WO2011029823A1

WO2011029823A1 - Monoclonal antibody reactive with cd63 when expressed at the surface of degranulated mast cells

Info

Publication number: WO2011029823A1
Application number: PCT/EP2010/063118
Authority: WO
Inventors: Thorsten Schaefer; Tamás Schweighoffer
Original assignee: Novartis Ag
Priority date: 2009-09-09
Filing date: 2010-09-07
Publication date: 2011-03-17

Abstract

The present invention relates to antibodies that specifically bind to degranulated mast cells and not to quiescent mast cells. The invention more specifically relates to antibodies that are specific to an isoform of CD63 which is expressed at the surface of degranulated mast cells but not at the surface of quiescent mast cells and methods of use for said antibodies to treat pathological disorders, such as rheumatoid arthritis, allergic disorders, asthma or other autoimmune and inflammatory disorders.

Description

MONOCLONAL ANTIBODY REACTIVE WITH CD63 WHEN EXPRESSED AT THE SURFACE

OF DEGRANULATED MAST CELLS

The present invention relates to antibodies that specifically bind to degranulated mast cells and not to quiescent mast cells. The invention more specifically relates to antibodies that are specific to an isoform of CD63 which is expressed at the surface of degranulated mast cells but not at the surface of quiescent mast cells and methods of use for said antibodies to diagnose or treat pathological disorders, such as rheumatoid arthritis, allergic disorders, asthma or other autoimmune and inflammatory disorders.

Mast cells are secretory immune cells with pivotal importance in allergic and inflammatory diseases, like asthma bronchiale, rheumatoid arthritis, or neurodermitis. They are a major source of immunoreactive substances, including histamine, proteases (tryptase, chymase), growth factors {NGF, TNF), neurotransmitters (serotonin, dopamine), chemokines and cytokines. These promote vasodilatation in inflamed tissue, attract and co-stimulate lymphocytes at infectious sites, and provide a primary defense line against pathogens and parasites in mucosa and epithelia.

The mediators are spatially compacted and temporarily stored in specialized intracellular granuli that are released upon cell activation. Degranulation commences with an antigen- induced crosslinking of IgE molecules at the surface of individual mast cells. Via the FCeRl receptor complex an intracellular signaling cascade is launched, at the end of which granuli and plasma-membrane fuse in a Ca2+-dependent manner. Also the secretion of entire granuli has been reported, although the molecular mechanisms underlying this pathway are less well understood.

Significantly less is known about how mast cells recover from degranulation and about their subsequent fate. One possibility is that similarly to the fate of other granular cells like neutrophils, mast cell degranulation is a terminal process, and ultimately coupled to cell death. Contradictory to this belief, the survival of post- degranulation state mast cells has recently been observed by time-lapse microscopy by Xiang.Z. et al., 2001 , Journal of Allergy and Clinical Immunology 108.1. A better understanding of the cell's life-cycle may thus lead to novel therapeutic approaches. In particular, novel epitopes specifically expressed at the surface of degranulated mast cells may provide new targets for anti-allergic therapy. The present invention describes a novel isoform of CD63, a broadly expressed transmembrane protein previously also detected on mast cells, specific of degranulated mast cells. CD63 belongs to the tetraspanin (TM4SF) family that comprises various members in different species (32 in mammals, 35 or more in drosophila, 21 in worms). CD63 is expressed on a variety of hematopoietic cell types (monocytes, macrophages, and T-cells), and was shown to interact with integrins, syntenin-1 and membrane metalloproteases when localized to TEMs. Processes like cell adhesion, cell motility, and phagocytosis/endocytosis are thus influenced by CD63. A substantial fraction of CD63 localizes to secretory vesicles, lysosomes and late endosomes (e.g. in platelets, dendritic cells, and melanosomes; Metzelaar et a!., 1991 , J.Biol.Chem. 266:3239 and Nishibori et al., 1993, J.Clin. Invest. 91 :1775). However, also this intracellular pool reaches the plasma membrane in response to cell activation and secretion. Also retrograde trafficking of CD63 has been demonstrated, which makes this protein a prime candidate to study the replenishment of granuli and the reactivation of granular immune.

The hitherto most advanced studies regarding the role of CD63 in mast cell functions were published by Kraft et al. 2005, The Journal of Experimental Medicine 201:385. They report that mouse-anti-rat antibodies can interfere in vitro with signalling, activation, mediator release, and degranulation of rat basophilic leukemia (RBL) cells. In addition, they show a partial (<40%) improvement in passive cutaneous anaphylaxis in rats. However, these antibodies do not recognize human CD63. The Cd63 molecule in rodents is much more different from the human counterpart than the name giving would suggest, for example mice have two homologues genes, with different genomic structure. Moreover, in humans several cell types express CD63. This means that any generic anti-CD63 antibody would cross-react with a number of cells, potentially causing life-threatening unwanted effects. Therefore for any therapeutic purpose, antibodies which only recognize specific, restricted epitopes are required. However, whether such epitopes exist at all on CD63, and what their characteristics would be, has not been addressed before. The present invention describes the first known restricted epitope on CD63, termed CD63var, which then satisfies the requirements for an antibody-mediated therapy.

Anti-CD63 antibodies have also been suggested for use in allergy test, such as for example, those described in Ebo et al. 2006, Allergy 2006: 61; 1028-1039, and WO2009/033691. Commonly used techniques to analyze and quantify in vitro activated basophils relies upon characterization of the cells by anti-lgE and assessment of their activation status by anti- CD63, commercially available from Buhlmann (Buhlmann Laboratories AG, Basel, Switzerland) and Orpegen {Orpegen Pharma GmBH, Heidelberg, Germany and BD (Pharmingen, Biosciences, Ermebodegen-Aalst, Belgium). However, there is a need for improved antibodies that would be more specific to granule containing cell types, such as mast cells and basophils but not reacting to other cells, such as platelets, and that would be more state-specific to activated cells. The invention further provides novel anti-CD63 monoclonal antibodies and the experimental evidence that two structurally distinct isoforms of the human CD63 protein exist, one characteristic of vesicles, and another expressed on the cell surface. Advantageously, the antibodies of the invention bind selectively to the vesicular CD63 isoform, which upon activation is transferred to the cell surface of degranulated mast cells and can be used not only as diagnostic markers of activated human mast cells, but also for therapy of allergic diseases or other autoimmune and inflammatory disorders.

Therefore, in one aspect, the invention provides an antibody or a functional protein comprising an antigen-binding portion of said antibody that binds to degranulated mast cells and not to quiescent mast cells.

In one embodiment, the antibody or functional protein binds to degranulated mast cells at least 100 fold more than to quiescent mast cells as measured by mean fluorescence intensity of antibody-stained cells in fluorescent activated cell sorting (FACS) analysis.

In one embodiment, the antibody or functional protein specifically binds to an isoform of human CD63 (SEQ ID NO:1) expressed at the surface of degranulated mast cells but not at the surface of one of the other CD63-expressing cell types selected among the group consisting of platelets, quiescent mast cells, quiescent myeloid-precursor-like THP-1 cells, the promonocyte cell line U937, or melanoma cells.

In one embodiment, the antibody or functional protein inhibits the degranulation of pre- activated mast cells in vitro, as measured by the enzymatic turnover of released reporter enzyme β-hexosaminidase. In a further specific embodiment, the mast cells are human mast cells.

In another embodiment, the antibody is a human, fully human or humanized lgG1 antibody.

The invention further relates to hybridomas expressing monoclonal antibody NIBR63/1 , NIBR63/2 or NIBR63/3 as deposited at DSMZ on September 8, 2009.

The invention also relates to antibodies that cross-block or are cross-blocked by one of the specific recombinant or monoclonal antibodies described above.

These antibodies of the invention can be used advantageously as a medicament or diagnostic tool.

In one embodiment, the antibodies of the invention are used for the treatment of a pathological disorder that is mediated by degranulation of mast cells or other CD63var expressing granulocytes, such as basophils, or that can be treated by killing or depleting degranulated mast cells or other CD63var expressing granulocytes, such as basophils, or that can be treated by preventing a secondary degranulation cycle to mast cells or other CD63var expressing granulocytes, such as basophils, which were previously degranulated. Such disorders include in particular some autoimmune and inflammatory disorders, such as rheumatoid arthritis, allergic disorders, asthma, multiple sclerosis or neurodermitis, or malignant proliferative diseases, such as systemic mastocytosis.

The invention further relates to nucleic acids encoding the antibodies of the invention and the corresponding cloning or expression vector and recombinant host cells comprising such nucleic acids.

The invention further relates to a process for the production of an antibody or functional protein, comprising culturing the recombinant host cell and isolating said antibody or functional protein.

The invention further relates to an in vitro method to detect FCERI degranulation of mast cells or other CD63var expressing granulocytes, such as basophils, comprising a) providing a biological sample containing potentially degranulated mast cells or other CD63var expressing granulocytes, such as basophils, b) incubating said biological sample in the presence of an antibody of the invention under suitable conditions for specific binding of said antibodies to its antigen expressed at the surface of degranulated mast cells, c) detecting binding of said antibodies to any degranulated mast cells, wherein said binding is indicative that said sample comprises degranulated mast cells or other CD63var expressing granulocytes, such as basophils.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "degranulated mast cells" refers to mast cells, that have undergone a release of their intracellular granuli upon activation. Physiologically this release is triggered by surface- bound IgE molecules that become cross-linked by their cognate antigen, which frequently is an allergen in human pathological conditions. For experimental purposes this process can be modeled in vitro by chemicals PMA (Phorbol 12-myristate 13-acetate) and ionomycin, or by cell coating with human IgE and subsequent exposure to respective, multivalent anti IgE antigen. The term "quiescent mast cells" refers to unstimulated (e.g. mock-treated) cord blood derived human mast cells, that were proliferated and differentiated according to standard procedures, but have not been exposed to PMA/ionomycin or IgE/antigen.

The term "pre-activated mast cells"" refers to human cord blood derived mast cells, that were once stimulated with IgE/antigen (e.gJw-8/NIP-BSA), and thus underwent an initial round of degranulation.

For ease of reading, the term "CD63var" refers to the novel epitope of an isoform of CD63 that has been identified in the present invention, expressed at the surface of degranulated granulocytes, such as mast cells but not at the surface of, at least, some other known CD63- expressing cell types selected among the group consisting of platelets, quiescent mast cells, quiescent myeloid-precursor-like THP-1 cells, the promonocytic cell line U937, and melanoma cells. Said epitope may comprise amino acid residues Tyr105-Val203 of SEQ ID NO:1 , corresponding to the external loop II region of human CD63. In one specific embodiment, it includes at least C170 and/or N172 residues of human CD63 isoform expressed at the surface of degranulated mast cells.

A "signal transduction pathway" or "signaling activity" refers to a biochemical causal relationship generally initiated by a protein-protein interaction such as binding of a growth factor to a receptor, resulting in transmission of a signal from one portion of a cell to another portion of a cell. In general, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in the series of reactions causing signal transduction. Penultimate processes typically include nuclear events, resulting in a change in gene expression.

The term CD63 refers to human CD63 as defined in SEQ ID NO: 1. PCT Patent Publications WO2005092377 refer to anti-CD63 antibodies in general.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i. e., "antigen-binding portion") or single chains thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various ceils of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a portion of CD63). it has been shown that the antigen- binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V_H and CH1 domains; a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a V_H domain; and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen- binding region" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds CD63var specific epitope is substantially free of antibodies that specifically bind CD63 epitopes other than CD63var specific epitope). An isolated antibody that specifically binds CD63var-specific epitope may, however, have cross-reactivity to other antigens, such as CD63 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia (AbM), etc. (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Al Lazikani et al. (1997) J. Mol. Bio. 273:927 948).

The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_H and V_L sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, "isotype" refers to the antibody class (e.g., IgM, IgE, IgG such as lgG1 or lgG2) that is provided by the heavy chain constant region genes.

The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen".

As used herein, an antibody that "binds to degranulated mast cells but not to quiescent mast cells" is intended to refer to an antibody that binds to degranulated mast cells at least 100 fold more, for example at least 1000 fold more or at least 10000 fold more than to quiescent mast cells as measured by mean fluorescent intensity of antibody-stained cells in FACS analysis. In some embodiments, said antibody does not show any significant binding to quiescent mast cells according to standard binding assays.

As used herein, an antibody that "specifically binds to an epitope specific of an isoform of human CD63 (SEQ ID NO: 1)" or "anti-CD63var antibody" is intended to refer to an antibody that binds to said specific epitope CD63var with a K_D of 1 μΜ or less, 100nM or less, 10nM or less. An antibody that "cross-reacts with a second epitope other than CD63var epitope" is intended to refer to an antibody that binds that second epitope with a K_D of of 10μΜ or less, 1μΜ or less, 100nM or less. In some embodiments, an antibody that "does not cross-react with a particular antigen" or "does not bind at the surface of another antigen" is intended to refer to an antibody that binds to that antigen, with a K_D of 10 μΜ or greater, or a KD of 100μΜ or 1 mM or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays.

The term "Kassoc" or "Ka", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K_dis" or "K_d," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term "K_D", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K_d to K_a (i.e. Kd Ka) and is expressed as a molar concentration (M). K_D values for antibodies can be determined using methods well established in the art. A method for determining the K_D of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore^® system.

As used herein, the term "Affinity" refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody "arm" interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

As used herein, the term "Avidity" refers to an informative measure of the overall stability or strength of the antibody-antigen complex. It is controlled by three major factors: antibody epitope affinity; the valence of both the antigen and antibody; and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antibody, that is, the likelihood that the particular antibody is binding to a precise antigen epitope.

As used herein, the term "CD63var antagonist antibody" is intended to refer to an antibody that reduces, decreases and/or inhibits CD63 induced signaling activity in pre-activated mast cells degranulation assay. An example of pre-activated mast cells degranulation assay is described in more details in the examples below, measuring the enzymatic turnover of released reporter enzyme β-hexominidase. In some embodiments, the antibodies reduce, decrease or inhibit CD63 induced degranulation of pre-activated mast cells to at least 10%, 20%, 30%, 40%, 50% or more.

As used herein, the term "ADCC" or "antibody dependent cellular cytotoxicity" activity refers to human B cell depleting activity.

In order to obtain a higher avidity probe, a dimeric conjugate (two molecules of an antibody protein coupled to a FACS marker) can be constructed, thus making low affinity interactions (such as with the germline antibody) more readily detected by FACS. In addition, another means to increase the avidity of antigen binding involves generating dimers, trimers or multimers of any of the constructs described herein of the anti-CD63var antibodies. Such multtmers may be generated through covalent binding between individual modules, for example, by imitating the natural C-to-N-terminus binding or by imitating antibody dimers that are held together through their constant regions. The bonds engineered into the Fc/Fc interface may be covalent or non-covalent. In addition, dimerizing or multimerizing partners other than Fc can be used in CD63 hybrids to create such higher order structures. For example, it is possible to use multimerizing domains such as trimerizing domain described in Borean (WO2004039841) or pentamerizing domain described in published patent application W098/18943. As used herein, the term "selectivity" for an antibody refers to an antibody that binds to a certain target polypeptide but not to closely related polypeptides.

As used herein, the term "high affinity" for an antibody refers to an antibody having a K_D of 1 nM or less for a target antigen. As used herein, the term "subject" includes any human or nonhuman animal.

The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

As used herein, the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a cell of Pichia, a cell of Trichoderma, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence. The optimized sequences herein have been engineered to have codons that are preferred in CHO mammalian cells; however optimized expression of these sequences in other eukaryotic cells is also envisioned herein. The amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

Various aspects of the invention are described in further detail in the following subsections.

Standard assays to evaluate the binding ability of the antibodies toward CD63 in general or CD63var are known in the art, including for example, ELISAs, western blots and RIAs. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore analysis. Assays to evaluate the effects of the antibodies on functional properties of mast cells or other CD63var expressing granulocytes, such as basophils (e.g., degranulatton, mobility, adhesion, signaling, differentiation, and survival of cells) are described in further detail in the Examples.

Accordingly, an antibody that "inhibits" one or more of these mast cells functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological activities, or the like) as determined according to methodologies known to the art and described herein, will be understood to relate to a statistically significant decrease in the particular activity relative to that seen in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present). An antibody that inhibits mast cells activity effects such a statistically significant decrease by at least 10% of the measured parameter, by at least 20%, 30%, 40% or 50%, and in certain embodiments an antibody of the invention may inhibit greater than 95%, 98% or 99% of mast cell functional activity.

The terms "cross-block", "cross-blocked" and "cross- blocking" are used interchangeably herein to mean the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to CD63var epitope in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent is able to interfere with the binding of another antibody or binding molecule to CD63var, and therefore whether it can be said to cross-block according to the invention, can be determined using standard competition binding assays. One suitable assay involves the use of the Biacore technology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using surface plasmon resonance technology. Another assay for measuring cross-blocking uses an ELISA-based approach.

Anti-CD63var antibodies that binds to degranulated mast cells and not to quiescent mast cells

The invention describes a novel epitope of human CD63, called herein CD63var, that is expressed at the surface of, at least, degranulated human mast cells expressing CD63 but not at the surface of quiescent mast cells. Accordingly, the invention provides an isolated antibody or a functional protein comprising an antigen-binding portion of an antibody that binds to degranulated mast cells and not to native quiescent mast cells. The invention further provides anti-CD63var antibodies.

Methods for screening and identifying anti-CD63var antibodies are described in the Examples. The Examples further describes specific monoclonal antibodies, NIBR63/1 , NIBR63/2 and NIBR63/2 as identified by the screening methods of the invention.

Additional antibodies can therefore be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard CD63 binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to human CD63 demonstrates that the test antibody can compete with that antibody for binding to human CD63; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on human CD63 as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein by their producing hybridomas. In a certain embodiment, the antibody that binds to the same epitope on human CD63 as the antibodies of the present invention is a human anti-CD63var antibody. Such anti-CD63var antibodies can be prepared and isolated as described in the screening flowchart of the Examples.

Monoclonal antibodies and their producing hvbridomas as deposited at DSMZ

Antibodies of the invention include the monoclonal antibodies, isolated as described, in the Examples. Examples of preferred full length heavy and light chain amino acid sequences of antibodies are those produced by the hybridomas NIBR63/1 , NIBR63/2 and NIBR63/3 and deposited by Novartis Pharma AG, Forum 1 , CH-4002 Basel, Switzerland, at DSMZ on September 8, 2009. Other antibodies of the invention include amino acids that have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDR regions depicted in the sequences of the antibodies described above. In some embodiments, it include mutant amino acid sequences wherein no more than 1 , 2, 3, 4 or 5 amino acids have been mutated by amino acid deletion, insertion or substitution in the CDR regions when compared with the CDR regions depicted in the sequences of the antibodies described above.

Other antibodies of the invention include amino acids or nucleic acids that have been mutated, yet have at least 60, 70, 80, 90 or 95 percent identity to the coding nucleotide sequences of the antibodies as produced by the hybridomas NIBR63/1 , NIBR63/2 and NIBR63/3.

Since each of these antibodies binds the same epitope, the V_H, V_u, full length light chain, and full length heavy chain sequences (nucleotide sequences and amino acid sequences) can be "mixed and matched" to create other anti-CD63var binding molecules of the invention. CD63var binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., ELISAs). When these chains are mixed and matched, a V_H sequence from a particular V_H V_L pairing should be replaced with a structurally similar V_H sequence. Likewise a full length heavy chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length heavy chain sequence. Likewise, a V_L sequence from a particular V_H V_L pairing should be replaced with a structurally similar V_u sequence. Likewise a full length light chain sequence from a particular full length heavy chain / full length light chain pairing should be replaced with a structurally similar full length light chain sequence.

Homologous antibodies

In yet another embodiment, an antibody of the invention has full length heavy and light chain amino acid sequences; full length heavy and light chain nucleotide sequences, variable region heavy and light chain nucleotide sequences, or variable region heavy and light chain amino acid sequences that are homologous to the amino acid and nucleotide sequences of the antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-CD63var antibodies of the invention.

For example, the invention provides an isolated recombinant antibody (or a functional protein comprising an antigen binding portion thereof) comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of the corresponding heavy chain variable region of NIBR63/1 , NIBR63/2 or NIBR63/3; the light chain variable region comprises an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of the corresponding light chain variable region of NIBR63/1 , NIBR63/2 or N(BR63/3 and the antibody specifically binds to CD63var. Optionally said antibody exhibits at least one of the following functional properties: it inhibits the degranulation of pre-activated mast cells in vitro, it competes with antibodies NIBR63/1-3 for association with degranulated human mast cells or other CD63var expressing cells, it interferes with growth and differentiation of cells, it inhibits their adhesion to protein substrates or other cells, it inhibits their migration, or it modulates their survival.

As used herein, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

Engineered and modified antibodies An antibody of the invention further can be prepared using an antibody having one or more of the V_H and/or V_L sequences shown herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i. e., V_H and/or V_L), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody- antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321 :522-525; Queen, C. et al., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Patent No. 5,225,539 to winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.)

Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L et al., 1994 Eur. J Immunol. 24:827-836.

An example of framework sequences for use in the antibodies of the invention are those that are consensus sequences and/or framework sequences used by monoclonal antibodies of the invention. The V_H CDR1, 2 and 3 sequences, and the V_L CDR1 , 2 and 3 sequences, can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101 ; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acid residues within the V_H and/or V_L CDR1 , CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation." Site- directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. Conservative modifications (as discussed above) can be introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

Grafting antigen-binding domains into alternative frameworks or scaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to CD63var. Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof (such as those disclosed elsewhere herein), and include immunoglobulins of other animal species, preferably having humanized aspects. Single heavy-chain antibodies such as those identified in camelids are of particular interest in this regard. Novel frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulin based antibodies using non-immunoglobulin scaffolds onto which CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for CD63var epitope. Such compounds are known herein as "polypeptides comprising a target-specific binding region". Examples of non-immunoglobulin framework are further described in the sections below (camelid antibodies and non-antibody scaffold).

Camelid antibodies

Antibody proteins obtained from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See WO 94/04678.

A region of the camelid antibody which is the small single variable domain identified as V_HH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See U.S. patent number 5,759,808 issued June 2, 1998; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261 ; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional. Accordingly, a feature of the present invention is a camelid antibody or nanobody having high affinity for CD63var epitope. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, i.e., is produced by the camelid following immunization with protein comprising the epitope of CD63var, using techniques described herein for other antibodies. Alternatively, the anti-CD63var camelid nanobody is engineered, i.e., produced by selection for example from a library of phage displaying appropriately mutagenized camelid nanobody proteins using panning procedures with CD63var as a target. In one embodiement, an antibody of the disclosure is camelized, having a camelid framework and V_H CDR1 , CDR2 and/or CDR3 regions of the antibodies NIBR63/1 or NIBR63/2 or NIBR63/3 as disclosed herein or corresponding humanized and/or camelized versions. Engineered nanobodies can further be customized by genetic engineering to have a half life in a recipient subject of from 45 minutes to two weeks. In a specific embodiment, the camelid antibody or nanobody is obtained by grafting the CDRs sequences of the heavy or light chain of the human antibodies of the invention into nanobody or single domain antibody framework sequences, as described for example in PCT/EP93/0221 .

Non-antibody scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AG, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(i) Fibronectin scaffold

The fibronectin scaffolds are based preferably on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (US 6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprises the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomization and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin- based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

(ii) Ankyrin - Molecular Partners

The technology is based on using proteins with ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel a-helices and a β-turn. Binding of the variable regions is mostly optimized by using ribosome display.

(iii) Maxybodies/Avimers - Avidia

Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, 20040175756; 20050053973; 20050048512; and 20060008844.

(vi) Protein A - Affibody

Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants (See e.g., US 5,831,012). Affibody® molecules mimic antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® molecules is similar to that of an antibody.

(v) Anticalins - Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, being just marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing "anticalins" is PCT WO 199916873.

(vi) Affilin - Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New Affilin™ molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulin proteins. Scil Proteins employs two Affilin™ scaffolds, one of which is gamma crystalline, a human structural eye lens protein and the other is "ubiquitin" superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of gamma crystalline derived proteins are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368

(vii) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2kDa) mimicking beta-hairpin secondary structures of proteins, the major secondary structure involved in protein-protein interactions.

Framework or Fc engineering

Engineered antibodies of the invention include those in which modifications have been made to framework residues within V_H and/or V_L, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et at.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6, 194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human lgG1 for FcyRI, FcvRII, FcvRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by; for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, EP 1 ,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in one embodiment, the antibodies of the invention are produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http://www.eurekainc.com/about us/ companyoverview.html). Alternatively, the antibodies of the invention can be produced in yeasts or filamentous fungi engineered for mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).

Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water- soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1- C10) alkoxy- or aryloxy- polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegyiated is an aglycosyiated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094.

Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0 486 525.

Nucleic acid molecules encoding antibodies of the invention

Another aspect of the invention pertains to nucleic acid molecules that encode the antibodies of the invention.

The nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences. In an embodiment, the nucleic acid is a cDNA molecule. The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from various phage clones that are members of the library. Once DNA fragments encoding V_H and V_L segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to an scFv gene. In these manipulations, a V_L- or V_H-encoding DNA fragment is operatively linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under control of a desired promoter.

The isolated DNA encoding the V_H region can be converted to a full-length heavy chain gene by operatively linking the V_H-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1 , CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., el al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an lgG1 , lgG2, lgG3, lgG4, IgA, IgE, IgM or IgD constant region. In some embodiments, the heavy chain constant region is selected among lgG1 isotypes. For a Fab fragment heavy chain gene, the V_H-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_L region can be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operatively linking the V_L-encoding DNA to another DNA molecule encoding the light chain constant region, C_L. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or a lambda constant region.

To create an scFv gene, the V_H- and V_L-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly - Ser)₃, such that the V_H and V_L sequences can be expressed as a contiguous single-chain protein, with the V_L and V_H regions joined by the flexible linker (see e.g., Bird et al., 1988 Science 242:423-426; Huston et at., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature 348:552-554). Generation of monoclonal antibodies of the invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, 1975 Nature 256: 495. Many techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.

An animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g.,. human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art. See e.g., U.S. Patent No. 5225539 to Winter, and U.S. Patent Nos. 5530101 ; 5585089; 5693762 and 6180370 to Queen et al. In a certain embodiment, the antibodies of the invention are humanized versions of NIBR63/1 , NIBR63/2 or NIBR63/3.

In a certain embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies directed against CD63var epitope can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

The HuMAb mouse^® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode un-rearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al., 1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG monoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N., 1994 Handbook of Experimental Pharmacology 1 13:49-101 ; Lonberg, N. and Huszar, D., 1995 Intern. Rev. Immunol.13: 65-93, and Harding, F. and Lonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546).

In another embodiment, human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice", are described in detail in PCT Publication WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CD63var antibodies of the invention. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used. Such mice are described in, e.g., U.S. Patent Nos. 5,939,598; 6,075,181 ; 6,1 14,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CD63var antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome, referred to as "TC mice" can be used; such mice are described in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., 2002 Nature Biotechnology 20:889-894) and can be used to raise anti- CD63var antibodies of the invention.

Human recombinant antibodies of the invention can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art or described in the examples below. See for example: U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571 ,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969, 108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521 ,404; 6,544,731 ; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.

Generation of hvbridomas producing human monoclonal antibodies

To generate hybridomas producing human monoclonal antibodies of the invention, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to one- sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 x 145 in flat bottom microtiter plates, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0:055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1X HAT (Sigma; the HAT is added 24 hours after the fusion). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened by ELISA for human monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 days. The antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD₂₈o using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80° C.

Generation of transfectomas producing monoclonal antibodies

Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full- length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V_H segment is operatively linked to the CH segment(s) within the vector and the V_L segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide {i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., poiyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA 1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, in particular mammalian host cells, is discussed because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today 6:12-13).

Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR selectable marker, e.g., as described in R.J. Kaufman and P A. Sharp, 1982 Mol. Biol. 159:601-621 , NSO myeloma cells, COS cells and SP2 cells). In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO 87/04462, WO 89/01036 and EP 338,841. In one embodiment, mammalian host cells for expressing the recombinant antibodies of the invention include mammalian cell lines deficient for FUT8 gene expression, for example as described in US6,946,292B2. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Bispecific molecules

In another aspect, the present invention features bispecific or multispecific molecules comprising an anti-CD63var antibody, or a fragment thereof, of the invention. An antibody of the invention, or antigen-binding regions thereof, can be denvatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for CD63var and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of CD63 different from the first target epitope. Another example is a bispecific molecule comprising at least one first binding specificity for CD63var and a second binding specificity for an epitope on mast cells that has regulatory properties, for example, cKit, or Fcgamma-receptor. Yet another example is a bispecific molecule comprising at least one first binding specificity for CD63var and a second binding specificity for an epitope present on a different immune or stromal cells, so that a forced interaction modifies the fate of both cells. For example, a chimeric or fusion antibody wherein one specificity may be directed against CD63var and another against CD3, CD8, CD28, TCR components, CD7, CD4, to induce T-cell targeting, or CD19, CD20, TNFRSF members like BAFFR to achieve coordinated regulation of B-cells.

Additionally, for the invention in which the bispecific molecule is multi-specific, the molecule can further include a third binding specificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')₂, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, the contents of which is expressly incorporated by reference. Other antibodies which can be employed in the bispecific molecules of the invention are fully human, murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each arm of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-l- carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, for example one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')₂ or ligand x Fab fusion protein. A bispecific molecule of the invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881 , 175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091 ,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.

Binding of the bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Multivalent antibodies

In another aspect, the present invention provides multivalent antibodies comprising at least two identical or different antigen-binding portions of the antibodies of the invention binding to CD63var. In one embodiment, the multivalent antibodies provide at least two, three or four antigen-binding portions of the antibodies. The antigen-binding portions can be linked together via protein fusion or covalent or non covalent linkage. Alternatively, methods of linkage have been described for the bispecific molecules. Tetravalent compounds can be obtained for example by cross-linking antibodies of the antibodies of the invention with an antibody that binds to the constant regions of the antibodies of the invention, for example the Fc or hinge region.

Pharmaceutical compositions

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention can comprise a combination of antibodies that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an anti-CD63var antibody of the present invention combined with at least one other antiinflammatory or another chemotherapeutic agent, for example, anti-htstamines or anti-allergic agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjuage, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al., 1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, one can include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatin.

Reviews on the development of stable protein (e.g., antibody) formulations may be found in Cleland et al. (1993) Crit. Reviews. Ther. Drug Carrier Systems 10(4):307-377 and Wei Wang (1999) Int. J. Pharmaceutics 185:129-88. Additional formulation discussions for antibodies may be found, e.g., in Daugherty and Mrsny (2006) Advanced Drug Delivery Reviews 58: 686-706, US 6171586; US4618486; US20060286103, WO06044908; WO07095337; WO04016286; Colandene et al. (2007) J. Pharm. Sci 96:1598-1608; Schulman (2001) Am. J. Respir. Crit. Care Med. 164:S6-S11 and other known references.

Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Sterile injectable solutions can be prepared by incorporating the antibody of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When a therapeutically effective amount of an antibody of the invention is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenteral^ acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to binding agents, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art. The pharmaceutical composition(s) of the present disclosure may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, from about 0.1 per cent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pg/ml and in some methods about 25-300 g/rnl.

Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half- life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-CD63var antibody of the invention can results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

A composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.

Alternatively, an antibody of the invention can be administered by a nonparenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poly gly colic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices shown in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which shows an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which shows a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which shows a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which shows a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which shows an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which shows an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais et al., 1995 Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., 1995 Am. J. Physiol.1233: 134); p120 (Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen, 1994 FEBSLett. 346:123; J.J. Killion; I.J. Fidler, 1994 Immunomethods 4:273.

Uses and methods of the invention

The antibodies of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. The term "subject" as used herein is intended to include human and non-human animals. Non- human animals include all vertebrates, e.g., mammals and non-mammals, such as non- human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles.

The methods are particularly suitable for treating, preventing or diagnosing disorders mediated by degranulation of mast cells or other CD63var expressing granulocytes, such as basophils, and/or autoimmune diseases, e.g., rheumatoid arthritis, allergic disorders, asthma, multiple sclerosis or neurodermitis. The invention also provides methods for depleting degranulated mast cells and/or other CD63var expressing granulocytes, such as basophils, in an animal, preferably depleting or killing human degranulated mast cell and/or other CD63var expressing granulocytes, such as basophils, by administering a composition comprising a therapeutically efficient dose of antibodies of the invention with appropriate ADCC or cell-killing activity.

As used herein, "disorders mediated by degranulation of mast cells or other CD63var expressing granulocytes, such as basophils" includes conditions associated with or characterized by aberrant degranulated mast cells levels and/or diseases or conditions that can be treated by depleting or killing degranulated mast cells. These include inflammatory conditions, allergies and allergic conditions, hypersensitivity reactions, autoimmune diseases, severe infections, and organ or tissue transplant rejection, neuropsychiatry conditions, stress, and certain malignant diseases.

For example, the antibodies of the invention may be used for the treatment of recipients of heart, lung, combined heart-lung, liver, kidney, pancreatic, skin or corneal transplants, including allograft rejection or xenograft rejection, and for the prevention of graft-versus-host disease, such as following bone marrow transplant, and organ transplant associated arteriosclerosis.

The antibodies of the invention are useful for the treatment, prevention, or amelioration of autoimmune disease and of inflammatory conditions, in particular inflammatory conditions with an etiology including an autoimmune component such as arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and rheumatic diseases, including inflammatory conditions and rheumatic diseases involving bone loss, inflammatory pain, spondyloarhropathies including ankylosing spondylitis, Reiter syndrome, reactive arthritis, psoriatic arthritis, and enterophathic arthritis, hypersensitivity (including both airways hypersensitivity and dermal hypersensitivity) and allergies. Specific auto-immune diseases for which antibodies of the invention may be employed include autoimmune haematological disorders (including e.g. hemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), acquired hemophilia A, cold agglutinin disease, cryoglobulinemia, thrombotic thrombocytopenic purpura, Sjogren's syndrome, systemic lupus erythematosus, inflammatory muscle disorders, polychondritis, sclerodoma, anti-neutrophil cytoplasmic antibody- associated vasculitis, IgM mediated neuropathy, opsoclonus myoclonus syndrome, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, pemphigus vulgaris, pemphigus foliacius, idiopathic sprue, autoimmune inflammatory bowel disease (including e.g. ulcerative colitis, Crohn's disease and Irritable Bowel Syndrome), endocrine ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis, neuromyelitis optica, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior, intermediate and posterior as well as panuveitis), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis and glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy), tumors, inflammatory disease of skin and cornea, myositis, loosening of bone implants, metabolic disorders, such as atherosclerosis, diabetes, and dislipidemia.

The antibodies of the invention are also useful for the treatment, prevention, or amelioration of asthma, bronchitis, pneumoconiosis, pulmonary emphysema, and other obstructive or inflammatory diseases of the airways.

The antibodies of the invention are also useful for treating diseases of bone metabolism including osteoarthritis, osteoporosis and other inflammatory arthritides, and bone loss in general, including age-related bone loss, and in particular periodontal disease.

The antibodies of the invention are also useful for the treatment of IgE-mediated disorders. IgE mediated disorders include atopic disorders, which are characterized by an inherited propensity to respond immunologically to many common naturally occurring inhaled and ingested antigens and the continual production of IgE antibodies. Specific atopic disorders includes allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastroenteropathy.

However, disorders associated with elevated IgE levels are not limited to those with an inherited (atopic) etiology. Other disorders associated with elevated IgE levels, that appear to be IgE-mediated and are treatable with the formulations of this present invention include hypersensitivity (e. g., anaphylactic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-lgE syndrome, ataxia- telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and graft- versus-host reaction.

The antibodies of the invention may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant to or in combination to, other drugs e.g. immunosuppressive or immunomodulating agents or other anti-inflammatory agents or other anti-allergic agents, e.g. for the treatment or prevention of diseases mentioned above. For example, the antibodies of the invention may be used in combination with DMARD, e.g. Gold salts, sulphasalazine, antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, glococorticoids; a calcineurin inhibitor, e.g. cyclosporin A or FK 506; a modulator of lymphocyte recirculation, e.g. FTY720 and FTY720 analogs; an mTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; an ascomycin having immuno-suppressive properties, e.g. ABT-281 , ASM981 , etc.; corticosteroids; cyclo-phos-phamide; azathioprene; methotrexate; mizoribine; mycophenolic acid; myco-phe no-late mofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogue or derivative thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD11a, CD25, CD28, CD40. CD45, CD52, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, e.g. a recombinant binding molecule having at least a portion of the extracellular domain of CTLA4 or a mutant thereof, e.g. an at least extracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4lg (for ex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y; adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-1 antagonists or VLA-4 antagonists; or a chemotherapeutic agent, e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin or 5- fluorouracil; anti TNF agents, e.g. monoclonal antibodies to TNF, e.g. infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII, e.g. Etanercept, PEG- TNF-RI; blockers of proinflammatory cytokines, IL-1 blockers, e.g. Anakinra or IL-1 trap, AAL160, ACZ 885, IL-6 blockers; chemokines blockers, e.g inhibitors or activators of proteases, e.g. metalloproteases, anti-IL4 antibodies, anti-IL-15 antibodies, anti-lL-6 antibodies, anti-IL-21 antibodies, anti-IL-12 antibodies, anti-p40 antibodies, anti-lL-17 antibodies, anti-CD20 antibodies, NSAIDs, such as aspirin or an anti-infectious agent (list not limited to the agent mentioned).

Additional therapeutic agent may also be selected from the group consisting of antiinflammatory, bronchodilatory, antihistamine or anti-tussive drug substances, particularly in the treatment of obstructive or inflammatory airways diseases, for example as potentiators of therapeutic activity of such drugs or as a means of reducing required dosaging or potential side effects of such drugs. A therapeutic agent of the invention may be mixed with the other drug substance in a fixed pharmaceutical composition or it may be administered separately, before, simultaneously with or after the other drug substance. Accordingly the invention includes a combination of an antibody of the invention as hereinbefore described with an anti-inflammatory, bronchodilatory, antihistamine or anti-tussive drug substance, said agent of the invention and said drug substance being in the same or different pharmaceutical composition.

Suitable anti-inflammatory drugs include without limitation steroids, in particular glucocorticosteroids such as budesonide, beclamethasone dipropionate, fluticasone propionate, ciclesonide or mometasone furoate.

Suitable antihistamine drug substances include wihtout limitation cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinastine, mizolastine and tefenadine.

Combinations of therapeutic agents of the invention and anticholinergic or antimuscarinic agents, steroids, beta-2 agonists, PDE4 inhibitors, dopamine receptor agonists, LTD4 antagonists or LTB4 antagonists may also be used. Other useful combinations of agents of the invention with anti-inflammatory drugs are those with other antagonists of chemokine receptors, e.g. CCR-1 , CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D, Takeda antagonists such as N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzocyclohepten-8- yl]carbonyl]amino]phenyl]-methyl]-tetrahydro-N,N-dimethyl-2H-pyran-4-aminium chloride (TAK-770) or CCR-5 antagonists.

The additional therapeutic agent may also be selected from the group consisting of other cytokine binding molecules, particularly antibodies of other cytokines, in particular a combination with an anti-IL4 antibody, an anti-IL5, an anti-lgE antibody, such as Xolair®, an anti-IL31 antibody, an anti-IL31 R antibody, an anti-TSLP antibody, an anti-TSLP receptor antibody, an antt-endoglin antibody, an anti-IL1 beta antibody or an anti-IL13 antibody.

In accordance with the foregoing the present invention provides in a yet further aspect:

A method as defined above comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of a anti-CD63var antibody, e.g., an antibody of the invention, and at least one second drug substance, said second drug substance being a immuno-suppressive / immunomodulatory, anti-inflammatory chemotherapeutic or antiallergic drug, e.g. as indicated above.

Or, a therapeutic combination, e.g. a kit, comprising of a therapeutically effective amount of a) a CD63var antibody of the invention, and b) at least one second substance selected from a immuno-suppressive / immunomodulatory, anti-inflammatory chemotherapeutic or antiallergic drug, e.g. as indicated above. The kit may comprise instructions for its administration.

Where the antibodies of the invention are administered in conjunction with other immunosuppressive / immunomodulatory, anti-inflammatory chemotherapeutic or anti-allergic therapy, dosages of the co-administered combination compound will of course vary depending on the type of co-drug employed, e.g. whether it is a DMARD, anti-TNF, IL-1 blocker or others, on the specific drug employed, on the condition being treated and so forth.

In one specific embodiment, the antibodies of the invention may be administered in combination with an anti human IgE antibody, e.g., Omalizumab/Xolair, an anti human IgE antibody with present approval in the treatment of asthma bronchiale (i.e. an IV injection every other week.

In other embodiment, the antibodies of the invention are administered only to patient population which is selected among patients suffering from allergic disorders and/or exhibiting an abnormal level of degranulated mast cells or other CD63var expressing granulocytes, such as basophils. Such patients can be identified for example by high serum IgE levels, or commercially available allergen tests, like RAST tests.

Methods for allergy diagnosis

In one embodiment, the antibodies of the invention can be used to detect levels of CD63var, or levels of degranulated mast cells, or other granulocytes that undergo FceRI mediated degranulation, such as basophils and that express CD63var at their surface. This can be achieved, for example, by contacting a sample (such as an in vitro sample) and a control sample with the anti-CD63var antibody under conditions that allow for the formation of a complex between the antibody and cells expressing CD63var. Any complexes formed between the antibody and cells are detected and compared in the sample and the control. For example, standard detection methods, well known in the art, such as ELISA and flow cytometric assays, can be performed using the compositions of the invention.

Accordingly, in one aspect, the invention further provides methods for detecting the presence of CD63var or cells expressing CD63var (e.g., human CD63var epitope) in a sample, or measuring the amount of cells expressing CD63var, comprising contacting the sample, and a control sample, with an antibody of the invention, or an antigen binding region thereof, which specifically binds to CD63var, under conditions that allow for formation of a complex between the antibody or portion thereof and CD63var. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of degranulated cells, e.g. degranulated mast cells or activated basophils or CD63var in the sample. The quantitation of cells expressing CD63var isoform in a biological sample can also be measured using fluorescent activated cell sorting technology (or flow cytometry), with a fluorescently labelled anti-CD63var antibody.

Those diagnostic methods are useful for example for detecting patients that suffers from allergic disorders, rheumatoid arthritis, neurodermitis, asthma bronchiale, allergic rhinitis, seasonal pollen allergy, systemic mastocytosis.

As used herein, "allergic disorders" refers to any disorders resulting from antigen activation of mast cells, or other CD63var expressing granulocytes, such as basophils, that results in an "allergic reaction" or state of hypersensitivity and influx of inflammatory and immune cells. Those disorders include without limitation,

- systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies;

- respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g. idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions);

- rhinitis, hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis;

- skin and dermatological disorders, including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma;

- vasculitis (e.g. necrotizing, cutaneous, and hypersensitivity vasculitis);

- spondyloarthropathies; and

- intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).

In one embodiment, the invention provides a method to determine the activation stage of a FcsRI mediated allergic reaction in granulocytes such as mast cells or basophils, comprising the steps of: a) providing a biological sample containing mast cells or other CD63 expressing granulocytes, such as basophils, b) incubating said sample with labelled anti-CD63var antibodies under appropriate conditions and time, c) measuring activated cells through labeling with anti-CD63var, wherein the relative or absolute amount of cells expressing CD63var is indicative of the activation stage of the cells.

In one other embodiment, the method uses anti-CD63 (non CD63var specific) as a control to determine the relative amount of cells expressing CD63var compared to the total amount of CD63 expressing cell types. The quantification step (step c) is preferably carried out by flow cytometric analysis. In one embodiment, said sample is an anti-coagulated human or animal whole blood sample or an anti-coagulated purified human or animal blood sample. In one specific embodiment, basophils and mast cells are first purified from human or animal blood sample.

In another embodiment, said sample is subjected to an activation step in the presence of a test substance or allergen to determine allergy sensitivity to said test substance. Examples of possible test substance are also described in WO2009/033691and include without limitation, pollen, house dust mite, latex, venoms, foods, neuromuscular blocking agents, drugs and related compounds, aspirin and non-steroidal anti-inflammatory drugs.

In some embodiments, additional antibodies are used to quantify and characterize activated granulocytes. Those additional antibodies include without limitation, anti-CCR3, anti-lgE, anti-FceRI, anti-CD45, anti-CD203c, anti-CD123, anti-HLA-DR, anti-CRTH2, anti-CD3, basophil specific monoclonal Ba103 antibody, and basophil specific monoclonal 212H6 antibody, non-CD63var specific anti-CD63 antibody, anti-CD13, anti-CD69, anti-CD164, anti- CD107a, anti-CD117 (c-kit), anti-CD9, anti-CD81 , anti-CD151.

The invention further relates to methods and kits to purify or sort cells expressing CD63var comprising a CD63var binding component covalently attached to a solid surface, such as resin or bead, including without limitation, magnetic or paramagnetic beads, purification, e.g affinity column chromatography. Said CD63var binding component comprises an antigen- binding portion of an anti-CD63var antibody of the invention. Columns or beads containing anti-CD63var antibodies are also part of the invention.

The invention further relates to labeled antibodies and their use for in vivo imaging for the detection of activated, degranulated mast cells or other expressing CD63var cells or hidden inflammatory herds.

Also within the scope of the invention are kits consisting of the compositions (e.g., antibodies, human antibodies and bispecific molecules) of the invention and instructions for use. The kit can further contain at least one additional reagent, or one or more additional antibodies of the invention (e.g., an antibody having a complementary activity which binds to an epitope on the target antigen distinct from the first antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. The kit may further comprise tools for diagnosing whether a patient belongs to a group that responds to an anti-CD63var antibody treatment, as defined above.

The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not meant to be further limiting. Brief Description of the Figures

Figure 1: Novel anti human CD63 mAbs with specificity for the activated mast cell state.

A) Generation of degranulation-specific anti human mast cell Abs. Human cord blood derived mast cells (HCB Cs) were either stimulated with PMA ionomycin to induce degranulation (open graphs) or were left untreated (black), and subsequently stained with selected degranulation-specific antibodies.

B) Verification of antibody targets through expression cloning. RBL cells were stably transfected with cDNAs of the human CD63 (upper row) and CD82 genes (lower panels), and stained with degranulation-specific anti human mast cell mAbs. Note selective surface staining only for CD63 transfected cells, but not for ceils expressing the related tetraspanin CD82. Non-transfected RBL cells are shown for reference (grey).

Figure 2: Degranulation-specific anti human CD63 mAbs do not cross-react with platelets, MalMe3, THP-1 or U937 cells.

A) Myeloid precursor-like THP-1 cells, promonocytic U937 cells, and melanoma cell line MalMe3 were surface stained either with a commercial anti CD63 Ab (Chemicon Int. CBL553, left) or the degranulation-specific anti human mast cell CD63 Ab NIBR63/1 (right). An isotype control (BD Pharmingen, Cat. No. 557273, mouse lgG1) is shown for reference (white). Although all three tested cell types clearly express tetraspanin CD63, virtually no surface staining with antibody NIBR61/1 is seen.

B) Degranulation-specific anti human mast cell Ab NIBR63/1 also does not bind to platelets. Human whole blood derived platelets were either incubated at 4°C to prevent cell activation (upper panels), or in a buffer supplemented with PMA/ionomycin to induce degranulation at RT (lower panels). Cell surface stained was performed with a commercial anti human CD63 Ab (Chemicon Int. CBL553, left), or a degranulation-specific anti human Mast Cell CD63 Ab (NIBR63/1 , right). Independent of cell activation, no significant staining with antibody NIBR61 1 is seen.

Figure 3: Subcellular localization of CD63 within native and degranulated human mast cells.

A) Human cord blood derived mast cells (left panels) and Jurkat control cells (right) were analyzed for expression and subcellular localization of CD63 on cytospin preparations using degranulation-specific mAbs.

B) Degranulation-dependent release of CD63 upon cell activation. Human cord blood derived mast cells were either incubated in mock buffer (left) or stimulated with PMA/ionomycin (right) to induce degranulation. The activation-dependent subcellular localization of CD63 was subsequently analyzed by APAAP cytospin staining.

C) Activation-dependent mAbs stain a sub-population of mast cell CD63. Human cord blood derived mast cells were either left untreated (native, top), or stimulated with PMA/ionomycin to induce degranulation (bottom), and subsequently stained with a conventional CD63 (Chemicon Int. CBL553, left), or with a degranulation-specific anti human CD63 mAb (NIBR63/1 , right). Whereas the commercial Ab interacts with native and degranulated mast cells, mAb NIBR63/1 reacts only with induced mast cells.

Figure 4: Mutant-based epitope mapping for degranulation-specific anti human mast cell CD63 mAbs.

A) A CD63 N 72Q single side mutation alters the binding characteristics of a degranulation- specific anti CD63 Ab. RBL cells were stably transfected either with a wild-type copy of the human CD63 gene (white), or a N172Q mutant allele (black), and subsequently stained with indicated Abs. Note a fluorescent intensity shift of mutant over wild-type cells, when surface labeled with degranulation specific anti human mast cell Ab NIBR63/1. In contrast, the staining efficiency of a commercial anti CD63 Ab (Chemicon Int. CBL553) is not affected by the N172Q mutation (left).

B) A C170S single site mutation selectively abrogates CD63 surface recognition by degranulation-specific Abs. Rat basophile leukemia (RBL) cells were stably transfected either with a wild-type copy of the human CD63 gene (upper panels), or a C170S mutant allele of the same protein (lower panels). Subsequent cell surface staining with a commercial (Chemicon Int. CBL553) or a degranulation-specific anti human CD63 Ab (NIBR63/1) revealed a selectively impaired surface interaction for the degranulation-specific Ab alone. Isotype control (BD Pharmingen, Cat. No. 557273, mouse lgG1) for reference.

Figure 5: Novel anti human CD63 mAbs selectively inhibit the degranulation of pre-activated mast cells.

A) Various commercial anti CD63 Abs impair human mast cell degranulation. IgE/antigen induced degranulation of human cord blood derived mast cells as followed by the enzymatic turnover of reporter enzyme β-hexosaminidase. Cells had been pre-incubated in presence (faint bars) or absence (dark bars) of indicated anti CD63 mAbs for 2.5h. Note significantly impaired degranulation efficiency in presence of the commercial mAbs #95 (Serotec MCA2142), #173 (Ancell 215-020), or #197 (MUV, aCD63-11 C9), but not for the state- specific anti human CD63 mAbs NIBR63/1-3. B) State-specific anti human CD63 mAbs selectively impair repeated mast cell degranuiation. Iterative mast cell degranuiation as induced with IgE/antigen systems Jw-8/Nip-BSA (degranuiation cycle No. 1 , left) and aPC17/PC17-BSA (degranuiation cycle No. 2, right). Spontaneous degranuiation of IgE-coated cells (dotted line), full-blown sequential degranuiation stimulus (solid line), iterative degranuiation in presence of state-specific anti human CD63 mAb NIBR63/1 and NIBR63/2 (dashed lines).

Figure 6: State-specific anti human CD63 mAbs inhibit repeated mast cell degranuiation.

A) CD63 expression-based fractionation of activated human mast cells. IgE-coated human mast cells were antigen-treated or not, and subjected to FACS after a degranuiation period of 1h. Based on CD63 surface expression low, intermediate and high content fractions were separated. Their homogeneity was verified by analytic separation (bottom three panels) and each specimen subjected to another round of cell activation.

B) State-specific and commercial anti CD63 Abs impair iterative mast cell degranuiation. Pre- activated, FACS-separated human mast cell fractions were stimulated with another IgE antigen system either in presence or absence of anti CD63 mAbs. Uninhibited degranuiation cycle No.2 (black), isotype control (grey), repeated mast cell degranuiation in presence of aCD63 mAbs (white bars). From left to right: commercial anti CD63 mAb (Ancell 215-020), NIBR63 1 , NIBR63 2.

Examples

Anti-CD63var antibodies

The following table shows the profile of 3 examples of the antibodies according to the invention.

Screening assays to identify anti-CD63var antibodies

Human anti CD63var antibodies can be obtained through immunization of mice with degranulated (i.e. PMA/ionomycin-treated) cord blood derived human mast cells, subsequent hybridoma fusion and standard antibody selection procedures.

Alternatively, human anti-CD63var antibodies may also be obtained through competitive phage display screening. This methodology may also be applied for an optimization of the antibody binding properties.

For example, the deposited antibody may be color-labeled, and alternative candidates screened for inhibiting its binding to activated human mast cells or adequate transfectants.

Characterization of anti-CD63var antibodies

Method of measuring binding to degranulated mast cells vs. quiescent mast cells

Degranulated human mast cells can be obtained from CBHMCs through incubation with PMA/ionomycin, IgE/antigen, or alternative stimuli. Degranulated or mock-treated, quiescent control cells are surface stained with 0^g/500.0O0cells of test antibodies in PBS (pH=7.5), 1 % BSA for 1h at 4'C. Excess antibody is removed and cells incubated in presence of 0.^g/500.000cells secondary anti mouse antibody (e.g. goat F(ab^*)2 anti mouse IgG, R-PE conjugate, Biosource Int., AMI4407) for 1h at 4°C. Significant binding is defined as a 100x MOI shift in FACS analysis over respective control cells. Method of measuring affinity to CD63var epitope of anti-CD63var antibodies

Affinity can be determined by using the above FACS method, where now a directly labelled NIBR63/1 antibody is used as reference. Different concentrations of an unlabelled test antibody, ranging from 10 to 0 ug/ml are preincubated with degranulated mast cells. After that binding of the labelled NIBR63/1 reference is evaluated. If the test antibody recognizes the CD63var epitope, an affinity-dependent displacement of the reference antibody will be observed. The relative affinity of the test antibody is determined by its required concentration.

Method of measuring inhibitory activity of secondary degranulaUon

For in vitro measurements preactivated, partially degranulated human mast cells can be obtained from CBHMCs through incubation with IgE, and subsequent crosslinking with its cognate antigen. These cells are then isolated, washed, and incubated with a test antibody. A second degranulation cycle is then induced through incubation with P A ionomycin, IgE/antigen, or alternative stimuli. β-Hexosaminidase assay is then carried out as described in the Methods section. If the test antibody had inhibitory activity, a proportional decline in enzyme activity will be detected.

Method of measuring inhibitory activity on migration of mast cells

Migration is evaluated using a 24-well, 5.0-pm pore size Transwell plate (Costar, Cambridge, MA). Cell are washed once with PBS and adjusted to 2-4 χ 10^Λ6 cells/ml in assay medium (RPM11640 without phenolred, 0.1%BSA). An aliquot (50 μΙ) of this cell suspension is placed in the top Transwell chamber. Migration inducing agents, like chemokines are diluted to optimal concentrations in assay medium (e.g. 500ng/ml of SLC/CCL21 , or 200ng/ml of MIP- 3B/CCL19, or 100ng/ml SDF 1a/CXCL12, or 100ng/ml SDF-1 fJ/CXCL12; all R&D Systems, Minneapolis MN, USA), and added to the bottom chamber. Antibodies are added to top, as well as to the bottom chamber. After 180 min incubation at 37°C in a 5% C02 atmosphere, the top chamber is removed, and the cells that migrated into the bottom chamber are counted.

Method of measuring inhibitory activity on differentiation and survival of mast cells

For differentiation assays, precursor cells, like cord blood derived mast cell precursors are placed into 24well plates (Costar, Cambridge, MA) at 2 * 10^A5 cells/ml in culture medium and are propagated at 37°C and 7.5% C02 in serum-free expansion medium (Stem Cell Technologies, #09650) supplemented with 100U/ml penicillin, 100ug/ml streptomycin, 2mM L-glutamate (Sigma, G1146), with a cytokine cocktail consisting of 100ng/ml rhSCF, 100ng/nl rhlL-6, and 30ng/ml rhlL-3 (Peprotech) in the presence of a test antibody (100ug/ml to 0.01 ug/ml concentration). After at least two weeks of cultivation, cultures are terminated. Cells analyzed for their expression of markers typical for mast-cells, such as for intracellular tryptase by cytospin stainings, and for surface markers, such as CD117, FceRIa, in FACS analysis. An inhibitory antibody is expected to cause loss of expression of these markers, proportionally to its activity.

For proliferation assays, mast cells are set into 96well plates (Costar, Cambridge, MA) at 2 χ 10M cells/well using the same media as above. Proliferation is measured after at least three days using an MTT/MTS kit (Promega, Madison, Wl, USA). An inhibitory antibody is expected to lower the amount of viable cells, proportionally to its activity.

Materials and Methods

Cell culture and transfectants: Human mast cells were differentiated from cord blood derived progenitor cells according to standard procedures (Dahl et ai. 2002. Journal of Immunological Methods 262:137). In brief, stem cells were isolated from heparinized human cord blood by Ficoll gradient centrifugation (Sigma, #46324) and magnetic enrichment for CD133 positive cells (CD133 Micro Bead Kit, Miltenyi Biotec, #130-050-801). The resultant mononuclear cell fraction was propagated at 37°C and 7.5% C02 in serum-free expansion medium (Stem Cell Technologies, #09650) supplemented with 100U/ml penicillin, 100μg ml streptomycin, 2mM L-glutamate (Sigma, G1146), and a cytokine cocktail consisting of 100ng/ml rhSCF, 100ng/nl rhlL-6, and 30ng/ml rhlL-3 (Peprotech) for three weeks. Cultivation was then continued in medium without IL-3 for two additional weeks. Finally, cultures were shifted to RPMI 1640 medium (Sigma, R8758) supplemented with 10% decomplemented FBS (Gibco, #16000-044), 100U/ml penicillin, 100μg/ml streptomycin, 2mM L-glutamate, 100ng/ml SCF, and 100ng/nl IL-6 for at least another six weeks before degranulation experiments were performed. RBL cells were cultivated in complete DMEM (Sigma, D6429) supplemented with 10% FBS (Gibco, #16000-044) and antibiotics. cDNAs of the human CD63 and CD82 genes were obtained from the IMAGE collection (clones IRAL10m10 and IRAL2m8, respectively) and recloned into a pEK series expression vector. CD63 mutants were generated by PCR site-directed mutagenesis. All constructs were verified by sequencing and transfected into rat basophile leukemia (RBL) cells using Fugene6 (Roche, #11814443001 ) reagent according to the manufacturer's instructions. Stable transfectants were selected using mCD8a as coexpressed marker by multiple rounds of magnetic sorting (BD I Mag, #55151 ), and final flow sorting on a FACS Aria cell sorter.

Generation of monoclonal antibodies: Pathogen-free Balb/c mice were immunized multiple times with a mix of 5E5 PMA/ionomycin treated CBHMCs homogenized in Titer-Max adjuvant (Sigma, H4397). Spleen cells were fused with SP2 0 mouse myeloma cells using standard techniques, and antibodies were purified from the culture supernatant on rProteinG FPLC columns (Amersham Biosciences, Uppsala, Sweden).

Degranulation experiments: CBHMC cells were incubated overnight with 5pg/ml coating antibody (Jw8, or anti phosphocholin) and 25ng/ml IL-4 in conditioned medium. Cells were washed the next day to remove access IgE, and resuspended in assay medium (RPMI 1640 w/o phenol red, 25mM HEPES pH 7.2, 0.5% BSA, 100U/ml penicillin, 100μg/ml streptomycin, 2mM L-glutamate) to an approximate cell density of 1E6/ml. Degranulation was induced by addition of respective antigens (100ng/ml NIP-BSA or phosphocholin-BSA) and the incubation continued for at least 2.5h at 37°C and 7.5% C02. For sequential degranulation experiments, cells were washed after the first round to remove access IgE/antigen, and resuspended in fresh medium supplemented with 25ng/ml IL-4 and 5pg/ml of the second coating antibody. The second degranulation cycle was induced essentially the same way as the first one described above. Total cell lysates were obtained by addition of 1% Triton X- 100 (Sigma, T8787). Alternatively, degranulation was also induced with 25ng/ml P A (Sigma, P1585) and 500 ng/ml ionomycin (Sigma, I3909). β-Hexosaminidase assay: 25μΙ of degranulation supernatants were transferred into 96-well microlite TCT plates (flat bottom, white) and supplemented with 75μΙ 1mM 4- Methylumbelliferyl N-acetyl-b-D-glucosaminide (Sigma, M2133) solution in 0.1 M sodium citrate buffer pH 4.5. Samples were mixed and incubated at 37°C and 7.5% C02 for up to 1h. Fluorescent emission was determined on a Tecan Ultra Spectrophotometer (excitation = 360nm / emission = 465nm) after 5, 15, 30 and 60min incubation times.

Cytospin stainings: Approximately 1xE5 CBHMCs were immobilized on poly-lysine coated glass slides and fixed with MeOH/Acetone (1:1 ) for 5min at RT. For detection of tryptase, cover slides were washed with TBS buffer, pH 7.6 and incubated o/n with an anti mast cell tryptase mAb (Dako cytomation, M7052, 1/500) at 4°C. The APAAP detection system (Dako Cytomation, K0670) was then used for color development. May-Grunwald-Giemsa staining was performed using standard solutions and protocols (Merck, #1.01424 and Merck, #1.09204).

Results

Degranulation-specific anti human mast cell antibodies

Monoclonal antibodies that selectively recognized degranulated, but not native CBDMCs (Fig.lA) have been generated. In order to identify the molecular target structures for these new cell-state specific antibodies, publicly available gene expression data (GEO entry numbers GSE1933 and GDS1520) were screened for candidate genes which were either up- regulated in response to mast cell degranulation or were generally over-expressed in CBDMCs (data not shown). Based on these criteria, rat basophile leukemia (RBL) cells were stably transfected with a selected collection of candidate genes, and screened for potential surface recognition by the novel antibodies. One surface protein successfully identified through this gene expression strategy was tetraspanin CD63. RBL transfectants stably expressing CD63, but not the structurally related tetraspanin CD82, were selectively recognized by state-specific anti human mast cell Abs NIBR63/1 , 12, and /3 (Fig.l B). As CD63 is known to be widely expressed on a number of cell types, and was implicated in the progression of melanoma (25,26), a thorough mapping of target and species selectivity was particularly important. In particular, myeloid precursor- 1 ike THP-1 cells, promonocyte U937 cells, and melanomal cell line MelMe3 were tested for surface expression of CD63 and for potential cross-reactivity with degranulation-specific anti human mast cell mAbs NIBR63/1-3 (Fig.2A). Although all tested cell lines clearly expressed tetraspanin CD63, virtually no surface staining with antibodies NIBR61/1-3 was found. We extended our analysis to the investigation of platelets, a cell type known to express CD63 in an activation dependent manner. Following cell stimulation with PMA ionomycin, a significant amount of CD63 became surface exposed and could be detected with commercial anti CD63 mABs, whereas no significant association of antibodies NIBR63/1-3 was found (Fig.2B). Similarly, no staining of Jurkat T cells, B cell LCL lines, or fresh PBMCs was seen. We therefore conclude that the novel degranulation-specific anti CD63 mAbs are highly selective and strongly associate with degranulated human mast cells alone.

Degranulation-specific anti human mast cell mAbs interact with a granular isoform of CD63

Mast cells have been reported before to express CD63 (Schernthaner et al. 2005, Allergy 60:1248), but the remarkable selectivity of the new anti CD63 mAbs towards granule-derived CD63 suggested that differently modified isoforms of this protein may be present within human mast cells.

The subcellular localization of DSaHMC-mAbs related epitopes was therefore examined by cytospin staining (Fig.3). In line with the preceding data, DSaHMC-linked epitopes are highly enriched in human mast cells, but absent from Jurkat control cells (Fig.3A). More importantly however, the DSaHMC-mAb signal is seen throughout the mast cell cytosol, which is in good agreement with a granular epitope. Cell activation on the other hand leads to diminished cytoplasmic CD63 signal and to a redistribution of reactivity. Interestingly, punctate staining was also found in the acellular extracellular space, suggesting that exosomes can carry this particular epitope with them (Fig.3B). Finally, the co-existence of differently modified CD63 variants within CBDMCs was also verified by FACS. Whereas commercially available anti human CD63 Abs stain native and degranulated mast cells, DSaHMC-mAb discriminate against quiescent CBDMCs (Fig.3C). Altogether, these data strongly suggest the coexistence of at least two CD63 variants within human mast cells, and an activation- dependent redistribution of a granular isoform of the protein.

A topological hot spot within a surface lobe of CD63

CD63 molecules sorted to granuli can easily be subjects of secondary modifications. For example, N-glycosylation had been reported to affect at least three residues within the CD63 protein chain (Engering et al. 2003, FEBS Journal 270:2412). Thus, an extensive mutant analysis was initiated in order to locate the epitope of specific DSaHMC antibody association. First the predicted CD63 glycosylation sites N130, N150, and N172 were successively replaced by glutamine, and the mutant alleles were stably transfected into RBL cells and subjected to FACS analysis. Whereas point mutations of the first two residues had no significant effect on antibody recognition the N172Q mutation clearly improved the reactivity of degranulation-specific antibody NIBR63/1 , while the interaction with conventional anti- CD63 Abs was not changed (Fig.4A). Improved NIBR63/1 surface staining was also seen with independently cloned double and triple glycosylation mutants, whenever position N172 was included.

Structure and sequence comparisons of CD63 with related tetraspanins indicated a cysteine cluster motif in the external loop II region of the protein. Cysteine C170 of this cluster is localized in utmost sequence neighborhood of the critical glycosylation site N172. We therefore decided to also create cysteine point mutants of CD63 and found that residue C170 significantly affects antibody recognition as well. While a C170S mutation was fully tolerated by the conventional anti-CD63 mAbs, binding of the DSaHMC-mAbs NIBR63/1 was completely abolished (Fig.4B). Thus, C170 and N172 are critical determinants of the degranulation-specific CD63 surface epitope.

Most human tetraspanins contain four universally conserved cysteine residues in their external loop II domain. As has been demonstrated for the previously crystallized CD81 protein, these residues are likely to engage in two structurally determining disulfide bridges. CD63 on the other hand contains six cysteine residues, strongly suggesting an additional disulfit bridge in this part of the protein. A series of mutants were thus cloned in which each cysteine was individually replaced by serine, expressed in RBL cells, and analyzed by FACS. In good agreement with the predicted disulfide interactions between C145-C191 and C146- C169, point mutations of each respective residue pair had comparable effects on CD63 protein expression and antibody recognition (data not shown). Clearly disfavoring an obligatory third cysteine bond however, point mutations C170S and C177S had remarkably dissimilar effects on CD63 antibody binding. Whereas the C170S allele completely abolished CD63 surface staining by DSaHMC-mAbs (see Fig.3B for comparison), the association of commercial and degranulation-specific antibodies was hardly affected by the C177S point mutant. In conclusion, residue C170 does not seem to participate in an obligatory cysteine bond, but rather contributes to a surface accessible epitope per se.

With experimental evidence for conserved cysteine bridges C145-C191 and C146-C169 and free residues C170 and C177, we calculated a homology model of the CD63 external loop II domain on the basis of the published crystal structure of tetraspanin CD81. Our calculation places both residues critical for DSaHMC antibody binding, C170 and N172, in surface- exposed juxtaposition. Both residues are part of a sequence insertion that protrudes from the CD63 protein, and apparently forms a signature structure for selective antibody recognition. Thus, antibodies NIBR63/1 , 12, and /3 recognize a characteristic surface feature of the human CD63 protein, of which residues C170 and N172 are essential constituents.

State-specific anti CD63var mAbs can modulate repeated mast cell degranulation

Although anti rat CD63 Abs have previously been shown to inhibit the degranulation of RBL cells (Kraft et al. 2005, The Journal of Experimental Medicine 201 :385) there was no reliable evidence if anti human CD63 Abs would influence the activation of human mast cells as well. Degranulation of CBDMCs was induced in vitro, either in presence or absence of anti human CD63 mAbs (Fig.5). Not unexpectedly, several commercially available anti-CD63 mAbs significantly impaired the degranulation efficiency of CBDMCs (Fig.SA). As expected on basis of their cell-state selectivity, degranulation-specific anti human mast cell CD63 mAbs NIBR63/1, 12 and /3 on the other hand had no effect on primary degranulation.

To test their potential applicability in repeated mast cell degranulation, an iterative mast cell activation assay was implemented. This assay uses sequential exposure of human cord blood derived mast cells to alternative IgE/antigen systems. Cells were first coated with the humanized IgE molecule Jw-8 and a degranulation response evoked by addition of its correspondign antigen, NIP-BSA. Following this first degranulation reaction, excess IgE was removed and the cells allowed to recover over night in presence of coating antibody No.2 (i.e. anti-phosphocholin). Following a pre-incubation with or without indicated antiCD63 mAbs, a second degranulation reaction was launched upon addition of its respective antigen, phosphocholin-BSA. Importantly, when added during a secondary degranulation cycle, the new anti-CD63var antibodies had a significant and reproducible inhibitory effect (Fig.5B).

In order to prove that indeed individual cells are capable of serial degranulation, we included a FACS sorting step after an initial round of degranulation to subdivide the cell population on basis of CD63 surface exposure (Fig.6A). Although essentially all cells had responded to the first activation cycle, the absolute amount of CD63 surface expression varied within the population. Cells were therefore separated into low, intermediate, and high CD63 expressing fractions. Each population was independently recovered, coated with IgE molecules required for the second cycle, and subjected to another round of degranulation (Fig.6B). Degranulation could be re-induced in all three fractions. Most remarkably, high responders of the first cycle degranulated also very efficiently in the second cycle, while low responders of the first round responded also lower thereafter. Thus, in spite of the intrinsic cell culture heterogeneity, persisting responsiveness to IgE/antigen stimuli is an inherent characteristic of CBDMC in vitro cultures.

We found no evidence for specialized mast cell subclasses, which preferably respond to one degranulation stimulus or the other. Instead, the addition of anti CD63 Abs to low, intermediate, and high CD63 expressing fractions reproducibly diminished the efficiency of repeated mast cell degranulation by 20-35% (Fig.6B). We also kept aliquots of low, intermediate and highly degranulated cells in culture, and observed them in regular intervals. While mature mast cells hardly proliferate, the majority of sorted cells survived for several weeks (data not shown). This is a further indication that degranulation is not inevitably coupled to cell death, and that in general native mast cells may undergo several activation cycles.

Claims

1. An isolated antibody, or a functional protein comprising an antigen-binding portion of an antibody, that binds to degranulated mast cells and not to quiescent mast cells.

2. The isolated antibody or functional protein of Claim 1 , that binds to degranulated mast cells at least 100 fold more than to quiescent mast cells as measured by mean fluorescent intensity of antibody-stained cells in FACS analysis.

3. The isolated antibody or functional protein of Claim 1 or 2, that specifically binds to an isoform of human CD63 (SEQ ID NO:1) expressed at the surface of degranulated mast cells but not at the surface of the CD63-expressing cell types selected among the group consisting of platelets, quiescent mast cells, quiescent myeloid-precursor-like THP-1 cells, promonocytic cell line U937, or melanomal cells.

4. The isolated antibody or functional protein of any one of Claims 1-3, that specifically binds to an epitope specific of an isoform of human CD63 (SEQ ID NO:1), said epitope including C170 and/or N172 residues of human CD63, said CD63 isoform being expressed at the surface of degranulated mast cells but not at the surface of the CD63-expressing cell type selected among the group consisting of platelets, quiescent mast cells, quiescent myeloid- precursor-like THP-1 cells, the promonocytic cell line U937, or melanomal cells.

5. The isolated antibody or functional protein of Claim 4, that binds to said epitope specific of an isoform of human CD63 expressed at the surface of degranulated mast cells, with a K_D of 1μΜ or less, 100nM or less, 1 nM or less, as measured with a Biacore assay.

6. The isolated antibody or functional protein of Claim 4 or 5, that inhibits the degranulation of pre-activated mast cells in vitro, as measured by the enzymatic turnover of released reporter enzyme β-hexosaminidase.

7. The isolated antibody or functional protein of Claim 6, wherein said mast cells are human mast cells.

8. The antibody according to any one of Claims 1-7, which is a fully human or humanized lgG1 antibody.

9. The antibody according to any one of Claims 1-8, which comprises a mutated or chemically modified amino acid Fc region, wherein said mutated or chemically modified Fc region provides increased ADCC activity when compared to wild type Fc region.

10. A hybridoma expressing a monoclonal antibody selected among the group consisting of NIBR63/1 , NIBR63/2 and NIBR63/3, as deposited at DSMZ on September 8, 2009.

11. The antibody or functional protein according to any one of Claims 1-9, which is cross- blocked from binding to CD63 isoform specific of human degranulated mast cells by at least one antibody of Claim 10.

12. The antibody or functional protein according to any of Claims 1-9 which cross-blocks at least one antibody of Claim 10 from binding to CD63 isoform specific of human degranulated mast cells.

13. The antibody or functional protein according to any one of claims 1-12, for use as a medicament.

14. The antibody or functional protein according to any one of Claims 1-13, for the treatment of a pathological disorder that is mediated by degranulation of mast cells or that can be treated by killing or depleting degranulated mast cells or that can be treated by preventing a secondary degranulation cycle to mast cells which were previously degranulated.

15. The antibody or functional protein according to any one of Claims 1-14, for the treatment of autoimmune and inflammatory disorders, such as rheumatoid arthritis, allergic disorders, asthma, multiple sclerosis or neurodermitis, or malignant proliferative diseases, such as systemic mastocytosis.

16. Use of an antibody or functional protein according to any one of Claims 1-12, in the preparation of a medicament for the treatment of autoimmune and inflammatory disorders, such as rheumatoid arthritis, allergic disorders, asthma, multiple sclerosis, neurodermitis, or malignant proliferative diseases, such as systemic mastocytosis.

17. A pharmaceutical composition comprising an antibody or functional protein according to any one of claims 1-12, in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

18. The pharmaceutical composition of Claim 17, additionally comprising other active ingredients selected among anti-allergic drugs, for example, anti-histaminic drugs or anti-igE or anti-FceRI drugs, for example Omalizumab, in the treatment of asthma bronchiale, or other auto-immune and inflammatory disorders.

19. An isolated nucleic acid encoding the antibody or functional protein according to any one of claims 1-12.

20. A cloning or expression vector comprising one or more nucleic acids according to claim 19.

21. A host cell comprising one or more cloning or expression vectors according to claim 20.

22. A process for the production of an antibody or functional protein of any one of claims 1- 12, comprising culturing the host cell of claim 21 and isolating said antibody or functional protein.

23. A method of preventing or inhibiting human degranulated mast cells from entering into a secondary degranulation cycle, said method comprising incubating a sample comprising degranulated mast cells with an antibody of any one of Claim 1-15.

24. An in vitro method to detect FceRI degranulation comprising a) providing a biological sample potentially containing degranulated cells, e.g.

degranulated mast cells or basophils, b) incubating said biological sample in the presence of an antibody of any one of Claims 1-12 under suitable conditions for specific binding of said antibodies to its antigen expressed at the surface of degranulated mast cells, c) detecting binding of said antibodies to any degranulated cells, e.g, degranulated mast cells or basophils, wherein said binding is indicative that said sample comprises FceRI degranulated cells.

25. The use of the isolated antibody or functional protein of any one of claims 1-15 for the in vitro selection or purification of degranulated human cells, e.g, degranulated mast cells or basophils.