US20100189722A1 - Anti cd37 antibodies - Google Patents

Anti cd37 antibodies Download PDF

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US20100189722A1
US20100189722A1 US12/672,378 US67237808A US2010189722A1 US 20100189722 A1 US20100189722 A1 US 20100189722A1 US 67237808 A US67237808 A US 67237808A US 2010189722 A1 US2010189722 A1 US 2010189722A1
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Karl-Heinz Heider
Eric Borges
Elinborg Ostermann
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Boehringer Ingelheim International GmbH
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to immunotherapies that are based on B cell depletion.
  • the present invention relates to anti-CD37 antibody molecules for use in such therapies, e.g. in the treatment of B cell malignancies and autoimmune conditions.
  • mAbs monoclonal antibodies
  • mAbs monoclonal antibodies
  • the role of monoclonal antibodies in therapies that are based on B cell depletion, e.g. in the treatment of B cell malignancies, has expanded since the introduction of rituximab (Rituxan®), an antibody that is directed against the CD20 antigen on the B cell surface.
  • Rituxan® rituximab
  • the CD37 antigen is a cell surface antigen that has, to date, not been considered as a target for B cell malignancies to the same extent as the B cell antigen CD20.
  • CD37 a member of the tetraspanin superfamily, is a heavily glycosylated cell surface molecule with four transmembrane domains and two extracellular loops. CD37 is almost exclusively expressed on mature B cells, with highest expression levels on peripheral blood B cells, reduced levels on plasma cells and non-detectable levels on CD10+ precursor B cells in the bone marrow. Low level expression of CD37 has also been reported on resting and activated T cells, granulocytes, and monocytes. In B cell neoplasm, CD37 expression is mainly observed in aggressive non-Hodgkin's lymphoma (NHL) and chronic lymphoid leukemia (CLL). High level of CD37 expression is also found on mantle cell lymphoma (MCL). This expression pattern makes CD37 an attractive target for antibody-mediated cancer therapy.
  • NHL non-Hodgkin's lymphoma
  • CLL chronic lymphoid leukemia
  • CD37 was first described in 1986 and characterized by the murine monoclonal antibody MB-1 (Link et al., 1986).
  • CD37 The physiological role of CD37 is unknown. Mice deficient for CD37 display no changes in development and cellular composition of lymphoid organs, but have reduced levels of IgG1 and attenuated T cell mediated immune reactions (Knobeloch et al., 2000). Studies with CD37 ⁇ / ⁇ T cells suggest a role for CD37 in T cell proliferation (van Spriel et al., 2004).
  • CD37 expression on malignant B cells of various diseases has been reported.
  • CD37 is expressed in the majority of mature B cell malignancies like Burkitt lymphoma, follicular lymphoma and lymphocytic lymphoma (Link et al., 1986).
  • High levels of CD37 expression have been observed in hairy cell leukemia and in samples of patients with chronic lymphocytic leukemia (CLL) and different subtypes of non-Hodgkin's lymphoma (NHL) including mantle cell lymphoma (MCL) (Schwartz-Albiez et al., 1988; Barrena et al., 2005).
  • CLL chronic lymphocytic leukemia
  • MCL mantle cell lymphoma
  • MCL mantle cell lymphoma
  • Binding of a CD37-specific mAb to cancer cells may trigger various mechanisms of action: First, after the antibody binds to the extracellular domain of the CD37 antigen, it may activate the complement cascade and lyse the targeted cell.
  • an anti-CD37 antibody may mediate antibody-dependent cell-mediated cytotoxicity (ADCC) to the target cell, which occurs after the Fc portion of the bound antibody is recognized by appropriate receptors on cytotoxic cells of the immune system.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody may alter the ability of B cells to respond to antigen or other stimuli.
  • anti-CD37 antibody may initiate programmed cell death (apoptosis).
  • Anti-CD37 mAb MB-1 was evaluated in two radio-immunotherapy trials in B-NHL patients (B-cell non-Hodgkin's lymphoma; Press et al., 1989; Kaminski et al., 1992).
  • Therapeutic doses of 131 I-MB-1 were administered to 6 relapsed NHL patients in one trial and all 6 patients achieved a clinical complete remission (CR) with a median duration of 7 months.
  • CR clinical complete remission
  • two of the six patients showed clinical regressions already after administration of only the tracer dose of MB-1 suggesting a direct anti-tumor effect of the antibody itself.
  • CD37-targeting is a promising approach for targeted anti-tumor therapy by induction of apoptosis and ADCC.
  • CD37 antigen is frequently expressed on tumor cells in several human B cell malignancies and on mature normal B lymphocytes and that anti-CD37-based therapy may be a promising approach for treating B cell malignancies.
  • the depletion of CD37-positive normal B cells is not considered critical since clinical data from numerous patients show that even prolonged depletion of B cells up to 6 months with an anti-CD20 mAb does not significantly reduce IgG serum levels or increases the risk of infections (Van der Kolk et al., 2002).
  • anti-CD37 antibodies or antibody-like molecules described above have shown anti-tumor efficacy in B-cell malignancies and the potential to target CD37, there is a need for alternate anti-CD37 inhibitors to improve therapies based on B-cell depletion.
  • anti-CD37 antibodies with improved effector functions.
  • the inventors sought to provide anti-CD37 mABs with antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • a murine monoclonal anti-CD37 antibody was used as a starting antibody for generating chimeric and humanized anti-CD37 antibodies that are useful in human therapy.
  • the present invention provides an antibody molecule that binds to human CD37 and that is derived from
  • an antibody that is “derived” from another antibody i.e. the starting antibody, means that said antibody has been generated by modification of the starting antibody as described below.
  • the antibody molecule is a chimeric or humanized antibody molecule derived from the starting antibody defined in a).
  • An antibody with a related sequence was designated G28.1 and described in WO 2005/017148.
  • the starting antibody of category b) may, for example, be selected from the CD37-specific antibodies that characterized, like G28.1, the CD37 antigen in the Third HLDA Workshop; these antibodies were designated HD28, HH1, BI14, F97-3G6 (Ling and MacLennan, 1987).
  • Other CD37-specific antibodies that have been described include RFB-7, Y29/55, MB-1, M-B371, M-B372 and IPO-24. According to Moldenhauer, 2000, and Schwartz-Albiez et al., 1988, all these antibodies (including G28.1) recognize the same or an overlapping or close CD37 epitope.
  • HH1 SantaCruz
  • RFB-7 Biodesign
  • Y29/55 Biogenesis
  • M-B371 BD Biosciences
  • M-B372 SantaCruz
  • IPO-24 AbCam
  • CD37-specific antibodies are S-B3 (Biosys), NMN46 (Chemicon), and ICO-66 (Bioprobe). Whether an antibody recognizes the same epitope as G28.1 can be determined by competitive binding assays or by cross inhibition radioimmunoassays as described by Moldenhauer et al., 1987, and Moldenhauer, 2000.
  • competitive binding may be determined in an ELISA, using plates coated with CD37 protein or CD37 peptides or with CD37 positive cells (Cell ELISA) and measuring binding of biotinylated antibody in the presence of a competitor candidate antibody.
  • Cell ELISA Cell ELISA
  • the binding of biotinylated G28.1 (or another antibody known to recognize the same epitope) is reduced in the case that the antibodies recognize a shared epitope.
  • fragments or short polypeptides or recombinant proteins derived from the CD37 sequence can be synthesized or produced and the binding of G28.1 to said peptides/polypeptides measured in an ELISA assay.
  • Competitive binding can also be determined by FACS analysis, as described in the Examples.
  • An antibody defined in b) may be used in an analogous manner as G28.1 as a starting antibody for the generation of chimeric or humanized antibody molecules.
  • a starting antibody of category b) may also be generated de novo by using peptides or protein fragments containing the relevant epitope, or DNA molecules encoding such peptides/fragments, respectively for immunization to obtain antibodies reactive with the same epitope as G28.1.
  • a starting antibody b) may also be obtained by immunization with whole cells carrying the relevant epitope; the thus obtained hybridoma cells are then screened for competitive binding of the secreted antibodies.
  • anti-CD37 antibody molecule encompasses anti-CD37 antibodies and anti-CD37 antibody fragments as well as conjugates with antibody molecules.
  • Antibodies include, in the meaning of the present invention, chimeric monoclonal and humanized monoclonal antibodies.
  • antibody which may interchangeably be used with “antibody molecule”, shall encompass complete immunoglobulins (as they are produced by lymphocytes and for example present in blood sera), monoclonal antibodies secreted by hybridoma cell lines, polypeptides produced by recombinant expression in host cells, which have the binding specificity of immunoglobulins or monoclonal antibodies, and molecules which have been derived from such antibodies by modification or further processing while retaining their binding specificity.
  • the anti-CD37 antibody molecule is a chimeric antibody defined by
  • variable regions of the non-human antibody are typically linked to at least a portion (F c ) of the immunoglobulin constant region of a human immunoglobulin.
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, preferably from immortalized B cells (see Kabat et al., 1991; and WO 87/02671).
  • the antibody molecules may contain all or only a portion of the constant region as long as they exhibit specific binding to the CD37 antigen and the Fc receptors. The choice of the type and length of the constant region depends on whether effector functions like complement fixation or antibody-dependent cell-mediated toxicity are desired, and on the desired pharmacological properties of the antibody molecule.
  • the antibody molecule of the invention is a chimeric CD37-specific antibody that has the heavy chain variable region of a non-human antibody defined in a) or b) fused to the human heavy chain constant region IgG1 and the light chain variable region of a non-human antibody defined in a) or b) fused to the human light chain constant region kappa.
  • the antibody molecule is a chimeric CD37-specific antibody that has the heavy chain variable region shown in SEQ ID NO:2 fused to the human heavy chain constant region IgG1 which is an IgG1 molecule with the sequence shown in SEQ ID NO:24 (coding DNA sequence: SEQ ID NO: 23) or a mutated IgG1 molecule derived therefrom and that has the light chain variable region shown in SEQ ID NO:4 fused to the human light chain constant region kappa shown in SEQ ID NO:26 (coding DNA sequence: SEQ ID NO: 25).
  • constant regions for chimerizing a non-human starting antibody defined in a) or b) are available to the person skilled in the art, e.g. IgG2, IgG3, IgG4, IgA, IgE or IgM (instead of IgG1) or lambda (instead of kappa).
  • the constant regions may also be chimeric, for example, a heavy chain IgG1/IgG2 or IgG1/IgG3 chimera.
  • the anti-CD37 antibody molecule is a humanized antibody that is defined by
  • Non-human antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding molecules with subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (from the recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementarity determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the sequences encoding CDRs of a non-human starting antibody defined in a) or b) have been grafted into the respective genes of human immunoglobulin heavy and light chains.
  • CDRs Complementarity determining regions of a monoclonal antibody are understood to be those amino acid sequences involved in specific antigen binding according to Kabat et al., 1991, in connection with Chothia and Lesk (1987). From the sequences of the variable regions as shown in SEQ ID NO:2 and SEQ ID NO:4, the CDR sequence can be routinely determined by searching the Kabat sequence database for sequence features.
  • Appropriate framework residues of the CDR-grafted antibody may be reverted to murine residues to improve binding affinity.
  • the expert knows how to obtain the CDRs from a given non-human antibody, to choose and obtain appropriate human immunoglobulin genes, to graft the CDRs into these genes, to modify selected framework residues, to express the CDR-grafted antibody in appropriate host cells, e.g. Chinese hamster ovary (CHO) cells, and to test the resulting recombinant antibodies for binding affinity and specificity.
  • appropriate host cells e.g. Chinese hamster ovary (CHO) cells
  • the antigen binding sites which are formed by the CDRs of the heavy chain and CDRs of the light chain, are excised from the DNA of cells secreting the rodent (murine) monoclonal antibody and grafted into the DNA coding for the framework of the human antibody.
  • non-human, in particular murine, anti-CD37 antibodies can be humanized by the so-called “resurfacing” technology, whereby the rodent frameworks are left unchanged with the exception of surface-exposed residues, as described in U.S. Pat. No. 5,639,641.
  • the invention relates to humanized antibodies having a variable heavy chain with a sequence shown in SEQ ID NO:6 and a variable light chain with a sequence selected from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
  • the invention relates to humanized antibodies having a variable heavy chain with a sequence shown in SEQ ID NO:8 and a variable light chain with a sequence selected from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
  • the invention relates to humanized antibodies having a variable heavy chain with a sequence shown in SEQ ID NO:10 and a variable light chain with a sequence selected from the sequences shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:22.
  • the humanized antibody has a human heavy chain constant region IgG1 and a human light chain constant region kappa.
  • the constant regions may be selected from other classes and subclasses.
  • the human constant heavy chain IgG1 is an IgG1 molecule with the sequence shown in SEQ ID NO:24 or a mutated IgG1 molecule derived therefrom and the human light chain constant region kappa has the sequence shown in SEQ ID NO:26.
  • Anti-CD37 antibody molecules of the invention may also be variants of the antibodies that are defined by the amino acid sequences shown in the sequence listing. Using routinely available technologies, the person skilled in the art will be able to prepare, test and utilize functional variants of the above-defined antibodies. Examples are variant antibodies with at least one position in a CDR and/or framework altered, variant antibodies with single amino acid substitutions in the framework region where there is a deviation from the germline sequence, antibodies with conservative amino substitutions, antibodies that are encoded by DNA molecules that hybridize, under stringent conditions, with the DNA molecules presented in the sequence listing encoding antibody variable chains.
  • amino acid substitutions i.e., “conservative substitutions”
  • conservative substitutions may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the respective amino acid.
  • the skilled person is familiar with commonly practiced amino acid substitutions, as described e.g. in WO 2007/042309, and methods for obtaining thus modified antibodies.
  • DNA molecules encoding variant antibodies with one or more conservative amino acid exchanges can be routinely designed and the respective antibodies readily obtained.
  • antibody variants encompassed by the invention have a sequence identity in the CDR regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%.
  • Preferred antibodies also have a sequence similarity in the CDR regions of at least 80%, more preferably 90% and most preferably 95%.
  • Preferred antibody variants have a sequence identity in the variable regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%.
  • Preferred antibodies also have a sequence similarity in the variable regions of at least 80%, more preferably 90% and most preferably 95%.
  • sequence identity between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences.
  • sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • a variant may also be obtained by using an antibody with a defined sequence as shown in the sequence listing as a starting point for optimization and diversifying one or more amino acid residues, preferably amino acid residues in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties. Diversification of one or more amino acid residues in CDR3 of the variable light chain, CDR3 of the variable heavy chain, CDR1 of the variable light and/or CDR2 of the variable heavy chain has been proven useful. Diversification can be done by methods known in the art, e.g. the so-called TRIM technology referred to in WO 2007/042309.
  • the anti-CD37 antibody molecule of the invention is an “affinity matured” antibody.
  • an “affinity matured” anti-CD37 antibody is an anti-CD37 antibody derived from an antibody with the sequences shown in the sequence listing, that has one or more alterations in one or more CDRs which result in an improvement in the affinity for the antigens, compared to the respective original non-matured antibody.
  • One of the procedures for generating such antibody mutants involves phage display (Hawkins et al., 1992; and Lowman et al., 1991). Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed mutants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
  • Affinity matured antibodies may also be produced by methods as described, for example, by Marks et al., 1992, (affinity maturation by variable heavy chain (VH) and variable light chain (VL) domain shuffling), or Barbas, et al., 1994; Shier et al., 1995; Yelton et al., 1995; Jackson et al., 1995; and Hawkins et al., 1992 (random mutagenesis of CDR and/or framework residues).
  • VH variable heavy chain
  • VL variable light chain domain shuffling
  • Barbas et al., 1994
  • Shier et al. 1995
  • Yelton et al. 1995
  • Jackson et al. 1995
  • Hawkins et al. 1992
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • the anti-CD37 antibody molecule of the invention is a “de-immunized” antibody.
  • a “de-immunized” anti-CD37 antibody is an antibody derived from a humanized or chimeric antibody with a sequence shown in the sequence listing, that has one or more alterations in its amino acid sequence which result in a reduction of immunogenicity of the antibody, compared to the respective original non-dehumanized antibody.
  • One of the procedures for generating such antibody mutants involves the identification and removal of T-cell epitopes of the antibody molecule (Baker and Jones, 2007).
  • the immunogenicity of the antibody molecule can be determined by several methods, e.g.
  • T-cell epitopes by in vitro determination of T-cell epitopes or in silico prediction of such epitopes, as has been described in the literature (Jones et al., 2004; Jones et al., 2005; Reche et al., 2004; Hertz et al., 2006). Once the critical residues for T-cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity (Jones et al., 2005; Tangri et al., 2005). Methods for introduction of mutations in proteins are well-known in the art, e.g. by overlapping PCR techniques.
  • the antibody is, in certain embodiments, a full length antibody or an antibody that contains a portion of the Fc region, the latter as long as the antibody exhibits specific binding both to the relevant portion of the antigen and to Fc receptors.
  • effector functions which are referred to as “effector functions”.
  • the choice of the type and length of the constant region depends on whether effector functions like complement fixation or antibody-dependent cell-mediated cytotoxicity are desirable features, and on the desired pharmacological properties of the antibody protein.
  • the anti-CD37 antibody is a chimeric or humanized antibody with an Fc region, or the relevant section thereof, that has been engineered to modulate effector functions, in particular to enhance binding of the antibody to one or more Fc receptors, thereby enhancing the effector function ADDC.
  • Engineering of the Fc region mediates the antibody's effector function in the presence of effector cells more effectively than that of the non-Fc-engineered parent antibody. In one embodiment, such antibody variant mediates ADCC that is greater than that mediated by the parent antibody.
  • the antibodies of the invention are Fc variants with amino acid substitutions at positions 332 and/or 239 and/or 236.
  • the antibodies of the invention have mutations in the Fc domain selected from the group of
  • the Fc variants in the antibodies of the present invention are defined according to the amino acid modifications that compose them.
  • I332E is an Fc variant with the substitution I332E relative to the parent Fc polypeptide.
  • S239D/I332E defines an Fc variant with the substitutions S239D and I332E and S239D/I332E/G236A defines an Fc variant with the substitutions S239D, I332E, and G236A relative to the parent Fc polypeptide.
  • the substituted positions 236, 239 and 332 correspond to positions 119, 122 and 215, respectively, of the IgG1 heavy chain depicted in SEQ ID NO:24. (In the full-length sequences of the heavy chains of antibodies A2, A4, B2 and B4 shown in SEQ ID NOs: 28, 32, 36 and 40, the substituted amino acids are at positions 235, 238 and 331).
  • the Fc variants of the invention are based on human IgG sequences, and thus human IgG sequences are used as the “base” sequences against which other sequences are compared.
  • the engineered Fc region is preferably IgG, in particular IgG1, but it may also be IgG2 or variant sequences from other immunoglobulin classes such as IgA, IgE, IgGD, IgM or chimeric versions of two or more immunoglobulin classes (e.g. IgG2/IgG1) and the like.
  • the variants of the present invention are engineered in the context of one parent IgG, the variants may be engineered in or “transferred” to the context of another, second parent IgG. This is done by determining the “equivalent” or “corresponding” residues and substitutions between the first and second IgG, typically based on sequence or structural homology between the sequences of the first and second IgGs. In order to establish homology, the amino acid sequence of a first IgG outlined herein is directly compared to the sequence of a second IgG.
  • the residues equivalent to particular amino acids in the primary sequence of the first Fc variant are defined. Regardless of how equivalent or corresponding residues are determined, and regardless of the identity of the parent IgG in which the IgGs are made, what is meant to be conveyed is that the Fc variants used in the present invention may be engineered into any second parent IgG that has significant sequence or structural homology with the Fc variant.
  • the variant antibody may be engineered, for example, in a human IgG2 parent antibody, a human IgA parent antibody (see WO 2007041635).
  • the antibodies of the invention target the antigen CD37, which may be advantageous over targeting CD20 in diseases in which the level of CD37 expression is higher than that of CD20, as e.g. in chronic lymphocytic leukemia, where samples have shown high levels of CD37 mRNA expression compared to low level expression of CD20 mRNA.
  • antibodies of the invention are superior to rituximab, a registered anti-CD20 antibody, with respect to ADCC activity on Ramos cells, normal B cell depletion in whole blood and Ramos Burkitt's lymphoma cell depletion.
  • the antibodies of the invention both the non-Fc engineered and the Fc-engineered ones
  • the antibodies with the mutated Fc region show a ca. 10 fold increase of B cell depletion activity as compared to rituximab ( FIG. 11B ).
  • CD37 antibodies of the invention show potent pro-apoptotic activity without cross-linking; in this respect, antibodies with this property are superior to the anti-CD37 SMIP Tru16.4, which does not show apoptosis without cross-linking (Zhao et al., 2007).
  • Induction of apoptosis without cross-linking is advantageous in the absence of a cross-linking agent in vivo (e.g. effector cells harboring Fc ⁇ receptors) or at low density of the target antigen CD37 (e.g. tumor cells with low level expression of CD37).
  • An antibody which induces apoptosis without cross-linking may still cause cell death, whereas an antibody dependent on cross-linking does not.
  • an anti-CD37 antibody molecule of the invention is an antibody fragment that is derived from a humanized or chimeric CD37 specific antibody according to the present invention.
  • antibody fragments e.g. Fab fragments
  • digestion can be accomplished by means of routine techniques, e.g. using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4,342,566.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, so-called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab′) 2 fragment that has two antigen combining sites and is still capable of cross-linking the antigen.
  • the Fab fragments obtained by digestion of the antibody also contain the constant domains of the light chain and the first constant domain (CH 1 ) of the heavy chain.
  • Fab′ fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the heavy chain CH 1 domain including one or more cysteines from the antibody hinge region.
  • Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab′) 2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them.
  • Antibody fragments can also be generated by molecular biology methods producing the respective coding DNA fragments.
  • the antibody molecule will typically be a tetramer consisting of two light chain/heavy chain pairs, but may also be dimeric, i.e. consist of a light chain/heavy chain pair, e.g. a Fab or Fv fragment, or it may be a monomeric single chain antibody (scFv; Johnson and Bird, 1991), a minibody, or a diabody.
  • the anti-CD37 antibody molecule may also be in the form of a conjugate, i.e. an antibody molecule that is chemically coupled to a cytotoxic agent, particularly a cytotoxic agent that induces cytotoxicity (e.g. apoptosis or mitotic arrest) of tumor cells.
  • a conjugate i.e. an antibody molecule that is chemically coupled to a cytotoxic agent, particularly a cytotoxic agent that induces cytotoxicity (e.g. apoptosis or mitotic arrest) of tumor cells.
  • cytotoxic agent e.g. apoptosis or mitotic arrest
  • an antibody employed in a drug conjugate an “immunoconjugate” contacts and binds to target cells only in limited amounts. Therefore, the cytotoxic agent employed in the conjugate must be highly cytotoxic such that sufficient cell killing occurs to elicit a therapeutic effect.
  • examples of such cytotoxic agents include taxanes (see, e.g.
  • DNA-alkylating agents e.g., CC-1065 analogs
  • anthracyclines e.g., tubulysin analogs
  • duocarmycin analogs e.g., doxorubicin
  • auristatin E e.g., ricin A toxin
  • cytotoxic agents comprising a reactive polyethylene glycol moiety
  • the cytotoxic agent is a maytansinoid, i.e. a derivative of maytansine (CAS 35846538), maytansinoids being known in the art to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. No. 5,208,020; and U.S. Pat. No. 6,441,163).
  • maytansinoid i.e. a derivative of maytansine (CAS 35846538)
  • maytansinoids being known in the art to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. No. 5,208,020; and U.S. Pat. No. 6,441,163).
  • Anti-CD37 antibody immunoconjugates may be designed and synthesized as described in WO 2007/077173 for anti-FAP immunoconjugates.
  • the anti-CD37 molecule of the invention may be radioactively labeled to form an radioimmunoconjugate, an approach suggested for the anti-CD37 antibody MB-1 (Buchsbaum et al., 1992, see above).
  • Radionuclides with advantageous radiation properties are known in the art, examples are Phosphorus-32, Strontium-89, Yttrium-90, Iodine-131, Samarium-153, Erbium-169, Ytterbium-175, Rhenium-188, that have been successfully and stably coupled to MAbs.
  • the anti-CD37 antibody molecules of the invention may be labeled with various radionuclides using direct labeling or indirect labeling methods known in the art, as described in U.S. Pat. No. 6,241,961.
  • direct labeling or indirect labeling methods known in the art, as described in U.S. Pat. No. 6,241,961.
  • An antibody molecule of the invention may also be bispecific, i.e. an antibody molecule that binds to two different targets, one of them being CD37, the other one being selected from e.g. surface antigens expressed by T cells, e.g. CD3, CD16 and CD56.
  • the present invention also relates to DNA molecules that encode the chimeric or humanized anti-CD37 antibody molecules of the invention.
  • the sequences encoding variable heavy chains of the antibody molecules of the invention are shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9.
  • the sequences encoding variable light chains of the antibody molecules of the invention are shown in SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO: 21.
  • Nucleic acid molecules coding for the light chain and the heavy chain may be synthesized chemically and enzymatically (PCR amplification) by standard methods.
  • suitable oligonucleotides can be synthesized with methods known in the art (e.g. Gait, 1984), which can be used to produce a synthetic gene.
  • Methods to generate synthetic genes from oligonucleotides are known in the art (e.g. Stemmer et al., 1995; Ye et al., 1992; Hayden et Mandecki, 1988; Frank et al., 1987).
  • the DNA molecules of the invention include, but are not limited to, the DNA molecules shown in the sequence listing. Accordingly, the present invention also relates to nucleic acid molecules that hybridize to the DNA molecules set forth in the sequence listing under high stringency binding and washing conditions, as defined in WO 2007/042309, where such nucleic molecules encode an antibody or functional fragment thereof that has properties equivalent or superior to an antibody encoded by a sequence shown in the sequence listing.
  • Preferred molecules are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90% and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein.
  • DNA variants that are within the scope of the invention may be defined with reference to the polypeptide they encode. These DNA molecules deviate with respect to their sequence from those depicted in the sequence listing, but encode, due to the degeneracy of the genetic code, antibodies with the identical amino acid sequences. By way of example, in view of expressing the antibodies in eukaryotic cells, the DNA sequences shown in the sequence listing have been designed to match codon usage in eukaryotic cells. If it is desired to express the antibodies in E. coli, these sequences can be changed to match E. coli codon usage. Variants of DNA molecules of the invention can be constructed in several different ways, as described e.g. in WO 2007/042309.
  • the DNA molecules encoding full-length light and heavy chains or fragments thereof are inserted into an expression vector such that the sequences are operatively linked to transcriptional and translational control sequences.
  • the skilled artisan may choose from a great variety of expression systems well known in the art, e.g. those reviewed by Kipriyanow and Le Gall, 2004.
  • Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like.
  • the expression vector and expression control sequences are selected to be compatible with the host cell.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors. In certain embodiments, both DNA sequences are inserted into the same expression vector.
  • Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above.
  • the constant chain is usually kappa or lambda for the antibody light chain, for the antibody heavy chain, it can be, without limitation, any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.
  • the recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the DNA encoding the antibody chain may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature antibody chain DNA.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein.
  • the DNA sequence encoding the antibody chain may already contain a signal peptide sequence.
  • the recombinant expression vectors carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals and other expression control elements that control the expression of the antibody chains in a host cell.
  • promoter sequences are promoters and/or enhancers derived from (CMV) (such as the CMV Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e. g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
  • CMV CMV Simian Virus 40
  • AdMLP adenovirus major late promoter
  • polyadenylation signals are BGH polyA, SV40 late or early polyA; alternatively, 3′UTRs of immunoglobulin genes etc. can be used.
  • the recombinant expression vectors may also carry sequences that regulate replication of the vector in host cells (e. g. origins of replication) and selectable marker genes.
  • Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of an anti-CD37 antibody, and vectors comprising these DNA molecules can be introduced into host cells, e.g. bacterial cells or higher eukaryotic cells, e.g. mammalian cells, according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjections, calcium phosphate precipitation, electroporation or transfer by viral vectors.
  • the DNA molecules encoding the heavy chain and the light chain are present on two vectors which are co-transfected into the host cell, preferably a mammalian cell.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells or the derivatives/progenies of any such cell line.
  • Other mammalian cells including but not limited to human, mice, rat, monkey and rodent cells lines, or other eukaryotic cells, including but not limited to yeast, insect and plant cells, or prokaryotic cells such as bacteria may be used.
  • the anti-CD37 antibody molecules of the invention are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody molecule in the host cells.
  • Antibody molecules are preferably recovered from the culture medium as a secreted polypeptide or it can be recovered from host cell lysates if for example expressed without a secretory signal. It is necessary to purify the antibody molecules using standard protein purification methods used for recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the antibody are obtained.
  • state-of-the art purification methods useful for obtaining the anti-CD37 antibody molecule of the invention include, as a first step, removal of cells and/or particulate cell debris from the culture medium or lysate.
  • the antibody is then purified from contaminant soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin.
  • the purified antibody molecule may be dried, e.g. lyophilized, as described below for therapeutic applications.
  • the present invention relates to a pharmaceutical composition containing, as the active ingredient, the anti-CD37 antibody molecule of the invention.
  • the anti-CD37 antibody is included into pharmaceutical compositions appropriate to facilitate administration to animals or humans.
  • Typical formulations of the anti-CD37 antibody molecule can be prepared by mixing the anti-CD37 antibody molecule with physiologically acceptable carriers, excipients or stabilizers, in the form of lyophilized or otherwise dried formulations or aqueous solutions or aqueous or non-aqueous suspensions. Carriers, excipients, modifiers or stabilizers are nontoxic at the dosages and concentrations employed.
  • buffer systems such as phosphate, citrate, acetate and other anorganic or organic acids and their salts; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone or polyethylene glycol (PEG); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, oligosacchari
  • Zn-protein complexes Zn-protein complexes
  • ionic or non-ionic surfactants such as TWEENTM (polysorbates), PLURONICSTM or fatty acid esters, fatty acid ethers or sugar esters.
  • organic solvents can be contained in the antibody formulation such as ethanol or isopropanol.
  • the excipients may also have a release-modifying or absorption-modifying function.
  • the anti-CD37 antibody molecules may also be dried (freeze-dried, spray-dried, spray-freeze dried, dried by near or supercritical gases, vacuum dried, air-dried), precipitated or crystallized or entrapped in microcapsules that are prepared, for example, by coacervation techniques or by interfacial polymerization using, for example, hydroxymethylcellulose or gelatin and poly-(methylmethacylate), respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), in macroemulsions or precipitated or immobilized onto carriers or surfaces, for example by pcmc technology (protein coated microcrystals).
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions or precipitated or immobilized onto carriers or surfaces for example by pcmc technology (protein coated microcrystals
  • formulations to be used for in vivo administration must be sterile; sterilization may be accomplished be conventional techniques, e.g. by filtration through sterile filtration membranes.
  • HCLF high concentration liquid formulation
  • the anti-CD37 antibody molecule may also be contained in a sustained-release preparation.
  • sustained-release preparations include solid, semi-solid or liquid matrices of hydrophobic or hydrophilic polymers, and may be in the form of shaped articles, e.g. films, sticks or microcapsules and may be applied via an application device.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or sucrose acetate butyrate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-( ⁇ )-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilization (e.g. as described in WO 89/011297) from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • Formulations that may also be used for the anti-CD37 antibody molecule of the invention are described in U.S. Pat. No. 7,060,268 and U.S. Pat. No. 6,991,790.
  • the CD37 antibody molecule can be incorporated also in other application forms, such as dispersions, suspensions or liposomes, tablets, capsules, powders, sprays, transdermal or intradermal patches or creams with or without permeation enhancing devices, wafers, nasal, buccal or pulmonary formulations, or may be produced by implanted cells or—after gene therapy—by the individual's own cells.
  • An anti-CD37 antibody molecule may also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g. to increase serum half-life or to increase tissue binding.
  • the preferred mode of application is parenteral, by infusion or injection (intraveneous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of application such as by inhalation, transdermal, intranasal, buccal, oral, may also be applicable.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • ⁇ g/kg to 40 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays, e.g. by determining the extent of B cell depletion (e.g. using flow cytometry).
  • the “therapeutically effective amount” of the antibody to be administered is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder.
  • the anti-CD37 antibody molecule of the invention and pharmaceutical compositions containing it are useful to deplete B cells that express CD37 on their surface and that cause cancerous or autoimmune/inflammatory disease.
  • the pharmaceutical composition of the invention is useful for the treatment of cancers, in particular any CD37-positive malignancies.
  • B cell malignancies include, without limitation, B cell lymphomas (e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenström's macroglobulinaemia (also called lymphoplasmacytic lymphoma or immunocytoma) or central nervous system lymphomas), leukemias (e.g. acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL; also termed B cell chronic lymphocytic leukemia BCLL), hairy cell leukemia and chronic myoblastic leukemia) and myelomas (e.g. multiple myeloma).
  • B cell lymphomas e.g. various forms of Hodgkin's disease, B cell non-Hodgkin's lymphoma (NHL) and related lymphomas (e.g. Waldenström's macroglobulinaemia (also called lymphoplasmacytic lymphom
  • Additional B cell malignancies include small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, grey zone lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.
  • MALT mucosa-associated lymphoid tissue
  • a pharmaceutical composition containing anti-CD37 antibodies is useful for the treatment of autoimmune and inflammatory diseases that involve B cells in their pathology.
  • Such diseases include, but are not limited to: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis,toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), subacute
  • the anti-CD37 antibody molecule of the invention may be used on its own or in combination with one or more additional therapeutic agents, in particular selected from DNA damaging or tubulin binding agents or therapeutically active compounds that inhibit angiogenesis, signal transduction pathways or mitotic checkpoints in cancer cells.
  • the additional therapeutic agent may be administered simultaneously with, optionally as a component of the same pharmaceutical preparation, or before or after administration of the anti-CD37 antibody molecule.
  • the additional therapeutic agent may be, without limitation, one or more inhibitors selected from the group of inhibitors of EGFR family, VEGFR family, IGF-1R, Insulin receptors, AuroraA, AuroraB, PLK and PI3 kinase, FGFR, PDGFR, Raf, KSP or PDK1.
  • additional therapeutic agents are inhibitors of CDKs, Akt, Src, Bcr-Abl, cKit, cMet/HGF, c-Myc, F1t3, HSP90, hedgehog antagonists, inhibitors of JAK/STAT, Mek, mTor, NFkappaB, the proteasome, Rho, an inhibitor of Wnt signaling or Notch signaling or an ubiquitination pathway inhibitor.
  • Aurora inhibitors are, without limitation, PHA-739358, AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735, SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237 and ENMD-2076.
  • PLK inhibitor An example for a PLK inhibitor is GSK-461364.
  • raf inhibitors are BAY-73-4506 (also a VEGFR inhibitor), PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFR inhibitor), XL-281, and Nevavar (also an inhibitor of the VEGFR).
  • KSP inhibitors examples include ispinesib, ARRY-520, AZD-4877, CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355, and EMD-534085.
  • Examples for a src and/or bcr-abl inhibitors are dasatinib, AZD-0530, bosutinib, XL-228 (also an IGF-1R inhibitor), nilotinib (also a PDGFR and cKit inhibitor), imatinib (also a cKit inhibitor), NS-187, KX2-391, AP-24534 (also an inhibitor of EGFR, FGFR, Tie2, Flt3), KM-80 and LS-104 (also an inhibitor of Flt3, Jak2).
  • An example for a PDK1 inhibitor is AR-12.
  • Rho inhibitor An example for a Rho inhibitor is BA-210.
  • PI3 kinase inhibitors examples include PX-866, PX-867, BEZ-235 (also an mTor inhibitor), XL-147, and XL-765 (also an mTor inhibitor), BGT-226, CDC-0941.
  • inhibitors of cMet or HGF are XL-184 (also an inhibitor of VEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor of VEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274, PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607, JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, NCB-028060, and E-7050.
  • c-Myc inhibitor is CX-3543.
  • Flt3 inhibitors are AC-220 (also an inhibitor of cKit and PDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl and Jak2), MC-2002, SB-1317, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC), TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor of cKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR, VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).
  • AC-220 also an inhibitor of cKit and PDGFR
  • KW-2449 also an inhibitor of bcr-abl and Jak2
  • MC-2002 SB-1317
  • lestaurtinib also an inhibitor of VEGFR, PDGFR, PKC
  • TG-101348 also an inhibitor of JAK2
  • HSP90 inhibitors examples include, tanespimycin, alvespimycin, IPI-504, STA-9090, MEDI-561, AUY-922, CNF-2024, and SNX-5422.
  • JAK/STAT inhibitors examples include CYT-997 (also interacting with tubulin), TG-101348 (also an inhibitor of Flt3), and XL-019.
  • Mek inhibitors are ARRY-142886, AS-703026, PD-325901, AZD-8330, ARRY-704, RDEA-119, and XL-518.
  • mTor inhibitors examples include temsirolimus, deforolimus (which also acts as a
  • VEGF inhibitor VEGF inhibitor
  • everolimus a VEGF inhibitor in addition
  • XL-765 also a PI3 kinase inhibitor
  • BEZ-235 also a PI3 kinase inhibitor
  • Aid inhibitors are perifosine, GSK-690693, RX-0201, and triciribine.
  • cKit inhibitors examples include masitinib, OSI-930 (also acts as a VEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR), tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also an inhibitor of VEGFR and PDGFR), sunitinib (also an inhibitor of Flt3, PDGFR, VEGFR), and XL-820 (also acts as a VEGFR- and PDGFR inhibitor), imatinib (also a bcr-abl inhibitor), nilotinib (also an inhibitor of bcr-abl and PDGFR).
  • masitinib, OSI-930 also acts as a VEGFR inhibitor
  • AC-220 also an inhibitor of Flt3 and PDGFR
  • tandutinib also an inhibitor of Flt3 and PDGFR
  • axitinib also an inhibitor of VEGFR and PDGFR
  • sunitinib
  • hedgehog antagonists examples include IPI-609, CUR-61414, GDC-0449, IPI-926, and XL-139.
  • CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (also inhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509, PHA-848125, and SCH-727965.
  • proteasome inhibitors examples include bortezomib, carfilzomib, and NPI-0052 (also an inhibitor of NFkappaB).
  • proteasome inhibitors/NFkappaB pathway inhibitors examples include bortezomib, carfilzomib, NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957, AVE-8680, and SPC-839.
  • An example for an inhibitor of the ubiquitination pathway is HBX-41108.
  • anti-angiogenic agents are inhibitors of the FGFR, PDGFR and VEGF(R), and thalidomides, such agents being selected from, without limitation, bevacizumab, motesanib, CDP-791, SU-14813, telatinib, KRN-951, ZK-CDK (also an inhibitor of CDK), ABT-869, BMS-690514, RAF-265, IMC-KDR, IMC-18F1, IMiDs, thalidomide, CC-4047, lenalidomide, ENMD-0995, IMC-D11, Ki-23057, brivanib, cediranib, 1B3, CP-868596, IMC-3G3, R-1530 (also an inhibitor of Flt3), sunitinib (also an inhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit), lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib
  • the additional therapeutic agent may also be selected from EGFR inhibitors, it may be a small molecule EGFR inhibitor or an anti-EGFR antibody.
  • anti-EGFR antibodies without limitation, are cetuximab, panitumumab, nimotuzumab, zalutumumab; examples for small molecule EGFR inhibitors are gefitinib, erlotinib and vandetanib (also an inhibitor of the VEGFR).
  • Another example for an EGFR modulator is the EGF fusion toxin.
  • EGFR and/or Her2 inhibitors useful for combination with an anti-CD37 antibody molecule of the invention are lapatinib, trastuzumab, pertuzumab, XL-647, neratinib, BMS-599626 ARRY-334543, AV-412, mAB-806, BMS-690514, JNJ-26483327, AEE-788 (also an inhibitor of VEGFR), AZD-8931, ARRY-380 ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769.
  • the additional drug may also be selected from agents that target the IGF-1R and insulin receptor pathways.
  • agents include antibodies that bind to IGF-1R (e.g. CP-751871, AMG-479, IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab IGFR and novel chemical entities that target the kinase domain of the IGF1-R (e.g. OSI-906 or BMS-554417, XL-228, BMS-754807).
  • IGF-1R e.g. CP-751871, AMG-479, IMC-A12, MK-0646, AVE-1642, R-1507, BIIB-022, SCH-717454, rhu Mab IGFR
  • novel chemical entities that target the kinase domain of the IGF1-R (e.g. OSI-906 or BMS-554417, XL-228, BMS-75
  • CD37 antibodies may be combined with inhibitors of other surface antigens expressed on leukocytes, in particular antibodies or antibody-like molecules, e.g. anti-CD2 (siplizumab), anti-CD4 (zanolimumab), anti-CD19 (MT-103, MDX-1342, SAR-3419, XmAb-5574), anti-CD22 (epratuzumab), anti-CD23 (lumiliximab), anti-CD30 (iratumunab), anti-CD32B (MGA-321), anti-CD38 (HuMax-CD38), anti-CD40 (SGN40), anti-CD52 (alemtuzumab), anti-CD80 (galiximab).
  • An antibody of the invention may also be combined with another CD37 antagonist, e.g. TRU-016.
  • agents to be combined with CD37 antibodies are immunotoxins like BL-22 (an anti-CD22 immunotoxin), inotuzumab ozogamicin (an anti-CD23 antibody-calicheamicin conjugate), RFT5.dgA (anti-CD25 Ricin toxin A-chain), SGN-35 (an anti-CD30-auristatin E conjugate), and gemtuzumab ozogamicin (an anti-CD33 calicheamicin conjugate), MDX-1411 (anti-CD70 conjugate), or radiolabelled antibodies like 90 Y-epratuzumab (anti-CD22 radioimmunoconjugate).
  • immunotoxins like BL-22 (an anti-CD22 immunotoxin), inotuzumab ozogamicin (an anti-CD23 antibody-calicheamicin conjugate), RFT5.dgA (anti-CD25 Ricin toxin A-chain), SGN-35 (an anti-CD30-auristatin E conjugate), and gemtuzum
  • anti-CD37 antibodies may be combined with immunomodulators, agents, e.g. antibodies, that induce apoptosis or modify signal transduction pathways like the TRAIL receptor modulators mapatumumab (a TRAIL-1 receptor agonist), lexatumumab (a TRAIL-2 receptor agonist), tigatuzumab, Apomab, AMG-951 and AMG-655; an anti-HLA-DR antibody (like 1D09C3), an anti-CD74, an osteoclast differentiation factor ligand inhibitor (like denosumab), a BAFF antagonist (like AMG-623a) or an agonist of a Toll-like receptor (e.g. TLR-4 or TLR-9).
  • TRAIL receptor modulators mapatumumab (a TRAIL-1 receptor agonist), lexatumumab (a TRAIL-2 receptor agonist), tigatuzumab, Apomab, AMG-951 and AMG-655; an anti-HLA-DR antibody (like 1D
  • LHRH agonists and antagonists e.g. goserelin acetate, leuprolide, abarelix, cetrorelix, deslorelin, histrelin, triptorelin
  • antimetabolites e.g.
  • antifolates like methotrexate, trimetrexate, pemetrexed, pyrimidine analogues like 5-fluorouracil, fluorodeoxyuridine, capecitabine, decitabine, nelarabine, 5-azacytidine, and gemcitabine, purine and adenosine analogues such as mercaptopurine, thioguanine, azathioprine, cladribine and pentostatin, cytarabine, fludarabine, clofarabine); antitumor antibiotics (e.g.
  • anthracyclines like doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin dactinomycin, plicamycin, splicamycin, actimomycin D, mitoxantrone, mitoxantroneidarubicin, pixantrone, streptozocin, aphidicolin); platinum derivatives (e.g. cisplatin, oxaliplatin, carboplatin, lobaplatin, satraplatin); alkylating agents (e.g.
  • vinca alkaloids like vinblastine, vindesine, vinorelbine, vinflunine and vincristine
  • taxanes like paclitaxel, docetaxel and their formulations, larotaxel; simotaxel, and epothilones like ixabepilone, patupilone, ZK-EPO); topoisomerase inhibitors (e.g.
  • epipodophyllotoxins like etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, banoxantrone, camptothecin) and miscellaneous chemotherapeutics such as retinoic acid derivatives, amifostine, anagrelide, interferon alpha, interferon beta, interferon gamma, interleukin-2, procarbazine, N-methylhydrazine, mitotane, and porflmer, bexarotene, celecoxib, ethylenemine/methyl-melamine, thriethyienemelamine, triethylene thiophosphoramide, hexamethylmelamine, and enzymes L-asparaginase, L-arginase and metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069,
  • the anti-CD37 antibody molecule is applied together with “CHOP” (a combination of cyclophosphamide, doxorubicin, vincristine and prednisone).
  • CHOP a combination of cyclophosphamide, doxorubicin, vincristine and prednisone.
  • the anti-CD37 antibody molecule of the invention may also be used in combination with other therapies including surgery, radiotherapy, endocrine therapy, biologic response modifiers, hyperthermia and cryotherapy and agents to attenuate any adverse effect (e.g. antiemetics), G-CSF, GM-CSF, photosensitizers such as hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, Npe6, tin etioporphyrin, pheoboride-a bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines
  • therapies including surgery, radiotherapy, endocrine therapy, biologic response modifiers, hyperthermia and cryotherapy and agents to attenuate any adverse effect (e.g. antiemetics), G-CSF, GM-CSF, photosensitizers such as hematoporphyrin derivatives, Photofrin®, benzop
  • Monoclonal antibodies show extremely antigen specificity and frequently only react with the human target antigen, but not with homologue proteins from animal species.
  • appropriate animal models for assessment of in vivo toxicity and pharmacodynamic behavior are desirable.
  • One possibility for an in vivo model is a transgenic mouse in which the endogenous target antigen is replaced by its human homologue (“knock-out/knock-in mouse”).
  • the murine CD37 gene can be replaced by the human CD37 gene. This can be achieved by constructing a targeting vector which contains the coding genomic sequence of the human CD37 gene flanked by non-translated sequences. This targeting vector can be used for homologous recombination using mouse ES cells.
  • Transgenic animals homozygous for human CD37 expression can be used to assess the pharmacodynamic effect of antibodies directed against human CD37, e.g. by monitoring the number of peripheral B cells after application of the antibodies. Alternatively, those mice can be used to investigate potential toxic effects of human CD37 specific antibodies, after i.v. application.
  • a surrogate antibody is an antibody which reacts with the homologous protein of an animal species which is relevant and useful for investigation of pharmacodynamic and toxic effects, e.g. the mouse or the cynomolgus monkey.
  • monoclonal antibodies are developed which are specific for macaque CD37 or mouse CD37, respectively Ideally, such a surrogate antibody should have similar binding and functional properties as the development antibody. This can be investigated by the use of assay systems which utilize macaque or mouse CD37 expressing cells as target cells, e.g. for binding, FACS Scatchard analysis, ADCC and apoptosis assays.
  • the surrogate antibody can be selected by virtue of its B cell depleting activity in macaque or mouse blood in vitro.
  • FIG. 1 Chimeric Antibody A0 specifically recognizes the CD37 antigen, determined by FACS competition assay
  • FIG. 2 Binding of humanized versions of A0 to cellular CD37 antigen, determined by FACS
  • FIG. 3 Binding of humanized versions of A0 to cellular CD37 antigen, determined by FACS
  • FIG. 4 Affinity of humanized versions of A0 to cellular CD37 antigen, determined by FACS scatchard analysis
  • FIG. 5 ADCC activity of humanized versions of A0 on Ramos cells
  • FIG. 6 Pro-apoptotic activity of humanized versions of A0 on Ramos cells
  • FIG. 7 ADCC activity of Fc-engineered versions of mAb A0 on Ramos cells
  • FIG. 8 ADCC activity of Fc-engineered versions of mAb B0 on Ramos cells
  • FIG. 9 Pro-apoptotic activity of mAb A0 and B0
  • FIG. 10 Pro-apoptotic activity of Fc-engineered versions of mAb A0
  • FIG. 11A Depletion of normal human B cells in a whole blood assay by Fc-engineered antibodies A2 and B2 in comparison to Rituximab
  • FIG. 11B Superior B cell depleting activity of antibodies after Fc-engineering in comparison to Rituximab
  • FIG. 11C Antibodies A2 and B2 do not deplete T cells and monocytes in whole blood assays
  • FIG. 12 Superior ADCC activity after Fc-engineering compared to Rituximab
  • FIG. 13 Depletion of Ramos Burkitt's lymphoma cells in a whole blood assay by Fc-engineered antibodies A2 and B2 in comparison to Rituximab
  • FIG. 14 In vivo tumor growth inhibition of Ramos xenograft tumors in nude mice by Fc-engineered antibodies A2 and B2
  • FIG. 15 Expression of CD37 on multiple myeloma cells
  • FIG. 16 ADCC activity of antibodies A2 and B2 on multiple myeloma cells
  • FIG. 17 Pro-apoptotic activity of antibodies A2 and B2 on patient-derived CLL cells
  • the corresponding DNA sequences are synthesized applying codon usage optimized for mammalian cells (GeneArt, Regensburg, Germany), adding at the 5′ end a HindIII and at the 3′ end a BamHI cloning site.
  • the synthesized DNA molecules are digested with HindIII and BamHI and the resulting DNA fragments (SEQ ID NO:1 and SEQ ID NO:3 plus restriction sites) are cloned into pcDNA3.1 based expression vectors coding for human IgG1 constant region and human kappa light chain constant region, respectively (SEQ ID NO:24 and SEQ ID NO:26).
  • EndoFree plasmid preparations are prepared and the heavy and light chain plasmids are co-transfected into HEK293 freestyle cells (Invitrogen) at a concentration of 1 mg/L of each plasmid according to the supplier's protocol. After 72 hours the supernatant is harvested and the IgG concentration is determined by ELISA. The resulting chimeric anti-CD37 antibody (designated A0) is purified on a modified protein A column (GE Healthcare), eluted into a citrate buffer then dialyzed in PBS.
  • Humanization of chimeric mAb A0, as obtained in a), is performed using a CDR grafting approach, as described e.g. in U.S. Pat. No. 5,225,539; U.S. Pat. No. 6,548,640; U.S. Pat. No. 6,982,321.
  • a structural template is chosen from the Protein Data Bank (PDB) of Brookhaven National Laboratory.
  • the VL domain from the murine monoclonal antibody entry “1KB5” is chosen with 88% sequence identity/81% similarity and 2.5 ⁇ resolution.
  • the same mouse monoclonal antibody structure “1KB5” with 90% sequence identity and 91% similarity is chosen as the main modeling template.
  • the best fit for human consensus framework is found to be of the type human Vkappa1 (hVK1) and human VH1 (hVH1).
  • a graft to the most stable human consensus domains hVK3 and hVH3 is chosen.
  • the mAb A0_VL and mAb A0_VH models are combined with human consensus domain models hVK1, hVK3, hVH1A and hVH3 and combined to produce to Fv-models.
  • Loop grafting is performed by embedding the murine mAb A0 CDR regions into the human antibody frameworks and the DNA molecules of the humanized chain constructs are synthesized.
  • the respective humanized variable regions are synthesized and cloned into immunoglobulin expression vectors and transiently expressed in the HEK 293 freestyle expression system (Invitrogen), as described in a), in the combinations of heavy and light chain sequences as shown in Table 1, and purified on protein A columns.
  • Fc mutants The generation of Fc mutants is performed as described by Lazar et al., 2006.
  • the resulting Fc-engineered heavy chain sequence is introduced into the expression vector pAD-CMV1 (described in EP 393 438) and co-transfected together with a plasmid containing the light chain encoding sequence into CHO-DG44 cells.
  • the antibody is harvested from cell culture media 5 to 7 days after transfection and purified via protein A chromatography, eluted into a citrate buffer then dialyzed in PBS.
  • the protein content of the samples is determined via Protein A HPLC, the endotoxin content is determined via Kinetic-QCL Kinetic Chromogenic Assay (Lonza).
  • the monomer content of the samples is determined by HP-SEC, all samples used for functional testing show a monomer content of >95%.
  • MAb A0 for cellular CD37 is tested in a FACS competition assay on Ramos Burkitt lymphoma cells (ATCC #CRL-1596).
  • Cells are grown in tissue culture flasks (175 cm 2 ) using RPMI-1640+G1utaMAX supplemented with 10% heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM non-essential amino acids as culture medium. Cells are cultivated with an initial density of 3 ⁇ 10 5 cells/ml at 37° C. and 5% CO 2 in a humidified atmosphere for three days.
  • the cultures are maintained at a cell concentration between 3 ⁇ 10 5 and 1.8 ⁇ 10 6 /ml by sub-cultivation in a ratio of 1:6 with fresh culture medium 2-3 times a week.
  • the CD37-specific mAb HH1 (Santa Cruz) directly labeled with phycoerythrin (PE) is used at a concentration of 1 ⁇ g/ml.
  • the antibody is preincubated with the unlabelled competitor antibody A0 for 10 min at 4° C. at the indicated molar ratio. Thereafter, 1 ⁇ 10 5 Ramos cells are incubated for 30 min with the antibody mixture on ice.
  • FIG. 1 Results of such an assay are shown in FIG. 1 .
  • Addition of a control human IgG1 antibody (Sigma IgG1 kappa) at 20-fold molar excess does not significantly reduce the mean fluorescence intensity (MFI) of Ramos cells.
  • Addition of either unlabelled HH1 antibody or A0 antibody at 20-fold molar excess almost completely abrogated binding of the directly labeled HH1 antibody. This indicates that A0 and HH1 antibodies recognize identical or similar epitopes on Ramos cells and compete for binding to cellular CD37 antigen.
  • Humanized versions of A0 are tested for their binding to cellular CD37 antigen by FACS analysis. Antibodies are added to Ramos cells at the indicated concentrations and allowed for binding for 30 min at 4° C. Thereafter, bound antibody is detected with PE-labelled goat-anti-human IgG antibody (Sigma), cells are washed twice with PBS, and thereafter cells are resuspended in FACS buffer and analyzed by FACS on a BD FACS Canto. Examples are shown in FIGS. 2 and 3 (antibodies A, B, C, D, I or A, H, I, J, K, L and M, respectively; see Table 1). Several of the humanized versions of A0 show similar binding to Ramos cells as the parental antibody A0, indicating that humanization does not reduce binding to cellular CD37 antigen.
  • FITC conjugated secondary antibody (conc. 15 ⁇ g/ml; mouse mAb anti-hu IgG all subclasses, Zymed 05-4211) is added, mixed, and incubated for 30 min on ice. 4 ml PBS ph7.2 containing 0.02% acid are added thereafter, cells are pelleted and resuspended in 300 ⁇ l PBS pH 7.2 and subjected to FACS analysis using a BD FACS Canto. All experimental steps are performed on wet ice, all antibody dilutions are made in PBS/0.5% BSA+0.02% acid. FACS calibration is performed using Quantum FITC MESF (Premix) High Level Beads (Bangs Laboratories).
  • FIG. 4 shows the MFI/antibody concentration relationship of several humanized variants of A0. The results show similar binding to Ramos cells of some of the humanized versions as the starting antibody, with dissociation constants (K d ) ranging from 2.15 to 4.90 nanomoles/liter.
  • Ramos cells As target cells and IL2-stimulated human PBMCs as effector cells.
  • Ramos cells (Burkitt's lymphoma; ATCC #CRL-1596) were purchased from ATCC. Cells are grown in tissue culture flasks (175 cm 2 ) using RPMI-1640+GlutaMAX supplemented with 10% heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM non-essential amino acids as culture medium.
  • Cells are cultivated with an initial density of 3 ⁇ 10 5 cells/ml at 37° C. and 5% CO 2 in a humidified atmosphere for three days.
  • the cultures are maintained at a cell concentration between 3 ⁇ 10 5 and 1.8 ⁇ 10 6 /ml by sub-cultivation in a ratio of 1:6 with fresh culture medium 2-3 times a week.
  • An aliquot of the cell culture at a cell density between 1.5 ⁇ 10 6 /ml and 1.8 ⁇ 10 6 /ml and growing in the log-phase is centrifuged (200 ⁇ g, i.e. 1000 rpm) for 10 min.
  • Cells are washed once with washing medium (RPMI 1640 w/o L-glutamine) and pelleted (200 ⁇ g, i.e. 1000 rpm; 10 min).
  • Cell pellet is resuspended in assay medium [1% BSA in RPMI w/o L-glutamine] and cell count is determined. Cell concentration is adjusted to 2 ⁇ 10 5 /ml
  • Approximately 50-80 ml whole blood drawn from healthy donors is used for the isolation of PBMC. 10 ml whole blood are diluted 1:3.6 with 26 ml HBSS (Hanks' Balanced Salt Solution w/o calcium and magnesium) in a 50 ml tube. 18 ml diluted whole blood is layered on top of 12 ml Lymphoprep (Nycomed Pharma) in a 50 ml tube and centrifuged at 370 ⁇ g (1400rpm) for 35 min. The mononuclear cells from the interface are aspirated and washed first with HBSS (750 ⁇ g, i.e. 1900 rpm; 10 min), then a second time with HBSS (300 ⁇ g, i.e.
  • the pelleted cells are gently resuspended in culture medium/assay medium (10% heat-inactivated human AB serum in RPMI 1640 w/o L-glutamine) using a pipette and the cell count is determined in the cell counter.
  • the PBMC concentration is adjusted to 1 ⁇ 10 7 /ml.
  • the freshly isolated PBMC (5 ⁇ 10 5 /ml) are maintained in culture medium (RPMI 1640 w/o L-glutamine supplemented with 10% human AB serum) in a tissue culture flask (75 cm 2 ) at 37° C. in CO 2 incubator over night.
  • IL-2 stimulated PBMC are separated from cell debris on a Lymphoprep gradient.
  • the purified IL-2 stimulated PBMC are suspended in culture medium/assay medium at a concentration of 1 ⁇ 10 7 /ml.
  • effector cells with target cells in presence of specific or unspecific antibody is performed in duplicates or triplicates in 96-well round-bottom microtiter plates in a final volume of 200 ⁇ l assay medium per well consisting of 10% human AB serum and 1% BSA in RPMI in 1:1 ratio.
  • First effector cells freshly isolated PBMC cells in 100 ⁇ l 10% human AB serum in RPMI per well
  • target cells and antibody solution diluted in 50 ⁇ l 1% BSA in RPMI.
  • effector cells are cultivated in assay medium alone (effector cell control) and target cells are cultivated either in assay medium alone (spontaneous lysis) or in assay medium supplemented with 1% Triton X-100 (maximal lysis).
  • the co-culture is incubated at 37° C. in a humid CO 2 incubator for 3 hours.
  • cells are removed from the culture medium by centrifugation (200 ⁇ g, i.e. 1000 rpm; 10 min) at room temperature.
  • Cell free supernatants 100 ⁇ l/well are transferred into corresponding wells of a 96-well flat-bottom plate.
  • Cytotoxicity Detection Kit (LDH; Roche) is used to measure ADCC activity. The detection of cytotoxicity is based on the measurement of LDH enzyme activity released from plasma membrane-damaged cells. LDH released into the culture supernatants reduces the tetrazolium salt from the kit to formazan. The absorption maximum of formazan dye is measured at 490 nm against a reference wavelength of 650 nm in an ELISA plate reader. To calculate percent cell mediated cytotoxicity five controls are performed in each experimental setup.
  • the average absorbance of the triplicates or duplicates is calculated and the background is subtracted according to the manufacturer's instructions.
  • FIG. 5 the results from an ADCC assay using an E:T ratio of 25:1 and Ramos target cells are shown. Antibodies are added in a concentration of 30 ng/ml. Both the starting mAb and humanized versions thereof display similar ADCC activity against the Ramos cells. In conclusion, humanization of anti-CD37 mAb A does not significantly alter its ADCC inducing capacity.
  • Ramos cells (Burkitt's lymphoma; ATCC #CRL-1596) are received from ATCC. Cells are grown in tissue culture flasks (175 cm 2 ) using RPMI-1640+G1utaMAX supplemented with 10% heat-inactivated fetal bovine serum, 12.5 mM HEPES, 1 mM sodium pyruvat, 1% MEM non-essential amino acids as culture medium.
  • Cells are cultivated with an initial density of 3 ⁇ 10 5 cells/ml at 37° C. and 5% CO 2 in a humidified atmosphere for three days.
  • the cultures are maintained at a cell concentration between 3 ⁇ 10 5 and 1.8 ⁇ 10 6 /ml by sub-cultivation in a ratio of 1:6 with fresh culture medium 2-3 times a week.
  • An aliquot of the cell culture at a cell density between 1.5 ⁇ 10 6 /ml and 1.8 ⁇ 10 6 /ml and growing in the log-phase is centrifuged (200 ⁇ g, i.e. 1000 rpm) for 10 min.
  • Cells are washed once with washing medium (RPMI 1640 w/o L-glutamine) and pelleted (200 ⁇ g, i.e.
  • Cell pellet is resuspended in culture medium and cell count is determined. Cell concentration is adjusted to 1 ⁇ 10 6 /ml. 100 ⁇ l cell suspension per well are plated into 96-well round bottom plates. Antibodies are diluted in cell culture medium containing 10% FBS and 100 ⁇ l antibody solution are added per well. Cells are incubated for 20 to 24 hrs at 37° C. in CO 2 incubator and thereafter stained with Vybrant apoptosis assay kit #2. Alexa Fluor 488 labelled Annexin V and propidium iodide solution are added to the cells and incubated for 15 min in the dark.
  • AnnexinV positive/PI negative cells are resuspended in 400 ⁇ l AnnexinV binding buffer and subjected to FACS analysis using a BD FACS Canto.
  • the percentage of AnnexinV positive/PI negative cells and AnnexinV/PI positive cells is determined in two-dimensional dot blots using FL1/FL2 channels.
  • An isotype matched non-binding antibody (Sigma human IgG1) is used as negative control.
  • FIG. 6 the pro-apoptotic effect of various humanized versions of mAb A on Ramos cells are shown.
  • Cells are incubated with antibody at 10 ⁇ g/ml for 24 hrs, the total percentage of AnnexinV positive cells (PI positive and PI negative) is displayed.
  • Parental mAb A shows potent pro-apoptotic activity.
  • humanized versions show a significantly reduced number of AnnexinV positive cells compared to the parental mAb A, indicative for altered pro-apoptotic activity of the humanized antibodies.
  • humanization of MAb A leads to a reduction of its pro-apoptotic activity in this experimental setting.
  • ADCC activity of Fc-engineered versions of mAb A0 (designated A1, A2, A3, A4; see Table 2) is assessed using Ramos cells as target cells.
  • ADCC assay is performed as described above (Example 5). The result of the experiment is shown in FIG. 7 .
  • Fc-engineered versions of A0 show clearly improved potency and efficacy compared to the parent mAb A0.
  • Certain Fc variants show improvement in maximal lysis of up to 100% compared to the parent mAb and improvement in EC 50 of up to 10-fold compared to the parent mAb.
  • introduction of specific Fc mutants strongly increases the ADCC activity of chimeric mAb A0.
  • ADCC activity of Fc-engineered versions of mAb B0 (designated B1, B2, B3, B4; see Table 2) is assessed using Ramos cells as target cells.
  • ADCC assay is performed as described above (Example 5).
  • Fc-engineered versions of B0 show clearly improved potency and efficacy compared to the parent mAb B0.
  • Certain Fc variants show improvement in maximal lysis of up to 80% compared to the parent mAb and improvement in EC 50 of up to 20-fold compared to the parent mAb.
  • introduction of specific Fc mutants strongly increases the ADCC activity of humanized mAb B0. The results of the experiments are shown in FIG. 8 .
  • the pro-apoptotic activity of mAbs A0 and B0 on Ramos cells before and after cross-linking with anti-IgG mAb is displayed in FIG. 9 .
  • the apoptotic assay is performed as described in Example 6, for antibody cross-linking an anti-human IgG antibody ( ⁇ -chain specific; Sigma) is added to the antibodies in a ratio of 1:1 and incubated for 15 min at 37° C. prior to adding to the target cells.
  • an anti-human IgG antibody ⁇ -chain specific; Sigma
  • the CD37-specific mAbs are added at a concentration of 1 ⁇ g/ml with and without cross-linking
  • Chimeric mAb A0 is a potent inducer of apoptosis even without cross-linking, this effect is significantly enhanced after cross-linking of the mAb.
  • the humanized mAb B0 is completely devoid of pro-apoptotic activity, however shows potent pro-apoptotic activity after cross-linking with anti-IgG Ab.
  • this experiment shows that pro-apoptotic activity of a humanized version of mAb A0 can be restored after antibody cross-linking.
  • the pro-apoptotic activity of Fc-engineered versions of chimeric mAb A0 on Ramos cells is assessed by AnnexinV/PI staining as described in Example 6.
  • Parent1 antibody A0 and Fc-engineered variants A2 and A4 are titrated over a concentration range from 0.1 to 10.000 ng/ml.
  • FIG. 10 all 3 antibodies show similar pro-apoptotic activity.
  • this experiment shows that Fc-engineering of mAb A0 does not alter its pro-apoptotic activity.
  • the efficacy and potency of depletion of normal B cells from human blood is assessed using a whole blood assay.
  • the test antibody is added to EDTA-treated samples of human blood from healthy individuals and subsequently, after 3 to 4 hrs incubation at 37° C., the number of B cells is quantitatively measured by a 4-color FACS assay.
  • the degree of B cell depletion by the test agent can be calculated. Due to the presence of human IgG levels and effector cells similar to the situation in human beings in vivo, this assay type is considered of high relevance for predicting the effect of the test antibodies in vivo.
  • a quantitative FACS assay is used to determine the number of B cells and/ or spiked Ramos cells in blood samples derived from healthy individuals.
  • Quantification is performed using BD Trucount tubes which contain a known number of fluorescent beads which serve as internal standard for quantification of the cell population of interest.
  • B cells are identified by 4-colour analysis using 4 different CD markers (CD3/CD14/CD19/CD45) in combination with FSC/SSC analysis.
  • 270 ⁇ l fresh blood per well is incubated in a 48 well plate together with 30 ⁇ l antibody dilution (in PBS) or PBS (buffer control) in duplicates. Samples are incubated for 4h at 37° C. and thereafter immediately placed on ice. 33 ⁇ l of CD marker master mix is added to Trucount tubes and 50 ⁇ l of the blood-antibody mixture is added. Samples are vortexed and incubated for 15 min at room temperature. Thereafter 450 ⁇ l of lysis buffer is added, vortexed, and incubated for additional 15 min at room temperature. Samples are placed on ice and immediately subjected to FACS analysis using a BD FACS CantoTM Flow Cytometer. Evaluation of data is performed using the BD FACSDiva software (Version 5.0.2).
  • Fc-engineered chimeric and humanized mAbs A2 and B2 show excellent potency on normal B cell depletion with EC 50 values ranging from 0.15 to 0.35 nM.
  • the degree of normal B cell depletion ranges from 57% to 65%.
  • Rituximab a registered antibody used for the treatment of B-NHL, is tested in parallel and yields significantly lower depletion of B-cells in this assay format ( FIG. 11A ).
  • the effect of mAbs on B cell depletion in human blood derived from healthy individuals is assessed as described in a).
  • the non Fc-engineered mAbs A0 and B0 show B cell depleting activity in the range from 13% to 26%, similar to Rituximab.
  • Fe-engineering results in a dramatic increase of B cell depleting activity for both mAbs, with a mean percentage of depletion of 75%. This clearly demonstrates the superiority of A2 and B2 compared to Rituximab ( FIG. 11B ).
  • A2 and B2 on T lymphocytes (CD3-positive) and monocytes (CD14-positive) is assessed in parallel to the effect on B lymphocytes. No significant change of either T cell numbers or monocyte numbers is observed, whereas a significant reduction of the number of B cells is seen ( FIG. 11C ). This indicates that A2 and B2 specifically deplete B cells from human blood.
  • ADCC activity of the Fc-engineered version A2 of mAb A0 is assessed using Ramos cells as target cells.
  • ADCC assay is performed as described above (Example 5).
  • the non Fc-engineered antibody A0 shows a maximal lysis of Ramos target cells which is inferior to Rituximab, an antibody specific for CD20 which is an approved treatment for patients suffering from B cell lymphomas.
  • Fc-engineering of A0 leads to a clearly improved potency and efficacy of A2 over Rituximab. This indicates, that at similar antigen densities of CD20 and CD37 on Ramos cells the Fc-engineered anti-CD37 mAb A2 shows clearly improved ADCC activity than Rituximab ( FIG. 12 ).
  • Ramos tumor cells are spiked in about a tenfold excess compared to endogenous B cells into the whole blood matrix, and their depletion is also monitored by FACS analysis.
  • Fc-engineered chimeric and humanized mAbs A2 and B2 show good potency on
  • Rituximab a registered antibody used for the treatment of B-NHL, is tested in parallel and yields significantly lower depletion of Ramos cells in this assay format ( FIG. 13 ).
  • the in vivo anti-tumor efficacy of mAbs A2 and B2 is assessed using a Ramos Burkitt's lymphoma model in nude mice.
  • CD37-positive Ramos cells are injected subcutaneously into the flank of the animals and i.v. treatment of the animals started when tumors are established.
  • a twice weekly treatment schedule is chosen (q3/4d), two different doses (8 mg/kg and 25 mg/kg) are tested in parallel.
  • Both mAbs show significant anti-tumor efficacy with T/C values ranging from 0.2% to 26%. No significant difference between the two dose levels and between the two antibodies are observed.
  • a correlation between the serum concentrations of A2 and B2 to their pharmacodynamic effect is established in mice using the Ramos tumor xenograft model.
  • These studies demonstrate that a dose of 8 mg/kg A2 and B2 (formulated in a citrate buffer: 25 mM Na-Citrate, 115 mM NaCl, 0.04% Tween 80, pH 6.0) causes significant retardation of tumor growth in this aggressive s.c. (subcutaneous) tumor model using a standard q3 or 4d antibody dosing schedule in mice, thus indicating continued activity throughout the dosing interval.
  • pharmacokinetic data are established for the same dose.
  • the estimated weekly dose for a 70 kg human is in the range of 86 to 184 mg.
  • the calculated estimated human weekly dose for B2 for a 70 kg human is 189 to 404 mg.
  • Antibodies A2 and B2 Show ADCC Activity on Multiple Myeloma Cells
  • CD37 on a panel of multiple myeloma cell lines is assessed by FACS analysis using antibodies specific for CD37.
  • Cells are either incubated with a directly fluorescently labeled anti-CD37 antibody or an unlabeled CD37-specific antibody followed by a second fluorescently labeled antibody directed against the primary antibody.
  • the fluorescence activity of the labeled cells is measured with a FACS Canto Flow Cytometer (BD Biosciences) and the fluorescence intensity is recorded as MFI using the FACS Diva Software. 6 out of 11 tested multiple myeloma demonstrate cell surface expression of CD37 ( FIG. 15 ).
  • One cell line (RPMI 8226) is subsequently tested in an ADCC assay as described in Example 5 using the CD37-specific antibodies A2 and B2. Both antibodies demonstrate potent ADCC activity on RPMI 8226 cells with EC 50 values in the range of 25 ng/ml and a maximal cell lysis of about 20% ( FIG. 16 ).
  • This example demonstrates that CD37-positive multiple myeloma cells are susceptible to ADCC mediated cell lysis using the CD37-specific mAbs A2 and B2.
  • FIG. 15 shows the FACS analysis of six multiple myeloma derived cell lines for CD37 expression.
  • the open curves indicate reactivity with the CD37-specific antibody, the filled curves represent the negative control antibody.
  • CLL patient-derived chronic lymphocytic leukemia
  • PBMCs Peripheral blood mononuclear cells
  • the primary CLL cells are purified from freshly collected blood according to Ficoll-Paque® plus procedure (StemCell Technologies, Meylan, France) and stored at 4° C. in RPMI 1640 culture medium containing 10% heat inactivated human AB serum (Sigma, France) until use.
  • the culture medium for primary CLL cells is RPMI 1640 supplemented with 2 mM L-glutamine and 10% of heat inactivated human AB serum.
  • CLL cells are counted in a hemocytometer and their viability is assessed by 0.25% trypan blue exclusion. The viability of CLL samples is more than 90%.
  • Cells are incubated at 37° C. for 24 hours with the antibodies at 30 ⁇ g/ml and thereafter the percentage of AnnexinV positive cells is determined as described in Example 6.
  • FIG. 17 Fc-engineered antibodies A2 and B2 show strong pro-apoptotic activity on the primary CLL cells with about 90% (A2) and 40% (B2) AnnexinV positive cells. Both mAbs are clearly superior to rituximab, a B cell-specific antibody approved for the treatment of B-NHL.
  • Mab A2 demonstrates also clearly superior activity compared to alemtuzumab, an antibody approved for the treatment of B-CLL.
  • a targeting vector is constructed which contains the coding sequence of human CD37 (BAC (bacterial artificial chromosome) IDs: RP11-433N13, RP11-50111) flanked by non-translated sequences.
  • This targeting vector (which contains, in addition, loxP sites flanking exons 3-4 and the neo selection marker flanked by frt sites) is then used for homologous recombination, using mouse ES cells and standard technology to replace exons 1-8 of the mouse genomic sequence with the human counterpart sequences,
  • the C57BL/6N ES cell line is grown on a mitotically inactivated feeder layer comprised of mouse embryonic fibroblasts (MEF) in DMEM High Glucose medium containing 20% FBS (PAN) and 1200 u/mL Leukemia Inhibitory Factor (Millipore ESG 1107).
  • ES clones are isolated on d8 and analyzed by Southern Blotting according to standard procedures, e.g. by the use of radiolabelled DNA probes specific for the target gene after expansion and freezing of clones in liquid nitrogen. Transgenic animals are then generated by standard procedures known in the art, e.g. by blastocyst injection and subsequent generation of chimeric animals.
  • Animals heterozygous and homozygous for human CD37 are obtained by conventional breeding of chimeric and heterozygous animals, respectively.
  • the successful knock-out of the murine CD37 gene and the knock-in of the human CD37 gene is monitored at the protein level using standard procedures, e.g. FACS analysis of peripheral blood lymphocytes or immunohistochemical analysis of tissue sections.
  • Monoclonal antibodies specific for macaque CD37 are generated by genetic immunization of mice and rabbits using the complete coding sequence of the macaque CD37 antigen (Acc. No. ENSMMUT00000020744). Specific antibodies are selected using recombinant HEK293 or CHO cells expressing the macaque CD37 antigen, e.g. by standard ELISA or FACS techniques. The variable heavy and light chain coding sequences of these antibodies are retrieved by PCR cloning and used for generation of chimeric antibodies (as described in Example 1) which harbor the VH and VL region derived from the murine or rabbit starting antibody and an Fc portion identical to that of an antibody of the invention, e.g. A2 or B2.
  • the binding and functional properties can be investigated by the use of assay systems which utilize macaque CD37 expressing cells as target cells, e.g. for binding, FACS, Scatchard analysis, ADCC and apoptosis assays.
  • the surrogate antibody is selected by virtue of its B cell depleting activity in Cynomolgus monkey blood in vitro.
  • the DNA molecule encoding the complete heavy chain e.g. with the sequence shown in SEQ ID NO: 27, 31, 35 or 39, respectively, is inserted into the eukaryotic expression vector designated pBI-26, encoding in addition the selection marker dihydrofolate reductase from hamster.
  • the DNA molecule encoding the complete light chain depicted in SEQ ID NO: 29, 33, 37 and 41, respectively, is inserted into the eukaryotic expression vector designated pBI-49, encoding in addition the selection marker neomycin phosphotransferase.
  • the DNA sequences of the entire heavy and light chains are sequenced completely.
  • the hamster cell line CHO-DG44 grown in suspension in chemically defined media, is co-transfected with the eukaryotic expression vectors for the heavy and for the light chain of the antibodies, as described above. Transfected cells are selected in medium without hypoxanthine and thymidine and in the presence of the antibiotic G418. Subsequently, cells are subjected to stepwise selection and amplification using increasing concentrations of methotrexate (MTX). From the 800 nM MTX amplification step, a single cell clone is selected based on growth performance and antibody production in spinner runs, and is cryopreserved in a Safety Cell Bank (SCB).
  • SCB Safety Cell Bank

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