIX in tumor sections and can thus be useful in syngeneic mouse models. MSC8, in addition, recognises only human CAIX but partially blocks its catalytic activity (see below).

[000186] Blocking CAIX catalytic activity is expected to have a beneficial impact on tumor growth. Functional analysis of CAIX activity includes measurement of pH-changes over time in membrane fragments of CAIX transfected HCT1 16 cells after addition of C0₂. Antibodies MSC5, MSC8, MSC 10 and MSC l 2 inhibited CAIX function partially in assays on membrane fragments. The most potent antibody MSC8 inhibited CAIX activity by up to 57% as Fab antibody. A significant, inhibition of CAIX activity was thus observed for MSC8 using this analysis (see FIGURE 19B). In addition, the affinity of MSC8 during these blocking assays (see FIGURE 19C) was much higher than that of any chemical compound (eg. acetazolamide (ATZ), a widely used broad-spectrum chemical small-molecule CA inhibitor, (42), (43), (44)) and confirmed our initial BIAcore data. The IC50 for ATZ has been reported to be in the range of 20- 30mM for CAIX (Innocenti A et al (2008) Bioorg Med Chem Lett 18(6): 1898-1903). Based on the data presented in Figure 19, the calculated IC50 for MSC8 is 2.2mM.

[000187] In conclusion, we have identified a number of novel CAIX antibodies. There is a particularly great potential for the MSC3 (human/mouse cross-reactive) and MSC8 (blocking CAIX activity) antibodies since both aspects of these unique antibodies, particularly antibody imaging of hypoxia in sygeneic mouse models (MSC3) and blocking of human CAIX activity (MSC8) has never been described for a monoclonal antibody in the past.

[0001881 MATERIALS AND METHODS

[000189] Immunoprecipitation of recombinant human CAIX (rhCAIX). The CAIX antibody M75 (Bayer USA) was incubated for 1 h at 4°C with rhCAIX in 300 μΐ of incubation solution containing 25 mM Tris, 0.15 M NaCl at pH 7.5. Immunoglobulin-binding Protein G beads (Dynabeads, Invitrogen) were washed three times with incubation solution, and incubated with the M75-CAIX complex for 1 h at 4°C. Beads were removed from suspension using a magnetic rack, washed four times with incubation solution, and resuspended in 700 μΐ of incubation solution. Accordingly, beads were loaded with CAIX antibody only.

[000190] Selection of antibodies by phage display. For selection of CAIX specific antibodies, a non-immunized phage library expressing antibody Fab fragments was used (20, 46, 47). l O¹³ Phages were blocked in 2% milk powder in PBS and preabsorbed with 700μ1 of the anti-CAIX IgG-coated protein G bead preparations. In this pre-absorption step, the library was cleared of phages that bind to either protein G or bound IgG. Pre-absorbed phages were then incubated with CAIX-containing immunoprecipitates for 1 h at RT, washed with 2% milk powder in PBS containing 0.3% Tween-20, and subsequently eluted with 100 niM triethylamine. Neutralized phages were amplified in E. coli TG-1 using M13K07 as helper phage. Three rounds of selection with decreasing antigen concentration were performed (round 1 with 90 μg CAIX antibody, 30 μg rhCAIX and 400 μΐ protein G beads; round 2 with 65 μg CAIX antibody, 15 μg rhCAIX and 250 μΐ protein G beads; round 3 with 45.5 μg CAIX antibody, 10 μg rhCAIX and 150 μΐ protein G beads).

[000191 ] Screening of supernatants flow cytometry. Supernatants of the output libraries after each selection round, and supernatants of individual bacterial clones after round three were screened for CAIX-binding phage by flow cytometry on HeLa cells induced with 1 mM DMOG or on SKRC 17 MW l cl4 cells. Positive clones were induced with I mM IPTG to produce soluble Fab and further screened for binding specificities using flow cytometry on human and murine cell lines as indicated in TABLE 1 above. Bound Fab antibody was detected with anti-myc tag antibody 9E 10, followed by an anti-mouse immunoglobulin-PE conjugate. For competition assays, biotinylated Fab antibodies were used and detected using a streptavidin-PE conjugate.

[000192] Expression of Fab fragments. Fab fragments were produced in E. coli TG-1 by induction with I mM IPTG for 4 hr at 30°C. Soluble Fab was released from the periplasmic fraction by incubation in PBS, pH 8 at 4°C o/n, purified using His-tag purification with TALON beads and analyzed by SDS-PAGE.

[000193] Determination of kinetic rate constants and affinity by surface Plasmon resonance. Binding analysis of Fabs to rhCAIX-coated or rmCAIX-coated (recombinant mouse

CAIX, R&D Systems) CM5 sensor chip was performed on a BIAcore T 100 instrument. rhCAIX or rmCAIX was immobilized at low density on a CM5 sensor chip using amine coupling chemistry. For analysis of the kinetics, various concentrations (5 to 240 nM) of Fabs in flow buffer (H BS-P+: 10 mM HEPES, pH 7.4, containing 0.05% v/v surfactant P20 and 0. 1 5 M NaCl) were injected at a flow rate of 30 μΐ/min at 25°C. Analysis of the binding curves and determination of rate constants was done using the nonlinear data analysis program BIAevaluation.

[000194] Epitope mapping by blocking assays. Blocking assays were performed by incubating cells with Fab MSC 1 - 12 for 15 min. at 4 °C. Then control IgG or biotinylated Fab (produced as published before (45)) was directly added w/o washing for another 15 min. The IgG or biotinylated Fab fragment.was detected by an anti-human-Fab-PE conjugate.

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35. Simi L, Venturini G, Malentacchi F, Gelmini S, Andreani M, Janni A, Pastorekova S, Supuran CT, Pazzagli M, Orlando C. Quantitative analysis of carbonic anhydrase IX mRNA in human non-small cell lung cancer. Lung Cancer, 52( 1 ), pp. 59-66 (2006).

36. Swinson DE, Jones JL, Richardson D, Wykoff C, Turley H, Pastorek J, Taub N, Harris AL, O'Byrne KJ. Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in nonsmall lung cancer J. Clini Oncol, 21 , pp473-82 (2003).

37. Kivela AJ, Parkkila S, Saarnio J, Karttunen TJ, Kivela J, Parkkila AK, Pastorekova S, Pastorek J, Waheed A, Sly WS, Rajaniemi H. Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours. Histochem Cell Biol, 1 14(3), pp 197-204 (2000). 38. Saarnio J, Parkkila S, Parkkila A , Haukipuro K, Pastorekova S, Pastorek J, airaluoma MI, arttunen TJ. Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am J Pathol, 153(1 ), pp 279-85 ( 1998).

39. Saarnio J, Parkkila S, Parkkila AK, Pastorekova S, Haukipuro K, Pastorek J, Juvonen T, Karttunen TJ.Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours. J Hepatol, 35(5), pp. 643-9 (2001 ).

40. Driessen A, Landuyt W, Pastorekova S, Moons J, Goethals L, Haustermans K, Nafteux P, Penninckx F, Geboes K, Lerut T, Ectors N. Expression of carbonic anhydrase IX (CA IX), a hypoxia-related protein, rather than vascular-endothelial growth factor (VEGF), a pro-angiogenic factor, correlates with an extremely poor prognosis in esophageal and gastric adenocarcinomas. Ann Surg, 243(3), pp 334-40 (2006).

41. Chrastina A, Zavada J, Parkkila S, Kaluz S, Kaluzova M, Rajcani J, Pastorek J, Pastorekova S. Biodistribution and pharmacokinetics of 1251-labeled monoclonal antibody M75 specific for carbonic anhydrase IX, an intrinsic marker of hypoxia, in nude mice xenografted with human colorectal carcinoma. Int J Cancer, 105(6), pp. 873-81 (2003).

42. De Fiore, A. et al. (2009) Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide. Proteins 74(1), 164-75

43. Alterio, V. et al. (2009) Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX. PNAS 106(38), 16233-8

44. Pastorekova et al. (2004) Carbonic Anhydrases: Current State of the Art, Therapeutic Applications and Future Prospects. Journal of Enzyme Inhibition and Medicinal Chemistry 19(3), 199-229

45. Held et al., (2004) Dissecting cytotoxic T cell responses towards the NY-ESO- 1 protein by peptide/MHC-specific antibody fragments. Eur J Immunol. 34(10), 2919-29

46. de Haard, H.J., van Neer, N., Reurs, A., Hufton, S.E., Roovers, R.C., Henderikx, P., de Bruine, A. P., Arends, J.W., Hoogenboom, H.R., 1999. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274, 18218- 18230.

47. Stewart-Jones, G., Wadle, A., Hombach, A., Shenderov, E., Held, G., Fischer, E., Kleber, S., Nuber, N., Stenner-Liewen, F., Bauer, S., cMichael, A., Knuth, A., Abken, H., Hombach, A. A., Cerundolo, V., Jones, E.Y., Renner, C, 2009. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A 106, 5784-5788.

EXAMPLE 2

Analysis and Sequencing Carbonic Anhydrase IX Specific Antibodies 1000196] The CAIX antibodies SC l through MSC l 2 were sequenced and variable region heavy chain and light chain nucleic acid and amino acid sequences, as well as the CDR regions

CDR1 , CDR2 and CDR3 for each of the heavy and light chains for each of antibodies, were determined. The web-based program IMGT/V-QUEST was used for determining the CDR regions. Amino acid numbering and CDRs were determined using the methods of Lefranc

(Lefranc, .-P. Et al (2003) Dev Comp Immunol 27:55-77; Lefranc, M.-P. ( 1999) The

Immunologist 7: 132- 136). The CAIX antibody sequences, MSC l , MSC2, MSC3, MSC4,

MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSCl 1 and MSC 12, with CDR regions depicted in blue, are set out in FIGURE 5 through FIGURE 16, respectively. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC l , with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 1 -4. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC2, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 5-

8. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC3, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in

SEQ ID NOS: 9- 12. Light and heavy chain nucleic acid and amino acid sequences for antibody

MSC4, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 13- 16. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC5, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 17-20. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC6, with protein sequence annotated to indicate the

CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 21 -24. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC7, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS:

25-28. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC8, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in

SEQ ID NOS: 29-32. Light and heavy chain nucleic acid and amino acid sequences for antibody

MSC9, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 33-36. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC10, with protein sequence annotated to indicate the CDR1 , 2 and 3 sequences, are provided respectively in SEQ ID NOS: 37-40. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC l 1 , with protein sequence annotated to indicate the CDRl, 2 and 3 sequences, are provided respectively in SEQ ID NOS: 41-44. Light and heavy chain nucleic acid and amino acid sequences for antibody MSC12, with protein sequence annotated to indicate the CDRl, 2 and 3 sequences, are provided respectively in SEQ ID NOS: 45-48.

[000197] The following TABLES provide the light chain CDR sequences CDRl, CDR2 and CDR3 (TABLE 3) and the heavy chain CDR sequences CDRl, CDR2 and CDR3 (TABLE 4) for each of the antibodies. Certain of the antibodies demonstrate related or similar heavy and light chain sequences and related CDR region sequences are notable from the below TABLES 3 and 4.

TABLE 3

CAIX Antibody Light Chain Variable Region CDR Sequences

Antibodv CDRl sequence CDR2 sequence CDR3 sequence

MSC1 SSNIGAGYD GNS QSYDSSLSGWV

MSC2 SSNIGAGYD RNT QSYDRSLSRWV

MSC3 VSNLGAGYE GNS QSYDRSLTE V

MSC4 RGAIGLSR DDD QSHDSDSWV

MSC5 SSNIGAGYD GNS QSYDSSLSA V

MSC6 SSNIGAGYD GNS QSYDSSLSAWV

MSC7 SSNIGAGYD GNS QSYDSSLSGSWV

MSC8 SGINVDTYM YKSESNQ MIWHSNTWV

MSC9 SSNIGAGYG GNN QSYDSSLSGWV

MSC10 SSNVGAGYD HNN QSYDTRSGWV

MSC11 QTVREY AAS QQYGTYPT

MSC12 SSNIGAGYD RNN QSYDRSLSRWV

TABLE 4

CAIX Antibodv Heavy Chain Variable Region CDR Sequences Antibody CDR1 sequence CDR2 sequence CDR3 sequence

SC1 GFTFSSYA ISGSGGST AKYCSSTSCWGAFDI

MSC2 GFTFSNYA INRSGGTA ASYGDYVSIFDY

MSC3 GFTFSSYA ISGSGGST AKGGGTGTTVI FDY

MSC4 GFTFSSYA ISGSGGST ARLGDYPNAFDI

SC5 GFTFSSYS ISSSGSYI ARVAGSSWRGASDY

SC6 GFTFSSYA ISGSGGST AKYGSSSWYDG

MSC7 GFTFSSYS ISSSSSYI AIIGYSSGSRGAFDI

MSC8 GGSFSGYY INHSGST ARGSGANYYDSSREPRAFDI

MSC9 GFTFSSYS ISSSSSYI ARVGATTGGAFDI

SC10 GFTFSSYS ISSSSSYI ARVGIGGAFDI

MSC11 GYTFTSYY INPSGGST ARVSSGSYSGFDY

SC12 GFTFSSYA ISGSGGST AKYCGGDTCYSGFDY

[000198J Further characterization of the antibodies includes additional epitope mapping studies. Two approaches are utilized. First, different CAIX fragments into expression vectors and the potential binding motifs are narrowed down. In parallel, overlaying peptide display is utilized to assess antibody binding, although this approach requires that the antibody(ies) tested bind to linear structures. The epitope mapping information is further extended by crystallography studies, including antibody and CAIX. The binding epitopes of the different antibodies could reveal important information on the structure and function of CAIX.

[0001991 The anti-CAIX (anti-CA9) antibodies ( SC8 in particular) have an effect on the biological activity of the targeted antigen (CA9). The inhibition of CAIX activity is further studied using in vitro and in vivo assays and analyses. These studies assess the inhibition of CAIX ativity with the novel blocking antibody (MSC8).

[000200] The effect of MSC3 and MSC8 on tumor cell growth in-vitro under normoxic and hypoxic conditions is assessed using different cell lines, VHL deficient or intact will be studied. For animal studies, BALB/c mice carrying syngeneic CT26 tumors are treated with MSC3 and control antibodies. For MSC8 studies, nude mice with human tumor xenografts are used. Tumour diameter is monitored to analyze the influence of selected antibodies on tumour growth. [000201 ] Labelling of antibody(ies) and functional assays in-vivo. The MSC3 antibody is labelled with radioactive moieties or Tumour Necrosis Factor (TNF), respectively. Labelling with radioactive moieties is used for biodistribution assays which allow imaging of hypoxia in tumours in-vivo. Antigen-dependent distribution of antibodies in a mouse model is analyzed with syngeneically established CT26 tumours. Biodistribution of radioactive labelled antibodies is analysed. At defined time points, blood- and organ-samples are collected and antibody up-take determined by the amount of radioactivity. Labelling of antibodies with TNF leads to specific accumulation of TNF in tumour tissue. TNF triggers tumouricidic mechanisms, e.g. induction of apoptosis, production of super oxide radicals and tissue factor, which is an early member of the activation cascade of endothelial cells to stimulate blood coagulation and thrombosis formation. Being coupled to hypoxia, targeting of Carbonic anhydrase IX allows one to enrich TNF-labelled anti-CAIX antibodies in hypoxic tissues. Thus, TNF-labelled antibodies can be used for targeting hypoxia.

EXAMPLE 3

Antibody Inhibiting Enzyme Activity of CAIX

[000202] Cancer progression involves major changes in cellular metabolism and the tumour microenvironment (Fang et al., 2008; Gillies et al., 2002). These adaptations provide a survival advantage to cancer cells (Gatenby and Gillies, 2004), and it has been proposed that inhibitors targeting these adaptive pathways may reduce the survival prospects of cancer cells. A key feature of cancer progression is hypoxia arising from oxygen depletion by elevated metabolism and inadequate oxygen delivery with blood (Kallinowski et al., 1989). Hypoxia feeds back on tumour physiology by regulating gene expression, principally via the hypoxia-inducible factor (HIF) (Semenza, 2003). An important hypoxia-induced protein is carbonic anhydrase IX (CAIX) (Wykoff et al., 2000), a member of a family of enzymes that catalyse the reaction

C0₂+H₂0^→H⁺+HC0₃ ^~.

[000203] This reaction involves H⁺-ions, the determinants of pH, and C0₂/HC0₃ ^~, a major pH buffering system. CAIX is membrane-tethered and its catalytic site has extracellular-facing topology. The enzyme has been shown to facilitate tumour growth (Chiche et al., 2009) through its role in regulating intracellular pH (Chiche et al., 2009; Swietach et al., 2008) and extracellular pH (Svastova et al., 2004; Swietach et al., 2009). Cancer cells release C0₂ (Holm et al., 1995), a substrate for CAIX, from the intracellular titration reaction between H -ions and HC0₃ ^~, from the pentose phosphate shunt or, in aerobic zones, from mitochondrial respiration. CAIX- catalysed extracellular C0₂ hydration helps to maintain a steep efflux gradient for C0₂ venting out of cells. Extracellular HCO3- that is produced by CAIX can be transported into cells to replenish intracellular HC0₃ ^~ supplies for pH buffering (Lee and Tannock, 1998). Extracellular H⁺-ions produced by CAIX can, in some cells, trigger intracellular signalling cascades via surface H⁺-receptors (Huang et al., 2008).

[000204] The proposed role for CAlX-catalysis in regulating cancer cell pH and the correlation between CAIX-expression and cancers with poor prognosis (Chia et al., 2001 ; Generali et al., 2006; Giatromanolaki et al., 2001 ; Pastorek et al., 1994) has evoked much interest in designing CAIX-inhibitors. Such inhibitors could be used experimentally to investigate the role of CAIX in cancer physiology and provide novel insights into the general importance of extracellular carbonic anhydrase isoforms in cellular physiology. Potentially, inhibitors of CAIX could be used therapeutically to target an important cancer-related protein. Since CAIX is strongly associated with cancer, CAIX-selectivity of such inhibitors would reduce unwanted side-effects on CAIX-negative non-cancerous tissues. A range of membrane-impermeant small- molecule inhibitors are now available for inhibiting extracellular carbonic anhydrase isoforms (Supuran, 2008). Recent studies have shown their ability to inhibit tumour growth in vivo (Ahlskog et al., 2009a). Although some chemical compounds have been designed to bind preferentially to CAIX (Supuran, 2008), it is disputed whether CAIX, among other extracellular isoforms, is being targeted selectively. A more selective approach could be achieved with inhibitory anti-CAIX antibodies. In this study, we have selected a number of CAIX-specific antigen-binding antibody fragments (Fab) by phage display. We have identified an inhibitory antibody, based on tests performed on CAlX-expressing cells and their membrane-fragments, and demonstrated a physiologically-relevant effect of the antibody in CAIX-expressing tissue- growths (spheroids).

[000205] We have selected antigen-binding antibody fragments (Fab) against human CAIX by phage-display, and tested these for inhibitory potency on CAIX catalytic activity. Inhibition was assessed from the kinetics of the CAIX-catalysed reaction, using assays performed on intact cells over-expressing CAIX, and their CAIX-containing membrane fragments. Inhibition was also assessed in multi-cellular tissue-models (spheroids) from the kinetics of C0₂ venting. The Fab antibody MSC8 and its corresponding full-length IgG inhibit CAIX by up to 57% and 76%, respectively, with half-maximal inhibition at 0.3 μg/ml. Incubation of CAIX-expressing cells with MSC8 IgG produced a lasting inhibitory effect. The inhibitory effect was prompt and was also observed in isolated membrane-fragments, suggesting that a direct inhibitory interaction takes place between the antibody and CAIX. The inhibitory effects in spheroids argue for a physiological relevance of the antibody. Biologically-active antibodies against CAIX can be used as selective, high-affinity inhibitors in experimental studies to dissect the role of CAIX and, therapeutically by targeting a catalytically-active cancer-related protein.

[000206] MATERIALS AND METHODS (0002071 Antibody selection by phage display

[000208] Immiinoprecipitation of recombinant human CAIX (rhCAIX).

[000209] The CAIX antibody M75 was incubated with rhCAIX (R&D Systems, Abingdon, UK) for 1 h at 4°C in 300 μΐ of incubation solution (containing 25 niM Tris, 1 50 mM NaCl at pH 7.5). Immunoglobulin-binding Protein G beads (Dynabeads, Invitrogen) were washed three times with incubation solution and then incubated with the M75-CAIX complex or M75 alone for 1 h at 4°C. Beads were removed from suspension using a magnetic rack, washed with incubation solution four times, and then resuspended in 700 μΐ of incubation solution.

[000210] Selection of antibodies by phage display.

[000211 ] A non-immunized phage library (kindly provided by Dyax, MA, USA) expressing Fab antibodies was used for selection of CAIX-specific antibodies (de Haard et al., 1999; Stewart- Jones et al., 2009). 10¹³ phages were blocked in 2% milk powder in PBS and pre- absorbed with 700 μΐ of M75-coated protein G bead preparations. In this pre-absorption step, the library was cleared of protein G and M75 specific phages. Pre-absorbed phages were then incubated with CAIX-containing immunoprecipitates for 1 h at room temperature, washed with 2% milk powder in PBS containing 0.3% Tween-20, and subsequently eluted with 100 mM triethylamine. Neutralized phages were amplified in E. coli TG-1 (Zymo Research, LucernaChem AG Switzerland, Luzern, Switzerland) using M 13K07 (New England Biolabs, Allschwil, Switzerland) as helper phage. Three rounds of selection with decreasing antigen concentration were performed (30 μg, 15 μg and 10 μg rhCAIX).

[000212] Expression of Fab antibodies 10002131 Fab antibodies were produced in E. coli TG- 1 (Zymo Research) by induction with 1 mM lsopropyl- -D-tiogalactopyranosid (Roche, Basel, Switzerland) for 4 h at 30°C. Cells were pelleted and frozen at -20°C, and soluble Fab antibody fragments were released from the periplasmic fraction by overnight incubation in PBS (pH 8, 4°C). Crude fractions were incubated with Talon® metal affinity resin (Clontech Laboratories, Saint-Germain-en-Laye, France) for 1 h at 4°C, washed with PBS containing 0.1 % Tween 20, and eluted with 100 mM imidazole. Fab antibodies were dialyzed against PBS and analyzed by SDS-PAGE.

[000214] IgG antibody cloning and production

(000215) Variable domains of selected Fab antibodies MSC3 and MSC8 were cloned into the pEE 12.4 backbone (LONZA) to obtain a fully human IgGl , stably expressed in NSO cells. The IgG antibody was purified by affinity chromatography on Protein A agarose (Invitrogen) from cell culture supernatant (Bauer et al., 2006).

[000216] Analysis of antibody binding by flow cytometry and ELISA

[000217] Fab and IgG antibodies were analyzed for binding specificity using flow cytometry. Fab binding was detected by an antibody cascade using anti-myc tag antibody 9E 10 (Novus Biological, Cambridge, UK) as primary antibody, followed by an anti-mouse immunoglobulin- Biotin and, finally, streptavidin-PE conjugate (both Jackson Immunoresearch, Suffolk, UK). Cell surface binding of IgG antibodies was detected with anti-huFab-PE conjugated polyclonal serum (Jackson Immunoresearch).

[000218] For ELISA, 96-well microtiter plates (MaxiSorp; Nunc, Langenselbold, Germany) were coated with 100 ng recombinant human CAIX (rhCAIX), recombinant murine CAIX (rmCAIX) or other recombinant human carbonic anhydrase (rhCA) isoforms (rhCAII, rhCAIV, rhCAXII or rhCAXIV) (R&D Systems, the recombinant proteins contain a His-tag), blocked with 2% milk powder in PBS and incubated with 20 μg/ml Fab or IgG antibody for 1 h at room temperature. Bound Fab antibodies were detected by anti-myc tag antibody 9E10 (Novus Biological), followed by an anti-mouse immunoglobulin-HRP antibody (Dako). Binding of IgG antibody was detected by an anti-human Fab-Biotin (Jackson Immunoresearch), followed by a Streptavidin-POD conjugate (Roche). Absorbance at 450 nm was measured to determine the amount of antibody binding.

[000219] M75 (Pastorek et al., 1994) and G250 (Oosterwijk et al., 1986) were used as control monoclonal antibodies against human CAIX. M- 100 (SantaCruz Biotechnology, Heidelberg, Germany) was used as a control serum against murine CAIX. Successful coating of plates used for ELISA binding studies was confirmed by staining of His-tagged carbonic anhydrase proteins using an anti-His antibody (Q1AGEN) followed by an anti-mouse immunoglobulin-HRP (Dako)

[000220] Determination of kinetic rate constants and binding affinity of CAIX-Fab interactions using surface plasmon resonance

[000221 ] Binding analysis of Fab antibodies to rhCAIX or rmCAIX was performed on a BIAcore T 100 instrument. CAIX was immobilized at low density on a CM5 sensor chip (Biacore AB) using amine coupling chemistry, in accordance to the manufacturer's instructions. Fab antibodies at different concentrations (5 to 240 nM in HBS-P+ buffer of 10 niM HEPES, pH 7.4, 0.15 M NaCl, 0.05% v/v Surfactant P20) were injected at a flow rate of 30 μΐ/min at 25°C to analyse binding kinetics. Analysis of the binding curves and the determination of rate constants were done using non-linear analysis with BIAevaluation software.

[000222] Screening membrane-fragments for carbonic anhydrase activity

[000223] Preparation of HCTl 16 membrane fragments.

[000224] Carbonic anhydrase activity was determined in membrane-fragments extracted from HCTl 16-CAlX and HCT l 16-EV tranfectants grown to 90%- 100% confluency. Cells were washed with ice cold PBS and scraped down to ice cold buffer containing 15 mM NaCl, 35 mM KG, 105 mM potassium gluconate, 20 mM HEPES, 20 mM MES, Complete protease inhibitors (Roche), pH adjusted to 8 at 2°C. The cell suspension was frozen, thawed and centrifuged at 5000 rpm for 5-7 min. The supernatant containing cytosolic proteins was discarded and replaced with ice cold buffer. The mixture was re-frozen, thawed and centrifuged again to obtain a more pure membrane protein suspension.

[000225] CAIX activity measurement.

[000226] A 0.67 ml aliquot of membrane-fragment suspension was added to a 2 ml well- stirred vessel cooled to 2°C. A narrow pH electrode (Biotrode, Hamilton, UK) was inserted to monitor solution pH at 1 Hz. To start the carbonic anhydrase-catalysed reaction, a 0.33 ml aliquot of 100% C0₂-saturated water (2°C) was added to the reaction vessel. C0₂ hydration yields H⁺- ions, which can be measured with the pH electrode. Cooling was necessary to bring the kinetics of reaction within the resolving power of the pH meter. The hydration rate constant was derived from the pH time-course using a fitting algorithm developed previously (Swietach et al., 2009). To determine the spontaneous C0₂ hydration rate, measurements were performed on membrane- free ("blank buffer") samples. The increase in ..hydration rate above this spontaneous rate is a measure of carbonic anhydrase-catalysis. Background carbonic anhydrase activity in HCT1 16 membranes was determined using HCT1 16-EV membrane preparations. Measured hydration rates were normalized to a scale on which HCT1 16-CAIX activity was one and HCT 1 16-EV activity was zero.

(000227) CAll activity measurement.

[000228] CAll was isolated from red blood cells by 1 : 10 dilution in ice cold buffer containing 15 mM NaCl, 35 mM KC1, 105 mM potassium gluconate, 20 niM HEPES, 20 ni MES, Complete protease inhibitors (Roche), pH adjusted to 8 at 2°C. The cell suspension was frozen, thawed and centrifuged at 5000 rpm for 5-7 min. The supernatant containing CAII was used to measure carbonic anhydrase activity according to the CAIX measurement.

[000229] Screening intact cells for extracellular carbonic anhydrase activity

[000230] HCT 1 16-CAIX cells were plated on a poly-L-lysine-coated chamber, fitted on an inverted Nikon microscope. Cells were then exposed for 10 s to medium containing 50 μΜ DHPE-fluorescein (Invitrogen, UK), a membrane-inserting dye that reports extracellular pH (pH_e) at the surface of cells, where CAIX catalysis takes place (Stock et al., 2007). The dye was excited at 484 nm by a monochromator (Cairn, UK) and emission was detected at 520 ± 20 nm with a photomultiplier (Electron Tubes, UK). Data were acquired at 100 Hz using Spike software. Solution (at 37°C) was delivered to the chamber at 2 ml/min to ensure continuous superfusion. Two test solutions were used: (i) normal Tyrode (NT: 125 mM NaCl, 4.5 mM KC1, 10 mM glucose, 12 mM NaHC0₃, 1 mM CaCl₂, 1 mM MgCl₂) and (ii) ammonium-containing NT (AmmNT: 95 mM NaCl, 30 mM NH₄C1, 4.5 mM KC1, 10 mM glucose, 12 mM NaHC0₃, 1 mM CaCl₂, 1 mM MgCl₂). Test solutions were bubbled with 5% C0₂/95% N₂ to maintain a pH of 7.2. Rapid change from NT to AmmNT superfusion and back produces surface pH_e transients that decrease in size with increasing extracellular carbonic anhydrase activity. CAIX activity was therefore estimated from the area under the surface pH_e transients, normalized to the measurement made in the presence of acetazolamide.

[000231] At the end of each experiment, the dye signal was calibrated into units of pH by superfusing with calibration solutions (125 mM NaCl, 4.5 mM KCl, 10 mM glucose, 10 mM

Hepes or 1 0 mM Mes, 1 mM CaCl₂, 1 mM MgCl₂) set at either pH 7.4 or 6.4.

[000232] Measuring carbonic anhydrase assisted CO₂ venting from cells in spheroid models 10002331 The rate of C0₂ efflux out of cells within a spheroid was measured by imaging pH, spatially during a rapid switch-over from solution containing 5% C0₂ to C0₂-free solution. Spheroids (radius 100- 150 μητ) were loaded with the pH reporter-dye, carboxy-SNARF- 1 (50 μΜ; Invitrogen, UK) for 45 min, and placed in a superfusion chamber mounted on a Leica inverted microscope coupled with a TCS NT confocal system. Extracellular dye was washed away by superfusion. Cells in the spheroid were imaged confocally for intracellular pH (x lO lens, 1 .5 Airy units pinhole, excitation 514 nm, dual-emission 580 nm and 640 nm). Spheroid fluorescence was averaged in ten non-overlapping, layered, concentric regions of interest (ROIs), each of mean thickness equal to a tenth of spheroid mean radius. ROl 1 represented the periphery. To improve signal-to-noise ratio at the spheroid core, signal in the ninth and tenth ROIs were averaged under ROI9. Fluorescence emissions in ROIs at 580 nm and 640 nm were background- offset, ratioed and calibrated into pH units using a standard curve obtained in separate experiments using the nigericin method (Swietach et al., 2009).

[000234] Spheroids were superfused in nominally C0₂-free Hepes-buffered solution (HepesNT: 125 mM NaCl, 20 mM Hepes, 4.5 mM KC1, 1 1 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂) and then exposed to 5% C0₂/HC0₃ ^~-buffered solution (BicarbNT: 103 mM NaCl, 22 mM NaHC0₃, 4.5 mM KC1, 1 1 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂; representing physiological solution) to drive C0₂ fluxes across the membranes of cells making up the spheroid. After 4 min, superfusion was switched back to HepesNT. The rate of alkalinisation in different regions of the spheroid was used as a measure of C0₂ efflux. Experiments were performed under control conditions and in the presence of Fab (exposure for 15 min or preincubation for at least 2 h) or IgG (pre-incubation during spheroid formation, 48-72 h).

[0002351 RESULTS

[000236] Selection and characterization of human CAIX Fab antibody fragments

[000237] Monoclonal antibodies specific to recombinant human CAIX (rhCAIX) were selected from a large human Fab antibody library by phage display. After three selection rounds, 19% of phages were able to bind to rhCAIX. Subsequent screening of 400 clones by flow cytometry and sequencing led to the identification of twelve distinct Fab antibodies (named MSC1 -MSC12, FIGURE 23). Binding data for two Fab antibodies, MSC3 and MSC8, are presented in FIGURE 17. Flow cytometry for CAIX-binding was performed on SKRC 17 cells transfected with the human ca9 gene to over-express CAIX (FIGURE 17A) and on HeLa cells cultured under hypoxic conditions to induce CAIX expression (FIGURE 1 7B). In both cell-types, all twelve Fab antibodies revealed a human CAIX-specific binding pattern, illustrated by the distribution of CAIX-positive cells relative to their CAIX-negative controls. In addition, MSC 1 (not shown) and MSC3 (FIGURE 17C) showed cross-reactivity with murine CAIX expressed by CT26 cells under hypoxia. ELISA studies have confirmed a restricted binding pattern to CAIX, but not to other membrane-tethered isoforms (CAIV, CAXII, CAXIV) or the cytosolic isoform CAII. A representative experiment on MSC 8 IgG is shown in FIGURE 18. Similar results were obtained using MSC 1 -MSC 12 Fab antibodies (data not shown).

[000238] Affinity measurements were performed on a low-density rhCAIX-coated chip using surface plasmon resonance. Binding affinity of different Fab antibodies to rhCAIX ranged from 1.9 nM (MSC 1 1 ) to 66 nM (MSC9). The binding affinity constants (EC₅₀) for two representative Fab antibodies MSC3 and MSC8 were 3.7 and 14 nM, respectively. Dissociation rate constants for CA1X-MSC3 and CAIX-MSC8 complexes were found to be 0.0035 and 0.012 s^"', respectively. Binding affinity of MSC3 Fab was 6.6 nM for rmCAIX, confirming cross-reactivity to the murine CAIX isoform (see FIGURE 17C).

[000239] A competition assay (not shown) revealed that binding of MSC3 Fab or MSC8 Fab to rhCAIX was not competitive with M75 or G250. These data indicate that the epitopes of these two previously characterized anti-CAIX antibodies do not overlap with the epitopes of the newly selected antibodies.

[000240] Screening membrane-fragments for carbonic anhydrase inhibition

[000241] A first screen for inhibitory antibodies was performed on membrane-fragments extracted from HCT 1 16-CAIX cells. Carbonic anhydrase activity was determined using an in vitro assay that measures C0₂ hydration by following the time-course of medium acidification. FIGURE 19A shows sample time-courses of medium pH obtained with membrane-fragments from HCT 1 16-CAIX or HCT1 16-EV cells, and also with membrane-free buffer. Carbonic anhydrase activity in HCT1 16-EV membranes was comparable to spontaneous hydration rate in membrane-free buffer and considerably lower than in HCT 1 16-CAIX membranes. This indicated that HCT 1 16-EV cells do not express significant surface carbonic anhydrase activity and that the carbonic anhydrase activity measured in HCT1 16-CAIX membranes was primarily due to CAIX protein. To compare data obtained on different days, carbonic anhydrase activity was normalized to a scale in which control HCT1 16-CAIX and HCT1 16-EV membrane activities were assigned values of one and zero, respectively (FIGURE 19B). The in vitro assays were repeated in the presence of 20 μ^ιηΐ CAIX-binding Fab antibodies (MSC 1 -MSC12), anti-CAIX G250 IgG (Grabmaier et al., 2000) and anti-CAII IgG as a non-binding negative control (kind gift from Dr Silvia Pastorekova, Bratislava). Inhibition was compared with the effect of 100 μΜ acetazolaniide, a broad-spectrum small-molecule carbonic anhydrase inhibitor.

[000242] Among the twelve MSC Fab antibodies tested, four were shown to reduce CAIX activity significantly. Among these, MSC8 Fab had the greatest inhibitory effect (FIGURE 19B). SC5, MSC 10 and MSC 12 Fab were also shown to be inhibitory, but their potency was much lower and, therefore, they were not investigated further. Anti-CAII and anti-CAIX (G250) antibodies did not have a significant effect on CAIX activity whereas acetazolaniide was able to inhibit CAIX activity of HCTl 16-CAIX membrane fragments completely (FIGURE 19B). MSC8 Fab did not block the activity of CAII extracted from lysed human red blood cells (P=0.94, FIGURE 24), consistent with the lack of MSC8 binding to CAII (see FIGURE 18).

[000243] Next, IgG antibodies were generated for MSC8 (an inhibitory Fab antibody) and SC3 (a non-inhibitory Fab antibody) to test whether full-length antibodies have the same effect on carbonic anhydrase-activity as their corresponding Fab antibody fragments. In agreement with Fab data, MSC8 IgG (but not MSC3 IgG) inhibited CAIX activity (FIGURE 19C). Normalized dose-response curves (FIGURE 19D) show maximal inhibition of 57% for MSC8 Fab and 76% for MSC8 IgG, with half-maximal inhibition (K_;) -0.33 Mg/ml, equivalent to 6.5 nM for MSC8 Fab and 2.2 nM for MSC8 IgG.

[000244] Inhibition of CAIX in intact cells

[0002451 Carbonic anhydrase activity was determined from the kinetics of extracellular pH changes at the surface (surface pH_e) of intact HCTl 16 cells to screen for CAIX inhibition under more physiological conditions and in the native cellular environment. The corresponding protocol (FIGURE 20A) and sample time-course (FIGURE 20B) is shown. Addition and subsequent removal of extracellular NH₄ ⁺-containing solution drives transmembrane NH3 flux.

NH3 influx deposits extracellular H⁺-ions at the cell-surface, lowering surface pH_e. The reverse flux depletes extracellular H⁺-ions from the cell-surface, raising surface pH_e (FIGURE 20C).

C0₂/HC0₃ ^~ buffer tends to reduce surface pH_e changes by taking-up or releasing H⁺-ions.

However, the ability of this buffer to "dampen" surface pH_e transients depends on extracellular carbonic anhydrase activity. In contrast, when C0₂/HC0₃ ^~ buffer was replaced with Hepes, a fast buffer that does not require carbonic anhydrase catalysis, surface pH_e transients were not measureable as surface pH_e equilibrated rapidly with bulk solution pH (FIGURE 25). Fig. 4B shows a specimen surface pH_e time-course measured in a HCT 1 16-CAIX cell, superfused with 5% CO2/ I 2 mM HCC>3^_ buffer. The bathing-solution manoeuvres displaced surface pH_e from the bulk solution pH of 7.2. These surface pH_e transients were considerably larger in the presence of the broad-spectrum carbonic anhydrase inhibitor, acetazolamide, illustrating the importance of carbonic anhydrase activity in pH buffering by C0₂/HC03^~.

[000246] FIGURE 2 1 shows mean surface pH_e time courses for HCT 1 1 6-EV and HCT 1 16- CAIX cells under control (drug-free) conditions and in the presence of acetazolamide. In HCT 1 1 6-EV cells, acetazolamide had no significant effect on surface pH_c transients (FIGURE 21 A), confirming negligible surface carbonic anhydrase activity in these transfectants. In contrast, surface pH_e transients measured in HCT 1 16-CAIX cells were 42% smaller than in the presence of acetazolamide, as expected from membrane CAIX activity (FIGURE 2 I B). Incubation of HCT 1 16-CAIX cells with 20 μ^ηιΐ MSC3 IgG for 1 h did not greatly affect surface CAIX activity (FIGURE 21 Ci). In contrast, following 1 h incubation with 20 >ig/ml MSC8 IgG, surface pH_e transients were only 16% smaller than in the presence of acetazolamide (FIGURE 21 Cii). This result suggests that a considerable fraction of membrane CAIX activity has been inhibited by SC8 IgG. MSC8 Fab, unlike its corresponding IgG antibody, failed to inhibit CAIX activity when incubated with HCT 1 16-CAIX cells for 1 h (FIGURE 21 Ciii). However, continuous exposure of cells to 1 μg/ml MSC8 Fab (dissolved in superfusates) was able to inhibit CAIX activity (FIGURE 21 Civ).

[000247] In summary, IgG MSC8 inhibited CAIX-activity by 61 % and MSC8 Fab was able to inhibit CAIX-activity by 48%, but only when the Fab antibody was present continuously in the experimental solutions throughout the measurement protocol (FIGURE 2 I D).

[0002481 Inhibitory effects of antibodies in multi-cellular tissue-growths

[000249] In a final test for inhibitory effects, CAIX physiology was investigated in multicellular tissue-growths (spheroids). A rapid switch-over from solution containing 5% C0₂ to CO"2-free solution leads to alkalinisation of the intracellular space of spheroids. Spheroid pHj was therefore monitored to provide a measure of the rate of C0₂ efflux from cells. |000250| An averaged pHj time-course at the periphery (ROI 1 ) and core (ROI9) of a control, CAIX-active spheroid is shown in FIGURE 22Ai. pHj increased in response to the rapid change of bulk extracellular C0₂ from 5% to 0%. Due to diffusional delays, pH_f changes were two-fold slower at the core. Acute exposure to 1 μg/ml MSC8 Fab in the superfusate for 15 min did not have a significant effect on alkalinisation (FIGURE 22Aii). However, pre-incubation of spheroids with 20 μg/ml MSC 8 Fab for 2-5 h reduced the rate of core-alkalinisation, which was now three-fold slower than periphery-alkalinisation (FIGURE 22Aiii). Similarly, pre-incubation with 20 μg/ml MSC8 IgG for 48-72 h slowed down core-alkalinisation (FIGURE 22Aiv). These data are summarised in FIGURE 22B in terms of the initial pHj slope. Incubation with MSC8 Fab (FIGURE 22Bi) or IgG (FIGURE 22Bii) reduced the rate of acid-equivalent efflux from cells. The inhibitory effect was most pronounced in the spheroid core (reduction by 43% with Fab, 36% with IgG) and significantly less in the spheroid periphery (reduction by 16% with Fab, 10% with IgG).

[000251 ] DISCUSSION

[0002521 Over the past several years, carbonic anhydrase IX (CA1X) has been established as an important prognostic and predictive marker for a variety of malignant diseases and serves as suitable target for immunotherapy (Chia et al., 2001 ; Generali et al., 2006; Giatromanolaki et al., 2001 ; Pastorek et al., 1994; Wykoff et al., 2000). The prognostic significance of up-regulated CA1X expression in hypoxic regions of many different tumours is demonstrated by reduced progression-free survival and overall survival (Winter et al., 2007). In addition, high CAJX expression is predictive of an unfavourable response to standard treatment, such as radiotherapy (Korkeila et al., 2009). Over the past two decades, CAIX-targeting antibodies, such as G250, have been developed, with excellent tumour targeting properties for renal cancer (Stillebroer et al., 2010). Chimeric G250 (cG250) antibody, when coupled to iodine- 124 could be used as a PET tracer to identify clear-cell renal carcinoma with a 100% positive predictive value, and to discriminate malignant from benign renal masses (Divgi et al., 2007). Recently, the first fully human anti-CAIX antibody was selected and shown to be useful for staining of CAIX ex vivo and in vivo (Ahlskog et al., 2009b).

[000253] CAIX-specific antibodies with an inhibitory effect on the target's carbonic anhydrase activity could expand the therapeutic use of antibodies by combining target specificity with biological effects on CAIX catalysis. The purpose of the present study was to identify antibodies with the aforementioned inhibitory effects on CAIX. These inhibitory agents would take advantage of the high selectivity and high affinity of CAIX-targeted antibodies. It is noteworthy, that unlike many other cancer-related proteins, CAIX harbours extracellular epitopes that are accessible to antibodies.

[000254) Twelve antigen-binding antibody fragments (Fab) were generated and screened for CAIX binding specificity and inhibitory effects. The binding selectivity for the CAIX isoform against other membrane-tethered isoforms and a ubiquitous intracellular isoform (CAII) was confirmed by ELISA (FIGURE 18). Anti-CAIX antibodies were then tested for inhibitory effects on carbonic anhydrase activity using an in vitro assay performed on membrane-fragments (FIGURE 19). This screen identified four Fab antibodies that reduce CAIX activity significantly (FIGURE 19B).

[000255] During the preparation of this report, an independent publication has reported on single chain Fv (scFv) fragments with inhibitory effects on human CAIX (Xu et al., 2010). The screening method was similar to the in vitro assay presented here (FIGURE 19). However, our assay was performed on cell-expressed CAIX rather than soluble CAIX-ECD-Fc, providing a more physiologically-accurate target for inhibition studies. The most potent of these other (Xhu et al) antibodies reduced carbonic anhydrase activity by a factor of two when tested on soluble CAIX fusion protein. In our study, the unique MSC8 Fab inhibited CAIX activity by up to 57% and the corresponding full-length IgG MSC8 antibody achieved up to 76% inhibition (FIGURE 19D).

[0002561 Both MSC8 Fab and IgG had a similar half-maximal inhibition dose ( j) of 0.3 μg/ml (FIGURE 19D), within the dose-range that is feasible for in vitro and in vivo antibody applications. The , is numerically close to the Fab's binding constant (EC₅₀) to recombinant human CAIX, estimated independently by surface plasmon resonance. These measurements suggest that the epitope-binding step corresponds to the event that inhibits enzymatic activity.

[0002571 The advantages of the in vitro assay are its ease of collecting data and relative high- throughput. However, this commonly-used assay for carbonic anhydrase activity has the major disadvantage of being performed under non-physiological conditions with respect to the ionic composition, alkaline pH and low temperature of experimental media. In addition, this assay was performed on CAIX extracted from its native cellular environment, i.e. lacking the cellular elements that may affect CAIX activity and its susceptibility towards inhibition. Findings based on this assay alone may not necessarily hold true for a more physiological setting. For instance, CAIX protein may undergo structural changes during membrane-extraction, which may impinge on its interactions with antibodies, possibly yielding a false-positive result for antibody inhibition. Furthermore, the preparation procedure removes intracellular components that may act as scaffolding or regulatory components, which may also affect carbonic anhydrase activity in the presence of antibody.

[000258] To propose a physiologically relevant inhibitory effect of MSC8 Fab or IgG on CAIX, it was necessary to test antibodies using an experimental protocol based on living, intact cells. This intact-cell assay, presented in FIGURE 20, was shown to have good resolving power for assessing carbonic anhydrase activity (FIGURE 21 A and 2 I B) in CAIX-transfected HCT 1 16 cells. MSC8 IgG was shown to inhibit CAIX by 61% (FIGURE 2 1 Cii), a level similar to that obtained with the in vitro assay on membrane fragments. MSC8 Fab inhibited CAIX activity on intact cells by 48%. MSC3 IgG, which was shown to be non-inhibitory using the in vitro assay (FIGURE 19C), was confirmed to have no inhibitory effect on CAIX on intact cells. The persistence of CAIX activity in MSC3 IgG-incubated cell argues against IgG-induced loss of surface CAIX-expression in the studied cell-preparation (FIGURE 21 Ci). This strengthens the case that MSC8 IgG reduces surface CAIX activity by a direct inhibitory effect, rather than by triggering an antibody-mediated change in surface protein expression, such as IgG-evoked protein internalization.

[000259] The intact-cell assay has pointed to a major difference in the inhibitory properties of Fab and IgG that could not be recognised solely from data obtained using the in vitro approach (FIGURE 19 and (Xu et al., 2010)). With the in vitro membrane-fragment or solubilised fusion- protein approach, antibodies were present continuously in the experimental medium. This technique could not, therefore, provide information about the strength or reversibility of antibody-CAIX interactions. In contrast, the intact cell assay for surface carbonic anhydrase activity was based upon a superfusion system. For experiments shown in FIGURE 21 Cii-iii, cells were pre-incubated with antibody before superfusion. The antibody was not, however, present in the superfusate solution throughout the measurement protocol. Cells pre-incubated with MSC8 IgG had significantly reduced surface carbonic anhydrase activity, indicating that the association between CAIX and the IgG persisted, even though extracellular antibody was wash-away with superfusion. In contrast, pre-incubation with MSC8 Fab did not produce a lasting inhibitory effect on CAIX. Presumably, superfusion with Fab-free solution reversed inhibition due to the dissociation of a more labile CAIX-Fab complex. The difference between Fab and IgG might be explained by the higher avidity of the latter which, because of its bivalent nature, may compensate for the fast dissociation rate of Fab antibodies and, therefore, significantly delay dissociation. CAIX inhibition by MSC8 Fab was, however, measured when cells were exposed continuously to the antibody added directly to all superfusate solutions (FIGURE 21Civ). This finding indicates that the onset of CAIX inhibition by MSC8 Fab is rapid and most likely due to a direct binding event to, or near, the CAIX catalytic site. Such differences in behaviour may be relevant therapeutically, as the IgG antibody is expected to have a longer lasting effect, even after a single dosage. Treatment with Fab antibody would, in contrast, require a more continuous infusion protocol to maintain constant plasma levels to slow Fab-CAIX complex dissociation.

[0002601 A further physiological test for the inhibitory effect of MSC8 Fab and IgG antibodies was done on spheroids grown from CAIX-transfected HCT1 16 cells. The CAIX- catalysed process under investigation was the rate of C0₂ efflux from cells, representing the loss of acid-equivalents. Since C0₂ is a principal metabolic acid-product, the kinetics of its diffusion is central to cellular pH homeostasis. Extracellular CAIX can facilitate C0₂ efflux across cell- membranes by hydrating extracellular C0₂, thereby maintaining a steeper efflux gradient (Swietach et al., 2008). This can be tested experimentally in a multi-cellular tissue-like structure that harbours a diffusionally-restricted extracellular space. Within such an unstirred compartment, C0₂ diffusion is impeded and C0₂ hydration can offer a feasible alternative to reducing extracellular C0₂ levels. This study provides experimental evidence that MSC8 Fab and IgG can have a relevant effect on tissue physiology, particularly in the core of spheroids (FIGURE 22). Since CAIX has been proposed to facilitate C0₂ diffusion (Swietach et al., 2008), the inhibitory effect of MSC8 antibodies is most likely due to CAIX inhibition. The lack of effect with acute (< 15 minute) Fab exposure in a multi-cellular tissue-model may be explained in terms of inadequate exposure time for antibody penetration into the spheroid core. A similar accessibility problem may take place in living tissues and tumours. Therefore, comparison of different antibody constructs like Fab and full length IgGs might be very important in context of pharmacokinetics.

[000261] All together, the data presented in this study (combining assays on membrane fragments, intact single cells and multi-cellular tissue-growths) provide a more robust quantification of CAIX inhibition by antibodies than other reported studies (Xu et al., 2010) and indicate physiological relevance of the selected antibodies. Therefore, the presently described fully human CAIX-specific IgG antibody MSC8 may extend beyond the use of previously developed CAlX-antibodies, by combining target-specificity with biological activity. In vivo experiments using an inducible ca9 gene silencing system demonstrated a 40% reduction of tumour growth (Chiche et al., 2009). Combining target specificity with enzymatic inhibition in one antibody molecule may have an additive effect on retarding tumour growth.

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Xu, C, Lo, A., Yammanuru, A., Tallarico, A.S., Brady, ., Murakami, A., Barteneva, N., Zhu, Q., Marasco, W.A., 2010. Unique biological properties of catalytic domain directed human anti- CAIX antibodies discovered through phage-display technology. PLoS One 5(3):e9625. EXAMPLE 4

Antibody Sequence Comparison

|000263| As above indicated, CAIX antibodies have been recently reported by another scientific group (Xu et al (2010) PLoS One 5(3):e9625). The antibodies reported by Xhu fail to demonstrate the potency and inhibitory activity as shown herein, including in comparison to antibody MSC8. It is also notable that the antibody CDR region sequences are distinct between and among all the CAIX antibodies. Table 5 and 6 provide a comparison of light chain variable region CDR sequences and heavy light chain variable region CDR sequences respectively between the MSC antibodies provided herein, particularly inhibitory antibodies MSC 5, SC8, MSC10 and MSC 12, and those reported by Xhu et al to have some CAIX activity.

TABLE 5

parison of Light Chain CDR Sequences for CAIX Inhibiting Antibodies

Antibody CDR1 sequence CDR2 sequence CDR3 sequence

MSC5 SSNIGAGYD GNS QSYDSSLSAWV

MSC8 SGINVDTYM YKSESNQ MIWHSNT V

MSC10 SSNVGAGYD HNN QSYDTRSGWV

MSC12 SSNIGAGYD RNN QSYDRSLSR V

G6 TGSRSNIGADYDVH ANNNRPS QSYDSSLRAWV

G39 TGSSSNIGRGYNVH DNTNRPS QSYDSGLR V

G37 TGSSSNIGAGYDVH GNSNRPS QSYDSSLSAWV

G36 TGSSSNIGAGFDVH GNTNRPS QSYDSRLSAWV

G125 GGDNIGRKSVH DDRDRPS QVWDSSSKHYV

TABLE 6

parison of Heavy Chain CDR Sequences for CAIX Inhibiting Antibodies

Antibody CDRl sequence CDR2 sequence CDR3 sequence

MSC5 GFTFSSYS ISSSGSYI ARVAGSSWRGASDY

MSC8 GGSFSGYY INHSGST ARGSGANYYDSSREPRAFDI MSC10 GFTFSSYS ISSSSSYI ARVGIGGAFDI MSC12 GFTFSSYA ISGSGGST AKYCGGDTCYSGFDY

G6 TYAMT AVSGSGGSTYYADSVKG GPVLRYGFDI

G39 SYAMS AISGSGGSTYYADSVKG IGRYSSSLGY

G37 SYAMS AISANGGTTYYADSVKG NGNYRGAFDI

G36 SYAMS AISANGGTTYYADSVKG NGNYRGAFDI

G125 SYGMH AISGSGGSTYYADSVKG AAVTRGAFP

EXAMPLE 5

Additional Antibody Studies

[000264] Further studies were undertaken to assess tumour staining and CAIX internalisation with the MSC antibodies. Influence on cell proliferation was also determined. Antibodies were coupled to toxic moieties such as TNF and cell survival determined.

[0002651 Materials and Methods

[000266] Immunofluorescence of tumour tissue

[000267] Sections of C51 tumours (kindly provided by S. Lehmann and M. Rudin, Zurich, Switzerland) were fixed in 4 % PFA for 10 min and stained with hypoxy-probe (1 : 100) and unpurified supematants of transiently transfected HEK 293 cells expressing MSC 3 IgG and MSC 1 1 IgG at a dilution of 1 :20, followed by anti-huFab-Cy5 ( 1 : 100). Incubations were performed in a volume of 300 μΐ in PBS containing 2 % NGS and 2 % Triton X-100. Between different antibodies, sections were washed three times for 10 min in PBS. Sections were embedded using ProLong®Gold antifade reagent with DAPI. Pictures were taken on an inverse SP2 Leica microscope with 40x magnification.

[000268] Internalization of CA IX detected by immunofluorescence

[000269] Cover slips were coated with 100 μ^ηιΐ collagen type I in PBS for 1 h at 4 °C. 5,000 cells were seeded o/n. CA IX expression was chemically induced using DMOG as described in the Proliferation Assay section below. Cells were stained with 20 μg/ml anti-CA IX antibodies (MSC 3, MSC 8) or negative control (ESC 1 1) for 1 h at 4 °C or 37 °C, respectively. Cells were fixed and permeabilized with BD Cytofix/Cytpoperm™ for 30 min at 4 °C. Bound IgG was detected with anti-huFab-biotin (1 :200) followed by Strep-DyLigth549 ( 1 μg/ml, 1 : 1 '800). Antibodies were dissolved in BD Perm/Wash and incubated for 1 h at 4 °C. Between different antibodies, cover slips were washed twice with BD Perm/Wash™. Cover slips were embedded with ProLong® Gold antifade reagent with DAPI. Pictures were taken on an inverse SP2 Leica microscope with 40x magnification.

[0002701 Proliferation assay

[000271 ] To test inhibition of proliferation by selected antibodies, 10,000 SKRC 52 cells were seeded in 96-well plates and next day different concentrations of Fab antibodies (up to Ι μΜ) were added. Alternatively 100,000 HeLa cells or MCF-7 cells were seeded in a 6-well plate, incubated for 24 h in an lnvivo₂ 400 hypoxic chamber (Ruskinn Technology) at I % 0₂ and next day 1 μΜ Fab antibody was added. To test inhibition by IgG, 1 ,000 HeLa, SKRC 17 and SKRC 17 MW 1 cl4 or SKRC 52 cells were seeded in a 96-well plate and incubated for 1-5 days in an Invivo₂ 400 hypoxic chamber (Ruskinn Technology) at 0.2 % 0₂ with up to 0.5 μΜ IgG. 1 - 10 days after addition of Fab antibodies or IgGs proliferation was determined using the EZ4U kit according to the manufacturer's instruction, by staining with crystal violet or by counting the cells. For crystal violet staining, cells were washed with PBS and incubated for 10 min at room temperature with crystal violet solution. Plates were washed in dH₂0 and dried. Crystal violet was dissolved in isopropanol/HCl and absorbance was measured at 560 nm in the Wallac Victor² 1420 Multilabel Counter (PerkinElmer). To assess significance a paired two- tailed T-test was performed. To test proliferation under conditions of low pH and low hydrogen carbonate, 1000 SKRC 17, SKRC 17 MW1 cl4 or SKRC 52 cells were seeded in 6-well-plates. After attachment of the cells to the plate, medium was changed to RPMI w/o hydrogen carbonate at pH 6.3 or 7.3 (R 10_6.3 or R10_7.3, respectively) and 20 μ /πι1 IgG or 100 μΜ acetazolamide (stock: 100 mM in DMSO) were added. Cells were incubated in an incubator at ambient C0₂- partial pressure for 24 h. Medium was changed to normal RPMI and cells were grown for another 7 d at 5 % C0₂. Colonies were counted.

[000272] TNF assay

[000273] 10,000 Wehi S cells were seeded in 96-well-plates in a volume of 100 μΐ. After attachment of the cells to the plate, 100 μΐ supernatant of HEK 293 cells transiently transfected with F(ab)₂-TNF constructs and serial dilutions of the same were added and incubated over night in the incubator. On the next day, cells were stained using the EZ4U kit according to the manufacturer's instruction (with only 10 μΐ of substrate added to 200 μΐ cell culture). Absorbance was measured at 450 nm in the Wallac Victor² 1420 Multilabel Counter (PerkinElmer) after 2 h. Significance was assessed using a paired, two-sided T-test.

[000274] RESULTS

[000275] Tumour staining using MSC 3 IgG and MSC 11 IgG

SC 3 IgG and MSC 1 1 IgG were used to stain tumour sections of C5 1 tumours (FIGURE 26). MSC 3 showed specific staining of tumour tissue corresponding to staining of the perfusion marker pimonidazole. Staining for MSC 1 1 was negative corresponding to the fact, that this antibody recognizes only human CA IX but not murine CA IX.

[000276[ Internalisation of CA IX by MSC 3 IgG and MSC 8 IgG

Both antibodies were then tested for their potential to trigger internalisation of bound CA IX protein. MSC 3 IgG as well as MSC 8 IgG led to internalisation of CA IX on stably transfected SKRC 17 MW 1 cl4 cells and HeLa cells chemically induced with 1 mM DMOG for 48 h when incubated at 37 °C for 2 h (data not shown). If incubation was performed at 4 °C, the cell membrane was stained and no internalisation was observed. In contrast, the corresponding Fab antibodies MSC 3 and MSC 8 did not induce internalisation, regardless of the incubation temperature. On murine CT26 cells chemically induced with 2 mM DMOG no internalisation was observed (data not shown). MSC 3 Fab and IgG both stained the cell membrane and did not induce internalisation. For the non-crossreactive antibody MSC 8 no signal could be detected on CT26. Control antibody ESC 1 1 did not led to any signal on the tested cell lines. The

internalization triggering demonstrated by these IgG antibodies, particularly of inhibiting antibody MSC8 IgG, may serve to further reduce CAIX activity on the cell surface, particularly of tumor or cancer cells.

[000277] Influence of selected antibodies on cell proliferation

[000278] Fab antibodies MSC 3, MSC 8 and MSC 1 1 as well as MSC 3 and MSC 8 IgG were tested for a possible antiproliferative effect on different cell lines expressing CA IX constitutively or after hypoxic induction. When SKRC 52 cells were incubated for 3 days with 1 μΜ MSC 3 or MSC 8 Fab antibody cell number was not decreased compared to cell number when incubated with media without any additives (data not shown). Cell number of SKRC 52 and HeLa cells induced in a hypoxic chamber at 0.2 % 0₂ decreased significantly when incubating the cells for 24

acetazolamide compared to incubation with medium without any additives. In contrast neither MSC 3 nor SC 8 IgG were able to do so at a concentration of 0.5 μΜ, which is more than ten times higher than the concentration of MSC 8 IgG needed for maximal inhibition of CA IX activity. In addition, none of the tested Fab antibodies or IgGs was able to influence proliferation of any of the other tested cell lines SKRC 17, SKRC 17 MW 1 cl4 or MCF-7 cells induced under hypoxia (data not shown).Under harsh conditions of low pH were cells have been proposed to depend on CA IX function to survive (Chiche, lie et al. 2009), survival of SKRC 17 MW 1 cl4 was not augmented compared to SKRC 17 cells (data not shown). Similarly, blocking by ATZ had no effect on cell survival, showing that survival under these conditions was indeed not CA IX dependent.

[000279] Coupling of antibodies to toxic moieties

[000280] Coupling to toxic moieties can be used to combine antibody specific targeting with functional effects of a toxic moiety. MSC 3 and MSC 1 1 were chosen for their high affinity on human CA IX with MSC 3 showing in addition cross-reactivity to murine CA IX. To increase a possible anti-tumour effect, TNF was cloned into the pEE 12.4 vectors expressing the respective antibodies, creating an F(ab)₂ fragment coupled to TNF. F(ab)₂-TNF constructs were transiently expressed in HEK 293 cells and tested in western blot and in flow cytometry (data not shown). All F(ab)₂-TNF constructs as well as the control MSC 3 IgG could be detected by an anti-hu-Fab- POX antibody in immuno blotting and anti-hu-Fab-PE in flow cytometry, whereas anti-huTNF and anti-muTNF stained only the F(ab)₂-TNF constructs containing the corresponding TNF. Functionality of the F(ab)₂-TNF constructs was confirmed by killing of TNF sensitive Wehi S cells in a dose dependent manner (data not shown). F(ab)₂-TNF constructs were able to significantly inhibit survival of Wehi S cells when supernatant of HEK 293 cells transiently transfected with F(ab)₂-TNF constructs was added undiluted. Inhibition was in the same range as with recombinant muTNF.

EXAMPLE 6

Animal Studies (0002811 To assess CAIX antibodies in animal models, xenografts are utilized because most of the antibodies described herein do not recognize mouse CAIX. Therefore, human tumor cells are grafted in nude/SCID mice and the activity of the antibody to block tumor growth is determined. Cell lines known to express CAIX constitutively such as most renal cancer cell lines of the S -RC type are used in xenograft studies. Alternatively or in addition, tumor cell lines known to up-regulate CAIX under hypoxia such as HT29 cells or colon cancer cell lines are useful. These xenograft models are similar to those utilized by Chiche et al to assess CAIX and CAXII contribution to tumor growth (Chiche J et al (2009) Cancer Res 69(l ):358-368). Xenograft studies using human head and neck carcinoma cells (for example SCCNij3), which were previously utilized in CAIX antibody labeling studies (using G250 antibody), are also useful (Hoeben BAW et al (2010) J Nuclear Med 51 (7): 1076- 1083).

[000282] Antibody for testing is administered either after establishment of xenografts and/or serially after introduction of cancer cells to nude/SCID mice and progression and/or maintenance of tumor growth or size is evaluated. Labeleled antibody is utilized to determine tumor uptake of antibody. In addition to CAIX single antibody studies, CAIX antibodies are evaluated in combination with one another, as well as with antibodies against other carbonic anhyrdrases (such as CAXI I), against HIF- 1 , or other inhibitors, such as ATZ and other sulfonamides, for their ability to induce significant tumor reduction. Tumor progression and tumor size is determined in xenograft animals.

[000283] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[000284] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1 . An isolated antibody molecule or fragment thereof which recognizes human CAIX and is selected from MSC 1 , MSC2, MSC3, MSC4, MSC5, MSC6, MSC7, MSC8, MSC9, MSC 10, MSC 1 1 and MSC 12.

2. The isolated antibody or fragment of claim 1 which also recognizes mouse CAIX.

3. The isolated antibody of claim 1 which also inhibits CAIX catalytic activity.

4. The isolated antibody or fragment of claim 1 which is an antibody or antibody fragment comprising CDR domain sequences as set out in any of FIGURES 5 through 16.

5. The isolated antibody or fragment of claim 1 which is an antibody or antibody fragment comprising a heavy chain and a light chain variable region comprising an amino acid sequence selected from the amino acid sequence set out in any of FIGURES 5 through 16, or highly homologous variants thereof comprising 1 to 3 amino acid substitutions in one or more CDR region of any of FIGURES 5 through 16, wherein said variants retain human CAIX reactivity.

6. An isolated antibody or fragment thereof which recognizes human and mouse CAIX comprising a heavy chain and a light chain variable region comprising an amino acid sequence set out in FIGURE 7.

7. The isolated antibody of claim 6 which comprises a heavy chain having a variable region amino sequence comprising the CDR region sequences of FIGURE 7 and a light chain having a variable region amino sequence comprising the CDR region sequences of FIGURE 7.

8. An isolated antibody or fragment thereof which recognizes human CAIX and inhibits CAIX catalytic activity comprising a heavy chain and a light chain variable region comprising an amino acid sequence set out in FIGURE 12.

9. The isolated antibody of claim 8 which comprises a heavy chain having a variable region amino sequence comprising the CDR region sequences of FIGURE 12 and a light chain having a variable region amino sequence comprising the CDR region sequences of FIGURE 12.

10. The isolated antibody or fragment of claim 1 which is an antibody or fragment thereof wherein said isolated antibody is the form of an antibody F(ab')2, scFv fragment, diabody, triabody or tetrabody.

1 1. The isolated antibody or fragment of claim 1 further comprising a detectable or functional label.

12. The isolated antibody of claim 1 1 , wherein said detectable or functional label is a covalently attached drug.

13. The isolated antibody of claim 1 1 , wherein said label is a radiolabel.

14. An isolated nucleic acid which comprises a sequence encoding an antibody or fragment of any of claims 1 through 5.

15. A method of preparing an antibody or fragment as defined in any one of claims 1 to 10 which comprises expressing the nucleic acid of claim 14 under conditions to bring about expression of said antibody or fragment, and recovering the antibody or fragment.

16. An antibody or fragment according to any one of claims 1 to 1 1 for use in a method of treatment or diagnosis of the human or animal body.

17. A method of treatment of epithelial cancer in a human patient which comprises administering to said patient an effective amount of an antibody or fragment as defined in any one of claims 1 to 1 1 .

18. A kit for the diagnosis or prognosis of cancer in which CAIX antigen is expressed, said kit comprising an antibody or fragment of any one of claims 1 to 1 1 , optionally with reagents and/or instructions for use.

19. A pharmaceutical composition comprising an antibody or fragment as defined in any one of claims 1 to 1 1 and a pharmaceutically acceptable vehicle, carrier or diluent.

20. A kit for the treatment of a tumor in a human patient, comprising a pharmaceutical dosage form of the pharmaceutical composition of claim 19, and a separate pharmaceutical dosage form comprising an additional anti-cancer agent selected from the group consisting of

chemotherapeutic agents, radioimmunotherapeutic agents, and combinations thereof.

21. A method for detecting the presence of cancer or determining the prognosis of cancer in a mammal wherein said cancer is measured by determining the presence and/or amount of CAIX:

A. contacting a biological sample from a mammal in which the presence of cancer is suspected with the antibody or fragment of any of claims 1 to 1 1 under conditions that allow binding of said CAIX to said antibody to occur; and

B. detecting whether binding has occurred between said CAIX from said sample and the antibody or determining the amount of binding that has occurred said CAIX from said sample and the antibody;

wherein the detection of binding indicates the presence of cancer in said sample and the amount of binding indicates the prognosis of cancer in said sample.

22. A method for targeting cancer in mammals, comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition of claim 19 or the kit of claim 20.