WO2004067569A1

WO2004067569A1 - Internalising human binding molecules against cd72

Info

Publication number: WO2004067569A1
Application number: PCT/EP2003/050004
Authority: WO
Inventors: Alexander Berthold Hendrik Bakker; Willem Egbert Marissen
Original assignee: Crucell Holland B.V.
Priority date: 2003-01-27
Filing date: 2003-01-27
Publication date: 2004-08-12
Also published as: AU2003209753A1; WO2004067569A8

Abstract

The present invention provides internalising human binding molecules that specifically bind to CD72, nucleic acid molecules encoding the internalising human binding molecules, compositions comprising the internalising human binding molecules and methods of identifying or producing the internalising human binding molecules. In a preferred aspect, the invention provides immunoconjugates comprising an internalising human binding molecules that specifically binds to CD72. The internalising human binding molecules and immunoconjugates comprising an internalising human binding molecule can be used in the diagnosis and treatment of B cell associated disorders.

Description

INTERNALISING HUMAN BINDING MOLECULES AGAINST CD72

FIELD OF THE INVENTION

The present invention relates to the field of medicine. The invention in particular relates to the identification of internalising human binding molecules capable of binding to CD72, to immunoconjugates comprising these binding molecules and to methods of obtaining such internalising binding molecules. The invention further relates to the use of such internalising binding molecules in medicine, in particular for the diagnosis and/or treatment of B cell associated disorders.

BACKGROUND OF THE INVENTION

CD72 is a B cell associated antigen that was initially discovered in mice as the B cell differentiation antigen Lyb-2 and was later re-discovered as the human CD72 molecule. The human CD72 protein is a 45 kD type-II transmembrane glycoprotein carrying a calcium dependent (C-type) -lectin-like domain in the extracellular region and two immunoreceptor tyrosine-based inhibition motifs (ITIM) in the cytoplasmic tail. CD72 is a marker on all B cells (i.e. a pan-B marker). It is constitutively expressed as a disulfide-linked homodimer at the very earliest (pro-B) stages of B cell differentiation and is lost only upon terminal differentiation to plasma cells (Gordon 1994) . This pattern of expression is observed both in humans and mice. In mice, no expression has been detected on spleen T cells, or in the thymus, liver, testis, or brain, indicating that CD72 is highly B cell restricted. In some mouse strains, CD72 is also expressed at low levels on subpopulations of T cells (Robinson et al . 1997). In humans, tonsil sections and B cells in germinal centers and in the follicular mantle are strongly positive for CD72. However, the majority of extra-follicular B cells show very weak CD72 expression. It was found that some tissue macrophages express CD72, as well as some follicular dendritic cells (FDC's). The high B cell restriction of CD72 and it's broad expression on all malignant human B cell lines, except those of plasma cell origin, and on all human malignant B cells reflecting pre-B and subsequent stages of maturation (Myers and Uckun 1995) , renders CD72 a very suitable target to specifically attack B cell associated disorders such as for instance B cell lymphomas .

In view of the above, antibodies that specifically bind to CD72 might be very useful in diagnosis and treatment of B cell associated disorders. Several murine monoclonal antibodies directed against CD72 are known in the art. Myers and Uckun (1995) have used a mouse anti-human CD72 monoclonal antibody called J3-109 to prepare an anti-CD72 immunotoxin by conjugating the monoclonal antibody to the ribosome- inactivating Pokeweed Antiviral Protein (PAP) . They have used this immunotoxin in the treatment of therapy-refractory B lineage Acute Lymphoblastic Leukemia.

However, murine antibodies, in naked or immunoconjugated format, are limited for their use in vivo due to problems associated with administration of murine antibodies to humans, such as short serum half life, an inability to trigger certain human effector functions and elicitation of an unwanted dramatic immune response against the murine antibody in a human (the "human antimouse antibody" (HAMA) reaction) (see Van Kroonenburgh and Pauwels (1988)).

In general, attempts to overcome the problems associated with use of fully murine antibodies in humans, have involved genetically engineering the antibodies to be more "humanlike". A first stage in the humanization process was preparing chimeric antibodies, i . e . antibodies in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived. Subsequently, domains between the variable domains which specify the antigen binding were replaced by their human counterparts leading to so-called humanized antibodies. A disadvantage of these chimeric and humanized antibodies is that they still retain some murine sequences and therefore still elicit an unwanted immune reaction, especially when administered for prolonged periods.

Two human antibodies binding to CD72 have been described in Van der Vuurst de Vries and Logtenberg (1999) . Both antibodies can be useful tools in biochemical, immunohistochemical and immunofluorescent analysis. However, these antibodies have not been shown to be able to internalise within cells containing CD72 and therefore their use in therapeutic and diagnostic applications relating to B cell associated disorders is unknown. In general, internalisation is considered to be an important prerequisite in certain therapeutic settings, since internalising antibodies can be coupled to several therapeutic or diagnostic agents for specific delivery of these agents inside the target cells. In view of their benefit in therapy of B cell associated disorders there is still a need for internalising human monoclonal antibodies against CD72.

The present invention provides internalising human monoclonal antibodies against CD72 that can be used in medicine, in particular for diagnosis and/or treatment of B cell associated disorders. DESCRIPTION OF THE FIGURES

Figure 1.

Binding of phage antibodies SC02-024 and SC02-025 to CD19+ B lymphocytes (Figure 1A; the x-axis as well as the Y-axis represent fluorescence intensity) and human CD72-transfected L929 cells (Figure IB; x-axis represents fluorescence intensity, the y-axis represents cell number) . In Figure IB, the white peaks relate to L929 control transfectants, while the black peaks relate to L929 human-CD72 transfectants.

Figure 2.

Binding of phage antibodies SC02-002, SC02-003, SC02-004 and SC02-057 to CD19+ B lymphocytes (Figure 2A; x-axis represents fluorescence intensity, the y-axis represents cell number) . and human CD72-transfected L929 cells (Figure 2B; x-axis represents fluorescence intensity, the y-axis represents cell number) . In Figure 2B, the white peaks relate to L929 control transfectants, while the black peaks relate to L929 human-CD72 transfectants .

Figure 3.

PCR strategy for the generation of variants of SC02-003. The upstream M13rev sense primer is shown in bold and underlined. An antisense primer encompassing the Xhol site in which the codons encoding the asparagine (N) and the serine (S) have been randomised is also shown in bold and underlined; the randomised codons are depicted by NNM with N being A or C and M being G or A or T or C) . Figure 4.

Binding of human anti-CD72 IgGl antibodies to Ramos Burkitt's lymphoma cells. The x-axis represents mean fluorescence intensity (MFI) , the y-axis represents antibody concentration in micrograms per ml .

Figure 5.

Internalisation of human anti-CD72 IgGl antibodies in Ramos Burkitt's lymphoma cells. The x-axis represents fluorescence intensity, the y-axis represents cell number.

Figure 6. Map of sc02-002.

Figure 7. Map of sc02-003.

Figure 8. Map of sc02-004.

Figure 9. Map of sc02-024.

Figure 10. Map of sc02-025.

Figure 11. Map of sc02-057.

Figure 12. Map of sc02-041.

Figure 13. Map of sc02-132.

Figure 14. Map of pgG102-002C01.

Figure 15. Map of pgG102-002C01 heavy chain ("heavy only" means heavy chain) . Figure 16. Map of pgG102-002C01 light chain ("light only" means light chain) .

Figure 17. Map of pgG102-004C01.

Figure 18. Map of pgG102-004C01 heavy chain.

Figure 19. Map of ρgG102-004C01 light chain.

Figure 20. Map of pgG102-024C01.

Figure 21. Map of pgG102-024C01 heavy chain.

Figure 22. Map of pgG102-025C01.

Figure 23. Map of pgG102-025C01 heavy chain.

Figure 24. Map of pgG102-025C01 light chain.

Figure 25. Map of pgG102-04lC01.

Figure 26. Map of pgG102-04lC01 heavy chain.

Figure 27. Map of pgG102-132C01.

Figure 28. Map of pgG102-132C01 heavy chain.

DESCRIPTION OF THE INVENTION

Herebelow follow definitions of terms as used in the invention DEFINITIONS

Amino acid sequence

The term "amino acid sequence" as used herein refers to naturally occuring or synthetic molecules and to a peptide, oligopeptide, polypeptide or protein sequence.

Binding molecule

As used herein the term "binding molecule" refers to an intact immunoglobulin including monoclonal antibodies, such as chimeric, humanised or human monoclonal antibodies, or to an antigen-binding and/or variable domain comprising fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e . g . CD72. Regardless of structure, the antigen-binding fragment binds with the same antigen that is recognised by the intact immunoglobulin. The term "binding molecule", as used herein also includes the immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, binding molecules can be divided into the five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes) , e.g., IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antigen-binding fragments include, in ter alia , Fab, F(ab' F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv) , bivalent single- chain antibodies, diabodies, triabodies, tetrabodies, (poly) peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly) peptide, etc. The above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineerd by recombinant DNA techniques. The methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988) , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, which is incorporated herein by reference. A binding molecule or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.

Complementary determining regions (CDR) '

The term "complementary determining regions" as used herein means sequences within the variable regions of binding molecules, such as immunoglobulins, that generate the antigen binding site which is complementary in shape and charge distribution to the epitope recognised on the antigen. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e . g. , by solubilization in SDS. Epitopes may also consist of post translational modifications of proteins.

Deletion

The term "deletion", as used herein, denotes a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to the parent, often the naturally occurring, molecule. Expression-regulating nucleic acid sequence

The term "expression-regulating nucleic acid sequence" as used herein refers to polynucleotide sequences necessary for and/or affecting the expression of an operably linked coding sequence in a particular host organism. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required. The expression-regulating nucleic acid sequences, such as inter alia appropriate transcription initiation, termination, promoter, enhancer sequences; repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion, can be any nucleic acid sequence showing activity in the host organism of choice and can be derived from genes encoding proteins, which are either homologous or heterologous to the host organism.

Functional variant

The term "functional variant", as used herein, refers to a binding molecule that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent binding molecule and that is still capable of competing for binding to the binding partner, e.g. CD72, with the parent binding molecule. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent binding molecule do not significantly affect or alter the binding characteristics of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e. the binding molecule is still able to recognize and bind its target. The functional variant may have conservative sequence modifications including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR- mediated mutagenesis.

Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains ( e . g. , lysine, arginine, histidine) , acidic side chains ( e . g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cystine, tryptophan) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Furthermore, a variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art. A mutation in a nucleotide sequence can be a single alteration made at a locus (a point mutation) , such as transition or transversion mutations, or alternatively, multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleotide sequence. The mutations may be performed by any suitable method known in the art.

Host

The term "host", as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic. It should be understood that this terms is intended to refer not only to the particular subject organism or cell, but to the progeny of such an organism or cell as well. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term "host" as used herein.

Human

The term "human", when applied to binding molecules as defined herein, refers to derived from a human, based upon a human sequence or derived from or based upon a human sequence and subsequently modified. In other words, the term human, when applied to binding molecules is intended to include binding molecules having variable and constant regions derived from human germline immunoglobulin sequences. The human binding molecules may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by for instance random or site-specific mutagenesis in vi tro or by somatic mutation in vivo) . Immunoliposome

The term "immunoliposome" refers to a liposome bearing a binding molecule, as defined herein, that acts as a targeting moiety enabling the liposome to specifically bind to the binding partner of the binding molecule. The binding partner may be present in solution or may be bound to the surface of a cell.

Insertion

The term "insertion", also known as the term "addition", denotes a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the parent, often the naturally occurring, molecule.

Internalising binding molecule

The term "internalising binding molecule" as used herein means a binding molecule as defined herein that is capable of being internalised within the target cells to which it binds. In other words, the binding molecule is taken up, i.e. transported from the outside (cell surface) of a target cell to the inside, e.g. into the endosomal compartment or other compartment or into the cytoplasm of the cell, by the target cells upon binding to the binding partner of the binding molecule .

Isolated

The term "isolated", when applied to binding molecules as defined herein, refers to binding molecules that are substantially free of other proteins or polypeptides, particularly free of other binding molecules having different antigenic specificities, and are also substantially free of other cellular material and/or chemicals. For example, when the binding molecules are recombinantly produced, they are preferably substantially free of culture medium, and when the binding molecules are produced by chemical synthesis, they are preferably substantially free of chemical precursors or other chemicals, i.e., they are separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Preferably, substantially free means that the binding molecule will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a sample, more usually about 95%, and preferably will be over 99% pure.

The term "isolated" when applied to nucleic acid molecules encoding binding molecules as defined herein, is intended to refer to nucleic acid molecules in which the nucleotide sequences encoding the binding molecules are free of other nucleotide sequences, particularly nucleotide sequences encoding binding molecules that bind binding partners other than CD72. Furthermore, the term "isolated" refers to nucleic acid molecules that are substantially separated from other cellular components that naturally accompany the native nucleic acid molecule in its natural host, e.g., ribosomes, polymerases, or genomic sequences with which it is naturally associated. Moreover, "isolated" nucleic acid molecules, such as a cDNA molecules, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Liposome

The term "liposome" as used herein refers to a small vesicle bounded by a layer composed of various types of lipids, preferably amphipathic lipids, phospholipids and/or surfactants and made artificially from these molecules by techniques known in the art such as sonication or removal of detergent from phospholipid-detergent complexes. The layer typically is a bilayer formed by molecules that comprise a hydrophobic portion and a hydrophilic portion, wherein hydrophobic portions associate in an aqueous medium to form an internal part of the layer, whereas hydrophilic portions remain in contact with the medium. The layer surrounds and encloses an interior, which may contain, wholly or partially, an aqueous phase, a solid, a gel, a gas phase, or a non- aqueous fluid. Liposomes are useful for delivery of one or more molecules such as nucleic acid molecules, binding molecules, proteins, toxic substances and other material or compounds into cells such as animal cells by liposome fusion with the plasma membrane, a process also called lipofection. The molecules may be contained within the interior of the liposome, in the lipid layer, or attached to the outer surface of the lipid layer.

Monoclonal antibody

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences or derived from completely synthetic sequences . Nucleic acid molecule

The term "nucleic acid molecule" as used in the present invention refers to a polymeric form of nucleotides and includes both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term also includes single- and double-stranded forms of DNA. In addition, a polynucleotide may include either or both naturally-occurring and modified nucleotides linked together by naturally-occurring and/or non-naturally occurring nucleotide linkages. The nucleic acid molecules may be modified chemically or biochemically or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages

(e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages

(e.g., alpha anomeric nucleic acids, etc.) The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplexed, triplex, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The complementary strand is also useful, e . g. , for antisense therapy, hybridization probes and PCR primers.

Operably linked

The term "operably linked" refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being "under the control of" the promoter. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

Pharmaceutically acceptable excipient

By "pharmaceutically acceptable excipient" is meant any inert substance that is combined with an active molecule such as a drug, agent, or binding molecule for preparing an agreeable or convenient dosage form. The "pharmaceutically acceptable excipient" is an excipient that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation comprising the drug, agent, or binding molecule. Specifically Binding

The term "specifically binding", as used herein, in reference to the interaction of a binding molecule, e.g. an antibody, and its binding partner, e.g. an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g. an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules. The binding may be mediated by covalent or non-covalent interactions or a combination of both.

Substitutions

A "substitution", as used herein, denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

Therapeutically effective amount

The term "therapeutically effective amount" refers to an amount of the binding molecule as defined herein that is effective for preventing, ameliorating or treating a disorder or disease wherein CD72 molecules play a role.

Treatment

The term "treatment" refers to therapeutic treatment as well as prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder wherein CD72 molecules play a role or are associated with as well as those in which the disease or disorder is to be prevented. Vector

The term "vector" denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed. In other words, a vector is capable of transporting a second nucleic acid molecule to which it has been linked. Cloning as well as expression vectors are contemplated by the term "vector", as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC) and vectors derived from bacteriophages or plant or animal viruses. Vectors comprise an origin of replication recognised by the proposed host and in case of expression vectors, promoter and other regulatory regions recognised by the host. A vector containing a second nucleic acid molecule is introduced into a cell by transformation, transfection, or by making use of viral entry mechanisms. Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication) . Other vectors can be integrated into the genome of a host upon introduction into the host, and thereby are replicated along with the host genome.

SUMMARY OF THE INVENTION

In the present invention several internalising human binding molecules have been identified and obtained by using phage display technology. Furthermore, methods of producing these binding molecules and the use of the binding molecules in B cell associated disorders and diseases has been described. DETAILED DESCRIPTION OF THE INVENTION

As a first aspect, the invention provides internalising human binding molecules capable of binding, preferably capable of specifically binding, to CD72 or a fragment thereof, the fragment at least comprising the antigenic determinant of CD72. The binding molecules may bind to soluble CD72 or CD72 bound or attached to a carrier or substrate. Furthermore, the binding molecules may bind to purified CD72 or non-purified CD72. Preferably, the human binding molecules bind to CD72 associated with cells, such as a CD72 positive cells or portions thereof comprising CD72 or a fragment thereof. Upon binding to CD72 present on the surface of target cells, the binding molecules as defined herein internalise. Internalisation of binding molecules can be assayed by known techniques that include, but are not limited to, specifically tracing internalised CD72-binding molecules that are labelled with a fluorochrome using flow cytometry or confocal scanning laser microscopy. The internalising human binding molecules of the invention is not the antibody characterised by a heavy chain CDR3 region having the amino acid sequence DYYVTYDSWFDS

(SEQ ID No. 5) and the V_H and V_L gene utilization 1-46 (DP7) and Vλ3, respectively. The nomenclature of the V_H segment above is according to Matsuda & Honjo (1996) .

The internalising human binding molecules can be intact immunoglobulin molecules such as monoclonal antibodies, in particular human monoclonal antibodies, or the binding molecules can be antigen-binding fragments including, but not limited to, Fab, F(ab').- F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies

(scFv) , bivalent single-chain antibodies, diabodies, triabodies, tetrabodies, and (poly) peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly) eptides . The internalising human binding molecules of the invention can be used in non-isolated or isolated form. Furthermore, the internalising human binding molecules of the invention can be used alone, in a mixture comprising more than one internalising human binding molecule (or variant thereof) according to the invention or in a mixture comprising at least one internalising human binding molecule according to the invention and at least one other therapeutic agent.

Typically, internalising human binding molecules according to the invention can bind to their binding partners with an affinity constant (K-value) that is lower than 0.2*10^" ⁴ M, l.Ono^-5 M, 1.0*10^~6 M, 1.0*10^"7 M, preferably lower than 1.0*10^~8 M, more preferably lower than 1.0*10^~9 M, more preferably lower than 1.0*10^-1 M, even more preferably lower than 1.0*10^-11 M, and in particular lower than 1.0*10^~12 M.

In a preferred embodiment, the internalising human binding molecules according to the invention comprise a CDR3 region, preferably a heavy chain CDR3 region, comprising the amino acid sequence ARRDTNLFDY (SEQ ID No. 3) .

In yet another embodiment, the internalising human binding molecules according to the invention comprise a heavy chain comprising the amino acid sequence of SEQ ID No 38. In a further embodiment, the internalising human binding molecules according to the invention comprise a heavy chain comprising the amino acid sequence of SEQ ID No 38 and a light chain comprising the amino acid sequence of SEQ ID No 50. Plasmids comprising DNA encoding the heavy chain and light chain of human IgGl antibodies directed against human CD72 (the antibodies being called 002, 004, 024, and 025), said plasmids being called pgG102-002C01, pgG102-004C01, pgG102-024C01 and pgG102-025C01 were deposited at the European Collection of Cell Cultures (ECACC) , CAMR, Salisbury, Wiltshire SP4 OJG, Great Britain on 15 January 2003, under (provisional) accession numbers 03011601, 03011602, 03011603 and 03011604, respectively.

Another aspect of the invention includes functional variants of internalising human binding molecules as defined herein. Molecules are functional variants of a binding molecule, when the variants are capable of competing for binding to CD72, preferably competing for the same binding site on CD72, with the parent binding molecules. In other words, when the functional variants are still capable of binding to CD72 or a portion thereof. Furthermore, the variants have to be capable of internalising upon binding to CD72 present on a cell. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, but contain modifications, chemical and/or biochemical, that are not found in the parent binding molecule. Such modifications include inter alia acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Alternatively, functional variants can be binding molecules as defined in the present invention comprising an amino acid sequence containing substitutions, insertions, deletions or combinations thereof of one or more amino acids compared to the amino acid sequences of the parent binding molecules. Functional variants according to the invention may have the same or different binding affinities compared to the parent binding molecule but are still capable of internalising upon binding to CD72 present on e.g. a cell. For instance, functional variants according to the invention may have increased or decreased binding affinities for CD72 compared to the parent binding molecules. Preferably, the amino acid sequences of the variable regions, including, but not limited to, framework regions, hypervariable regions, in particular the CDR3 regions, are modified. Generally, the light chain and the heavy chain variable regions comprise three hypervariable regions, comprising three CDRs, and more conserved regions, the so-called framework regions (FRs) . The hypervariable regions comprise amino acid residues from CDRs and amino acid residues from hypervariable loops. Functional variants intended to fall within the scope of the present invention have at least 50%, preferably at least 60%, at least 70%, at least 75%, more preferably at least 80%, at least 85%, even more preferably at least 90%, at least 95%, and in particluar at least 97%, at least 98%, at least 99% amino acid sequence homology with the parent binding molecules as defined herein. Computer algorithms known to a person skilled in the art can be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants can be obtained by altering the parent binding molecules or parts thereof by general molecular biology methods known in the art including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis and site-directed mutagenesis.

In case the internalising human binding molecules as defined in the present invention are slowly internalising and, before internalisation, stay bound to the surface of target cells for a prolonged period of time, they may even be used in the format of naked binding molecules to support possible effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) . ADCC refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound binding molecules on a target cell and subsequently cause lysis of the target cell. CDC refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a binding molecule complexed with a cognate antigen. Alternatively, the naked binding molecules may inhibit or block the binding of another molecule such as a ligand normally binding to CD72, such as inter alia CD5 or CD100.

Furthermore, slowly internalising binding molecules may be useful in antibody-directed enzyme-prodrug therapy (ADEPT) . In this technique binding molecule-enzyme conjugates are administered and bind to the binding partner of the binding molecule. After clearance of the conjugates from the circulation, prodrugs are administered, which are converted into active drugs by the enzyme of the conjugates. Passive uptake of the active drugs into the target cells will then occur. In yet a further aspect, the invention includes immunoconjugates, i.e. molecules comprising at least one internalising human binding molecule as defined herein and further comprising at least one tag, such as a therapeutic moiety that inhibits or prevents the function of cells and/or causes destruction of cells. Also contemplated in the present invention are mixtures of immunoconjugates according to the invention or mixtures of at least one immunoconjugates according to the invention and another molecule, such as a therapeutic agent or another antibody. The internalising human binding molecules to be used in the immunoconjugates of the invention comprise a CDR3 region, preferably a heavy chain CDR3 region, comprising an amino acid sequence selected from the group consisting of ARRDTNLFDY (SEQ ID No. 3) and DYYVTYDSWFDS (SEQ ID No. 5) . Within this group the amino acid sequence ARRDTNLFDY (SEQ ID No. 3) is preferred. In another embodiment, the internalising human binding molecules of the immunoconjugate of the invention comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID No 38 and SEQ ID No 42, with the amino acid sequence SEQ ID No 38 being preferred. In yet another embodiment, the internalising human binding molecules of the immunoconjugate of the invention comprise a heavy chain comprising the amino acid sequence of SEQ ID No 38 and a light chain comprising the amino acid sequence of SEQ ID No 50 or they comprise a heavy chain comprising the amino acid sequence of SEQ ID No 42 and a light chain comprising the amino acid sequence of SEQ ID No 54. The combination of SEQ ID No 38 with SEQ ID No 50 is a preferred embodiment. Alternatively, the immunoconjugates can comprise more than one tag. The tags can be the same or distinct from each other and can be joined/conjugated non-covalently to the internalising human binding molecules. The tags can be joined/conjugated directly to the internalising human binding molecules through covalent bonding, including, but not limited to, disulfide bonding, hydrogen bonding, electrostatic bonding, recombinant fusion and conformational bonding. Alternatively, the tags can be joined/conjugated to the internalising human binding molecules by means of one or more linking compounds. Techniques for conjugating tags to binding molecules, are well known, see, e . g. , Arnon et al . , Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, p. 243-256 in Monoclonal Antibodies And Cancer Therapy (1985) , Edited by: Reisfeld et al . , A. R. Liss, Inc.; Hellstrom et al . , Antibodies For Drug Delivery, p. 623-653 in Controlled Drug Delivery, 2^nd edition (1987), Edited by: Robinson et al . , Marcel Dekker, Inc.; Thorpe, Antibody Carriers Of Cytotoxic Agents, p. 475-506 In Cancer Therapy: A Review, in Monoclonal Antibodies ' 84 : Biological And Clinical Applications (1985) , Edited by: Pinchera et al . ; Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, p. 303-316 in Monoclonal Antibodies For Cancer Detection And Therapy (1985), Edited by: Baldwin et al . , Academic Press. Tags according to the invention include, but are not limited to, toxic substances, radioactive substances, liposomes, enzymes, polynucleotide sequences, plasmids, proteins, peptides or combinations thereof. Toxic substances include, but are not limited to, cytotoxic agents, such as small molecule toxins or chemotherapeutic agents, or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. Examples of cytotoxic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonate such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines such as altreta ine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiehin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromoinycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; macrolide antibiotics such as geldanamicin and maytansin, anti-metabolites such as methotrexate and 5-fluorouracil; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as a inoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; platinum analogs such as cisplatin and carboplatin; triazenes; epipodophyllotoxins; platinum coordination complexes; maytansinoids; and taxoids, such as paclitaxel and doxetaxel . In general, suitable chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 18^th edition (1990), Edited by: A.R. Gennaro, Mack Publishing Co., Philadelphia and in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7^th edition (1985), Edited by: A.G. Gilman, L.S. Goodman, T.W. Rail and F. Murad. MacMillan Publishing Co., New York. Suitable chemotherapeutic agents that are still in the experimental phase are known to those of skill in the art and might also be used as toxic substances in the present invention. Often the conjugation of cytotoxic agents to binding molecules converts them to inactive prodrugs . Activation of the prodrugs involves release of the cytotoxic agents from the binding molecules . This occurs primarily inside the cells comprising the binding partners of the binding molecules following binding of the binding molecules to the binding partners and subsequent internalisation of the binding molecules.

Examples of enzymatically active toxins of bacterial, fungal, plant or animal origin include, but are not limited to, ricin A chain, modeccin A chain, abrin A chain, Pseuc.o-rio.nas exotoxin and endotoxin A chain, shiga toxin A, anthrax toxin lethal factor, diphteria A chain, nonbinding active fragments of diphtheria toxin, staphylococcal enterotoxin A, the human ribonuclease angiogenin, Aleuri tes fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S) , momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, saporin, alpha-sarcin, and fragments or derivatives thereof. Fusion proteins comprising enzymatically active toxins and binding molecules of the immunoconjugate of the invention can be produced by methods known in the art such as, e . g. , recombinantly by constructing nucleic acid molecules comprising nucleotide sequences encoding the binding molecules in frame with nucleotide sequences encoding the enzymatically active toxin and then expressing the nucleic acid molecules. Alternatively, fusion proteins can be produced chemically by conjugating, directly or indirectly via for instance a linker, binding molecule as defined herein to enzymatically active toxins .

Also contemplated within the present invention are binding molecules of the immunoconjugate of the invention that are labeled with radionuclides. Suitable radionuclides include, but are not limited to, radionuclides that emit alpha radiation such as inter alia ²¹²bismuth, ²¹³bismuth and ²¹¹astatine; radionuclides that emit beta radiation such as inter alia ¹³¹iodine, ⁹⁰yttrium, ¹⁸⁶rhodium and ¹⁸⁸rhodium; and radionuclides that emit gamma radiation such as inter alia ¹³¹iodine, ¹⁸⁶rhodium and ¹⁸⁸rhodium. Suitable radionuclides further include, but are not limited to, Auger-electron- emitting radionuclides such as in ter alia iodine, iodine, iodine, iodine, iodine, indium, bromine, and other radiolabeled halogens. The skilled man will appreciate that other suitable radionuclides can also be identified as suitable in the present invention. The choice of radionuclide will be dependent on many factors such as, e.g., the type of disease to be treated, the stage of the disease to be treated, the patient to be treated and the like. Binding molecules can be attached to radionuclides directly or indirectly via a chelating agent by methods well known in the art.

In another embodiment, the binding molecules of the immunoconjugate of the invention can be conjugated to liposomes to produce so-called immunoliposomes. A liposome may be conjugated to one or more binding molecules, the binding molecules being either the same or different. A variety of methods are available for preparing liposomes. These methods are well known in the art and include, but are not limited to, sonication, extrusion, high pressure/homogenization, microfluidisation, detergent dialysis, calcium-induced fusion of small liposome vesicles, and ether-infusion methods. The liposomes may be multilamellar vesicles, but preferably the liposomes are unilamellar vesicles such as small unilamellar (200 - 500 A) or large unilamellar vesicles (500 - 5000 A) . After preparation, the liposomes which have not been sized during formation may be sized by methods known in the art to achieve a desired size range and relatively narrow distribution of liposome sizes. The methods of loading drugs into liposomes are well known to those of skill in the art. The most common methods include the encapsulation technique and the transmembrane potential loading method. In the encapsulation technique, the drugs and liposome components are dissolved in an organic solvent or mixture of solvents in which all species are miscible, and then concentrated to a dry film. A buffer is then added to the dried film and liposomes are formed having the drugs incorporated into the vesicle walls. This method has been described in detail in U.S. Patent Numbers 4,885,172, 5,059,421, and 5,171,578, the contents of which are incorporated herein by reference. The transmembrane potential loading method has been described in detail in U.S. Patent Numbers 4,885,172, 5,059,421, 5,171,578, 5,316,771 and 5,380,531, the contents of which are also incorporated herein by reference. As will be understood, the loading techniques are not limited to these two general loading techniques. The drugs that can be loaded into liposomes include, but are not limited to, the toxic substances mentioned above. Liposomes having loaded different drugs and different liposomes, each liposome having loaded one kind of drug, may be alternative embodiments of liposomes that can be used and these embodiments are therefore also contemplated in the present invention. Internalising human binding molecules may be attached at the surface of the liposomes or to the terminus of polymers such as polyethylene glycol that are grafted at the surface of the liposomes using conventional chemical- , coupling techniques. An advantage of immunoliposomes is the ability to deliver several tens of thousands of drug molecules with a few tens of binding molecules per liposome resulting in high drug to binding molecule ratios. Following binding of the immunoliposomes to the target cells the drug can either, in case of binding molecules that are slowly internalised, be gradually released from the immunoliposomes and taken up by the cells as a free drug using standard uptake mechanisms or, in case of binding molecules that are rapidly internalised, the immunoliposomes themselves are taken up by the target cells by receptor-mediated endocytosis and the drugs are gradually released within the cells.

In yet another embodiment, the internalising human binding molecules of the invention may be linked to water- soluble, biodegradable polymers, such as for instance polymers of hydroxypropylmethacrylamine (HPMA) . The polymers have toxic substances linked on separate sites of the polymers with the use of appropriate degradable spacers to allow for release of the toxic substances. The above described polymers are also called immunopolymers .

Alternatively, the binding molecules as described in the present invention can be conjugated to tags and be used for detection and/or analytical and/or diagnostic purposes. The tags used to label the binding molecules for those purposes depend on the specific detection/analysis/diagnosis techniques and/or methods used such as in ter alia immunohistochemical staining of tissue samples, flow cytometric detection, scanning laser cytometric detection, fluorescent immunoassays, Western blotting applications, etc. For immunohistochemical staining of tissue samples preferred labels are enzymes that catalyze production and local deposition of a detectable product. Enzymes typically conjugated to binding molecules to permit their immunohistochemical visualization are well-known and include, but are not limited to, alkaline phosphatase, P- galactosidase, glucose oxidase, horseradish peroxidase, and urease . Typical substrates for production and deposition of visually detectable products include, but are not limited to, o-nitrophenyl-beta-D-galactopyranoside (ONPG) , o- phenylenediamine dihydrochloride (OPD) , p-nitrophenyl phosphate (PNPP) , p-nitrophenyl-beta-D-galactopryanoside (PNPG) , 3^?, 3 'diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC) , 4-chloro-l-naphthol (CN) , 5-bromo-4-chloro-3-indolyl- phosphate (BCIP) , ABTS, BluoGal, iodonitrotetrazolium (INT) , nitroblue tetrazolium chloride (NBT) , phenazine methosulfate (PMS) , phenolphthalein monophosphate (PMP) , tetramethyl benzidine (TMB) , tetranitroblue tetrazolium (TNBT) , X-Gal, X- Gluc, and X-glucoside. Other substrates that can be used to produce products for local deposition are luminescent substrates. For example, in the presence of hydrogen peroxide, horseradish peroxidase can catalyze the oxidation of cyclic diacylhydrazides such as luminol. Next to that, binding molecules of the immunoconjugate of the invention can also be labeled using colloidal gold or they can be labeled with radioisotopes, such as ³³p, ³²p, ³⁵S, ³H, and ¹²⁵I. When the binding molecules of the present invention are used for flow cytometric detections, scanning laser cytometric detections, or fluorescent immunoassays, they can usefully be labeled with fluorophores . A wide variety of fluorophores useful for fluorescently labeling the binding molecules of the present invention include, but are not limited to, Alexa Fluor and Alexa Fluor&commat dyes, BODIPY dyes, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein isothiocyanate (FITC) , allophycocyanin (APC) , R-phycoerythrin (PE) , peridinin chlorophyll protein (PerCP) , Texas Red, fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-Cy5, PE- Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7. When the binding molecules of the present invention are used for secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the binding molecules may be labeled with biotin.

Next to that, the binding molecules of the immunoconjugate of the invention may be conjugated to photoactive agents or dyes such as fluorescent and other chromogens or dyes to use these immunoconjugates in photoradiation, phototherapy, or photodynamic therapy. The photoactive agents or dyes include, but are not limited to, photofrin.RTM, synthetic diporphyrins and dichlorins, phthalocyanines with or without metal substituents, chloroaluminu phthalocyanine with or without varying substituents, O-substituted tetraphenyl porphyrins, 3,1-meso tetrakis (o-propionamido phenyl) porphyrin, verdins, purpurins, tin and zinc derivatives of octaethylpurpurin, etiopurpurin, hydroporphyrins, bacteriochlorins of the tetra (hydroxyphenyl) porphyrin series, chlorins, chlorin eβ, mono-1-aspartyl derivative of chlorin eβ, di-1-aspartyl derivative of chlorin e₆, tin (IV) chlorin e₆, meta- tetrahydroxyphenylchlor- in, benzoporphyrin derivatives, benzoporphyrin monoacid derivatives, tetracyanoethylene adducts of benzoporphyrin, dimethyl acetylenedicarboxylate adducts of benzoporphyrin, Diels-Adler adducts, onoacid ring "a" derivative of benzoporphyrin, sulfonated aluminum PC, sulfonated AlPc, disulfonated, tetrasulfonated derivative, sulfonated aluminum naphthalocyanines, naphthalocyanines with or without metal substituents and with or without varying substituents, anthracenediones, anthrapyrazoles, aminoanthraquinone, phenoxazine dyes, phenothiazine derivatives, chalcogenapyrylium dyes, cationic selena and tellurapyrylium derivatives, ring-substituted cationic PC, pheophorbide derivative, naturally occurring porphyrins, hematoporphyrin, ALA-induced protoporphyrin IX, endogenous metabolic precursors, 5-aminolevulinic acid benzonaphthoporphyrazines, cationic imminium salts, tetracyclines, lutetium texaphyrin, tin-etio-purpurin, porphycenes, benzophenothiazinium and combinations thereof.

When the immunoconjugates of the invention are used for in vivo diagnostic use, the binding molecules can also be made detectable by conjugation to e.g. magnetic resonance imaging (MRI) contrast agents, such as gadolinium diethylenetriaminepentaacetic acid, to ultrasound contrast agents or to X-ray contrast agents, or by radioisotopic labeling.

Furthermore, the binding molecules of the immunoconjugate of the invention can also be attached to solid supports, which are particularly useful for immunoassays or purification of the binding partner. Such solid supports might be porous or nonporous, planar or nonplanar and include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene supports. The binding molecules can also for example usefully be conjugated to filtration media, such as NHS-activated Sepharose or CNBr- activated Sepharose for purposes of immunoaffinity chromatography. They can also usefully be attached to paramagnetic microspheres, typically by biotin-streptavidin interaction. The microspheres can be used for isolation of cells that express or display CD72 or fragments thereof. As another example, the antibodies of the present invention can usefully be attached to the surface of a microtiter plate for ELISA.

Another aspect of the present invention concerns nucleic acid molecules as defined herein encoding internalising human binding molecules of the present invention. In yet another aspect, the invention provides nucleic acid molecules encoding at least the internalising human binding molecules, specifically binding to CD72, of the immunoconjugate of the invention. In a preferred embodiment, the nucleic acid molecules are isolated or purified.

The skilled man will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parent nucleic acid molecules. Preferably, the nucleic acid molecules encode internalising human binding molecules comprising a CDR3 region, preferably a heavy chain CDR3 region, comprising an amino acid sequence selected from the group consisting of ARRDTNLFDY (SEQ ID No. 3) and DYYVTYDSWFDS (SEQ ID No. 5), with the amino acid sequence ARRDTNLFDY (SEQ ID No. 3) being more preferred. Even more preferably, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID No 38 and SEQ ID No 42, with the amino acid sequence SEQ ID No 38 being preferred. In yet another embodiment, the nucleic acid molecules encode binding molecules comprising a heavy chain comprising the amino acid sequence of SEQ ID No 38 and a light chain comprising the amino acid sequence of SEQ ID No 50 or they encode a heavy chain comprising the amino acid sequence of SEQ ID No 42 and a light chain comprising the amino acid sequence of SEQ ID No 54. The combination of SEQ ID No 38 with SEQ ID No 50 is a preferred embodiment. Particularly preferred are nucleic acid molecules containing a nucleotide sequence selected from the group consisting of SEQ ID No 37 or SEQ ID No 41, with the nucleotide sequence of SEQ ID No 37 being preferred. In another preferred embodiment, the nucleic acid molecules contain the nucleotide sequences of SEQ ID No 37 and SEQ ID No 49 or the nucleotide sequences of SEQ ID No 41 and SEQ ID No 53, with the combination of SEQ ID No 37 and SEQ ID No 49 being preferred.

It is another aspect of the invention to provide vectors, i.e. nucleic acid constructs, comprising one or more nucleic acid molecules according to the present invention. Vectors can be derived from plasmids such as inter alia F, RI, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu, Pi, P22, Q_β, T-even, T-odd, T2, T4, T7, etc; plant viruses such as inter alia alfalfa mosaic virus, bromovirus, capillovirus, carlavirus, carmovirus, caulivirus, clostervirus, comovirus, cryptovirus, cucumovirus, dianthovirus, fabavirus, fijivirus, furovirus, geminivirus, hordeivirus, ilarvirus, luteovirus, machlovirus, marafivirus, necrovirus, nepovirus, phytorepvirus, plant rhabdovirus, potexvirus, potyvirus, sobemovirus, tenuivirus, tobamovirus, tobravirus, tomato spotted wilt virus, tombusvirus, tymovirus, etc; or animal viruses such as inter alia adenovirus, arenaviridae, baculoviridae, birnaviridae, bunyaviridae, calciviridae, cardioviruses, coronaviridae, corticoviridae, cystoviridae, Epstein-Barr virus, enteroviruses, filoviridae, flaviviridae, Foot-and-Mouth disease virus, hepadnaviridae, hepatitis viruses, herpesviridae, immunodeficiency viruses, influenza virus, inoviridae, iridoviridae, orthomyxoviridae, papovaviruses, paramyxoviridae, parvoviridae, picornaviridae, poliovirus, polydnaviridae, poxviridae, reoviridae, retroviruses, rhabdoviridae, rhinoviruses, Semliki Forest virus, tetraviridae, togaviridae, toroviridae, vaccinia virus, vescular stomatitis virus, etc. Vectors can be used for cloning and/or for expression of the binding molecules of the invention and might even be used for gene therapy purposes. Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression-regulating nucleic acid molecules are also covered by the present invention. The choice of vector is dependent on the recombinant procedures followed and the host used. Introduction of vectors in host cells can be effected by inter alia calcium phosphate transfection, virus infection, DEAE- dextran mediated transfection, lipofectamin transfection or electroporation. Vectors may be autonomously replicating or may replicate together with the chromosome into which they have been integrated. Preferably, the vectors contain one or more selection markers. Useful markers are dependent on the host cells of choice and are well known to persons skilled in the art. They include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from Herpes simplex virus (HSV-TK) , dihydrofolate reductase gene from mouse (dhfr) . Vectors comprising one or more nucleic acid molecules encoding the internalising human binding molecules as described above operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the internalising human binding molecules are also covered by the invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase . Hosts containing one or more copies of the vectors mentioned above are an additional subject of the present invention. Preferably, the hosts are host cells. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Bacterial cells include, but are not limited to, cells from Gram positive bacteria such as several species of the genera Bacillus, Streptomyces and Staphylococcus or cells of Gram negative bacteria such as several species of the genera Escheri chia and Pseudomonas . In the group of fungal cells preferably yeast cells are used. Expression in yeast can be achieved by using yeast strains such as Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha . Furthermore, insect cells such as cells from Drosophila and Sf9 can be used as host cells. Besides that, the host cells can be plant cells such as inter alia cells from crop plants such as forestry plants, or cells from plants providing food and raw materials such as cereal plants, or medicinal plants, or cells from ornamentals, or cells from flower bulb crops. Transformed (transgenic) plants or plant cells are produced by known methods, for example, Agrobacterium-mediated gene transfer, transformation of leaf discs, protoplast transformation by polyethylene glycol- induced DNA transfer, electroporation, sonication, microinjection or bolistic gene transfer. Additionally, a suitable expression system can be a baculovirus system. Expression systems using mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells or Bowes melanoma cells are preferred in the present invention. Mammalian cells provide expressed proteins with posttranslational modifications that are most similar to natural molecules of mammalian origin. Since the present invention deals with molecules that may have to be administered to humans, a completely human expression system would be particularly preferred. Therefore, even more preferably, the host cells are human cells, such as HeLa, 911, AT1080, A549, 293 or PER.C6™ (PER.C6 is a trademark owned by Crucell Holland B.V.) . In preferred embodiments, the producing human cells comprise at least a functional part of a nucleic acid sequence encoding an adenovirus El region in expressible format. In even more preferred embodiments, said host cells are derived from a human retina and immortalised with nucleic acids comprising adenoviral El sequences, such as PER.Cβ™ cells and derivatives thereof. Production of recombinant proteins in host cells can be performed according to methods well known in the art. The use of PER.C6™ cells as a production platform for proteins of interest has been described in WO 00/63403 the disclosure of which is incorporated herein by reference.

As a further aspect of the invention can be seen a method of internalising a tag into a cell expressing CD72 on its surface, the method comprising the step of contacting a conjugate of the tag and a human internalising binding molecule capable of binding to CD72 with one or more cells expressing CD72 on their surface under conditions that allow internalisation of the conjugate. Preferably, the tag and/or the human internalising binding molecule are/is a tag and/or human internalising binding molecule as defined herein. The cell expressing CD72 can be any cell expressing CD72, preferably human CD72, but is preferably a B cell, more preferably a disease associated B cell. The contacting of the conjugate and the cells expressing CD72 on their surface can be performed in vi tro and/or in vivo .

It is another aspect of the invention to provide a method of producing internalising human binding molecules, preferably internalising human binding molecules according to the present invention or internalising human binding molecules of the immunoconjugates of the invention. The method comprises the steps of a) culturing a host as described above under conditions conducive to the expression of the internalising human binding molecules, and b) optionally, recovering the expressed internalising human binding molecules. The expressed internalising human binding molecules can be recovered from the cell free extract, but preferably they are recovered from the culture medium. Methods to recover proteins, such as binding molecules, from cell free extracts or culture medium are well known to the man skilled in the art. Internalising human binding molecules as obtainable by the above described method are also a part of the present invention.

Alternatively, next to the expression in hosts, such as host cells, the internalising human binding molecules as defined herein can be produced synthetically by conventional peptide synthesizers or in cell-free translation systems using RNA' s derived from DNA molecules according to the invention. Internalising human binding molecule as obtainable by the above described synthetic production methods or cell-free translation systems are also a part of the present invention.

In yet another alternative embodiment, internalising human binding molecules according to the present invention, preferably binding molecules specifically binding to CD72 or fragments thereof, may be generated by transgenic non-human mammals, such as for instance transgenic mice or rabbits, that express human immunoglobulin genes. Preferably, the transgenic non-human mammals have a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of the internalising human binding molecules as described above. The transgenic non-human mammals can be immunized with a purified or enriched preparation of CD72 or fragment thereof and/or cells expressing CD72. Protocols for immunizing non-human mammals are well established in the art. See Using Antibodies: A Laboratory Manual, Edited by: E. Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York and Current Protocols in Immunology, Edited by: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober (2001), John Wiley & Sons Inc., New York, the disclosures of which are incorporated herein by reference. Immunization protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, but may also include naked DNA immunizations. In another embodiment, the internalising human binding molecules are produced by B cells or plasma cells derived from the transgenic animals. In yet another embodiment, the internalising human binding molecules are produced by hybridomas which are prepared by fusion of B cells obtained from the above described transgenic non-human mammals to immortalized cells. B cells, plasma cells and hybridomas as obtainable from the above described transgenic non-human mammals and internalising human binding molecules as obtainable from the above described transgenic non-human mammals, B cells, plasma cells and hybridomas are also a part of the present invention. In yet another embodiment, internalising human binding molecules of the present invention can also be produced in transgenic, non-human, mammals such as inter alia rabbits, goats or cows, or in for instance milk thereof .

In a further aspect, the invention provides a method of identifying binding molecules, preferably internalising human binding molecules such as internalising human monoclonal antibodies or fragments thereof, according to the invention or nucleic acid molecules according to the invention and comprises the steps of a) contacting a phage library of human binding molecules with material comprising CD72 or a part thereof, b) selecting at least once for a phage binding to the material comprising CD72 or a part thereof, and c) separating and recovering the phage binding to the material comprising CD72 or a part thereof. The selection step according to the present invention is preferably performed in the presence of at least part of CD72, e.g. cells transfected with CD72 expression plasmids, isolated CD72, the extracellular part thereof, fusion proteins comprising such, and the like. Phage display methods for identifying and obtaining binding molecules, e.g. antibodies, are by now well-established methods known by the person skilled in the art. They are e.g. described in US Patent Number 5,696,108; Burton and Barbas, 1994; and de Kruif et al . , 1995b. For the construction of phage display libraries, collections of human monoclonal antibody heavy and light chain variable region genes are expressed on the surface of bacteriophage, preferably filamentous bacteriophage, particles, in for example single chain Fv (scFv) or in Fab format (see de Kruif et al . , 1995b). Large libraries of antibody fragment-expressing phages typically contain more than 1.0*10⁹ antibody specificities and may be assembled from the immunoglobulin V regions expressed in the B lymphocytes of immunized- or non-immunized individuals. Alternatively, phage display libraries may be constructed from immunoglobulin variable regions that have been partially assembled in vi tro to introduce additional antibody diversity in the library (semi-synthetic libraries) . For example, in vi tro assembled variable regions contain stretches of synthetically produced, randomized or partially randomized DNA in those regions of the molecules that are important for antibody specificity, e.g. CDR regions. Antigen specific phage antibodies can be selected from the library by immobilising target antigens such as CD72 or fragements thereof on a solid phase and subsequently exposing the target antigens to a phage library to allow binding of phages expressing antibody fragments specific for the solid phase- bound antigen. Non-bound phages are removed by washing and bound phages eluted from the solid phase for infection of Escherichia coli (E . coli ) bacteria and subsequent propagation. Multiple rounds of selection and propagation are usually required to sufficiently enrich for phages binding specifically to the target antigen. Phages may also be selected for binding to complex antigens such as complex mixtures of proteins or whole cells such as cells transfected with CD72 expression plasmids or cells naturally expressing CD72. Selection of antibodies on whole cells has the advantage that target antigens are presented in their native configuration, i.e. unperturbed by possible conformational changes that might have been introduced in the case where an antigen is immobilized to a solid phase. Antigen specific phage antibodies can be selected from the library by incubating a cell population of interest, expressing known and unknown antigens on their surface, with the phage antibody library to let for example the scFv or Fab part of the phage bind to the antigens on the cell surface. After incubation and several washes to remove unbound and loosely attached phages, the cells of interest are stained with specific fluorescent labeled antibodies and separated on a Fluorescent Activated Cell Sorter (FACS) . Phages that have bound with their scFv or Fab part to these cells are eluted and used to infect Escheri chia coli to allow amplification of the new specificity. Generally, one or more selection rounds are required to separate the phages of interest from the large excess of non-binding phages. Monoclonal phage preparations can be analyzed for their specific staining patterns and allowing identification of the antigen being recognized (De Kruif et al . , 1995a; Lekkerkerker and Logtenberg, 1999). The phage display method can be extended and improved by subtracting non-relevant binders during screening by addition of an excess of non-target molecules that are similar but not identical to the target, and thereby strongly enhance the chance of finding relevant binding molecules (see US Patent Number 6,265,150 which is incorporated herewith by reference).

A method of obtaining a human binding molecule, preferably an internalising human binding molecule as described herein or a nucleic acid molecule according to the invention, wherein the method comprises the steps of a) performing the above described method of identifying binding molecules, preferably internalising human binding molecules such as internalising human monoclonal antibodies or fragments thereof according to the invention, or nucleic acid molecules according to the invention, and b) isolating from the recovered phage the human binding molecule and/or the nucleic acid encoding the human binding molecule, is another part of the present invention. Once a new monoclonal phage antibody has been established or identified with the above mentioned method of identifying binding molecules or nucleic acid molecules encoding the binding molecules, the DNA encoding the scFv or Fab can be isolated from the bacteria or phages and combined with standard molecular biological techniques to make constructs encoding bivalent scFv's or complete human immunoglobulins of a desired specificity (e.g. IgG or IgA). These constructs can be transfected into suitable cell lines and complete, human monoclonal antibodies can be produced (Huls et al . , 1999; Boel et al . , 2000).

In a further aspect, the invention provides compositions comprising one or more internalising human binding molecules as defined in the invention, one or more immunoconjugates according to the invention, or combinations thereof. Next to the binding molecules, the compositions may comprise in ter alia stabilising molecules, such as albumin or polyethylene glycol, or salts.

In yet a further aspect, the invention provides compositions comprising one or more nucleic acid molecules as defined in the present invention. The compositions may comprise aqueous solutions such as aqueous solutions containing salts (e.g., NaCl) , detergents ( e . g. , SDS) and/or other components .

Furthermore, the present invention pertains to pharmaceutical compositions comprising one or more internalising human binding molecules according to the invention, one or more immunoconjugates according to the invention, one or more nucleic acid molecules according to the invention, one or more compositions according to invention, or combinations thereof. The pharmaceutical compositions further comprise one or more pharmaceutically acceptable excipients. The internalising human binding molecules, immunoconjugates, nucleic acid molecules or compositions of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable excipient before or at the time of delivery. Alternatively, they can be in solution and the appropriate pharmaceutically acceptable excipient can be added and/or mixed before or at the time of delivery to provide a unit dosage injectable form. The choice of the optimal route of administration of the pharmaceutical compositions will be influenced by several factors including the physico-chemical properties of the active molecules within the compositions, the urgency of the clinical situation and the relationship of the plasma concentrations of the active molecules to the desired therapeutic effect. The routes of administration can be divided into two main categories, oral and parenteral administration. These two categories include, but are not limited to, inhalation, intra-arterial, intradermal, intramedullary, intramuscular, intranasal, intraocular, intraperitoneal, intraplaque, intrapulmonary, intrasynovial, intrathecal, intratumoral, intra-uterine, intravenous, intraventricular, rectal, subcutaneous, sublingual, topical, transdermal, and transmucosal administration. Oral dosage forms can be formulated inter alia as tablets, troches, lozenges, aqueous or oily suspensions, dispersable powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels, or slurries. These formulations can contain pharmaceutically excipients including, but not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as calcium stearate, glyceryl behenate, hydrogenated vegatable oils, magnesium stearate, mineral oil, polyethylene glycol, sodium stearyl, fumarate, stearic acid, talc, zinc stearate; preservatives such as n-propyl-p- hydroxybenzoate; colouring, flavouring or sweetening agents such as sucrose, saccharine, glycerol, propylene glycol or sorbitol; vegetable oils such as arachis oil, olive oil, sesame oil or coconut oil; mineral oils such as liquid parrafin; wetting agents such as benzalkonium chloride, docusate sodium, lecithin, poloxamer, sodium lauryl sulfate, sorbitan esters; and thickening agents such as agar, alginic acid, beeswax, carboxymethyl cellulose calcium, carageenan, dextrin or gelatin.

The pharmaceutical compositions of the present invention can also be formulated for parenteral administration. Formulations for parenteral administration can be inter alia in the form of aqueous or non-aqueous isotonic sterile non- toxic injection or infusion solutions or suspensions. Preferred parenteral administration routes include intravenous, intraperitoneal, epidural, intramuscular and intratumoral injection. The solutions or suspensions may comprise agents that are non-toxic to recipients at the dosages and concentrations employed such as 1, 3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils such as synthetic mono- or diglycerides or fatty acids such as oleic acid, local anaesthetic agents, preservatives, buffers, viscosity or solubility increasing agents, antioxidants such as ascorbic acid, chelating agents such as EDTA, and the like.

The binding molecules, preferably the internalising human binding molecules such as internalising human monoclonal antibodies according to the invention, the immunoconjugates according to the invention, the nucleic acid molecules according to the invention, the compositions according to the invention or the pharmaceutical compositions according to the invention can be used as medicaments. The above mentioned molecules or compositions may be employed in conjunction with other molecules useful in diagnosis and/or treatment. The internalising human binding molecules and/or immunoconjugates are typically formulated in the compositions and pharmaceutical compositions in a therapeutically effective amount. The molecules and compositions according to the present invention are preferably sterile. Methods to render these molecules and compositions sterile are well known in the art.

Internalising human binding molecules and/or immunoconjugates comprising internalising human binding molecules are particularly useful, and often preferred, when to be administered to human beings as in vivo diagnostic or therapeutic agents, since recipient immune response to the administered antibody will often be substantially less than that occasioned by administration of a monoclonal murine, chimeric_, or humanized binding molecule. Preferably, the molecules or compositions are used in the diagnosis, treatment, or combination thereof, of a B cell associated disorder and/or disease. The B cell associated disorders and/or diseases are selected from the group consisting of B cell associated cancer, autoimmune disorders and graft vs. host incompatibility disorders associated with transplantation. Preferred disorders and/or diseases that can be diagnosed and/or treated are B lineage Acute Lymphoblastic Leukemia, B cell Chronic Lymphocytic Leukemia, non-Hodgkin Lymphoma, Mantle Cell Lymphoma, Rheumatoid Arthritis, Systemic Lupus Erythematosus, Acute and Chronic Graft versus Host Disease and transplant rejection.

Alternatively, cells that are genetically engineered to express the binding molecules of the invention are administered to patients in vivo . Such cells may be obtained from an animal or patient or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are genetically engineered in vi tro using recombinant DNA techniques to introduce the nucleic acid molecules of the invention into the cells. Preferably, the binding molecules are secreted from the cells. The engineered cells which express and preferably secrete the internalising human binding molecules as described herein can be introduced into the patient for example systemically, e.g., in the circulation, or intraperitoneally. In other embodiments, the cells can be incorporated into a matrix or can be encapsulated and implanted in the body.

In another aspect, the invention concerns the use of binding molecules, preferably internalising human binding molecules such as internalising human monoclonal antibodies as described above, immunoconjugates according to the invention, nucleic acid molecules according to the invention, compositions or pharmaceutical compositions according to the invention in the preparation of a medicament for the diagnosis, treatment, or combination thereof, of a B cell associated disorder and/or disease as mentioned above.

Next to that, kits comprising one or more binding molecules, preferably internalising human binding molecules such as internalising human monoclonal antibodies according to the invention, one or more immunoconjugates according to the invention, one or more nucleic acid molecules according to the invention, one or more compositions according to the invention, one or more pharmaceutical compositions according to the invention, one or more vectors according to the invention, one or more hosts according to the invention or a combination thereof are also a part of the present invention. Optionally, the above described components of the kits of the invention are packed in suitable containers and labeled for diagnosis and/or treatment of the indicated conditions. The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts . Associated with the kits can be instructions customarily included in commercial packages of therapeutic products, that contain information about for example the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic products.

EXAMPLES

To illustrate the invention, the following examples are provided. These examples are not intended to limit the scope of the invention. Example 1

Selection of phages carrying single chain Fv fragments specifically recognizing the human CD72 receptor using human tonsillar mononuclear cells

Antibody fragments were selected using antibody phage display libraries and Abstract™ technology, essentially as described in US Patent Number 6,265,150 and in WO 98/15833. All procedures were performed at room temperature unless stated otherwise. In this example phage selection experiments were performed using tonsillar B cells with the aim of identifying phage antibodies recognizing different subsets of B lymphocytes including IgD^~/CD38^~ ^memory' B cells (see van der Vuurst de Vries and Logtenberg (1999) ) . An aliquot of phage library (500 μl, approximately 10¹³ cfu) was blocked with 2 ml RPMI/10%FCS/1%NHS for 15' at room temperature. Inflamed tonsils were obtained from patients undergoing routine tonsillectomy . Tonsils were minced and the mononuclear cell (MNC) fraction was isolated by density centrifugation. Tonsil MNC (~10*10⁶ cells) were added to the blocked phage library and incubated for 2.5 hr while slowly rotating at 4°C. Subsequently, the cells were washed twice and were resuspended in 500μl RPMI/10%FCS and incubated with a FITC-conjugated anti-IgD antibody (Southern Biotechnology Associates) and a phyco-erythrin-conjugated anti-CD38 antibody (Becton Dickinson) for 15 min. on ice. The cells were washed once and transferred to a 4 ml tube. Cell sorting was performed on a FACStar fluorescence-activated cell sorter (Becton Dickinson) , ~10⁵ cells of the IgD^~/CD38^" B cell population were sorted. The sorted cells were spun down, the supernatant was saved and the bound phages were eluted from the cells by resuspending the cells in 500μl 50mM glycin pH2.2 followed by incubation for 5 min. at room temperature. The mixture was neutralized with 250μl 1M Tris-HCl pH 7.4 and added to the rescued supernatant.

The eluted phages were added to 500 μl 1M Tris-HCl pH 7.4. To this mixture, 3.5 ml of exponentially growing XL-1 blue bacterial culture was added. The tubes were incubated for 30 min at 37°C without shaking. Then, the bacteria were plated on 2 trypton yeastextract (TY) agar plates containing ampicillin, tetracycline and glucose. After overnight incubation of the plates at 37°C, the colonies were scraped from the plates and used to prepare an enriched phage library, essentially as described by De Kruif et al. (1995a) . Briefly, scraped bacteria were used to inoculate 2TY medium containing ampicillin, tetracycline and glucose and grown at a temperature of 37 °C to an OU6oonm of -0.3. Helper phages were added and allowed to infect the bacteria after which the medium was changed to 2TY containing ampicillin, tetracycline and kanamycin. Incubation was continued overnight at 30 °C. The next day, the bacteria were removed from the 2TY medium by centrifugation after which the phages were precipitated using polyethylene glycol (PEG) 6000/NaCl. Finally, the phages were dissolved in a small volume of PBS-1% BSA, filter-sterilized and used for a next round of selection. The selection/reinfection procedure was performed twice. After the second round of selection, individual E. coli colonies were used to prepare monoclonal phage antibodies. Essentially, individual colonies were grown to log-phase in 96 well plate format and infected with helper phages after which phage antibody production was allowed to proceed overnight. PEG/NaCl- precipitated and filter-sterilized phage antibodies were tested using flow cytometry (FacsCalibur, Becton Dickinson) for binding to human B lymphocytes. From this selection a large panel of phage antibodies was obtained that demonstrated either broad binding to B cells or binding to particular subsets of B lymphocytes . The phage antibodies that demonstrated broad reactivity with B lymphocytes were analyzed for binding to CD72 using a cell line that was transfected with a CD72 encoding cDNA. For this purpose L929 cells had been transfected with a plasmid carrying a cDNA sequence encoding human CD72 and stable transfectants were selected using standard techniques known to a person skilled in the art

(Current Protocols in Immunology, Edited by: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober

(2001), John Wiley & Sons Inc., New York). For flow cytometry analysis, phage antibodies were first blocked in an equal volume of PBS, 4% milkprotein (MPBS) for 15 min. at 4°C prior to the staining of the CD72 transfected L929 cells or the untransfected L929 control cell line. The binding of the phage antibodies to the cells was visualized using a biotinylated anti-M13 antibody (Santa Cruz Biotechnology) followed by streptavidin-phyco-erythrin (Caltag) . As shown in Figure 1A, the selected phage antibodies SC02024 and SC02025 (see table 1) bound to all peripheral blood CD19⁺ B lymphocytes and they selectively stained the L929-human CD72 transfectant (see Figure IB, black peaks) while they did not bind the L929 control transfectant (see Figure IB, white peaks) .

Example 2

Selection of phage carrying single chain Fv fragments specifically recognizing human CD72 using peripheral blood mononuclear cells (PBMNC)

Phage selection experiments were performed as described supra, now using peripheral blood B lymphocytes as target. An aliquot of the phage library (500 μl, approximately 10¹³ cfu) were blocked with 2 ml RPMI/10%FCS/1%NHS for 15 min. at room temperature. PBMNC (~10*10⁶ cells) were added to the blocked phage library and incubated for 2.5 hr while slowly rotating at 4°C. Subsequently, the cells were washed twice and were resuspended in 500μl RPMI/10%FCS and incubated with a phyco- erythrin-conjugated anti-CD19 antibody (Pharmingen) for 15 min. on ice. The cells were washed once and transferred to a 4 ml tube. Cell sorting was performed as supra and ~30.000 CD19+ B cells were sorted. The sorted cells were spun down, the supernatant was saved and the bound phages were eluted from the cells by resuspending the cells in 500μl 50mM glycin pH2.2 followed by incubation for 5 min. at room temperature. The mixture was neutralized with 250μl 1M Tris-HCl pH 7.4 and added to the rescued supernatant. Collectively these phages were used to prepare an enriched phage library as described earlier. The selection/re-infection procedure was performed twice. After the second round of selection, monoclonal phage antibodies were prepared and tested for binding to peripheral blood CD19⁺ B cells. Selected phage antibodies that met this criterium were subsequently tested for binding to CD72- transfected L929 cells. The results in Figure 2A show that the selected phages sc02-002, sc02-003, sc02-004 and sc02-057 (see table 1) selectively bind to peripheral blood CD19⁺ B cells and figure 2B shows that they bind specifically to the human CD72 L929 transfectant (see Figure 2B, black peaks) .

Example 3

Characterization of the human CD72-speclfic scFv's From the selected CD72-specific scFv clones plasmid DNA was obtained and nucleotide sequences were determined according to standard techniques . Nucleotide sequences of the scFv's sc02-002, sc02-003, sc02-004, sc02-024, sc02-025, and sc02-057 are listed in table 1 and have the SEQ ID Nos 7, 9, 11, 13, 15 and 17, respectively. Amino acid translations of the nucleotide sequences of the scFv' s sc02-002, sc02-003, sc02-004, sc02-024, sc02-025, and sc02-057 are also listed in table 1 and have the SEQ ID Nos 8, 10, 12, 14, 16 and 18, respectively. The V_H and V_L gene identity and heavy chain CDR3 regions (see SEQ ID Nos 1 - 6) of the above mentioned anti- human CD72 scFv's are also depicted in table 1.

Example 4

Generation of scFv variants

ScFv sc02-003 expressed within its V_H CDR3 region an N- linked glycosylation sequence (SHSNMSFDY (SEQ ID No 2)) that results in glycosylation of the CDR3 loop upon expression in eukaryotic cells and hence will abrogate the binding to the CD72 receptor. To avoid this phenomenon a novel small phage library was constructed by PCR based site directed mutagenesis using the original sc02-003 encoding plasmid as a template (Figure 3) . To this purpose a PCR reaction was performed with an upstream M13rev sense primer and an anti-sense primer encompassing the Xhol site in which the codons encoding the asparagine (N) and the serine (S) have been randomized. The resulting PCR fragment was digested with Ncol and Xhol and was used to replace the original Ncol - Xhol fragment in the parental sc02-003-encoding vector. The ligation mixture was used to transform XLl-Blue cells and the resulting colonies were scraped from the plates and used to prepare a phage sublibrary.

This sublibrary was used in a stringent selection protocol using CD72-transfected L929 cells with the aim of generating variant scFv's that lacked the glycosylation sequence. To this purpose aliquots of the sublibrary was blocked as supra and were incubated with 2xl0⁶ CD72-transfected L929 cells in duplicate for 1.5 hours at room temperature. The CD72- transfected cells with attached phages were either directly washed ten times with PBS, 0.1% Tween-20 prior to phage elution as described supra or after a subsequent incubation period at 37°C for 30 min. The rescued phages were used to reinfect XLl-Blue cells as described supra. Subsequently, single colonies were used to prepare monoclonal phage antibodies that were tested for binding to peripheral blood lymphocytes and CD72-transfected L929 cells. ScFv that bound to peripheral B lymphocytes and the L929-CD72 transfectant were sequenced and characterized as supra. In addition to the parental SC02-003 sequence, two variant scFv's lacking the N- linked glycosylation site were obtained: SC02-041 (see table 1; SHSNMAFDY (SEQ ID No 19)) and SC02-132 (see table 1; (CVK) SHSNMAFDY (SEQ ID No 20)). SC02-132 contained an additional subtitution A—>V just outside the heavy chain CDR3 region. The amino acid sequence of the heavy chain CDR3 regions are listed in table 1 as SEQ ID Nos 19 and 20, respectively. The nucleotide sequences of the scFv's SC02-041 and SC02-132 are listed in table 1 as SEQ ID Nos. 21 and 23, respectively and the amino acid translations of the nucleotide sequences of the scFv's SC02-041 and SC02-132 are listed in table 1 as SEQ ID Nos. 22 and 24, respectively. The V_H and V_L gene identity and heavy chain CDR3 compositions of these two anti-human CD72 scFv's are also depicted in table 1. Example 5

Construction of fully human immunoglobulin molecules from the selected anti -human CD72 single chain Fv fragments

To use the selected antibody fragments that recognize human CD72 for therapeutic applications in humans it is desirable to generate human immunoglobulin molecules. The engineering and production of the human IgGl monoclonal antibodies is essentially performed as described in detail by Boel et al . (2000) . In detail, scFv were recloned in IgG expression vector C01 (pCRU-KOl) . To that purpose, V_H and V_L regions were PCR amplified using designated primers to append restriction sites and restore complete human frameworks. The PCR fragments were cloned in pTOPO (Invitrogen) , the integrity of the per fragments was verified by sequencing and thereafter the inserts were sequentially cloned (EcoRI - J3atz.HI for V_H and Xhol - Notl for V ) into the IgG expression vector C01.

ScFv 5'VH oligo3'VH oligo5'V oligo3'V oligo

02-002 5H-B 3H-B 5K-B 3K-B

02-004 5H-B 3H-B 5K-C 3K-C

02-024 5H-A 3H-B 5K-C 3K-C

02-025 5H-A 3H-B 5L-A 3L-B

02-041 5H-C 3H-B 5K-C 3K-C

02-132 5H-C 3H-B 5K-C 3K-C

primer sequences:

5H-A: acctgtcttgaattctccatggcccaggtgcagctggtgcagtctgg (SEQ ID

No. 25)

5H-B : acctgtcttgaattctccatggccgaggtgcagctggtggagtctg (SEQ ID

No. 26) ^r5H-C : acctgtcttgaattctccatggcccaggtgcagctggtqgagtctgg (SEQ ID

No. 27)

3H-B : gctcgcggatccactcacctgaggagacggtcaccagggtgccctggcccc (SEQ

ID No. 28)

5K-B : acctgtctcgagttttccatggctgacatccagatgacgcagtc (SEQ ID No.

29)

5K-C : acctgtctcgagttttccatggctgacatccagatgacccagtctccatcctccc

(SEQ ID No. 30)

5L-A:acctgtctcgagttttccatggcttcctccgagctgactcaggaccctgctg (SEQ

ID No. 31)

3K-B : ttttccttagcggccgcaaagtgcacttacgtttgatttccactttggtgccctg

(SEQ ID No. 32)

3K-C : ttttccttagcggccgcaaagtgcacttacgtttgatctccaccttggtcccttg

(SEQ ID No. 33)

3L-B : ttttccttagcggccgcgactcacctaggacggtcagcttggtc (SEQ ID No.

34)

The resulting expression constructs pgG102-002C01, pgG102- 004C01, pgG102-024C01, pgG102-025C01, pgG102-041C01 and pgG102-132C01 encoding the human IgGl antibodies directed against human CD72 were transiently expressed in PER.C6™ cells and supernatants containing IgGl antibodies were obtained. The nucleotide sequences of the heavy chains of the antibodies called 002, 004, 024, 025, 041 and 132 are shown in SEQ ID Nos 35, 37, 39, 41, 43 and 45, respectively. The amino acid sequences of the heavy chains of the antibodies called 002, 004, 024, 025, 041 and 132 are shown in SEQ ID Nos 36, 38, 40, 42, 44 and 46, respectively. The nucleotide sequences of the light chains of the antibodies called 002, 004, 024, 025, 041 and 132 are shown in SEQ ID Nos 47, 49, 51, 53, 55 and 57, respectively. The amino acid sequences of the light chains of the antibodies called 002, 004, 024, 025, 041 and 132 are shown in SEQ ID Nos 48, 50, 52, 54, 56 and 58, respectively. Subsequently, the antibodies were purified over size-exclusion columns and protein-A columns using standard purification methods used generally for immunoglobulins (see for instance WO 00/63403) .

The anti-CD72 IgGl antibodies were validated for their ability to bind to Ramos Burkitt's lymphoma cells. To this purpose 2.10⁵ Ramos cells were stained with IgGl antibodies at concentrations ranging from 0 to 100 μg/ml at 4°C. Binding of the antibodies called 002, 004, 024 and 025 was visualized using biotinylated goat-anti-human IgG (Fc specific, Caltag) followed by streptavidin-phyco-erythrin (Caltag) . The stained cells were analyzed by flow cytometry. All antibodies bound to Ramos cells (figure 4), and they specifically recognized the CD72 molecule on CD72-transfected L929 cells while they did not bind the untransfected L929 cell line (data not shown) .

Example 6 Immunohistochemistry

The anti-CD72 IgG's 004 and 025 are analysed for their ability to bind to normal tissues by immunohistochemistry. To this purpose, frozen sections of the following normal tissues: adrenal gland; bladder; brain (cerebellum and cerebrum) ; blood vessels (aorta and coronary artery) ; fallopian tube; oesophagus; stomach (antrum and body) ; duodenum; ileum; colon; heart; kidney; liver; lung; lymphnode; ovary; pancreas; parathyroid; peripheral nerve; pituitary gland; placenta; prostate; salivary gland; skin; spinal cord; spleen; striated muscle; testis; tonsil; thyroid; ureter and uterus (cervix and endometrium) are cut, mounted on glass slides and are dried at room temperature. The sections are blocked for endogenous peroxidase with 50 mM sodiumazide containing 0.03% H₂0₂ for 20 min., followed by blocking for endogenous biotin according to the provided protocol (X0590, Dako) . Subsequently, the sections are blocked with PBS containing 4% BSA and 10% normal human serum prior to incubation with the biotinylated anti- human CD72 IgG' s for 60 min. at room temperature. To detect bound IgG molecules the sections are incubated with streptavidin coupled-Horse Radish Peroxidase (Dako) followed by incubation with diaminobenzidine (Sigma) resulting in a local deposition of brown crystals. The sections are counterstained with hematoxilin to visualize nucleated cells within the sections. Prior to analysis the sections are dehydrated and the slides are sealed with eukitt (BDH) .

Example 7

Affinity measurement human CD72 immunoglobulin molecules

For affinity measurement purposes the CD72 molecule is prepared from the human Burkitt's lymphoma cell line Ramos by affinity purification using the mouse anti-human CD72 antibody J4-117. Alternatively, a recombinant soluble CD72 molecule is produced in human 293T cells. The gene encoding the human CD72 molecule can be found under Genebank accession number NM_001782. References directed to the human CD72 molecule are Von Hoegen et al . (1990), Van de Velde et al . (1991) and Von Hoegen et al . (1991) . To the purpose of producing a recombinant soluble human CD72 molecule a cDNA construct is generated that is comprised of the human HAVT20 leader sequence that is linked in frame to a nucleotide sequence encoding a strech of 6 histidines that is linked in frame to a nucleotide sequence encoding the extracellular domain of the human CD72 molecule. This construct is cloned by methods known to a skilled person in the art into a vector e.g. pCDNA3.1

(Invitrogen) that is suitable for expression in eukaryotic cells. Subsequently, this construct is transfected into 293T cells and the HIS-tagged human CD72 that is secreted into the supernatant is purified via standard metal chelating methodology known to a skilled person in the art.

The affinities of the human anti-CD72 IgG' s called 002, 004, 024 and 025 are determined using Biacore 2000. For K_Qf_f- ranking, the purified human IgGl antibodies are applied directly to CM5 sensor chips coupled with 10.000 resonance units (RU) of purified human CD72 using l-ethyl-3 (3- dimethylaminopropyl) -carbodiimide (EDC) - H-hydroxysuccinimide

(NHS) coupling chemistry. Alternatively, NTA sensor chips are coupled with the purified HIS-tagged soluble human CD72. Antibody affinity data are based on measuring several different preparations of antibody at 7 concentrations on sensor chips coated with different concentrations (500-4000 RU) of human CD72.

Example 8

Internalisation of CD72 receptors upon binding of human anti -

CD72 immunoglobulin molecules

Internalization assays with Ramos Burkitt's lymphoma cells were performed in order to determine the internalising capacity of the anti-human CD72 IgGl antibodies called 002, 004, 024 and 025. The anti-human CD72 IgGl antibodies called 002, 004, 024 and 025 were stained with Oregon Green-480-SE (Molecular Probes) labeling dye as follows. 0.1 mg of dye was dissolved in 10 microliters of DMSO, added to 0.4 mg of antibody in a final volume of 0.4 ml PBS, and incubated at room temperature for 1 h. Subsequently, this mixture was loaded onto a Sephadex G25 column equilibrated in PBS. The labeled antibody was eluted with PBS and the coloured fraction was collected. Aliquots of 5 x 10⁵ Ramos cells were loaded with the appropriate antibody at a saturating concentration on ice for 30 min. Unbound antibody was removed by three washes with ice-cold RPMI 1640, 10% FBS medium. Subsequently, cells were resuspended in 50 μl of medium and incubated for 1 h at either 4 °C (no internalisation) or 37 °C to allow internalisation of the antibodies. Following three washes with ice-cold PBS, cell surface-bound antibodies were stripped off the cells with 2.5 mg/ml subtilisin for 1 h at 4 °C. Cells were washed again with ice-cold PBS-1% BSA and samples were analyzed by flow cytometry. Figure 5 shows that the two human anti-CD72 antibodies called 004 and 025 internalize. When cells were incubated at 4 °C (a temperature at which no internalization occurs) the cell-bound IgGs could be stripped off the cells (top panel) as shown by loss of fluorescence (open histograms: no treatment; filled histograms: subtilisin treated). On the other hand, when cells were incubated at 37 °C to allow internalization of antibodies (lower panel) , the cells remained fluorescent after eliminating antibodies bound to CD72 molecules at the cell surface by stripping the cells using subtilisin. This indicates that the anti-CD72 IgG' s 004 and 025 did internalize into cells and have become resistant to protease treatment. In contrast, the negative control antibody, anti-CD20 (which does not internalise, see Ghetie et al . (2001)), could be stripped of the cells at both temperatures indicating that the antibody binds to its target, but does not internalise into the cell (data not shown) . The anti-CD72 IgG' s 002 and 024 did not internalise and have lost their binding at 37°C. In conclusion, of the panel of four anti-CD72 antibodies, the 004 and 025 antibodies do internalise upon binding to the CD72 antigen. We further conclude that weak binders are less likely to have the capacity to internalise.

Example 9

Prepara tion of immunoconj uga tes

In this example the conjugation of anti-CD72 antibodies or fragments thereof to toxins or to liposomes which encapsulate toxins is described. The internalising anti-CD72 antibodies 004 and 025 are conjugated, essentially as described in WO 98/19705, EP 0 665 020, EP 0 624 377, EP 0 328 147, EP 0 317 957, EP 0 439 095 or US 5,314,995 which are all incorporated by reference herein, to a toxin like auristatin E or deglycosylated ricin A through a protease-sensitive linker or they are chemically linked to a fixed amount of doxorubicin molecules .

Additionally, the internalising anti-CD72 antibodies 004 and 025 or fragments thereof are conjugated, essentially as described in US 6,027,726 which is incorporated by reference herein, to liposomes. Briefly, liposomes are formulated from a mixture of cholesterol and one or more phospholipids such as for instance phosphatidylcholine/sphingomyelin according to methods known to a person skilled into the art. The lipid composition of the liposomes is among others dependent on the drug to be encapsulated. It is well within the reach of a person skilled in the art to select the appropriate lipid composition of the liposomes. Subsequently, a cytotoxic drug (i.e. doxorubicin or other members of the anthracycline family, or vincristine or other members of the alkaloid family) are loaded into the liposomes via a pH gradient-driven process. Immunoliposomes are prepared by coupling suitable antibody fragments of the anti-CD72 antibodies 004 and/or 025, such as for instance Fab' fragments, to a sulfhydryl-reactive lipid that is inserted into the liposome. The generation of antibody fragments of the anti-CD72 antibodies, such as for instance Fab' fragments, is performed using standard methodology known to a person skilled in the art. Alternatively, whole antibodies can be coupled to liposomes containing reactive lipids after thiolation of amino groups. The coupling efficiency is quantified using fluorimetric or radioactive assays known in the art. These methods enable studies to determine the effect of antibody fragment density on immunoliposomal targeting and binding and are useful in determining the optimal conditions for the coupling reaction. Both the anti-CD72 immunoconjugates are analyzed in vitro for their ability to internalise upon binding to CD72-expressing cell lines. Furthermore, the anti-CD72 immunoconjugates and the anti-CD72 immunoliposomes are tested for their cytotoxic potential towards CD72-expressing cell lines.

Example 10

Anti -tumor efficacy of anti-CD72 immunocoj ugates in animal models of human B cell lymphoma

The anti-tumor efficacy of the different anti-CD72 immunoconjugates and/or immunoliposomes are analyzed in animal models of human B cell lymphoma. Xenograft experiments using SCID mice and the human Burkitt's lymphoma cell lines Namalwa and/or Daudi are performed as described by Liu et ai . (1996) and Newton et al . (2001). In short, 5 x 10⁵ Namalwa or Daudi cells are injected intravenously in groups of 5-10 female CB- 17 SCID mice which are 6 to 8 weeks of age. At day 1, 3 and 5 the anti-CD72 immunoconjugates or immunoliposomes are administered in vi tro diluted in phosphate buffered saline to a maximal total dose that equals approximately 50% of the maximal tolerated dose (the maximal dose that can be administered to tumor-bearing mice without resulting in drug- related death) . The endpoints of the studies are animal survival (duration in days) . Animals are either monitored daily for the development of hind limb paralysis, which occurs approximately 3 to 4 days before death, and are sacrificed as soon as hind limb paralysis develops or animals are checked daily and animals that show deteriorating and moribund condition are euthanised with for instance C0₂.

Table 1

REFERENCES

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Claims

1. An internalising human binding molecule capable of specifically binding to CD72, with the proviso that the internalising human binding molecule is not the antibody characterised by a heavy chain CDR3 region having the amino acid sequence DYYVTYDSWFDS (SEQ ID No. 5) and the V_H and V_L gene utilization 1-46 (DP7) and Vλ3, respectively.

2. An internalising human binding molecule according to claim 1, wherein the internalising human binding molecule comprises at least a CDR3 region comprising the amino acid sequence ARRDTNLFDY (SEQ ID No. 3) .

3. An internalising human binding molecule according to claim 1 or 2, wherein the internalising human binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 38.

4. A functional variant of an internalising human binding molecule according to any one of the claims 1 - 3, wherein the functional variant is capable of competing for specifically binding to CD72 with the internalising human binding molecule and wherein the functional variant is capable of internalising.

5. An immunoconjugate comprising an internalising human binding molecule specifically binding to CD72 and further comprising at least one tag.

6. An immunoconjugate according to claim 5, wherein the tag is selected from the group consisting of a toxic substance, a radioactive substance, a liposome, an enzyme and combinations thereof .

7. A nucleic acid molecule encoding an internalising human binding molecule or functional variant thereof according to any one of the claims 1 - 4.

8. A nucleic acid molecule encoding at least the internalising human binding molecule specifically binding to CD72 of an immunoconjugate according to claim 5 or 6.

9. A vector comprising at least one nucleic acid molecule according to claim 7 or 8.

10. A host comprising at least one vector according to claim 9.

11. A host according to claim 10, wherein the host is a cell derived from a human cell .

12. A method of internalising a tag into a cell expressing CD72 on its surface, the method comprising the step of contacting a conjugate of the tag and a human internalising binding molecule capable of binding to CD72 with the cell expressing CD72 on its surface under conditions that allow internalisation of the conjugate.

13. A method of producing an internalising human binding molecule according to any one of the claims 1 - 4 or the internalising human binding molecule specifically binding to CD72 of the immunoconjugate according to claim 5 or 6, wherein the method comprises the steps of: a) Culturing a host according to claim 10 or 11 under conditions conducive to the expression of the internalising human binding molecule, and b) Optionally recovering the expressed internalising human binding molecule .

14. An internalising human binding molecule as obtainable by the method according to claim 13.

15. A method of identifying a human binding molecule specifically binding to CD72 or a nucleic acid molecule encoding a human binding molecule specifically binding to CD72, wherein the method comprises the steps of: a) contacting a phage library of human binding molecules with material comprising CD72 or a part thereof, b) Selecting at least once for a phage binding to the material comprising CD72 or a part thereof, and c) Separating and recovering the phage binding to the material comprising CD72 or a part thereof.

16. A method of obtaining a human binding molecule specifically binding to CD72 or a nucleic acid molecule encoding a human binding molecule specifically binding to CD72, wherein the method comprises the steps of: a) Performing the method according to claim 15, and b) Isolating from the recovered phage the human binding molecule and/or the nucleic acid molecule encoding the human binding molecule.

17. A composition comprising an internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, or an immunoconjugate according to claim 5 or 6.

18. A composition comprising a nucleic acid molecule according to claim 7 or 8.

19. A pharmaceutical composition comprising an internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8 or a composition according to claim 17 or 18, said pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

20. An internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8, a composition according to claim 17 or 18 or a pharmaceutical composition according to claim 19 for use as a medicament.

21. An internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8, a composition according to claim 17 or 18 or a pharmaceutical composition according to claim 19, for use in the diagnosis, treatment, or combination thereof, of a B cell associated disorder or disease.

22. An internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8, a composition according to claim 17 or 18 or a pharmaceutical composition according to claim 19, for use in the diagnosis, treatment, or combination thereof, of a B cell associated cancer or a B cell-associated auto immune disorder.

23. Use of an internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8, a composition according to claim 17 or 18 or a pharmaceutical composition according to claim 19 in the preparation of a medicament for the diagnosis, treatment, or combination thereof, of a B cell associated disorder or disease .

24. Use according to claim 23, wherein the B cell associated disorder or disease is selected from the group consisting of B cell associated cancer and B cell associated auto immune disorder.

25. A kit comprising an internalising human binding molecule according to any one of the claims 1 - 4, an internalising human binding molecule according to claim 14, an immunoconjugate according to claim 5 or 6, a nucleic acid molecule according to claim 7 or 8, a composition according to claim 17 or 18, a pharmaceutical composition according to claim 19, a vector according to claim 9, a host according to claim 10 or 11 or a combination thereof.