WO2003093319A1 - Novel humanized anti-vap-1 monoclonal antibody - Google Patents

Abstract

Chimeric anti-VAP-1 antibodies and fragments thereof are disclosed. Nucleic acids encoding anti-VAP-1 antibodies or fragments thereof, as well as expression vectors and host cells incorporating these nucleic acids for the recombinant expression of anti-VAP-1 antibodies are also given. Pharmaceutical compositions comprising said antibodies and therapeutic uses thereof are also disclosed.

Description

NOVEL HUMANIZED ANTI-VAP-1 MONOCLONAL ANTIBODY

FIELD OF THE INVENTION

[0001] The present invention relates to nucleic acid sequences encoding humanized monoclonal antibodies recognizing a human endothelial cell adhesion protein, VAP-1 , chimeric anti-VAP-1 antibodies and particularly to a chimeric monoclonal antibody, designated BTT-1002, which recognizes a functional epitope of VAP-1.

BACKGROUND OF THE INVENTION

[0002] Generally, antibodies share a common Y-shape structure composed of two identical light chains and two identical heavy chains. These four polypeptide subunits are assembled so that the two heavy chains are linked, and a light chain is attached to each heavy chain by disulfide bonds. Each polypeptide constituting the antibody consists of a variable and a constant region. [0003] The variable region is located in the arms of the Y-shaped antibody, and determines the antigen-binding specificity of the antibody. This region contains short amino acid sequences, which are responsible for the binding of the antibody to its antigen. These regions are called complementarity determining regions (CDRs). The remaining parts of the variable regions are important for the conformation of the antigen-binding pocket as a whole.

[0004] The constant region of an antibody is located at the base of the heavy chains, and determines the antibody's ability to activate immune reactions through interactions with specific receptors. These regions are generally highly conserved, and variability is limited to five basic isoforms, IgA, IgD, IgE, IgG and lgM.

[0005] Vascular adhesion protein-1 (VAP-1) is a non-classical, in- flammation-inducible, adhesion molecule expressed on vascular endothelial cells, where it mediates leukocyte-subtype specific rolling under physiological shear. VAP-1 belongs to a particular molecular class of cell surface amine oxi- dases. The amine oxidase enzymatic reaction itself, and the biologically active end products (including aldehydes and hydrogen peroxide), have multiple potentials to regulate the adhesive status of the vessel wall. Thus, VAP-1 is an ectoenzyme, which has interrelated adhesive and enzymatic functions in regulating leukocyte trafficking and inflammation. In this role it contributes towards lymphocyte re-circulation through high endothelial venules (HEV's) of secondary lymphoid tissue as part of the normal process of immune surveillance.

[0006] However, under inflammatory conditions, VAP-1 promotes the infiltration of leukocytes into inflamed tissue, thereby contributing to and maintaining the inflammatory response. This infiltration can in itself be damaging in chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and many other diseases. In other settings, the massive infiltration of pro-inflammatory cells into tissue after the severe tissue damage resulting from myocardial infarction, stroke and other diseases con- tributes to the tissue destruction seen in these acute inflammatory responses. Reducing the infiltration of cells into sites of inflammation by preventing VAP-1 function with blocking antibodies is likely to lead to an improvement in these diseases.

[0007] US Patent 5580780 describes a monoclonal antibody, 1 B2, which recognizes VAP-1 and which can block lymphocyte binding to tonsillar HEV in a frozen section assay. MAb 1 B2 is a murine IgM-antibody and is specific for VAP-1.

[0008] The use of murine mAbs as therapeutics has a limited potential, since the human immune system recognizes murine antibodies as foreign material and produces human anti-mouse antibodies (HAMA) to clear them from the body. This immune reaction is a major limitation to the use of murine antibodies in long-term therapy when repeated administration is needed. The use of murine anti-VAP-1 antibodies in the clinic might have to be limited to patients treated with immunosuppressants, and thus less prone to HAMA reac- tions, and to treatment regimens, where once only administration of the antibodies is feasible, such as in ischaemia, reperfusion injury in acute infarction or acute respiratory distress syndrome.

[0009] One further disadvantage related to the use of murine anti- VAP-1 antibodies in therapy is the unfavorable kinetic profile of such antibod- ies, i.e., the short half-life, which render them unsuitable for use in chronic disorders, such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and many other diseases.

[0010] Several methods of creating less immunogenic monoclonal antibodies are known in the art. Preferred approaches include "humanizing" the antibodies. Frequently used strategies are to create chimeric mAbs, humanized mAbs or fully human mAbs. Chimeric mAbs are antibodies wherein the variable region is murine derived whereas the constant region is of human origin. US Patent No. 5169939 describes a method of producing recombinant chimeric monoclonal antibodies and US Patent No. 5530 101 describes methods for producing humanized immunoglobulins having one or more comple- mentarity determining regions (CDR's) from a donor immunuglobulin and a framework region from an accepting human immunoglobulin. US Patent No. 5202238 describes a process for producing chimeric antibodies using recombinant DNA vectors and homologous recombination in vivo. Fully human mAbs are currently derived from human immunoglobulin genes expressed in trans- genic mice. Human mAbs can also be produced from human hybridomas or immortalized human B-lymphocyte cell lines.

[0011] International patent publication WO99/58572 describes a recombinant monoclonal antibody, wherein the constant domains are derived from a human lgG2 antibody (G2Δa). The modification in G2Δa is such that the resulting antibodies should not activate complement and have reduced binding to certain Fc receptors. This should assist in avoiding the unwanted antiglobu- lin response and protect the target cells from antibody-mediated destruction.

[0012] There is good evidence that replacing mouse constant regions with human constant regions reduces HAMA-responses. However, there is as yet no clear evidence to suggest that fully humanizing the variable regions will reduce the immunogenicity further. In contrast, there is good evidence that such a process can result in disadvantages such as loss of antibody affinity (Riechmann et al., Nature 332, 323-327 (1988)).

[0013] The present invention provides anti-VAP-1 therapeutic anti- bodies suitable for use in the treatment of chronic inflammation disorders. A preferred embodiment provides a chimeric anti-VAP-1 antibody consisting of mouse variable regions, which are sufficiently homologous with human variable regions, fused to modified human constant regions. The^' chimeric anti- VAP1 antibodies of the present invention thus possess many desirable charac- teristics for the therapeutic applications envisaged.

BRIEF DESCRIPTION OF THE INVENTION

[0014] The present invention is broadly directed to novel chimeric anti-VAP-1 antibodies, methods of producing such antibodies and uses of the antibodies. [0015] An object of the present invention is to provide nucleic acids encoding chimeric anti-VAP-1 antibodies or fragments thereof, as well as expression vectors and host cells incorporating these nucleic acids for the recombinant expression of anti-VAP-1 antibodies are also given. [0016] Another embodiment of the present invention is directed to methods for producing the chimeric anti-VAP-1 antibodies according to the present invention by recombinant production methods.

[0017] Pharmaceutical compositions comprising said antibodies and therapeutic uses thereof are also disclosed. [0018] A further aspect of the present invention is to provide a chimeric monoclonal antibody for use in vivo diagnostics and/or therapy, which does not elicit a immune response in the patients and which has a favourable kinetic profile for therapeutic purposes.

BRIEF DESCRIPTION OF THE FIGURES [0019] In the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached figures, in which

[0020] Figure 1 shows a comparison of amino acid sequences of anti-VAP-1 variable regions. Amino acid sequences of the heavy chain (A) and the light chain (B) variable regions of the mouse antibodies Mo2D10, MoTKδ- 14 and Mo1 G6 deduced from the cloned mouse cDNAs are aligned. The three CDRs in each chain are shown in bold. Residues are numbered according to the Kabat system;

[0021] Figure 2 shows the variable genes of mouse anti-VAP-1 an- tibodies cloned into pSV-vectors. A schematic presentation of the heavy chain, pSV-gpt and the light chain, pSV-hyg expression constructs is shown. The PCR-amplified /-//ndlll-BamHI fragments are indicated.

[0022] Figure 3 represents an SDS-PAGE analysis of chimeric anti- VAP-1 antibodies. Protein-A purified chimeric antibodies (2μg/lane) were run on a SDS-polyacrylamide gel (12.5% acrylamide) under reducing conditions and stained with Coomassie Brilliant Blue. Lanes: 1 , BTT-1005; 2, BTT-1006; 3, BTT-1002. Molecular size markers (in kD) are indicated on the left;

[0023] Figure 4 shows the results of VAP-1 binding activity of the chimeric antibodies. The binding of the chimeric antibodies and an irrelevant control antibody (ctrAb) to Ax cells expressing human VAP-1 or mock control cells was analysed by flow cytometry. Results are expressed as percentage of maximal mean channel fluorescence (MCF);

[0024] Figure 5 shows the functional characterization of the mouse and chimeric monoclonal antibodies in human tissue. Tonsil sections were stained with biotinylated MoTK8-14 (A), BTT-1006 (B), Mo2D10 (C), and BTT- 1002 (D) and negative control antibody (E) followed by streptavidin- phycoerythrin. In each of panels A-D, two HEVs are arrowed (GC, germinal center). Original magnification x50;

[0025] Figure 6 illustrates the inhibition of the lymphocyte binding by anti-VAP-1 antibodies. The abilities of the murine and chimeric antibodies to block the binding of lymphocytes to HEV were analysed using an in vitro frozen section assay. Sections were treated with anti-VAP-1 antibodies or with negative control antibodies (irrelevant monoclonal antibody 3G6 or human immunoglobulin). Results of two experiments are presented with their standard er- rors.

[0026] Figure 7 shows the results of flow cytometric analysis of antibody binding to human Fcj/RI. B2KA cells expressing human FcγR\ on their surface were incubated with the mouse (A) and chimeric (B) anti-VAP-1 antibodies followed by appropriate detection reagents. A human lgG1 antibody was used as a positive control in B.

[0027] Figure 8 shows the serum concentrations after i.v. injection of chimeric anti-VAP-1 antibody, BTT-1002 (panel A) and murine anti-VAP-1 antibody (panel B);

[0028] Figure 9 shows the clinical score of BTT-1002 treated (panel A) and placebo treated (panel B) animals;

[0029] Figure 10 shows the survival of rhesus monkeys in the CIA study.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention is directed to humanized, preferably chimeric, recombinantly produced monoclonal antibodies specifically recognizing human Vascular Adhesion Protein-1 , VAP-1. The chimeric MAbs according to the present invention have improved kinetic properties compared to corresponding murine antibodies and are thus useful for treating a number of inflammatory conditions and diseases of connective tissue, skin, and the gastro- intestinal tract, central nervous system, and pulmonary systems, including conditions, such as chronic arthritis, inflammatory bowel diseases, and chronic dermatoses. The chimeric VAP-1 antibodies are further useful for in vitro and in vivo diagnostic applications, including in vivo immunoscintigraphic imaging of inflammation sites. [0031] The present invention establishes stable cell lines which provide a source of chimeric antibodies that can be purified in a standard one-step protocol. All of the chimeric antibodies according to the present invention were shown to bind specifically to the surface-expressed human VAP-1. The VAP-1 binding properties of the antibodies of the present invention were comparable to those of their respective mouse monoclonal antibodies or, in the case of BTT-1005, improved due to the heterogeneous nature of the murine antibody preparation. Moreover, both the mouse and chimeric antibodies stained the different cell types and structural compartments of tonsils in a similar manner. The functional characteristics of the chimeric antibodies were studied by ana- lyzing their capacity to block the adhesive function of VAP-1 and thereby lymphocyte binding to HEV. The original mouse monoclonal antibodies used in the preparation of the chimeric antibodies according to the present invention are known to inhibit lymphocyte binding to HEV. The chimeric anti-VAP-1 antibodies according to the present invention caused similar levels of inhibition in in vitro adhesion assays. Additionally, the present antibodies lack the disadvantages of their murine counterparts, in having a substantially longer half-life and not eliciting Fc-receptor binding. These results strongly suggest that these chimeric antibodies are invaluable tools for therapeutic purposes.

[0032] In therapeutic applications using VAP-1 antibodies, harmful side effects could be caused by the complement and Fc-receptor binding activities of the Fc region. For this reason, the chimeric antibodies were constructed using a modified human lgG2 constant regions, called G2Δa, which has been engineered to lack natural effector functions. The preparation of G2Δa is disclosed by Armous et al. in Eur. J. Immunol. 29:2613-2624, 1999 and International Patent publication WO99/58572. The G2Δa constant region is not only deficient in binding to FcγRI and in activating human monocytes but in that the anti-Rh version could specifically inhibit the triggering of human monocytes by RhD⁺ red blood cells in the presence of human alloimmune anti- RhD sera. Therefore, the human constant region G2Δa appears to be useful when constructing therapeutic antibodies lacking natural effector functions. [0033] The present invention provides chimeric anti-VAP-1 antibodies comprising an amino acid sequence having much less than 30% of amino acids, preferably less than 20% or less than 10%, that would be classed as non-human. The present invention further provides chimeric anti-VAP-1 anti- bodies having advantageous Fcy'RI binding activity in comparison with their murine counterparts. Unlike all the murine antibodies, the chimeric anti-VAP-1 antibodies according to the present invention did not show binding to the receptor even at the highest concentration tested. In addition to their nondestructive nature, the antibodies according to the present invention should be less immunogenic in man. They are not likely to cause inflammation because complement activation and antibody dependent cell-mediated cytotoxicity triggered through FcγR binding should not occur.

[0034] A further aspect of the present invention is to provide a chimeric antibody wherein the variable region of the heavy chain comprises a mouse anti-VAP-1 Mab amino acid sequences SEQ ID NO:s 11 to 13 or functional variants thereof, and wherein the variable region of the light chain comprises a mouse anti-VAP-1 Mab amino acid sequences SEQ ID NO:s 17 to 19 or functional variants thereof.

[0035] The chimeric antibody molecules and chains of the present invention may comprise: a complete natural antibody molecule, having full length heavy and light chains; a fragment thereof, such as a Fab, Fab', F(ab').sub.2 or Fv fragment; a light chain or heavy chain monomer or dimer; or a single chain antibody, e.g. a single chain Fv in which heavy and light chain variable regions are joined by a peptide linker; or any other recombinant, chi- meric or CDR-grafted molecule with the same specificity as the original murine antibodies. Similarly the heavy and light chain variable region may be combined with other antibody domains as appropriate.

[0036] The chimeric antibodies of the present invention are preferably of lgG2-type. The constant domain is preferably an lgG2 and of a form such that it does not elicit Fc-binding or complement activation. Most preferably the constant domain is G2Δa. Alternative constant domains useful in preferred embodiments of the present invention include other human IgG constant domains.

[0037] The chimeric antibodies of the present invention have a longer half-life in comparison to their corresponding murine counterparts, and two studies in non human primates indicate that when using the chimeric anti- bodies of the present invention an effective concentration of antibody will be achieved by once weekly administration, or even at much longer intervals between administration, of antibody at doses in the range of 1 and 10 mg/kg. Murine 1 B2 at doses of the same range, however, will necessitate daily dosing in order to achieve effective concentrations of antibody in the serum.

[0038] Preferably there is provided a specific chimeric antibody, BTT-1002, wherein the heavy chain consists of the amino acid sequence depicted in SEQ ID NO: 15 and the light chain consists of the amino acid sequence depicted in SEQ ID NO: 21. [0039] According to another aspect the present invention provides a

DNA molecule encoding a chimeric, humanised anti-VAP-1 antibody heavy chain, comprising the DNA sequence depicted in SEQ ID NO: 5 or fragments thereof, and a DNA molecule encoding a chimeric, humanised anti-VAP-1 antibody light chain, comprising the DNA sequence depicted in SEQ ID NO: 10 or fragments thereof. There is further provided expression vectors incorporating said DNA molecules and host cells transformed with said expression vectors.

[0040] Yet another aspect of the present invention provides a method of preparing the chimeric anti-VAP-1 antibodies according to present invention by a process which comprises transforming a host cell with a first expression vector comprising a DNA sequence encoding a chimeric, humanised anti-VAP-1 antibody heavy chain, and with a second expression vector comprising a DNA sequence encoding a chimeric, humanised anti-VAP-1 antibody light chain, and propagating said host cell under such conditions that each chain is expressed and isolating said expressed and assembled chimeric anti-VAP-1 antibody or biologically active derivatives thereof from the culture. The expression vector according to the present invention may also be such, that it comprises a construct encoding both heavy and light chain. General methods by which the vectors may be constructed, transfection methods and culture methods are well known per se. [0041] Any suitable host cell/vector system may be used for expression of the DNA sequences coding for the chimeric and humanised antibody heavy and light chains. Bacterial e.g. Escherichia coli, and other microbial systems may be used, in particular for expression of antibody fragments such as Fab and F(ab').sub.2 fragments, and especially Fv fragments and single chain antibody fragments e.g. single chain Fv's. Eucaryotic e.g. plant, yeast or mammalian host cell expression systems or transgenic plants and animals may be used for production of larger chimeric antibody products, including complete antibody molecules, and/or if glycosylated products are required. Suitable mammalian host cells include CHO cells and myeloma or hybridoma cell lines. Preferred host cells are CHO cells. [0042] The present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, a chimeric anti-VAP-1 antibody according to the present invention. The composition of the present invention contains chimeric anti- VAP-1 antibodies according to the present invention in amounts sufficient to antagonize (fully or partially) the patient's native VAP-1 binding to the biological ligands of VAP-1 in patients in need of such antagonizing, and specifically to VAP-1 ligands presented on leukocytes.

[0043] Amounts and regimens for the administration of chimeric anti-VAP-1 antibodies according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating inflammation- related disorders. Generally, the dosage of the chimeric anti-VAP-1 antibody treatment will vary depending on considerations such as: age, gender and general health of the patient to be treated; kind of concurrent treatment, if any; frequency of treatment and nature of the effect desired; extent of tissue dam- age; duration of the symptoms; and other variables to be adjusted by the individual physician. A desired dose can be administered in one or more applications to obtain the desired results. Pharmaceutical compositions according to the present invention may be provided in unit dosage forms.

[0044] The pharmaceutical compositions according to the present invention can be administered in any appropriate pharmacological carrier for administration. They can be administered in any form that effects prophylactic, palliative, preventive or curing conditions of VAP-mediated medical conditions in human or animal patients.

[0045] Pharmaceutical compositions of the chimeric anti-VAP-1 an- tibodies according to the present invention for parenteral administration include sterile aqueous or non-aqueous solvents, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, including saline and buffered medial parenteral vehi- cles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replen- ishers, such as those based on Ringer's dextrose and the like. Aqueous compositions according to the invention may comprise suitable buffer agents, such as sodium and potassium phosphates, citrate, acetate, carbonate or glycine buffers depending on the targeted pH-range. The use of sodium chloride as a tonicity adjuster is also useful. Compositions may include other excipients, such as stabilizing agents or preservatives. Useful stabilizing excipients include surfactants (polysorbate 20 & 80, poloxamer 407), polymers (polyethylene glycols, povidones), carbohydrates (sucrose, mannitol, glucose, lactose), alcohols (sorbitol, glycerol propylene glycol, ethylene glycol), suitable proteins (albumin), suitable amino acids (glycine, glutamic acid), fatty acids (ethanola- mine), antioxidants (ascorbic acid, cysteine etc.), chelating agents (EDTA salts, aspartic acid) or metal ions (Ca, Ni, Mg, Mn). Among useful preservative agents are benzyl alcohol, chlorbutanol, benzalkonium chloride and possible parabens.

[0046] The pharmaceutical composition according to the present invention may be provided in concentrated form or in form of a powder to be reconstituted on demand. In such cases formulations of powder for solution for injection/infusion excipients mentioned above may be used. In case of lyophi- lizing, certain cryoprotectants are preferred, including polymers (povidones, polyethylene glycol, dextran), sugars (sucrose, glucose, lactose), amino acids (glycine, arginine, glutamic acid) and albumin. If solution for reconstitution is added to the packaging, it may consist e.g., of pure water for injection or sodium chloride solution or dextrose or glucose solutions. [0047] The composition of the invention is suitable for diagnosing or treating any condition involving an inflammatory reaction in which VAP-1 adhesion plays a role. Thus, the composition is useful for diagnosing or treating such conditions as arthritis, dermatoses, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, autoimmune diseases, psoriasis, atopic eczema, lichen ruber planus, etc.

[0048] The therapeutically useful chimeric anti-VAP-1 antibodies according to the present invention may be conjugated, either chemically or by genetic engineering, to fragments or other agents, which provide targeting of the antibodies to a desired site of action. Alternatively, other compounds may be conjugated, either chemically or by genetic engineering, to the antibodies according to the present invention, so as to enhance or provide additional properties to the antibodies, especially properties, which enhance the antibodies' ability to promote alleviation of harmful effects mediated by VAP-1 binding. [0049] The chimeric anti-VAP-1 antibodies according to the present invention may by labelled, either chemically or by genetic engineering, to pro- vide detectable antibodies. Such labelled antibodies will be useful tools for imaging inflammatory sites in humans, especially for in vivo immunoscintigraphic imaging of inflammation sites. This type of imaging may replace the more cumbersome and expensive leukocyte imaging-method currently used. For imaging purposes, the use of antibody fragments will be preferable to the whole antibody approach to anti-inflammatory therapy but fragments derived from chimeric antibodies should still be safer than their mouse equivalents. [0050] In another aspect, the present invention is directed to a method of lessening or treating inflammation, in vivo, in the human body, by administering, to a human patient in need of such treatment, efficacious levels of the chimeric anti-VAP-1 antibody according to the present invention. The term "treatment" or "treating" is intended to include the administration of chimeric anti-VAP-1 antibodies to a subject for purposes which may include prophylaxis, amelioration, prevention or cure of disorders mediated by VAP-1 adhesion events. The particular anti-VAP-1 antibodies that are the subject of the methods of the in- vention are purified recombinant chimeric anti-VAP-1 antibodies of the present invention.

[0051] By an "efficacious level" of a chimeric anti-VAP-1 antibody is meant a level in which the harmful effects of VAP-1 mediated events are, at a minimum, ameliorated. An efficient amount of the antibody of the present invention is one that is sufficient to block, or partially block, the endothelial binding of leukocytes in order to inhibit leukocytic infiltration in inflammation sites, where such infiltration is harmful or undesired. Amounts and regimens for the administration of chimeric anti-VAP-1 antibodies can be determined readily by those with ordinary skill in the clinical art of treating inflammation-related disorders such as arthritis and tissue injury. Preferably, the chimeric anti-VAP-1 antibodies according to the present invention are provided intravenously at intervals ranging between once weekly to once every three months at doses in the range of 0.1 to 20 mg/kg, more preferably in the range of 0.2 to 10 mg/kg, most preferably 0.5 to 5 mg/kg. [0052] The following examples are given to further clarify the invention in more detail but are not intended to restrict the scope of the present invention. Fur- ther applications and uses are readily apprehended by a person skilled in the art, i.e., clinicians familiar with inflammatory disorders and treatment thereof.

EXAMPLES

Example 1 Cloning of mouse Fd and light chain cDNAs

[0053] We have generated a number of well-characterized and cloned hybridoma cell lines producing mouse monoclonal antibodies specific to human VAP-1 and they have been successfully used for in vitro and in vivo characterization of VAP-1 (Kurkijarvi et al. in J. Immunology, 161 :1549-1557, 1998). In order to clone the variable region cDNAs of the murine antibodies, total RNA was first isolated from the different hybridoma cell lines, using Ul- traspec™ RNA Isolation kit according to manufacturer's protocol (Biotecx Laboratories, Inc., Houston, Texas) and first-strand cDNAs were synthesized with M-MLV reverse transcriptase (Gibco BRL) by priming 7 μg RNA with 100 pmol of oligo-dT primer in a 20-μl volume. The cDNAs encoding the mouse Fd fragment and the entire light chain were specifically amplified by a polymerase chain reaction (PCR) using 200 pmol of degenerate 5'- and 3'-primers described by Kettleborough et al. in Eur. J. Immunol. 23:206-211 , 1993, yielding DNA molecules of approximately 630 bp. Similarly, using the seven degener- ate kappa chain 5' primers and the 3' reverse kappa primer, light chain cDNAs of about 660 bp were amplified.

[0054] The PCR-amplified DNA fragments were purified using a Qi- aex™ II kit (Qiagen GmbH, Germany) and blunt-end cloned into phosphatase- treated Sma I -cut pUC18 using a SureClone™ Ligation kit (Amersham Phar- macia Biotech) according to the manufactures' procedures. The resulting clones were sequenced by the dideoxy method.

[0055] Based on the sequence information combined with the knowledge of the properties of the original antibodies, three mouse antibodies, Mo2D10, MoTK8-14 and Mo1G6, were selected for construction as chimeric antibodies.

[0056] Figure 1 shows the predicted amino acid sequences of the variable regions of these three particular anti-VAP-1 antibodies. Comparison with the sequences in Kabat et al. (in Sequences of proteins of immunological interest, 5^th Ed., National Institutes of Health, Bethesda, U.S.A.) revealed that the Vh genes of 2D10, TK8-14 and 1 G6 belong to mouse subgroups IB, IIB and IB, respectively. The Vk genes of 2D10 and 1G6 are members of the mouse subgroup V whereas the Vk gene of TK8-14 belongs to mouse subgroup IV.

[0057] Alignment of VH and VK sequences of the different mouse anti-VAP-1 antibodies with the available human germline V- and J-segment sequences which have been compiled into the V-Base directory (V BASE Sequence Directory, Tomlinson et al., MRC Centre for Protein Engineering, Cambridge, U.K.) demonstrated that the mouse variable region sequences used for constructing chimeric anti-VAP-1 antibodies were very homologous to human antibody sequences (data not shown).

Example 2

Construction and production of chimeric antibodies

[0058] In order to construct chimeric antibodies, the cDNAs encoding mouse heavy and light chains variable regions obtained in Example 1 were subcloned into separate mammalian expression vectors containing sequences for either human heavy chain constant regions or a kappa light chain constant. The Fc region of an antibody is known to determine the ability of antibody/antigen complexes to direct immune responses. Our aim was to produce therapeutic antibodies, which would block the binding of leukocytes to vascular endothelium and not recruit any effector functions. Therefore, we decided to use the constant region of human lgG2, which has been modified to lack complement activation and FcγR-binding properties (G2Δa) described by Armour et al. (supra).

[0059] The cDNAs encoding mouse variable regions of anti-VAP-1 antibodies, 2D10, TK8-14 and 1 G6, were joined to the modified human lgG2 constant regions and to the kappa constant regions. The cDNAs of the VH and VK were modified by PCR with specific primers

2D1 OVhBack, 5'-TCCACAGGTGTCCACTCCCAGGTCCAGCTGAAGGAG-3';

2D1 OVhFor, 5'-GAGGTTGTAAGGACTCACCTGAGGAGACGGTGACTGA-3'; 2D1 OVhBack, 5'-TCCACAGGTGTCCACTCCGACATCCAGATGACCCAG-3';

2D1 OVkFor, 5'-GAGGTTGTAAGGACTCACGTTTCAGCTCCAGCCTGGTC-3';

TK8-l4VhBack, 5'-TCCACAGGTGTCCACTCCCAGGTCCAGCTGCAGCAAC-3';

TK8-14VhFor, 5'-GAGGTTGTAAGGACTCACCTGAGGAGACTGTGAGAGT-3';

TK8-l4VkBack, 5'-TCCACAGGTGTCCACTCCCAGATTGTGCTGACACAG-3'; TK8-14VkFor, 5'-GAGGTTGTAAGGACTCACGTTTGATCTCCAGC'ITGTT-3'; 1 GΘVhBack, 5'-TCCACAGGTGTCCACTCCCAGGTCCAGCTGAAGGAG-3'; IGδVhFor, same as TK8-l4VhFor;

IG6VkBack, 5'-TCCACAGGTGTCCACTCCGACATCCAGATGACCCAG -3'; IGΘVkFor, same as TK8-l4VkFor.

[0060] The variable region cDNAs were modified for expression in the genomic style constructs. The variable region DNAs were each joined, at their 5' end, to DNA providing a mouse immunoglobulin heavy chain promoter and signal peptide sequences and, at their 3' end, to DNA representing the 5' end of the VH-CH intron as well as the appropriate splice site. Both of these DNA segments were originated from M13VHPCR1 (Orlandi et al., Proc. Natl. Acad. Sci. U.S.A., 86:3833-3837, 1989).

[0061] These variable region expression cassettes were constructed by sequential overlap extension PCR, using Pwo polymerase (Boe- hringer Mannheim). In the first step, the variable regions were amplified from the pUC18 plasmids containing the Fd and light chain cDNAs. The 18 nucleotides at the 3'-end of each of the PCR primers matched the ends of the variable regions but the 5'ends of the oligonucleotides effectively added 18 nucleotides to each end of the PCR products. These extra nucleotides were ho- mologous to the M13VHPCR1 -derived DNAs. These primers alter the nucleotide and amino acid sequences at the beginning of the variable regions. The N- terminal amino acid residues of each variable region were determined by the sequence of the PCR primers used in their isolation and the genuine amino acid found in the mouse antibody is not known. The changes made here intro- duce the amino acids residues typical of similar variable regions of the murine heavy chain variable region subgroup IB and kappa chain variable region subgroup V (Kabat et al., 1991 ). In 2D10 the amino acid changes are D1→Q in the VH and V2→l, K3→Q in the VK.

[0062] The leader and 3'-region sequences, containing the se- quences for a mouse immunoglobulin heavy chain promoter and signal peptide as well as the appropriate splicing sites, were amplified from M13VHPCR1 , as described by Orlandi et al. (supra) and respectively added to the 5' and 3'-ends of the VH and VK CDNA molecules. H/ndlll-SamHI fragments carrying sequences for each leader-VH-3'region were cloned into a pSV-gpt expression vector containing the modified lgG2 constant region gene, described in International Patent publication W099/58572 on page 37. The equivalent VK frag- ments were placed in a pSV-hyg expression vector containing a kappa constant region gene (see International Patent publication W099/58572, page 39. See Figure 1. DNA sequences were confirmed by sequencing as above.

[0063] In chimeric antibodies, approximately 70% of the rodent anti- body molecule is usually replaced with corresponding human sequences whilst maintaining the rodent antigen-binding sites with their particular specificities and affinities. However, due to the large homology between the mouse and the human sequences of the variable regions of the antibodies of the present invention, much less than 30% of the chimeric antibody sequences would be classed as non-human, removing the need for complete humanization.

[0064] Non-secreting rat myeloma cells (YB2/0), described by Kil- martin et al. in J. Cell. Biol. 93:576-582, 1992, were transfected by electroporation with 10 μg of the heavy-chain and 20 μg of the light-chain construct linearized by Pvul digestion (Boehringer Mannheim). In the case of the 1 G6 light chain construct, 30 μg of non-linearized DNA was used due to the presence of a Pvul site internal to the VK. For transfections 10⁷ YB2/0 cells were harvested and resuspended in 0.5 ml IMDM (Sigma). The DNAs were added and the mixture was incubated on ice for 5 min. Electroporation was carried out using a Gene Pulser™ (Bio-Rad) and conditions of 960 μF and 250 V. The resulting time constants were approximately 40 ms. After a 15-min incubation on ice, transfected cells were transferred into 20 ml IMDM with 10% fetal calf serum (FCS). Selection with 0.8 μg/ml mycophonolic acid and 250 μg/ml xanthine was applied 24-48 h later and the cells were grown in 96-well plates. After two weeks, well supematants were screened for the presence of whole antibodies by ELISA.

[0065] For the ELISA, microtiter wells were coated with 1 //g/ml of anti-human IgG (Fc-specific, Sigma) in 50 mM bicarbonate buffer for 1 h at +37°C. After washing the wells with PBS containing 0.5% (v/v) Tween™-20, samples of supernatant, diluted 5-fold, were allowed to react for 1 h at +37°C and thereafter the bound antibodies were detected by peroxidase conjugated goat anti-human kappa antibody (0.2 μg/ml, Seralab). Color development was obtained by adding o-phenylenediamine/H₂0₂ substrate solution, the reaction was stopped with 0.3 M H₂S0₄ and absorbance values were measured at 492 nm. Stable transfectants giving the highest absorbance values were expanded into 2-I roller bottles with 400 ml IMDM, 2% FCS for large-scale production and purification of chimeric antibodies. [0066] The chimeric antibodies, designated BTT-1002, BTT-1006 and BTT-1005, were purified from the culture medium by Protein A-agarose (Sigma) affinity chromatography, with a yield 1 - 7 mg/l. The supernatant containing secreted antibodies was cleared by centrifugation and TrisHCI, pH 8.0 was added to a final concentration of 0.1 M and NaN₃ to 0.05% (v/v). 0.1 g of ProteinAagarose, pre-swollen in PBS, was added to the supernatant. Antibodies were allowed to bind to ProteinA for 18 h at +4°C. Thereafter ProteinAagarose was loaded into a column and washed with 10 bed volumes of 0.1 M TrisHCI, pH 8.0 followed by the same amount of 10 mM TrisHCI, pH 8.0. Anti- bodies were eluted in 1 ml fractions of 0.1 M glycine, pH 3.0 and immediately neutralized with 100 μl of 1 M TrisHCI, pH 8.0. Fractions containing eluted antibodies (measured by A₂₈o) were pooled and dialyzed against PBS for 18 h at +4°C. The antibody preparation was then filter-sterilized and its protein content determined by measuring A₂so {e = 0.714). [0067] The human IgG antibody concentration was confirmed by

ELISA as above. The purity of the antibodies was judged by Coomassie- stained SDS-polyacrylamide gels according to Laemmli. Following SDS-PAGE under reducing conditions, the Coomassie™-stained gel, shown in Figure 3, showed two bands for each purified antibody: bands at approx. 50 kD-band for the heavy chain and 22 kD-band for the light chain. When the production levels were low some additional protein bands, presumably bovine immunoglobulins, were detected.

Example 3

Binding properties of the chimeric antibodies [0068] The binding of the chimeric antibodies to human VAP-1 was analyzed by immunofluorescence staining and flow cytometry. Anti-VAP-1 antibodies were biotinylated by dialyzing against 200 mM NaHCO₃, 81 mM Na₂C0₃ at +4°C for 2 h, incubating with biotinamidocaproate N-hydroxy- succinimide ester (Sigma, 120 μg/mg antibody) at room temperature for 2 h and re-dialyzing against PBS at +4°C for 16 h. For immunohistochemistry, acetone fixed frozen sections of different human tissues were overlaid with a biotinylated mouse anti-VAP-1 antibody, its chimeric derivatives or a class- matched negative control antibody, followed by incubation with Streptavidin- Phycoerythrin (Becton-Dickinson). After washing, the coverslips were attached with Fluoromount™ (Southern Biotechnology Associates, Inc., Alabama, U.S.A.) and sections were analyzed under a fluorescence microscope (Olympus).

[0069] For flow cytometry, transfected cells from a rat endothelial cell line (Ax cells), either expressing human VAP-1 cDNA or transfected with the vector containing human VAP-1 cDNA in an inverse orientation (mock control) (Bono et al., J.Immunol. 160:5563-5571 , 1998) were used. These were grown in 175 cm³ flasks in RPMI 1640 supplemented with 20% FCS, 2 mM L- glutamine, 1 mM Na-pyruvate, 10 μM β-ME, 1 % non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.75 mg/ml Geneticin™. In order to release the cells, they were washed twice with PBS and incubated in 10 ml Cell dissociation buffer (GibcoBRL) for 3 min at +37°C. After addition of 10 ml of medium, the cells were pelleted (5 min, 1000 g, at room temperature), resuspended in Wash buffer [PBS, 0.1 % (w/v) BSA, 0.1 % (v/v) NaN₃] at 10⁶ cells/ml and kept on ice. [0070] Cell suspension (100 μl/well) was transferred into 96-well plates, the cells pelleted (4 min, 1000 g, +10°C) and 100 /I aliquots of antibody solution added into the wells. After a 30-min incubation on ice, the cells were washed trice with 150 μl wash buffer/well and then incubated with 100 μl of 39 μg/ml FITC-conjugated anti-human IgG (Fc-specific, Sigma) for 30 min on ice. Finally, the cells were washed as earlier and fixed by adding 100 μl of Wash Buffer with 1 % (v/v) formaldehyde and kept at +4°C until analysis in a flow cy- tometer.

[0071] All flow cytometry samples were analyzed on a FACScan™ (Becton Dickinson, Oxford, U.K.). For each sample, data for a minimum of 10 000 gated events were collected and the geometric mean channel of fluorescence calculated using Lysys II software.

[0072] All of the chimeric antibodies bound specifically to human VAP-1 in that staining of transfected Ax cells which express VAP-1 on their surface, but not of mock-transfected cells, was detected. See Figure 4. An an- tibody with the same human constant regions and irrelevant variable regions did not bind to either transfectant. This indicates that conversion of the antibodies to chimeric molecules caused no detectable change in their ability to recognize the human VAP-1 molecule. In a comparison between the different chimeric antibodies, BTT-1002 showed the best VAP-1 binding properties. It was determined that about 0.1 μg/ml BTT-1002 was needed to achieve 50% saturation of mean channel fluorescence (MCF) compared to 0.2 μg/ml BTT- 1005 and 0.8 μg/ml BTT-1006. These figures equate to approximate dissociation constants (Kd) for the avidities of the three antibodies of 0.6 nM, 1.3 nM and 5.0 nM, respectively.

[0073] We have also compared the binding properties of the chi- meric antibodies to those of the original mouse antibodies (Mo2D10, M0TK8- 14 and M0I G6) in competition binding experiments (data not shown). A constant amount of biotinylated mouse antibody was mixed with increasing amounts of unlabelled murine or chimeric competitor antibodies and the inhibition of VAP-1 binding by the labeled antibody was analyzed by flow cytometry. [0074] BTT-1002 and BTT-1006 competed for binding to human

VAP-1 in a similar manner to their mouse counterparts but the BTT-1005 showed better inhibition of the binding of biotinylated mouse antibody than M0IG6. In the preparation of the hybridoma cell line producing M0I G6, NS-1 cells were used as a fusion partner. NS-1 cells are known to produce irrelevant antibody light chains, making it likely that the M0I G6 preparation contains antibodies with two different types of light chains whereas BTT-1005 has VAP-1 specific light chains only.

Example 4

Functional characteristics of the chimeric antibodies [0075] To test the recognition properties of the chimeric antibodies in human tissue displaying several cell types and structural compartments, tonsil sections were stained with both the chimeric and original antibodies. An in vitro frozen section assay was used to test the capacity of the chimeric and mouse monoclonal antibodies to inhibit lymphocyte binding to HEV, special- ized vessels through which lymphocyte extravasation takes place in lymphatic tissues. For this experiment only Mo2D10 and MoTK8-14 antibodies and their chimeric counterparts were chosen because M0IG6 contains a heterogeneous mixture of light chains as discussed above.

[0076] Human tonsils were obtained from elective tonsillectomies. Freshly cut 8-μm sections of frozen tonsils were first incubated for 30 min with 100 μl of RPMI 1640 medium supplemented with 5% AB-serum (Finnish Red Cross) and 10 mM HEPES, pH 7.2, containing 100 μg/ml of different anti-VAP- 1 antibodies or class-matched negative control antibodies. Thereafter, the sections were overlaid with 3 x 10⁶ Ficoll™-purified peripheral blood lymphocytes. Under constant rotation, lymphocytes were allowed to bind to vascular endo- thelium in the sections for 30 min at +7°C. Following this, the non-adherent cells were gently tilted off, and the adherent cells were fixed to the sections overnight in ice-cold PBS containing 1 % (v/v) glutaraldehyde. At least 100 high endothelial venules (HEV) per sample were analyzed under dark field illumina- tion. The results are expressed as percentage of maximal binding where binding of cells in the presence of the appropriate negative control defines 100 % binding.

[0077] The staining patterns for the TK8-14 and 2D10 antibodies are shown in Figure 5. In common with the mouse antibodies, the chimeric an- tibodies stained HEV and a subpopulation of flat walled venules in the tonsil. In addition, follicular dendritic cells and vascular smooth muscle cells were positive with both antibody series. In contrast, neither the chimeric antibodies nor the original monoclonal antibodies stained lymphocytes, epithelial cells or other structures. Thus these results demonstrate that the engineering of the antibod- ies produced no detectable alteration in their recognition patterns.

[0078] All of the mouse anti-VAP-1 antibodies were able to prevent lymphocyte adherence to vascular endothelium. However, the extent of the blocking by the original monoclonal antibodies varies somewhat. This is likely to be due to the differences in the epitopes recognized by each individual mouse antibody. Treatment of the tonsil sections with the chimeric and parental antibodies resulted in comparable inhibition of lymphocyte binding to HEV (Figure 6), indicating that the chimeric antibodies have the same function- blocking properties as their murine counterparts.

Example 5 Fc/ 1 binding

[0079] The chimeric antibodies contain constant domains of human lgG2 which have been modified to reduce their effector function efficiency. One property of this constant region is a lack of binding to human FcγRI and an inability to activate cells via this receptor. [0080] The binding of mouse and chimeric antibodies to FcγRI was measured by flow cytometry. Transfectants expressing FcγRI cDNA were obtained as single cell suspensions in wash buffer following treatment with cell dissociation buffer as described in example 3. Cells were pelleted at 10⁵ cell/well in 96-well plates, resuspended in 100 μl dilutions of the anti-VAP-1 antibodies or control human IgG antibody and incubated on ice for 30 min. Cells were washed as above and similarly incubated with 20 μg/ml biotin- conjugated goat anti-human K-chain antibody (Sigma) or biotin-conjugated goat anti-mouse IgG (Fab-specific, Sigma) as appropriate. Cells were then incubated with 20 μg/ml ExtrAvidin™-FITC (Sigma). After the final wash, cells were fixed as above. Surface expression of FcγRI was confirmed by staining with CD64 monoclonal antibody (Serotec, Oxford, U.K.) and FITC-conjugated goat anti-mouse IgG antibody (Sigma).

[0081] The results of this experiment are shown in Figure 7. The chimeric antibodies did not show any binding to FcγRI whereas the parental mouse monoclonal antibodies recognized human FcγRI to a degree determined by their isotypes. The MoTK8-14 and Mo1G6, both of the lgG2a,κ iso- type, gave binding curves with midpoints very similar to that of the human lgG1 ,κ antibody which was acting as a positive control for the anti-human reagents used in the immunostaining. This agrees with existing data which show these two isotypes of antibody to have similar abilities in binding to human FcγRI. The Mo2D10 has an lgG1 ,κ isotype and is expected to show weaker binding to the receptor.

Example 6

Improved kinetic properties of the chimeric antibodies [0082] Single dose pharmacokinetics of 1 B2, a murine IgM-antibody against VAP-1 , and chimeric BTT-1002 antibody was examined in the cynomolgus monkey and in the rhesus monkey, respectively.

[0083] In the pharmacokinetic study on chimeric BTT-1002 antibody in the rhesus monkey, the antibody at a dose of 10 mg/kg was intravenously administered to three animals. Serum was collected for antibody concentration analyses at 0, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 7 h, 12 h, 1 d, 2 d, 3 d, 4 d, 7 d, 10 d. The sensitivity level of BTT-1002 quantification by the analytical method was approximately 50 ng/ml. The analysis revealed that the antibody has two phase pharmacokinetics (see Table 1 and Fig. 8, panel A). The initial dilution volume was close to plasma volume as anticipated. At day 10 the antibody concentrations in serum were 0.1 , 0,2, and 0.4 μg/ml. The distribution phase lasts approximately one day. The elimination half-life was approximately three days. In the rhesus monkey, steady state is reached in 2 - 3 weeks using twice weekly administration.

Table 1 : Pharmacokinetic factors for BTT-1002 in the rhesus monkey.

[0084] In the pharmacokinetic study on the murine 1 B2 anti-VAP-1 mAb in the cynomolgus monkey, the antibody was intravenously administered to three animals at a dose of 2 mg/kg. Serum samples for antibody analysis were taken at 0 min, 2, 5, 10, 20, 40, 60, 120, 180, 240, 360, 540, and 720 minutes after the 1 B2 injection (Fig. 8, panel B). The concentration analyses were performed using a fluoro immunoassay. The maximum antibody concentrations seen either at 2 min, 5 min, or 10 min varied between 44 μg/ml and 70 μg/ml. The elimination half-life (group average) of 1 B2 was 59 minutes. The volume of distribution was approximately 0.04 l/kg supporting the concept that the antibody exists mainly in the plasma compartment.

[0085] The ED₅₀ values of chimeric BTT-1002 antibody and 1 B2 murine antibody for saturation of recombinant VAP-1 are 35 ng/ml and 85 ng/ml, respectively. In animals administered with 10 mg/kg of chimeric BTT- 1002 antibody, the concentrations of antibody in serum were approximately 10 times higher than the ED₅₀ values at day 10. In animals treated with 2 mg/kg of 1 B2 antibody, the concentrations 10 times higher than the ED₅₀ values were observed approximately 6 h after the antibody injection. [0086] The data of these two studies in non human primates indicate that using the chimeric BTT-1002 antibody an effective concentration of antibody will be achieved by once weekly or once per two weeks administration of antibody at doses in the range of 1 and 10 mg/kg. Concerning 1 B2 at doses of the same range, effective concentrations of antibody in the serum will necessitate daily dosing. This conclusion is based on the assessment of saturation of recombinant human VAP-1 with chimeric BTT-1002 antibody and 1 B2, and on the results of the pharmacokinetic studies in monkeys indicating an elimination half-life of approximately three days for chimeric BTT-1002 antibody and one hour for 1 B2.

Example 7

Pharmaceutical composition

[0087] Examples of a pharmaceutical composition comprising a chimeric anti-VAP-1 antibody according to the present invention, suitable for parenteral administration, given as a solution for injection or for infusion or as a concentrate for such a solution.

Percentual amount of ingredient to 1 ml

Chimeric anti-VAP-1 antibody 0.1 - 10%

Sodium chloride 0.5 - 1.5%

Disodium hydrogen phosphate dihyi drate 0.1 - 2%

Sodium dihydrogen phosphate dihyJ drate

Sucrose 0.5 - 10%

Polysorbate 20 or 80 0.01 - 1 %

Water for injection to 1 ml

Percentual amount of ingredient to 1 ml

Chimeric anti-VAP-1 antibody 0.1 - 10%

Sodium chloride 0.5 - 1.5%

EDTA 0.01 - 0.05%

Glycine 0.01 - 2%

Sodium citrate 1 - 5%

Water for injection to 1 ml

Example 8

In vivo animal data

[0088] The effect of chimeric BTT-1002 antibody was assessed in the model of collagen-induced arthritis (CIA) in the rhesus monkey aiming at gathering data on the usefulness of chimeric BTT-1002 antibody in arthritic indications.

[0089] Adult rhesus monkeys, negative in terms of MHC A26, which have CIA incidence of 99%, immunised with bovine type II collagen, were selected to the study. Ten animals were divided into two groups of five animals. Arthritis was induced by injecting 3-5 mg of bovine collagen in Freund's complete adjuvant in 10 spots in the animal's back.

[0090] Using this approach arthritis is clinically evident at 3 - 5 weeks after immunisation and lasts normally 7-9 weeks. [0091] The intravenous treatment of four weeks with chimeric BTT-

1002 antibody at a dose of 5 mg/kg twice weekly was started when CRP level > 20 mg/l had been detected in two consecutive recordings. Vehicle solution was administered to control animals. The condition of the animals was evaluated using an overall clinical score (0=no clinical signs of arthritis, 0.5=fever (> 0.5°C), 1=apathy, decreased mobility and loss of appetite, 2=weight-loss, warm extremities and/or joints, pain but without soft tissue swelling (STS), 3=moderate redness and STS of joints, normal flexibility of extremities, 4=severe redness and STS of joints, with joint stiffness, 5=thus severe arthritis that euthanasia is indicated), and scoring the severity of CIA (the severity of soft tissue swelling, flexibility and crepitation scored on a scale of - to +++: - none, ± doubtful, + moderate, ++ severe, +++ extreme). All these parameters resulted in an overall clinical score of CIA severity which is a semiquantitative scale.

[0092] In the chimeric BTT-1002 antibody test all animals survived the four weeks duration of treatment. Two animals out of five showed minor or no signs of disease (Figure 9). All animals of this group were regarded as re- sponders to CIA as they had the same high level of anti-collagen IgM antibody. In control group, three out of five animals had to be euthanized rather soon after the start of the treatment period (see Fig. 10) (two animals due to extreme apathy, one due to severity of arthritis). One placebo animal with no signs of arthritis was regarded as non-responder.

[0093] The chimeric BTT-1002 antibody treated animals, which were also treated with pain killer, were active. However, the three animals in the placebo group that had to be euthanized were also treated with analgesics but still developed such severe symptoms that euthanasia was required, in contrast to the BTT-1002 treated animals where euthanasia was not indicated.

[0094] In conclusion, these examples demonstrate that the chimeric anti-VAP-1 antibodies retained the specific recognition patterns of their murine counterparts, lacked human FcγRI binding activity and blocked the VAP-1 de- pendent binding of lymphocytes to HEV. [0095] It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

Claims

1. An isolated nucleic acid molecule, comprising one or more of the nucleotide sequences depicted in SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3 or functional variants thereof, wherein said sequences encode a CDR region of a mouse anti-VAP-1 antibody heavy chain.

2. The nucleic acid molecule according to claim 1, comprising the nucleotide sequence depicted in SEQ ID NO: 4 or a functional variant thereof, wherein said sequence encodes a variable region of an anti-VAP-1 antibody heavy chain.

3. The nucleic acid molecule according to claim 2, comprising the nucleotide sequence depicted in SEQ ID NO: 5 or a functional variant thereof, wherein said sequence encodes an anti-VAP-1 antibody heavy chain.

4. An isolated nucleic acid molecule, comprising one or more of the nucleotide sequences depicted in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID

NO: 8 or functional variants thereof, wherein said sequences encode a CDR region of an anti-VAP-1 antibody light chain.

5. The nucleic acid molecule according to claim 1 , comprising the nucleotide sequence depicted in SEQ ID NO: 9 or a functional variant thereof, wherein said sequence encodes a variable region of an anti-VAP-1 antibody light chain.

6. The nucleic acid molecule according to claim 2, comprising the nucleotide sequence depicted in SEQ ID NO: 10 or a functional variant thereof, wherein said sequence encodes an anti-VAP-1 antibody light chain.

7. An anti-VAP-1 antibody heavy chain polypeptide, comprising one or more of the amino acid sequences depicted in SEQ ID NO: 11 , SEQ ID NO:

12 and SEQ ID NO: 13 or functional variants thereof.

8. The polypeptide according to claim 7, comprising the amino acid sequence depicted in SEQ ID NO: 14 or a functional variant thereof.

9. The polypeptide according to claim 8, comprising the amino acid sequence depicted in SEQ ID NO: 15 or a functional variant thereof.

10. An anti-VAP-1 antibody light chain polypeptide, comprising one or more of the amino acid sequences depicted in SEQ ID NO: 17, SEQ ID NO:

18 and SEQ ID NO: 19 or functional variants thereof.

11. The polypeptide according to claim 10, comprising the amino acid sequence depicted in SEQ ID NO: 20 or a functional variant thereof.

12. The polypeptide according to claim 11 , comprising the amino acid sequence depicted in SEQ ID NO: 21 or a functional variant thereof.

13. A heavy chain of a chimeric anti-VAP-1 antibody or a fragment thereof, wherein the heavy chain comprises a variable region according to any of the claims 7 to 9 and further comprising a constant region of a heavy chain.

14. A light chain of a chimeric anti-VAP-1 antibody or a fragment thereof, wherein the light chain comprises a variable region according to any of the claims 10 to 12, further comprising a constant region of a light chain.

15. A mouse-human chimeric anti-VAP-1 antibody, comprising an amino acid sequence wherein less than 30% of the amino acids are derived from corresponding mouse antibody.

16. The antibody according to claim 15, comprising a variable region of a heavy chain and a variable region of a light chain of a chimeric antibody or a fragment thereof, wherein said variable region of the heavy chain comprises the amino acid sequence from the Gin at position numbered 20 to the Ser at the position numbered 136 of SEQ ID NO: 15 and said variable region of the light chain comprises the amino acid sequence from the Asp at position num- bered 20 to the Arg at the position numbered 127 of SEQ ID NO: 21.

17. The antibody according to claim 16, wherein said antibody further comprises a constant region of a heavy chain and a constant region of a light chain.

18. The antibody according to claim 17, wherein said constant re- gion of said heavy chain comprises a sequence depicted in SEQ ID NO: 16 and said constant region of said light chain comprises a sequence depicted in SEQ ID NO: 22.

19. The antibody according to any of claims 15 and 16, wherein said antibody fragment is a Fab, Fab', F(ab')2, FV or single chain FV.

20. An expression vector, comprising a nucleic acid sequence according to any of claims 1 to 6.

21. A host cell or organism comprising an expression vector according to claim 20.

22. A method of producing a chimeric anti-VAP-1 antibody, comprising the steps of a) transforming a suitable host with at least one expression vector according to claim 20; b) culturing said host cell under conditions favoring expression, and c) purifying assembled chimeric antibodies from the culture medium.

23. A pharmaceutical composition comprising a chimeric anti-VAP-1 antibody according to any of claims 15 to 19.

24. A method of treating a VAP-1 mediated inflammatory disorder in a patient in need thereof, said method comprising providing to said patient an efficient amount of a pharmaceutical composition according to claim 23.