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WO2004067569A1 - Molecules de liaison humaines d'internalisation dirigees contre cd72 - Google Patents

Molecules de liaison humaines d'internalisation dirigees contre cd72 Download PDF

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Publication number
WO2004067569A1
WO2004067569A1 PCT/EP2003/050004 EP0350004W WO2004067569A1 WO 2004067569 A1 WO2004067569 A1 WO 2004067569A1 EP 0350004 W EP0350004 W EP 0350004W WO 2004067569 A1 WO2004067569 A1 WO 2004067569A1
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Prior art keywords
internalising
human
binding molecule
human binding
nucleic acid
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PCT/EP2003/050004
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English (en)
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WO2004067569A8 (fr
Inventor
Alexander Berthold Hendrik Bakker
Willem Egbert Marissen
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Crucell Holland B.V.
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Priority to PCT/EP2003/050004 priority Critical patent/WO2004067569A1/fr
Priority to AU2003209753A priority patent/AU2003209753A1/en
Publication of WO2004067569A1 publication Critical patent/WO2004067569A1/fr
Publication of WO2004067569A8 publication Critical patent/WO2004067569A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • 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.
  • 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) .
  • CD72 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 .
  • CD72 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.
  • PAP Pokeweed Antiviral Protein
  • 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)).
  • HAMA human antimouse antibody
  • 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
  • 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) .
  • Figure IB the white peaks relate to L929 control transfectants, while the black peaks relate to L929 human-CD72 transfectants.
  • 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) .
  • the x-axis represents mean fluorescence intensity (MFI)
  • the y-axis represents antibody concentration in micrograms per ml .
  • the x-axis represents fluorescence intensity
  • the y-axis represents cell number
  • FIG. 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) .
  • FIG. 1 Map of pgG102-004C01 heavy chain.
  • FIG. 21 Map of pgG102-024C01 heavy chain.
  • FIG. 23 Map of pgG102-025C01 heavy chain.
  • FIG. 24 Map of pgG102-025C01 light chain.
  • FIG. 25 Map of pgG102-04lC01.
  • FIG. 26 Map of pgG102-04lC01 heavy chain.
  • FIG. 27 Map of pgG102-132C01.
  • FIG. 28 Map of pgG102-132C01 heavy chain.
  • amino acid sequence refers to naturally occuring or synthetic molecules and to a peptide, oligopeptide, polypeptide or protein sequence.
  • 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.
  • binding molecule as used herein also includes the immunoglobulin classes and subclasses known in the art.
  • 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.
  • 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.
  • CDR Complementary determining regions
  • complementary determining regions 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 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 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.
  • 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.
  • 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) .
  • polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cystine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, me
  • 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 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 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.
  • 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) .
  • immunosorbome 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 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.
  • binding molecule means a binding molecule as defined herein that is capable of being internalised within the target cells to which it binds.
  • 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 .
  • substantially free 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.
  • the binding molecules when they 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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 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.
  • a polynucleotide may include either or both naturally-occurring and modified nucleotides linked together by naturally-occurring and/or non-naturally occurring nucleotide linkages.
  • 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
  • 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
  • nucleic acid sequence encompasses its complement unless otherwise specified.
  • 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 refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other.
  • 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.
  • two nucleic acid sequences when 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 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.
  • 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.
  • binding 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.
  • 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.
  • substitution denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • terapéuticaally 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 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 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.
  • 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.
  • 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.
  • the binding molecules may bind to purified CD72 or non-purified CD72.
  • 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.
  • the binding molecules as defined herein internalise.
  • 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
  • V H segment (SEQ ID No. 5) and the V H and V L gene utilization 1-46 (DP7) and V ⁇ 3, respectively.
  • DP7 V H and V L gene utilization 1-46
  • V ⁇ 3 V ⁇ 3
  • 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
  • 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.
  • 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.
  • K-value affinity constant
  • 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) .
  • 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.
  • ECACC European Collection of Cell Cultures
  • 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.
  • 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.
  • functional variants according to the invention may have increased or decreased binding affinities for CD72 compared to the parent binding molecules.
  • the amino acid sequences of the variable regions including, but not limited to, framework regions, hypervariable regions, in particular the CDR3 regions, are modified.
  • 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.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • 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.
  • FcRs Fc receptors
  • 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.
  • 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.
  • binding molecules may be useful in antibody-directed enzyme-prodrug therapy (ADEPT) .
  • ADPT antibody-directed enzyme-prodrug therapy
  • binding molecule-enzyme conjugates are administered and bind to the binding partner of the binding molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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 .
  • radionuclides include, but are not limited to, radionuclides that emit alpha radiation such as inter alia 212 bismuth, 213 bismuth and 211 astatine; radionuclides that emit beta radiation such as inter alia 131 iodine, 90 yttrium, 186 rhodium and 188 rhodium; and radionuclides that emit gamma radiation such as inter alia 131 iodine, 186 rhodium and 188 rhodium.
  • 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.
  • Auger-electron- emitting radionuclides such as in ter alia iodine, iodine, iodine, iodine, iodine, indium, bromine, and other radiolabeled halogens.
  • 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 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • the internalising human binding molecules of the invention may be linked to water- soluble, biodegradable polymers, such as for instance polymers of hydroxypropylmethacrylamine (HPMA) .
  • HPMA hydroxypropylmethacrylamine
  • 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 .
  • 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.
  • 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 ?
  • DAB 3-amino-9-ethylcarbazole
  • AEC 3-amino-9-ethylcarbazole
  • CN 4-chloro-l-naphthol
  • BCIP 5-bromo-4-chloro-3-indolyl- phosphate
  • INT iodonitrotetrazolium
  • NBT nitroblue tetrazolium chloride
  • PMS phenazine methosulfate
  • PMP phenolphthalein monophosphate
  • TMB tetramethyl benzidine
  • TBT tetranitroblue tetrazolium
  • X-Gal X- Gluc
  • X-glucoside X-glucoside
  • luminescent substrates For example, in the presence of hydrogen peroxide, horseradish peroxidase can catalyze the oxidation of cyclic diacylhydrazides such as luminol.
  • binding molecules of the immunoconjugate of the invention can also be labeled using colloidal gold or they can be labeled with radioisotopes, such as 33 p, 32 p, 35 S, 3 H, and 125 I.
  • radioisotopes such as 33 p, 32 p, 35 S, 3 H, and 125 I.
  • 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.
  • 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,
  • 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.
  • MRI magnetic resonance imaging
  • 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.
  • 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.
  • the antibodies of the present invention can usefully be attached to the surface of a microtiter plate for ELISA.
  • nucleic acid molecules as defined herein encoding internalising human binding molecules of the present invention.
  • the invention provides nucleic acid molecules encoding at least the internalising human binding molecules, specifically binding to CD72, of the immunoconjugate of the invention.
  • the nucleic acid molecules are isolated or purified.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ , 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
  • plasmids such as inter alia F, RI, RP1, Col, pBR322, TOL, Ti, etc
  • cosmids
  • 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.
  • 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.
  • 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.
  • 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 .
  • Gram positive bacteria such as several species of the genera Bacillus, Streptomyces and Staphylococcus
  • Gram negative bacteria such as several species of the genera Escheri chia and Pseudomonas .
  • yeast cells are used in the group of fungal cells. Expression in yeast can be achieved by using yeast strains such as Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha .
  • insect cells such as cells from Drosophila and Sf9 can be used as host cells.
  • 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.
  • 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.C6TM (PER.C6 is a trademark owned by Crucell Holland B.V.) .
  • the producing human cells comprise at least a functional part of a nucleic acid sequence encoding an adenovirus El region in expressible format.
  • said host cells are derived from a human retina and immortalised with nucleic acids comprising adenoviral El sequences, such as PER.C ⁇ TM cells and derivatives thereof.
  • adenoviral El sequences such as PER.C ⁇ TM 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.C6TM 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.
  • a method of internalising a tag into a cell expressing CD72 on its surface 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.
  • 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 .
  • 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.
  • 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.
  • internalising human binding molecules according to the present invention may be generated by transgenic non-human mammals, such as for instance transgenic mice or rabbits, that express human immunoglobulin genes.
  • 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.
  • 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.
  • the internalising human binding molecules are produced by B cells or plasma cells derived from the transgenic animals.
  • 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.
  • 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 .
  • 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.
  • Phage display methods for identifying and obtaining binding molecules 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.
  • 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 .
  • 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) .
  • 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.
  • 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 .
  • 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).
  • 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).
  • 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.
  • the compositions may comprise in ter alia stabilising molecules, such as albumin or polyethylene glycol, or salts.
  • 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 .
  • 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.
  • 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 benzalkon
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells can be incorporated into a matrix or can be encapsulated and implanted in the body.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 1 To illustrate the invention, the following examples are provided. These examples are not intended to limit the scope of the invention. Example 1
  • Antibody fragments were selected using antibody phage display libraries and AbstractTM 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.
  • 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 ⁇ l, approximately 10 13 cfu) was blocked with 2 ml RPMI/10%FCS/1%NHS for 15' at room temperature.
  • Tonsils were minced and the mononuclear cell (MNC) fraction was isolated by density centrifugation. Tonsil MNC ( ⁇ 10*10 6 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 500 ⁇ l 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.
  • MNC mononuclear cell
  • 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) , ⁇ 10 5 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 500 ⁇ l 50mM glycin pH2.2 followed by incubation for 5 min. at room temperature.
  • the mixture was neutralized with 250 ⁇ l 1M Tris-HCl pH 7.4 and added to the rescued supernatant.
  • the eluted phages were added to 500 ⁇ l 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) .
  • 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 bacteria were removed from the 2TY medium by centrifugation after which the phages were precipitated using polyethylene glycol (PEG) 6000/NaCl.
  • PEG polyethylene glycol
  • 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.
  • 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
  • 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) .
  • MPBS milkprotein
  • 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) .
  • PBMNC peripheral blood mononuclear cells
  • Phage selection experiments were performed as described supra, now using peripheral blood B lymphocytes as target.
  • An aliquot of the phage library (500 ⁇ l, approximately 10 13 cfu) were blocked with 2 ml RPMI/10%FCS/1%NHS for 15 min. at room temperature.
  • PBMNC ⁇ 10*10 6 cells
  • PBMNC ⁇ 10*10 6 cells
  • the cells were washed twice and were resuspended in 500 ⁇ l 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 500 ⁇ l 50mM glycin pH2.2 followed by incubation for 5 min. at room temperature. The mixture was neutralized with 250 ⁇ l 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.
  • 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.
  • 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.
  • SHSNMSFDY SEQ ID No 2
  • 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) .
  • 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.
  • aliquots of the sublibrary was blocked as supra and were incubated with 2xl0 6 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.
  • SC02-041 see table 1; SHSNMAFDY (SEQ ID No 19)
  • 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.
  • 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.
  • 5K-B acctgtctcgagttttccatggctgacatccagatgacgcagtc (SEQ ID No.
  • 3K-B ttttccttagcggccgcaaagtgcacttacgtttgatttccactttggtgccctg
  • 3K-C ttttccttagcggccgcaaagtgcacttacgtttgatctccaccttggtcccttg
  • 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.C6TM 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.
  • 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.
  • 2.10 5 Ramos cells were stained with IgGl antibodies at concentrations ranging from 0 to 100 ⁇ g/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) .
  • the anti-CD72 IgG's 004 and 025 are analysed for their ability to bind to normal tissues by immunohistochemistry.
  • 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% H 2 0 2 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) .
  • BDH eukitt
  • 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.
  • 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) .
  • 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
  • the affinities of the human anti-CD72 IgG' s called 002, 004, 024 and 025 are determined using Biacore 2000.
  • 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
  • NTA NTA
  • 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.
  • the labeled antibody was eluted with PBS and the coloured fraction was collected. Aliquots of 5 x 10 5 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 ⁇ l 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.
  • FIG. 5 shows that the two human anti-CD72 antibodies called 004 and 025 internalize.
  • 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).
  • the 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.
  • the anti-CD72 IgG' s 004 and 025 did internalize into cells and have become resistant to protease treatment.
  • the negative control antibody, anti-CD20 which does not internalise, see Ghetie et al . (2001)
  • anti-CD20 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.
  • 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.
  • anti-CD72 antibodies or fragments thereof conjugated to toxins or to liposomes which encapsulate toxins.
  • 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 .
  • 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.
  • a cytotoxic drug i.e.
  • 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.
  • suitable antibody fragments of the anti-CD72 antibodies 004 and/or 025 such as for instance Fab' fragments
  • 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.
  • 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.
  • 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).
  • 5 x 10 5 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.
  • 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 C0 2 .
  • Van Kroonenburgh MJ and Pauwels EK (1988) Human immunological response to mouse monoclonal antibodies in the treatment or diagnosis of malignant diseases. Nucl. Med. Commun. 9:919-930. Van der Vuurst de Vries A-R and Logtenberg T (1999) , Dissecting the human peripheral B-cell compartment with phage display-derived antibodies. Immunol. 98:55-62.
  • the B-cell surface protein CD72/Lyb-2 is the ligand for CD5. Nature 351: 662-665.

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Abstract

La présente invention concerne des molécules de liaison humaines d'internalisation qui se lient spécifiquement à CD72, des molécules d'acides nucléiques codant ces molécules de liaison humaines d'internalisation, des compositions comportant ces molécules de liaison humaines d'internalisation et des procédés d'identification ou de production de ces molécules de liaison humaines d'internalisation. Dans un mode de réalisation préféré, l'invention se rapporte à des immunoconjugués comportant une molécule de liaison humaine d'internalisation qui se lie spécifiquement à CD72. Les molécules de liaison humaines d'internalisation et des immunoconjugués comprenant une molécule de liaison humaine d'internalisation peuvent être utilisés pour le diagnostic et le traitement de troubles associés aux lymphocytes B.
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US7763244B2 (en) 2002-07-01 2010-07-27 Human Genome Sciences, Inc. Antibodies that specifically bind to Reg IV
WO2010132532A1 (fr) * 2009-05-15 2010-11-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps réagissant à la surface des lymphocytes b
CN107072858A (zh) * 2014-09-19 2017-08-18 佩尔莫比尔公司 用于电动轮椅的底盘装置以及包含该底盘装置的电动轮椅
WO2022036287A1 (fr) * 2020-08-14 2022-02-17 Cero Therapeutics, Inc. Récepteurs chimériques anti-cd72 et utilisations de ceux-ci
US11708423B2 (en) 2017-09-26 2023-07-25 Cero Therapeutics, Inc. Chimeric engulfment receptor molecules and methods of use
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7763244B2 (en) 2002-07-01 2010-07-27 Human Genome Sciences, Inc. Antibodies that specifically bind to Reg IV
US7741443B2 (en) * 2003-06-25 2010-06-22 Crucell Holland B.V. Binding molecules for the treatment of myeloid cell malignancies
US8268966B2 (en) 2003-06-25 2012-09-18 Crucell Holland B.V. Binding molecules for the treatment of myeloid cell malignancies
US8632985B2 (en) 2003-06-25 2014-01-21 Crucell Holland, B.V. Binding molecules for the treatment of myeloid cell malignancies
WO2010132532A1 (fr) * 2009-05-15 2010-11-18 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anticorps réagissant à la surface des lymphocytes b
US20120121504A1 (en) * 2009-05-15 2012-05-17 The United States of America, as represented by the Secretary, Dept. of Health and Human Svcs. B cell surface reactive antibodies
US8877199B2 (en) 2009-05-15 2014-11-04 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services B cell surface reactive antibodies
CN107072858A (zh) * 2014-09-19 2017-08-18 佩尔莫比尔公司 用于电动轮椅的底盘装置以及包含该底盘装置的电动轮椅
US11708423B2 (en) 2017-09-26 2023-07-25 Cero Therapeutics, Inc. Chimeric engulfment receptor molecules and methods of use
US12303551B2 (en) 2018-03-28 2025-05-20 Cero Therapeutics Holdings, Inc. Cellular immunotherapy compositions and uses thereof
WO2022036287A1 (fr) * 2020-08-14 2022-02-17 Cero Therapeutics, Inc. Récepteurs chimériques anti-cd72 et utilisations de ceux-ci

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