EP1397387A1 - Antibodies specific for cd44v6 - Google Patents

Antibodies specific for cd44v6

Info

Publication number
EP1397387A1
EP1397387A1 EP02735364A EP02735364A EP1397387A1 EP 1397387 A1 EP1397387 A1 EP 1397387A1 EP 02735364 A EP02735364 A EP 02735364A EP 02735364 A EP02735364 A EP 02735364A EP 1397387 A1 EP1397387 A1 EP 1397387A1
Authority
EP
European Patent Office
Prior art keywords
seq
antibody
acid sequence
nucleic acid
biwa
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP02735364A
Other languages
German (de)
English (en)
French (fr)
Inventor
Günther Adolf
Elinborg Ostermann
Erik Patzelt
Marlies Sproll
Karl-Heinz Heider
John J. Miglietta
Augustinus Antonius Maria Silvester Van Dongen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Boehringer Ingelheim Pharmaceuticals Inc
Original Assignee
Boehringer Ingelheim International GmbH
Boehringer Ingelheim Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boehringer Ingelheim International GmbH, Boehringer Ingelheim Pharmaceuticals Inc filed Critical Boehringer Ingelheim International GmbH
Priority to EP02735364A priority Critical patent/EP1397387A1/en
Publication of EP1397387A1 publication Critical patent/EP1397387A1/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention belongs to the field of oncology.
  • the invention relates to antibodies with specified sequence which are specific for an epitope which is coded by the variant exon v6 of the CD44 gene and to derivatives of said antibody.
  • the invention also provides nucleic acid molecules encoding said antibody proteins.
  • the invention furthermore pertains to methods for producing said antibody proteins.
  • the invention also provides pharmaceutical compositions comprising said antibody proteins.
  • the invention furthermore is concerned with the use in the manufacture of a medicament for the treatment of cancer.
  • CD44 surface glycoprotein CD44
  • the standard form CD44s or CD44std
  • CD44v, CD44var certain CD44 splice variants
  • the CD44 variants are generated by alternative splicing in a way that the sequences of ten exons (vl-vlO) are completely excised in CD44s but can appear in the bigger variants in different combinations (Screaton et al., 1992; Tolg et al, 1993; Hofmann et al, 1991).
  • the variants differ in that different amino acid sequences are inserted at a certain site of the extracellular part of the protein. Such variants can be detected in various human tumor cells as well as in human tumor tissue. So, the expression of CD44 variants in the course of colorectal carcinogenesis has recently been investigated (Heider et al, 1993a).
  • CD44 variants are absent in normal human colon epithelium, and only a weak expression is detectable in the proliferating cells of the crypts. In later stages of the tumor progression, e.g. in adenocarcinomas, all malignancies express variants of CD44. Tissue expression of variant CD44 on a high level has also been shown in aggressive Non-Hodgkin lymphomas (Koopman et al, 1993).
  • v6 appears to play a special role especially in the course of metastatic spread (Rudy et al, 1993).
  • antibodies against v6 specific epitopes could prevent the settlement of metastatic cells and the growth of metastases (sammlungr et al, 1993).
  • colon carcinomas v6 expression correlates with tumor progression (Wielenga et al, 1993).
  • gastric carcinomas v6 expression is an important diagnostic marker to distinguish tumors of the intestinal type from those of the diffuse type (Heider et al, 1993b). In the latter two publications, v6 expression has been determined using antibodies against v6 specific epitopes.
  • CD44v6 has been shown to be a tumor-associated antigen with a favorable expression pattern in human tumors and normal tissues (Heider et al., 1995; Heider et al., 1996), it has been subject to antibody-based diagnostic and therapeutic approaches, (Heider et al., 1996; WO 95/33771; WO 97/21104).
  • chimeric antibodies although significantly better than mouse antibodies, can still elicit an anti-chimeric response in humans (LoBuglio A. F., Wheeler R. H., Trang J., Haynes A., Rogers K., Harvey E. B., Sun L., Ghrayeb J. and Khazaeli M. B. (1989) Mouse/human chimeric monoclonal antibody in man: Kinetics and immune response. Proc. Natl Acad. Sci. 86: 4220).
  • CDRs complementarity determining regions
  • an antibody humanised by CDR-grafting may still be able to elicit some immune reactions, such as an anti-allotype or an anti-idiotypic response, as seen even with natural human antibodies, the CDR-grafted antibody will be significantly less immunogenic than a mouse antibody thus enabling a more prolonged treatment of patients.
  • CDR-grafted antibodies have relatively poor binding characteristics as compared to their parent non-human antibodies because more amino acids than those within the CDR's are involved in antigen binding. In consequence, CDR-grafted antibodies with poor binding affinity are not regarded to be useful in therapy. Therefore, attempts have been made to create antibodies which combine the low immunogenicity of CDR-grafted antibodies with the good binding characteristics of the non-human parent antibodies. The concept was developed that, in addition to CDR-grafting, one to several amino acids in the humanized framework region have to be retained as residues of rodent donor origin for retaining binding affinity (Queen et al, (1989)Proc. Natl Acad. Sci. 86: 10029-10033).
  • the problem underlying the present invention was to provide an antibody with significantly better properties as compared to the known CD44v6 specific antibodies.
  • the present inventors have designed and generated a CD44v6 specific humanised antibody called BIWA8, which was both CDR-grafted and framework-mutated and had low immunogenicity combined with high affinity.
  • the present invention belongs to the field of oncology.
  • the invention relates to antibodies with specified sequence which are specific for an epitope which is coded by the variant exon v6 of the CD44 gene and to derivatives of said antibody.
  • the invention also provides nucleic acid molecules encoding said antibody proteins.
  • the invention furthermore pertains to methods for producing said antibody proteins.
  • the invention also provides pharmaceutical compositions comprising said antibody proteins.
  • the invention furthermore is concerned with the use in the manufacture of a medicament for the treatment of cancer.
  • Figure 1 Evaluation of relative binding affinities tested in a competitive cell ELISA.
  • IC50 concentrations of cMAb and hMAbs at which binding of mMAb BIWA 1 to attached A431 cells is reduced by 50%.
  • IC50 values relative to BIWA 2 are indicated.
  • FIG. 1 Biodistributions of co-injected 125 I- and 131 I-labeled CD44v6-specific MAbs (10 ⁇ Ci, 50 ⁇ g) in HNX-OE xenograft-bearing mice at 3 or 4 days p.i.
  • mice were bled, sacrificed, dissected and the radioactivity levels (%ID/g ⁇ s.e.m.) of tumor, blood and several organs were assessed.
  • %ID/g ⁇ s.e.m. radioactivity levels of tumor, blood and several organs were assessed.
  • Bid blood, Turn: tumor, Liv: liver, Spl: spleen, Kid: kidney, Hit: heart, Stm: stomach, Urn: ileum, Cln: colon, Blr: bladder, Str: sternum, Msc: muscle, Lng: lung, Skn: skin, Tng: tongue).
  • FIG. 3 Therapeutic efficacy of m Re-labeled CD44v6-specific MAbs in HNX-OE xenograft-bearing nude mice.
  • Mice received 300 ⁇ Ci I86 Re-U36 ( - * -, Fig A), 300 ⁇ Ci 186 Re-BIWA 1 (-*>-, Fig A), 300 ⁇ Ci 186 Re-BIWA 4 (- *-, Fig. B), 300 ⁇ Ci 186 Re-BIWA 2 (- 4 -, Fig. B), 400 ⁇ Ci 18 ⁇ Re-BIWA 4 (- * -, Fig. C), 400 ⁇ Ci Re-BIWA 8 (- ? -, Fig.
  • Fig A and B are the same.
  • the tumor size is expressed as the average tumor volume ( ⁇ s.e.m.) during treatment relative to the average tumor volume at the start of therapy.
  • Figure 4 Relationship between MAb dose administered and the AUC observed following BIWA 4 intravenous infusion to 10 patients in Part A of the study.
  • antibody molecule or “antibody protein” or “antibody” as used herein shall be considered equivalent.
  • “Complementarity determining regions of a monoclonal antibody” are understood to be those amino acid sequences involved in specific antigen binding according to Kabat (Kabat E. A., s Wu T. T., Perry H. M., Gottesman K. S. and Foeller C. (1991) Sequences of Proteins of Immunological Interest (5th Ed.). NIH Publication No. 91-3242. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.) in connection with Chothia and Lesk (Chothia and Lesk (1987) J. Mol Biol 196:901-917).
  • framework modifications refers to the exchange, deletion or addition of single or multiple amino acids in the variable regions surrounding the individual complementarity determining regions. Framework modifications may have an impact on the immunogenicity, producibility or binding specificity of an antibody protein.
  • the present invention provides an antibody molecule comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof. Both antibodies BIWA4 and BIWA 8 comprise the variable region of the heavy chain as characterized in amino acid sequence SEQ ID No. 1.
  • a according to the invention is a shorter antibody molecule, i.e. any polypeptide subset, characterized in that it is encoded by a shorter nucleic acid molecule than disclosed below, however still retains its antibody binding activity.
  • a bornfunctional variant of the antibody molecule according to the invention is an antibody molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar substrate specificity or cleavage of the substrate.
  • the term “more functional variant” also includes locallya fragment", locallyan allelic variant” faceda functional variant", pitchvariant based on the degenerative nucleic acid code” or towardschemical derivatives".
  • Such a yorkfunctional variant e.g. may carry one or several point mutations, one or several nucleic acid exchanges, deletions or insertions or one or several amino acid exchanges, deletions or insertions.
  • Said functional variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
  • a notedfunctional variant of the antibody molecule according to the invention is a antibody molecule which possesses a biological activity (either functional or structural) that is substantially similar to the antibody molecule according to the invention, i.e. a substantially similar target molecule binding activity.
  • the term “bulfunctional variant” also includes swirla fragment", frequentlyan allelic variant” passing a functional variant", usually human advant protein, or synthetic analogs.
  • An allelic variant is a variant due to the allelic variation, e.g. differences in the two alleles in humans. Said variant is still retaining its biological activity such as antibody target binding activity, at least in part or even going along with an improvement said biological activity.
  • a suspiciousvariant based on the degenerative of the genetic code is a variant due to the fact that a certain amino acid may be encoded by several different nucleotide triplets. Said variant is still retaining its biological activity such as antibody binding activity, at least in part or even going along with an improvement said biological activity.
  • a suspicious molecule may be the antibody molecule according to the invention fused to e.g. a reporter such as a radiolabel, a chemical molecule such as a toxin or a fluorescent label or any other molecule known in the art.
  • a reporter such as a radiolabel
  • a chemical molecule such as a toxin or a fluorescent label or any other molecule known in the art.
  • a wrinkle derivative is an antibody molecule according to the invention chemically modified or containing additional chemical moieties not normally being part of the molecule.
  • Such moieties may improve the molecule's activity such as target destruction (e.g. killing of tumor cells) or may improve its solubility, absorption, biological half life etc.
  • a molecule is substantially similar" to another molecule if both molecules have substantially similar structures or biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical.
  • CD44v6-specific antibody proteins according to the invention consist of the variable regions of both chains which are held together by the adjacent constant region. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced in the mean time by genetic engineering.
  • an antibody protein according to the invention is an F(ab')2 fragment, which may be prepared by proteolytic cleaving with pepsin.
  • an CD44v6-specific antibody molecule according to the invention is such an Fv fragment. Since these Fv-fragments lack the covalent bonding of the two chains by the cysteines of the constant chains, the Fv fragments are often stabilised. It is advantageous to link the variable regions of the heavy and of the light chain by a short peptide fragment, e.g. of 10 to 30 amino acids, preferably 15 amino acids.
  • an antibody protein of this kind is known as a single-chain-Fv (scFv).
  • scFv single-chain-Fv
  • Examples of scFv-antibody proteins of this kind known from the prior art are described in Huston et al. (1988, PNAS 16: 5879-5883). Therefore, in another preferred embodiment an CD44v6-specific antibody protein according to the invention is a single-chain-Fv protein (scFv).
  • scFv as a multimeric derivative. This is intended to lead, in particular, to recombinant antibodies with improved pharmacokinetic and biodistribution properties as well as with increased binding avidity.
  • scFv were prepared as fusion proteins with multimerisation domains.
  • the multimerisation domains may be, e.g. the CH3 region of an IgG or coiled coil structure (helix structures) such as Leucin-zipper domains.
  • the interaction between the VH/VL regions of the scFv are used for the multimerisation (e.g. di-, tri- and pentabodies).
  • an antibody protein according to the invention is an CD44v6-specific diabody antibody fragment.
  • diabody the skilled person means a bivalent homodimeric scFv derivative (Hu et al, 1996, PNAS 16: 5879-5883).
  • the shortening of the Linker in an scFv molecule to 5- 10 amino acids leads to the formation of homodimers in which an inter-chain VH/VL-superimposition takes place.
  • Diabodies may additionally be stabilised by the incorporation of disulphide bridges. Examples of diabody-antibody proteins from the prior art can be found in Perisic et al. (1994, Structure 2: 1217-1226).
  • minibody means a bivalent, homodimeric scFv derivative. It consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, most preferably IgGl as the dimerisation region which is connected to the scFv via a Hinge region (e.g. also from IgGl) and a Linker region. The disulphide bridges in the Hinge region are mostly formed in higher cells and not in prokaryotes.
  • an antibody protein according to the invention is an CD44v6-specific minibody antibody fragment. Examples of minibody-antibody proteins from the prior art can be found in Hu et al. (1996, Cancer Res. 56: 3055-61).
  • triabody means a: trivalent homotrimeric scFv derivative (Kortt et al.
  • miniantibodies which have a bi-, tri- or tetravalent structure and are derived from scFv.
  • the multimerisation is carried out by di-, tri- or tetrameric coiled coil structures (Pack et al., 1993 Biotechnology 11:, 1271-1277; Lovejoy et al. 1993 Science 259: 1288-1293; Pack et al., 1995 J. Mol. Biol. 246: 28-34).
  • CD44v6-specific multimerised molecule based on the abovementioned antibody fragments and may be, for example, a triabody, a tetravalent miniantibody or a pentabody.
  • the invention relates to an antibody molecule wherein the variable region of the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 1.
  • the invention relates to an antibody molecule comprising a variable region of the light chain as characterized by the amino acid sequence as defined in
  • Antibody BIWA4 as used herein comprises the variable region of the light chain as defined in amino acid sequence SEQ ID No. 2.
  • the invention relates to an antibody molecule wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 2.
  • the invention relates to an antibody molecule comprising a variable region of the light chain as characterized by the amino acid sequence as defined in
  • Antibody BIWA 8 comprises the variable region of the light chain as characterized in amino acid sequence SEQ ID No. 3.
  • the invention relates to an antibody molecule wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 3.
  • the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 and comprising a variable region of light chain as characterized by the amino acid sequence as defined in SEQ ID No. 2 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA4 comprises the variable region of the heavy chain as characterized in amino acid sequence SEQ ID No. 1 and variable region of the light chain as defined in amino acid sequence SEQ ID No. 2.
  • the invention relates to an antibody molecule according to the invention wherein the variable region of the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 1 and wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ED No. 2.
  • the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 1 and comprising a variable region of the light chain as characterized by the amino acid sequence as defined in SEQ ID No. 3 or a fragment, allelic variant, functional variant, glycosylation variant, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA8 comprises the variable region of the heavy chain as characterized in amino acid sequence SEQ ID No. 1 and variable region of the light chain as defined in amino acid sequence SEQ ID No. 3.
  • the invention relates to an antibody molecule according to the invention wherein the variable region of the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 1 and wherein the variable region of the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 3.
  • the invention relates to an antibody molecule comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Both antibodies BIWA4 and BIWA 8 comprise the variable region of the heavy chain as characterized in nucleic acid sequence SEQ ID No. 4.
  • the invention relates to an antibody molecule wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4.
  • the invention relates to an antibody molecule comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA4 as used herein comprises the variable region of the light chain as defined in nucleic acid sequence SEQ ID No. 5.
  • the invention relates to an antibody molecule wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 5.
  • the invention relates to an antibody molecule comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 6 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA 8 comprises the variable region of the light chain as characterized in nucleic acid sequence SEQ ID No. 6.
  • the invention relates to an antibody molecule wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 6.
  • the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 5 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA4 comprises the variable region of the heavy chain as characterized in nucleic acid sequence SEQ ID No. 4 and variable region of the light chain as defined in nucleic acid sequence SEQ ID No. 5.
  • the invention relates to an antibody molecule according to to the invention wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 5.
  • the invention relates to an antibody molecule according to the invention comprising a variable region of the heavy chain encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and comprising a variable region of the light chain encoded by the nucleic acid sequence as defined in SEQ ID No. 6 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA8 comprises the variable region of the heavy chain as characterized in nucleic acid sequence SEQ ID No. 4 and variable region of the light chain as defined in nucleic acid sequence SEQ ID No. 6.
  • the invention relates to an antibody molecule according to the invention wherein the variable region of the heavy chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 4 and wherein the variable region of the light chain is encoded by the nucleic acid sequence as defined in SEQ ID No. 6.
  • variable regions of the antibody proteins of the present invention are typically linked to at least a portion of the immunoglobulin constant region (F c ), typically that of a human immunoglobulin.
  • F c immunoglobulin constant region
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B cells (see Kabat et al., supra, and WO 87/02671).
  • the antibody proteins of the invention may contain all or only a portion of the constant region as long as they exhibit specific binding to the CD44v6 antigen.
  • the choice of the type and extent of the constant region depends on whether effector functions like complement fixation or antibody dependent cellular toxicity are desired, and on the desired pharmacological properties of the antibody protein.
  • the antibody 5 protein of the invention will typically be a tetramer consisting of two light chain/heavy chain pairs, but may also be dimeric, i.e. consisting of a light chain/heavy chain pair, e.g. a Fab or Fv fragment.
  • the invention relates to antibody proteins according to the o invention, characterised in that they have a variable light chain region and a variable heavy chain region, each joined to a human constant region.
  • the variable region of the light chain was joined to a human kappa constant region and the variable region of the heavy chain was joined to a human gamma- 1 constant region.
  • Other human constant regions for chimerizing light and heavy chains are also available to the expert.
  • s Humanization of the variable region of a murine antibody may be achieved employing methods known in the art.
  • EP 0239400 discloses grafting of the CDRs of a murine variable region into the framework of a human variable region.
  • WO 90/07861 discloses methods of reshaping a
  • 92/11018 discloses methods of producing humanized Ig combining donor CDRs with an 0 acceptor framework that has a high homology to the donor framework.
  • WO 92/05274 discloses the preparation of framework mutated antibodies starting from a murine antibody.
  • the invention relates to an antibody molecule according to s the invention characterised in that each of said variable region of the light chain and said variable region of the heavy chain region is separately joined to a human constant region.
  • the invention relates to an antibody molecule . according to the invention, wherein said human constant region of the light chain is a human kappa constant region. o In another more preferred embodiment, the invention relates to an antibody protein according to the invention, wherein said human constant region of the heavy chain is a human IgGl constant region. Preferred are also antibodies comprising the heavy chain as characterized by the amino acid sequence of SEQ ID No. 7 and/or the light chain as characterized by the amino . acid sequence of SEQ ID No. 8 or as characterized by the amino acid sequence of SEQ ID No. 9.
  • another important embodiment is an antibody molecule according to the invention comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ED No.
  • Antibody BIWA4 comprises the heavy chain as characterized in amino acid sequence SEQ ED No. 7 and variable region of the light chain as defined in amino acid sequence SEQ ED No. 8.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ED No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 8.
  • Antibody BIWA4 consists of the sequences as disclosed in amino acid sequence SEQ D No. 7 (heavy chain) and amino acid sequence SEQ ID No. 8 (light chain).
  • BIWA4 is a CDR-grafted antibody without framework modifications. Surprisingly, this antibody has, despite lower binding affinity, superior therapeutic efficacy, better biodistnbution and tumor uptake over the framework-mutated antibody BIWA8 (see example).
  • Another important embodiment is an antibody molecule according to the invention comprising a heavy chain as characterized by the amino acid sequence as defined in SEQ ID No. 7 and comprising a light chain as characterized by the amino acid sequence as defined in SEQ ED No.
  • Antibody BIWA8 comprises the heavy chain as characterized in amino acid sequence SEQ ID No. 7 and variable region of the light chain as defined in amino acid sequence SEQ ED No. 9.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 7 and wherein the light chain consists of the amino acids as characterized by the amino acid sequence of SEQ ID No. 9.
  • Antibody BIWA8 consists of the sequences as disclosed in amino acid sequence SEQ ED No. 7 (heavy chain) and amino acid sequence SEQ ED No. 9 (light chain).
  • BIWA8 is a CDR-grafted antibody with framework modifications. This antibody has significant higher binding affinity than BIWA4 (see example).
  • Preferred are also antibodies comprising the heavy chain as encoded by the nucleic acid sequence of SEQ ID No. 10 and/or the light chain as characterized by the nucleic acid sequence of SEQ ID No. 11 or as characterized by the nucleic acid sequence of SEQ ID No. 12. Said sequences include non-translated sequences and the leader sequence as cloned in vector ⁇ AD-CMVl/ ⁇ AD-CMV19.
  • an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No.
  • Antibody BIWA4 comprises the heavy chain as encoded by nucleic acid sequence SEQ ED No. 10 and variable region of the light chain as encoded by nucleic acid sequence SEQ ED No. 11.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ED No. 10 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ID No. 11.
  • antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 10 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ED No. 12 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA8 comprises the heavy chain as encoded by nucleic acid sequence SEQ ED No. 10 and variable region of the light chain as encoded by nucleic acid sequence SEQ ID No. 12.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID No.
  • antibodies comprising the heavy chain as encoded by the nucleic acid sequence of SEQ ID No. 13 and/or the light chain as characterized by the nucleic acid sequence of SEQ ED No. 14 or as characterized by the nucleic acid sequence of SEQ ED No. 15.
  • Said sequences include the leader sequence as cloned in vector N5KGlval.
  • an antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ID No. 13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ID No. 14 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA4 comprises the heavy chain as encoded by nucleic acid sequence SEQ ID No. 13 and variable region of the light chain as encoded by nucleic acid sequence SEQ ED No. 14.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ED No. 13 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ED No. 14.
  • antibody molecule according to the invention comprising a heavy chain as encoded by the nucleic acid sequence as defined in SEQ ED No. 13 and comprising a light chain as characterized by the nucleic acid sequence as defined in SEQ ED No. 15 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • Antibody BIWA8 comprises the heavy chain as encoded by nucleic acid sequence SEQ ED No. 13 and variable region of the light chain as encoded by nucleic acid sequence SEQ ED No. 15.
  • the invention relates to an antibody molecule according to the invention wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ED No. 13 and wherein the light chain is encoded by the nucleic acid sequence of SEQ ED No. 15.
  • antibody protein comprising the heavy and light chain as encoded by the nucleic acid sequence of SEQ ED No. 16.
  • Said sequence includes the leader sequence as cloned in vector N5KGlval.
  • an antibody molecule according to the invention comprising a heavy and light chain as encoded by the nucleic acid sequence as defined in SEQ ED No. 16 or a fragment, allelic variant, functional variant, variant based on the degenerative nucleic acid code, fusion molecule or a chemical derivative thereof.
  • BIWA4 comprises the heavy and light chain as encoded by nucleic acid sequence SEQ ED No.
  • the invention relates to an antibody molecule according to the invention wherein the heavy and light chain is encoded by the nucleic acid sequence of SEQ ED
  • the antibody proteins of the invention provide a highly specific tool for targeting therapeutic agents to the CD44v6 antigen. Therefore, in a further aspect, the invention relates to antibody proteins according to the invention, wherein said antibody protein is conjugated to a therapeutic agent.
  • therapeutic agents selected from the group consisting of radioisotopes, toxins, toxoids, inflammatogenic agents, enzymes, antisense molecules, peptides, cytokines, and chemotherapeutic agents are preferred.
  • radioisotopes gamma, beta and alpha-emitting radioisotopes may be used as a therapeutic agent, ⁇ -emitting radioisotopes are preferred as therapeutic radioisotopes.
  • radioisotopes selected from the group consisting of 186 Rhenium, 188 Rhenium, 131 Iodine and 90 Yttrium are particularly preferred as therapeutic agents conjugated to the antibody proteins of the invention.
  • a method as disclosed in WO 93/05804 may be employed for the radioiodination of an antibody of the invention.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherem said therapeutic agent is a therapeutic agent selected from the group consisting of radioisotopes, toxins, toxoids, pro-drugs and chemotherapeutic agents.
  • the formulae of said linkers are as follows:
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said therapeutic agent is linked to the antibody protein via MAG-2 GABA.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is a ⁇ -emitting radioisotope.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is selected from the group consisting of 186 Rhenium,
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is 186 Rhenium.
  • a further aspect of the present invention pertains to antibody proteins according to the invention, characterised in that they are labelled.
  • Such an CD44v6-specific labelled antibody allows for the localisation and/or detection of the CD44v6 antigen in vitro and/or in vivo.
  • a label is defined as a marker that may be directly or indirectly detectable.
  • An indirect marker is defined as a marker that cannot be detected by itself but needs a further directly detectable o marker specific for the indirect marker.
  • Preferred labels for practicing the invention are detectable markers.
  • a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin is most preferred.
  • a more preferred aspect of the present invention is an antibody protein according to the s invention, characterised in that it is labelled. More preferred is the antibody protein according to the invention, wherein said label is a detectable marker. Also more preferred is the antibody protein according to the invention, wherein the detectable marker is a detectable marker selected from the group consisting of enzymes, dyes, radioisotopes, digoxygenin, and biotin.
  • a further aspect of the present invention relates to antibody proteins according to the 0 invention, characterised in that they are conjugated to an imageable agent.
  • a more preferred aspect of the present invention is an antibody protein to the 5 invention conjugated to an imageable agent.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein the imageable agent is a radioisotope.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said radioisotope is a ⁇ -emitting radioisotope.
  • a more preferred aspect of the present invention is an antibody protein according to the invention, wherein said o radioisotope is 125 I.
  • a more preferred aspect of the present invention is an antibody protein conjugated to a radioisotope as described above, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi/mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.
  • Another preferred embodiment of the present invention is a pharmaceutical composition containing an antibody according to the invention and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of an AMPA glutamate receptor agonist, antagonist or modulator.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients (see also e.g. Remington's Pharmaceutical Sciences (1990), 18th ed. Mack Publ., Easton).
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.
  • an intravenous or other route e.g. systemically, locally or topically to the tissue or organ of interest, depending on the type and origin of the disease or problem treated, e.g. a tumor.
  • a systemic mode of action is desired when different organs or organ systems are in need of treatment as in e.g. systemic autoimmune diseases, or allergies, or transplantations of foreign organs or tissues, or tumors that are diffuse or difficult to localise.
  • a local mode of action would be considered when only local manifestations of neoplastic or immunologic action are expected, such as, for example local tumors.
  • compositions comprising antibody proteins of the present invention may be applied by different routes of application known to the expert, notably intravenous injection or direct injection into target tissues.
  • routes of application known to the expert
  • intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred.
  • a more local application can be effected subcutaneously, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue).
  • pharmaceutical antibody compositions may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
  • suitable pharmaceutical compositions comprising antibody preparations for the applications described above
  • the expert may use known injectable, physiologically acceptable sterile solutions.
  • aqueous isotonic solutions such as e.g. saline or corresponding plasma protein solutions are readily available.
  • the pharmaceutical compositions may be present as lyophylisates or dry preparations, which can be reconstituted with a known injectable solution directly before use under sterile conditions, e.g. as a kit of parts.
  • the final preparation of the antibody compositions of the present invention are prepared for injection, infusion or perfusion by mixing purified antibodies according to the invention with a sterile physiologically acceptable solution, that may be supplemented with known carrier substances or/and additives (e.g. serum albumin, dextrose, sodium bisulfite, EDTA).
  • a sterile physiologically acceptable solution that may be supplemented with known carrier substances or/and additives (e.g. serum albumin, dextrose, sodium bisulfite, EDTA).
  • carrier substances or/and additives e.g. serum albumin, dextrose, sodium bisulfite, EDTA.
  • the amount of the antibody applied depends on the nature of the disease.
  • the applied dose of a 'naked' antibody which is comprised in the pharmaceutical composition according to the invention may be between 0.1 and 100 mg/m 2 , preferably between 5 and 50 mg/m 2 per application, preferably 10 mg/m 2 to about 40 mg/m 2 , preferably 10 mg/m 2 to about 30 mg/m 2 , also preferably 20 mg/m 2 to about 30 mg/m 2 , and most preferably about 25 mg/m 2 body surface area. Also most preferred is an antibody protein dose of about 50 mg/m 2 body surface area.
  • the dose of radioactivity applied to the patient per administration has to be high enough to be effective, but must be below the dose limiting toxicity (DLT).
  • DLT dose limiting toxicity
  • MTD maximally tolerated dose
  • Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Clin. Oncol. 12: 1193-1203).
  • Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials should be administered at longer intervals, i.e., 4-24 weeks apart, preferable 12-20 weeks apart.
  • the artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.
  • the applied radioactivity dose will be in accordance with the guidelines outlined below.
  • the radioactivity dose per administration will be between 30 and 75 mCi/m 2 body surface area (BSA).
  • the amount of radiolabelled antibody in the pharmaceutical composition according to the invention preferably labelled with 186 Rhenium, 188 Rhenium, 99m Technetium, 13 iodine, or 90 Yttrium, most preferably labelled with 186 Rhenium, to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi/m 2 , preferably 50 mCi/m 2 .
  • the invention relates to a pharmaceutical composition, wherein the dose of said radiolabelled antibody according to the invention is MTD, preferably 50 mCi/m 2 . This is extensively exemplified in clinical studies as set out in examples 3 to 6.
  • a pharmaceutical composition according to the invention comprising an antibody protein conjugated to a radioisotope according to the invention as defined supra, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi/mg.
  • Preferred also is a pharmaceutical composition according to the invention comprising an antibody protein conjugated to a radioisotope according to the invention as defined supra, wherein said antibody or antibody derivative is in an aqueous solution at pH of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg/ml.
  • a preferred embodiment is a pharmaceutical composition according to the invention, further comprising one or more radioprotectants selected from the group of ascorbic acid, gentisic acid, reductic acid, erythrorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2-5-dihydroxy-l,4-benzenedisulfonic acid, povidone, inositol, and/or citrate.
  • radioprotectants selected from the group of ascorbic acid, gentisic acid, reductic acid, erythrorbic acid, p-aminobenzoic acid, 4-hydroxybenzoic acid, nicotinic acid, nicotinamide, 2-5-dihydroxy-l,4-benzenedisulfonic acid, povidone, inositol, and/or citrate.
  • Another preferred embodiment is a pharmaceutical composition according to the invention, wherein said antibody protein comprises an antibody molecule selected from the group of antibody molecules BIWA4 or BIWA8 as described supra linked to 186 Rhenium via MAG-2 GABA further comprising the radioprotectant ascorbic acid.
  • Another preferred embodiment of the present invention is the use of an antibody protein according to the invention in the manufacture of a medicament for treatment of cancer.
  • the present invention relates to the use of antibody proteins according to the invention conjugated to a therapeutic agent as described above for the treatment of cancer.
  • Cancer includes any disease associated with malignant growth such as solid tumors, sarcomas and leukemias. A necessary precondition for such diseases is the expression of CD44v6.
  • Cancer according to the invention includes, but is not limited to:
  • epithelial carcinomas including breast, lung, colorectal, head and neck, pancreatic, ovarian, bladder, gastric, skin, endometrial, ovarian, testicular, esophageal, prostatic and renal origin;
  • Bone and soft-tissue sarcomas Osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma (MFH), leiomyo sarcoma;
  • Hematopoietic malignancies Hodgkin's and non-Hodgkin's lymphomas, leukemias;
  • Neuroectodermal tumors Peripheral nerve tumors, astrocytomas, melanomas;
  • cancerous disease states associated with solid tumors include, but are not limited to: colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancers of the brain.
  • a preferred embodiment is the use of an antibody protein according to the invention wherein said cancer is selected from the group consisting of colorectal cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, pancreatic cancer and metastatic cancers of the brain.
  • an antibody protein according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the amount of antibody protein per application is between 0.1 and 100 mg/m 2 preferably between 5 and 50 mg/m 2 , preferably 10 mg/m 2 to about 40 mg/m 2 , preferably 10 mg/m 2 to about 30 mg/m 2 , also preferably 20 mg/m 2 to about 30 mg/m 2 , and most preferably about 25 mg/m 2 body surface area. Also most preferred is an antibody protein dose of about 50 mg/m 2 body surface area.
  • BSA body surface area
  • the invention relates to the use of an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein to antibody dose is 10, 20, 30, 40, 50 or 60 mCi/m 2 , most preferably 50 mCi/m 2 .
  • antibody dose is 10, 20, 30, 40, 50 or 60 mCi/m 2 , most preferably 50 mCi/m 2 .
  • an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein the antibody protein has specific activity of from about 0.5 to about 15 mCi/mg, or from about 0.5 to about 14 mCi mg, preferably about 1 to about 10 mCi/mg, preferably about 1 to about 5 mCi/mg, and most preferably 2 to 6 mCi/mg or 1 to 3 mCi mg.
  • an antibody protein conjugated to a radioisotope according to the invention as defined supra in the manufacture of a medicament for treatment of cancer, wherein said antibody or antibody derivative is in an aqueous solution at pH of from about 7 to about 8, and at a concentration of from about 0.5 to about 2.0 mg/ml.
  • the invention further relates to a method of cancer treatment, wherein an antibody protein according to the invention is administered once to several times to an individual in need thereof, said antibody protein selectively binds to CD44v6, destroys tumor cells via the therapeutic agent linked to the antibody protein and the therapeutic success is monitored.
  • Said antibody protein may be present as naked/unmodified antibody protein, modified antibody protein, such as e.g. fusion protein, or antibody protein conjugated to a therapeutic agent, which comprises contacting the tumor with an effective amount of said antibodies.
  • the method s of treating tumors as described above may be effective in vitro or in vivo. Cancer is any cancer as described above.
  • the applied dose of a 'naked' antibody depends on the nature of the disease.
  • the applied dose of a 'naked' antibody maybe between 0.1 and 100 mg/m 2 , preferably between 5 and 50 mg/m 2 per application, preferably 10 mg/m 2 to about 40 mg/m 2 , preferably 10 mg/m 2 o to about 30 mg/m 2 , also preferably 20 mg/m 2 to about 30 mg/m 2 , and most preferably about 25 mg/m 2 body surface area.
  • an antibody protein dose of about 50 mg/m 2 body surface area.
  • the dose of radioactivity applied to the patient per administration has be high enough to be effective, but must be below the dose limiting toxicity (DLT).
  • DLT dose limiting toxicity
  • MTD maximally tolerated dose
  • Application of radiolabeled antibody to cancer patients may then be carried out by repeated (monthly or weekly) intravenous infusion of a dose which is below the MTD (See e.g. Welt et al. (1994) J. Clin. Oncol 12: 1193-1203).
  • Multiple administrations are preferred, generally at weekly intervals; however, radiolabelled materials o should be administered at longer intervals, i.e., 4-24 weeks apart, preferable 12-20 weeks apart.
  • the artisan may choose, however, to divide the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging, e.g. from 1 day to 1 week.
  • the applied radioactivity dose will be in accordance with the guidelines outlined s below.
  • the radioactivity dose per administration will be between 30 and 75 mCi m 2 body surface area (BSA).
  • the amount of radiolabelled antibody, preferably labelled with 186 Rhenium, 188 Rhenium, 99m Technetium, 13 iodine, or 90 Yttrium, most preferably labelled with 186 Rhenium, to be applied to a patient is 10, 20, 30, 40, 50 or 60 mCi m 2 , preferably 50 mCi/m 2 .
  • the invention relates to a method of treatment, wherein o the radiolabelled antibody as described above is administered to a patient suffering from cancer, wherein the dose of said radiolabelled antibody is MTD, preferably 50 mCi/m 2 , whereby said cancer is prevented or treated. This is extensively exemplified in clinical studies as set out in examples 3 to 6.
  • the invention relates to a method according to the invention, wherein the tumor is a tumor selected from the cancer group consisting of colorectal cancers, non-small cell lung cancers, breast cancers, head and neck cancer, ovarian cancers, lung cancers, bladder cancers, pancreatic cancers and metastatic cancers of the brain.
  • a further aspect of the present invention is a nucleic acid, characterised in that it codes for an antibody protein according to the invention.
  • Said nucleic acid may be RNA or preferably DNA.
  • Said DNA molecule may be chemically synthesized.
  • suitable oligonucleotides can be synthesized with methods known in the art (e.g. Gait,M.J., 1984, Oligonucleotide Synthesis. A Practical ApproackJKL Press, Oxford, UK), which can be used to produce a synthetic gene. Methods to generate synthetic genes are known in the art (e.g. Stemmer et al.
  • a nucleic acid according to the invention is characterised in that it contains 5' or 3' or 5' and 3' untranslated regions.
  • the nucleic acid according to the invention may contain other untranslated regions upstream and/or downstream.
  • the untranslated region may contain a regulatory element, such as e.g. a transcription initiation unit (promoter) or enhancer.
  • Said promoter may, for example, be a constitutive, inducible or development-controlled promoter.
  • the constitutive promoters of the human Cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpes simplex promoter are examples of the constitutive promoters of the human Cytomegalovirus (CMV) and Rous sarcoma virus (RSV), as well as the Simian virus 40 (SV40) and Herpes simplex promoter.
  • CMV Cytomegalovirus
  • RSV40 Simian virus 40
  • Inducible promoters according to the invention comprise antibiotic-resistance promoters, heat-shock promoters, hormone-inducible maybeMammary tumour virus promoter" and the metallothioneine promoter.
  • a nucleic acid according to the invention is characterised in that it codes for a fragment of the antibody protein according to the invention. This refers to part of the polypeptide according to the invention.
  • a nucleic acid according to the invention is a nucleic acid as disclosed in SEQ ID SEQ ID No. 4, 5, 6, 10, 11, 12, 13, 14, 15, and/or 16. Most preferred, said nucleic acid is a nucleic acid of SEQ ID No. 16.
  • Another important aspect of the present invention is a recombinant DNA vector, characterised in that it contains a nucleic acid according to the invention.
  • said vector contains a nucleic acid as characterized in SEQ ID No. 4, 5, 6, 10, 11, 12, 13, 14, 15, and/or 16.
  • said vector contains the nucleic acid as characterized in SEQ ID No. 16.
  • examples are viral vectors such as e.g. Vaccinia, Semliki-Forest- Virus and Adenovirus.
  • Vectors for use in COS-cells have the SV40 origin of replication and make it possible to achieve high copy numbers of the plasmids.
  • Vectors for use in insect cells are, for example, E. coli transfer vectors and contain e.g. the DNA coding for polyhedrin as promoter.
  • Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is an expression vector.
  • Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is vector pAD-CMV or a functional derivative thereof. Such derivatives are e.g. pAD-CMVl, pAD-CMV19 or pAD-CMV25.
  • Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is the of SEQ ED No. 17 or a functional derivative thereof.
  • Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is the of SEQ ID No. 18 or a functional derivative thereof.
  • said vectors comprise one or several of the nucleic acid molecules as characterized in SEQ ID No. 4, 5, 6, 10, 11, 12, 13, 14, 15, and/or 16.
  • a vector as disclosed in US 5648267 A or US US 5733779 A comprising a nucleotide sequence according to the invention.
  • said vector comprises one or several of the nucleic acid molecules as characterized in SEQ ED No. 4, 5, 6, 10, 11, 12, 13,
  • Another preferred aspect of the present invention is a recombinant DNA vector according to the invention, characterized in that it is vector N5KGlVal or a derivative thereof.
  • Another important aspect is a host, characterised in that it contains a vector according to the invention.
  • the eukaryotic host cells according to the invention include fungi, such as e.g. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, insect cells (e.g. from Spodopterafrugiperda Sf-9, with a Baculovirus expression system), plant cells, e.g. from Nicotiana tabacum, mammalian cells, e.g. COS cells, BHK, CHO or myeloma cells.
  • fungi such as e.g. Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma
  • insect cells e.g. from Spodopterafrugiperda Sf-9, with a Baculovirus expression system
  • plant cells e.g. from Nicotiana tabacum
  • mammalian cells e.g. COS cells, BHK, CHO or myeloma cells.
  • the antibody proteins according to the invention are particularly well folded and glycosylated.
  • Mammalian host cells preferably CHO or COS cells are preferred, e.g. a CHO
  • Another preferred aspect is a host according to the invention according to the invention, characterised in that it is a BHK, CHO or COS cell, most preferred
  • CHO DG44 or CHO-K1 ATCC CCL-61 cells.
  • Another preferred aspect is a host according to the invention, characterised in that it is a bacteriophage.
  • Another preferred aspect is a host according to the invention, characterised in that it is a prokaryotic host cell.
  • prokaryotic host cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.
  • the invention further relates to a process for preparing an antibody protein according to the invention, characterized in that it comprises the following steps: a host according to the invention is cultivated under conditions in which said antibody protein is expressed by said host s cell and said antibody protein is isolated.
  • the antibody according to the invention may be produced as follows. Nucleic acid molecules coding for the light chain and the heavy chain may be synthesised chemically and enzymatically by standard methods. First, suitable oligonucleotides can be synthesized with methods known in the art (details supra). Methods to generate synthetic genes from oligonucleotides are known in the art (details supra).
  • the host cell preferably is a malian host cell (details supra), e.g. a COS, CHO, or BHK cell, more preferably a Chinese hamster ovary (CHO) cell, The host cell then is cultured in a suitable culture medium under s conditions where the antibody is produced, and the antibody is then isolated from the culture according to standard procedures. Procedures for production of antibodies from recombinant DNA in host cells and respective expression vectors are well-known in the art (see e.g.
  • the invention preferably relates to a process according to the invention, characterised in that o said host is a mammalian cell, preferably a CHO or COS cell.
  • the invention preferably relates to a process according to the invention, characterised in that said host cell is co-transfected with two plasmids which carry the expression units for the light or the heavy chain.
  • the following examples serve to further illustrate the present invention; but the same should 5 not be construed as limiting the scope of the invention disclosed herein.
  • the epitope recognized by BIWA 1 has been mapped to amino acids 360-370 in domain v6 of CD44 (numbering according to Kugelman et al. (1992)).
  • the batch used for the present studies was obtained after purification on protein- G-Sepharose and dialysis against PBS.
  • MAb U36 (IgGl) was derived after immunization of mice with the HNSCC cell line UM-SCC- 22B and recognised a different epitope within CD44v6 as BIWA 1.
  • U36 was purified from a concentrated tissue culture supernatant by affinity chromatography on protein-A-Sepharose and further purified on Q-Sepharose.
  • mRNA was isolated from the BIWA 1 hybridoma cell line by use of the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden).
  • cDNA from the variable heavy (VH) and variable light (V ) chain was generated by RT-PCR.
  • the fragments were cloned into the TA cloning vector pCR II (Invitrogen, Groningen, The Netherlands) and sequenced.
  • Two expression vectors derived from the plasmid pAD CMVl (Himmler et al., 1990) were constructed carrying the constant region of human gamma- 1 and the constant region of the human kappa light chain, respectively.
  • the V H and V fragments of BIWA 1 were cloned into the corresponding expression vectors in front of the constant regions.
  • the chimeric antibody was named cMAb BIWA 2.
  • Humanized versions of the BIWA 1 heavy and light chain variable regions were cloned in front of the immunoglobulin constant regions of the above mentioned expression vectors.
  • the human variable regions used were derived for the heavy chain from the human immunoglobulin fragment accession number S31669 of databank GenPept and for the light chain from the human immunoglobulin HUMIGKAX (rearranged anti-myelin kappa chain), Genbank accession number M29469.
  • the resulting MAbs were named hMAb BIWA 4 and BIWA 8, respectively.
  • BIWA 8 contained two amino acids of the murine parent antibody within the light chain framework 2 while BIWA 4 did not contain murine residues in the framework.
  • Recombinant MAbs were stably expressed in dihydrofolate reductase deficient Chinese hamster ovary cells by electroporation with heavy and light chain expression plasmids. Cells were seeded into 96 well microtiter plates at densities of 500 and 100 cells/well in selection medium ( ⁇ -MEM with 10% dialyzed fetal calf serum).
  • cMAb BIWA 2, hMAb BIWA 4 and hMAb BIWA 8 (prediluted to 10 ⁇ g/ml) were applied in 1:2 serial dilutions (8 steps) in 100 ⁇ l/well in PBS/0.5%BSA/0.05% Tween 20 (assay buffer) and incubated for 30 min at room temperature.
  • Control samples contained prediluted samples only, without BIWA 1 (0% control) or BIWA 1 only without any competing antibodies (100% control). After washing three times with PBS/0.05% Tween 20 (washing buffer), 100 ⁇ l of the secondary antibody (peroxidase-conjugated goat anti-mouse Fc, diluted 1:15,000 in assay buffer, DAKO Copenhagen, Denmark) was added for detection of mMAb BIWA 1, and plates were incubated for 1 h at room temperature on an orbital shaker. After washing three times with washing buffer, the plates were developed with 100 ⁇ l/well tetramethylbenzidine substrate solution (Kierkegaard and Perry Laboratories, Gaithersburg, USA). The reaction was stopped after 15 min with 50 ⁇ l/well 1 M phosphoric acid. Absorbance was measured in an ELISA plate reader at 450 nm (reference 610-690 nm).
  • Rhenium-186-Iabeled MAbs were prepared according to a multistep procedure using the chelate S-benzoylmercaptoacyltriglycine (S-benzoyl- MAG3) as previously described (Van Gog et al., 1997a). In this procedure a solid-state synthesis for the preparation of 186 Re-MAG3 is followed by esterification with 2,3,5,6- tetrafluorophenol (TFP) and conjugation of the reactive 1S6 Re-MAG3-TFP ester to the MAb. After conjugation the 186 Re-labeled MAb was purified on a PDlO-column. After removal of unbound 186 Re the radiochemical purity always exceeded 98%.
  • S-benzoyl- MAG3 2,3,5,6- tetrafluorophenol
  • Binding-assay for radiolabeled antibodies In vitro binding characteristics of the labeled MAbs used in the biodistribution and therapy studies were determined in an immunoreactivity assay essentially as described previously (Van Gog et al, 1997a). To test the binding of iodinated or 186 Re-labeled MAbs, UM-SCC-llB cells fixed in 0.1% glutaraldehyde were used. UM-SCC-llB cells were kindly provided by Dr. T.E. Carey, University of Michigan, Ann Arbor, MI. Five serial dilutions (ranging from 5 x 10 6 cells per tube to 3.1 x 10 5 cells per tube) were prepared with 1% BSA in PBS.
  • HNSCC-bearing nude mice Biodistribution studies in HNSCC-bearing nude mice.
  • nude mice bearing subcutaneously implanted human HNSCC xenografts HNX-OE
  • Female mice Hsd: Athymic nu/nu, 25-32 g, Harlan CPB, Zeist, The Netherlands
  • Three biodistribution experiments were conducted with mice bearing 1 or 2 tumors ranging from 30 to 470 mm 3 .
  • the antibody dose (total dose 100 ⁇ g per mouse) was chosen high enough to prevent rapid isotype-related elimination of the MAb from the blood (Sharkey et al., 1991, Van Gog et al., 1997b), and low enough to prevent antigen o saturation in the tumor.
  • mice were anaesthetized, bled, killed and dissected. Besides the tumors, the following organs were removed: liver, spleen, kidney, heart, stomach, ileum, colon, bladder, sternum, muscle, lung, skin and tongue.
  • radioactivity in tumors blood and organs was counted in a dual-isotope gamma counter (LKB- s Wallace 1282 CompuGamma), with automatic correction for the 131 I-comptons in the 125 I window setting. Radioactivity uptake in these tissues was calculated as the percentage of the injected dose per gram of tissue (%ED/g).
  • mice were routinely housed under specific-pathogen-free conditions, in sterile cages in a humidity- and temperature controlled clean room, classification 0 2000 according to the Federal Standard 209d.
  • mice were transported to a Radio Nuclide Center, and sterile radioimmunoconjugates were administered under aseptic conditions in a laminar flow hood.
  • Radioimmunotherapy studies in nude mice Animal RIT studies were performed to s compare the therapeutic efficacy of the different MAbs labeled with 186 Re. The immunoreactive fractions of the conjugates always exceeded 75 %.
  • Three therapy experiments were conducted with mice bearing 1 or 2 HNX-OE tumors ranging from 45 to 195 mm 3 .
  • the 186 Re doses were chosen at the maximum tolerated dose (MTD) level (i.e. 400 ⁇ Ci) or lower (300 ⁇ Ci).
  • MTD level is defined as the dose resulting in 5-15% body weight loss.
  • mice were given a single i.v.
  • mMAb U36 and hMAb BIWA 4 have lower k a and higher k , resulting in markedly lower dissociation constants (factors 35.0 and 10.5, respectively).
  • hMAb BIWA 8 containing murine residues in the light chain framework region 2, shows a marked decrease of kd resulting in increased affinity.
  • Murine BIWA 1 1.3 x 4.2 x lO "5 3.2 x lO "10 1.0
  • cMAb BIWA 2 was the most effective competitor, followed by hMAb BIWA 8 and hMAb BIWA 4. Similar results (not shown) were obtained with two other human HNSCC cell lines (FaDu and LICR-LON-HN5).
  • mMAb U36 has a 35.0 fold lower affinity than mMAb BIWA 1 (experiment 1)
  • hMAb BIWA 4 has a 14.0- fold lower affinity than cMAb BIWA 2 (experiment 2)
  • hMAb BIWA 4 has a 4.0-fold lower affinity than hMAb BIWA 8 (experiment 3).
  • the immunoreactive fractions of all iodinated MAbs were at least 74% after extrapolation (Table 2).
  • biodistributions in experiment 1 were determined at day 1, 2, 3 and 7 after injection; biodistributions in experiments 2 and 3 were determined at day 1, 2, 4 and 7 days after injection.
  • the calculated average %ID/g of tumor and blood of all three experiments are given in Table 3.
  • hMAb BIWA 4 while having the lower affinity, showed a significantly higher tumor uptake (p ⁇ 0.001) than cMAb BIWA 2 and hMAb BIWA 8 at all time points (Table 3).
  • MAb levels in blood and normal tissues were similar for these pairs of MAbs at 1, 2, and 4 days p.i..
  • BIWA 2 and BIWA 8 levels in blood and most of the organs were significantly lower (p ⁇ 0.05) than BIWA 4 levels, indicating more rapid clearance of these MAbs from the blood/body.
  • a 45% higher tumor uptake of BIWA 4 in comparison with BIWA 2 is illustrated by Figure 2B, while a 20% higher tumor uptake of BIWA 4 in comparison with BIWA 8 is illustrated by Figure 2C, for the 4 days post injection time points.
  • mice were treated with either 400 ⁇ Ci 186 Re-BIWA 4 or BIWA 8, which resulted in a decrease of the relative tumor volume to a minimum of 80 ⁇ 62 % and 98 ⁇ 81%, respectively, at day 19. Thereafter, tumors started regrowth. J These data indicate that the low affinity MAb BIWA 4 is more effective in RIT than the high affinity Mabs cBIWA 2 and BIWA 8.
  • glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65: 13-24 (1991).
  • Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc. Natl Acad. Sci. U.S.A. 89: 12160-12164 (1992).
  • This example shows the details of sequences, e.g. the position of cloning sites, leaders and untranslated regions.
  • V ss Apparent volume of distribution under steady-state conditions
  • V z Apparent volume of distribution during the terminal phase
  • the general aim of the present study was to assess the safety and tolerabiUty of intravenously administered 99m Tc and 186 Re-labelled hMAb BIWA 4, to confirm preferential accumulation in the tumour of 99m Tc-labelled hMAb BIWA 4, to determine the maximum tolerated radiation dose of 18 ⁇ Re-labelled hMAb BIWA 4 and to propose a safe dose for phase II development.
  • the study was divided into two parts:
  • the MTD for single dose treatment was to be identified first before more patients were entered to define the MTD for a second treatment with 186 Re-labelled hMAb BIWA 4.
  • Part A evaluated the optimal dose of cold BIWA 4 and quantified tumour uptake while Part B investigated the maximum tolerated dose of 186 Re- BIWA 4. o 3.1.1 Part A:
  • RadiolabeUed antibody was injected and the patient was observed for occurrence of adverse events. Radioimmunoscintigraphic scans were performed 21 hours post infusion (p.i.) prior to 5 surgery. Patients underwent surgery 48 hours (hrs) after the infusion of the radioactively labelled hMAb BIWA 4. The pathologist investigated the neck dissection specimen to determine the exact tumour load. Moreover, the amount of 99m Tc in biopsies from tumour site(s) and normal tissues in the surgical specimen was measured. Tumour sites and tumour infiltrated nodes were examined for the presence of CD44v6 antigen by immunohistochemical o techniques.
  • the first three patients were administered 2 mg hMAb BIWA 4 labelled with 20 mCi 99m Tc combined with 23 mg unlabelled hMAb BIWA 4.
  • the second group of three evaluable patients was administered 2 mg hMAb BIWA 4 labeUed with 20 mCi 99m Tc combined with 48 mg unlabelled hMAb BIWA 4 and the third group was administered 2 mg of the labelled antibody combined with 98 mg of unlabelled antibody. 5 Pharmacokinetic assessments were done at the specified timepoints.
  • This part of the clinical trial was an open uncontroUed, dose escalation study. It was designed to assess the safety and tolerabiUty of 186 Re-labelled hMAb BIWA 4, to determine the maximum tolerated dose (MTD) of intravenously administered 186 Re-labelled hMAb BIWA 4 o and to determine the preUminary therapeutic effects of 186 Re-labelled hMAb BIWA 4 in head s and neck cancer patients for whom no curative options were available. In addition the pharmacokinetic profile of 186 Re-labelled hMAb BIWA 4 was assessed.
  • hMAb BIWA 4 All patients entering into this part of the trial received the dose of hMAb BIWA 4, which was selected basing on the results of Part A of the study.
  • the hMAb BIWA 4 was labelled with escalating doses of 186 Re. At the lower dose levels (toxicity observed did not exceed grade 1) o two patients were entered per dose group and at the higher levels (> grade 2 toxicity) a minimum of three patients were entered per dose group. AU patients were evaluated to determine the safety of the administered hMAb BIWA 4.
  • tumour s locations were routinely investigated by the Department of Otolaryngology to determine the extent of the tumour. This included physical examination and CT or MRI scanning of tumour s locations.
  • Re-labelled antibody was injected with escalating radiation doses: patients in the first dose group received a radiation dose of 20 mCi/m 2 , after which the dose for patients in the subsequent dose groups was escalated by 10 mCi/m 2 increments until the MTD was reached. Patients were observed for occurrence of adverse events. Radioimmunoscintigraphic scans 0 were performed.
  • Blood samples for pharmacokinetics were drawn at the end of the infusion and at 5, 10, 30 minutes and 1, 2, 4, 16, 21, 48, 72 hrs after the end of the infusion, and on day 7 (144-hrs p.i. including a serum sample for HAHA and soluble CD44v6 assessment).
  • Urine for pharmacokinetics was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs and 12-24 hrs during the first 24 hrs and a 24-hr sample until 48 hrs p.i.
  • the patient was operated at 48 hrs p.i. and stayed in the hospital for post-operative care.
  • Urine for pharmacokinetics was collected from 0-4 hrs, 4-8 hrs, 8-12 hrs and 12-24 hrs during o the first 24 hrs and in 24-hr samples until 96 hrs p.i.
  • Planar images of the head and neck region were made at 21, 48 (optional), 72 and 144 hrs p.i. and optionally at two weeks p.i., if counting statistics permitted.
  • s SPECT imaging was only performed seventy-two hours after the infusion.
  • Part A o Patients visited the outpatient clinic six weeks after the infusion. A physical examination was performed, safety blood and urine samples collected, body weight and vital signs measured and adverse events recorded. Blood samples for pharmacokinetics, HAHA assessment and soluble CD44v6 were also obtained. A pregnancy test was required for women with childbearing potential. Follow-up of adverse events was recorded. Any changes in the concomitant therapy 5 were recorded.
  • the aim of the present study was to assess the preferential accumulation of 99m Tc-labelled BIWA 4 in the tumour (Part A) and to evaluate the maximum tolerated dose of 186 Re-BIWA 4 (Part B) as well as the pharmacokinetics of BIWA 4 in patients suffering from advanced head s and neck cancer (Part A and B).
  • the dose of BIWA 4 administered was based on previous results with mMAb BIWA 1 indicating that a dose of 50 mg yielded a high and selective uptake in tumour tissue with a low uptake in non-tumour tissues and the results from Part A of the trial (see also section 3.4.4.1).
  • Part A Patients destined for surgery by means of a neck dissection (Part A) or:
  • tumour deposits had to be measurable either clinically or by one or more radiological technique(s) (CT, MRI, bone scintigraphy). Because RIT was expected to be more effective in smaller size tumour deposits, patients with lesions measuring ⁇ 3 cm in greatest dimension were preferred (Part B).
  • White blood cell count ⁇ 3000/mm 3 granulocyte count ⁇ 1500/mm 3 or platelet count ⁇ 100,000 /mm 3 .
  • the infusion had to be terminated immediately if the patient developed tachycardia (pulse rate greater than 120 per minute), hypotension (blood pressure less than 100 mm Hg systolic), respiratory distress, chest pain, or any symptoms intolerable to the patient.
  • tachycardia pulse rate greater than 120 per minute
  • hypotension blood pressure less than 100 mm Hg systolic
  • respiratory distress chest pain, or any symptoms intolerable to the patient.
  • Patients in Part A were administered 99m Tc-BIWA 4 at a radioactivity dose of 20 mCi.
  • the dose of BIWA 4 administered was 25 mg, 50 mg or 100 mg for three patients each.
  • the drug was administered intravenously as a single dose.
  • Patients in Part B received 50 mg BIWA 4 labelled with Rhenium 186.
  • the lowest radioactivity dose was 20 mCi/m 2 which was increased in dose tiers of 10 mCi/m 2 .
  • the trial drug was administered intravenously as a single dose.
  • Chiffre number BIWA 4 LOI 99 ID 1 A
  • BIWA 4 was administered as radioconjugate Unked with 99m m ⁇ Tc.
  • Linker molecule was MAG3.
  • MAG3 was purchased from Mallinckrodt, Petten, The Netherlands.
  • BIWA 4 was administered as radioconjugate linked with 186 Re.
  • Linker molecule was MAG3.
  • MAG3 was purchased from Mallinckrodt, Petten, The Netherlands.
  • hMAb BIWA 4 Reactivity of hMAb BIWA 4 was found to be essentiaUy restricted to squamous epitheUa. As demonstrated by the previous RIS study with murine monoclonal antibody (mMAb) BIWA 1, s reactivity with normal squamous epithelium was not a limiting factor for utility in tumour targeting with respect to tumour uptake.
  • mMAb murine monoclonal antibody
  • the antibody was labelled with 99m Tc or 186 Re according to a method described by Fritzberg et al. (R96-2106: See protocol Appendix IV) which was modified according to Visser et al. 0 (R96-2094) and Van Gog et al. (R96-2111).
  • the preparation for infusion was re-evaluated in a second binding assay.
  • the antibody preparation was labeUed with 131 I.
  • the immunoreactive fraction in at least one of both assays had to be larger than 60% for each patient to be evaluable and for the study to continue with the next patient.
  • BIWA 4 used for radiolabelling with 99m Tc and 186 Re in this study is a well-characterised monoclonal antibody product which was produced by Chinese hamster ovary (CHO)-cell culture fermentation.
  • a master ceU bank was established under Good Manufacturing Practice (GMP) conditions and thoroughly examined for microbiological status (bacteria, fungi, mycoplasma) as well as viral status (adventitious viruses, retroviruses). With the exception of endogenous retroviruses, which were known to be present in most CHO cells, no contaminants were detected.
  • GMP Good Manufacturing Practice
  • the final hMAb BIWA 4 to be used for further radiolabelling was analysed in great detail and was proven to be a highly pure (SDS page, isoelectric focusing), sterile solution containing minimal endotoxin levels ( ⁇ 0.01 EU/mg). Results of testing for pyrogenicity were conform to European Pharmacopoea standard. The equality of the BIWA 4 product from the manufacture for pre-clinical and clinical supplies was demonstrated by analytical results.
  • Radiolabelling of hMAb BIWA 4 with 99m Tc or 186 Re was performed by the department of Otolaryngology and Head & Neck Surgery of the Vrije Universiteit University Medical Center according to Standard Operating Procedures (SOPs). Sterility of the final product was guaranteed. Absence of endotoxins was tested during validation runs. For drug administration to a patient in the University Hospital Nijmegen the proper amount of radiolabelled hMAb BIWA 4 was transported in a special container directly after preparation to the study centre in Nijmegen. Twenty-four hours were allowed between labelling of the compound and administration to the patient. s See also appendix 16.1.6 for an allocation of the individual Rhenium batches to the patients.
  • BIWA 4 was supplied by Boehringer Ingelheim The Netherlands. It was produced by Boehringer Ingelheim, Germany using a GMP manufacturing and purification process and filled in vials as a sterile, non-pyrogenic solution containing 25 mg hMAb BIWA 4 in 5 mL o isotonic PBS, pH 7.2. Examples of the vial labels of the native and also of the labelled antibody were included in the Clinical Trial Manual.
  • the unlabelled hMAb BIWA 4 had to be stored in the hospital pharmacy in a limited access area for study materials at a monitored temperature between +2 and +8°C.
  • hMAb BIWA 4 Since hMAb BIWA 4 had never been administered to patients before, it was essential to be 5 informed about its safety and biodistribution before starting RIT trials. Its biodistribution might strongly depend on the MAb dose used for tumour targeting and would need careful consideration. On the basis of MAb protein dose escalation studies with the low affinity anti- CD44v6 mMAb U36 and the high affinity anti-CD44v6 mMAb BIWA 1, it was anticipated that the optimal dose was in the range of 25-100 mg, with 50 mg being optimal for mMAb 0 U36.
  • hMAb BIWA 4 dose of 50 mg was calculated as most suited for development. Part A of this study was performed to confirm the tumour preferential uptake of hMAb BIWA 4 at the three dose levels tested (25 mg, 50 mg and 100 mg). It was expected that the tumour uptake (expressed as percent injected dose per kilogram, % ID/kg) and tumour to non-tumour uptake ratio for these three dose levels would not differ much. If this was the case the hMAb BIWA 4 dose to be used for Part B of the study was 50 mg. However, if there was a clinicaUy relevant difference between these dose levels, which favoured one over the other levels, the dose level with the best pattern of biodistribution would have been selected.
  • the hMAb BIWA 4 dose selected in Part A of the study was labelled with escalating doses of 186 Re.
  • the dose was escalated with 10 mCi/m 2 increments. Two evaluable patients were entered at the lower dose levels. When > grade 2 drug-related toxicity according to the CTC was observed a minimum of three patients were treated per dose level. s Before entering patients at a next higher dose level it had to be sure that the patients at the ongoing dose level did not experience dose limiting toxicity (DLT) defined as: drug-related CTC grade 3 non-haematologic toxicity or drug-related CTC grade 4 haematologic toxicity • excluding nausea and vomiting without adequate antiemetic treatment. For this purpose aU patients at such an ongoing dose level had to be observed long enough to ensure that possibly o induced toxicity was reversible.
  • DLT dose limiting toxicity
  • Anaphylaxis was considered to be the most serious potential side effect and would have mandated immediate cessation of antibody infusion and the institution of appropriate resuscitative measures. Precautions to be taken were: Resuscitation equipment, within reach: anti-histaminics, corticosteroids and epinephrine. Any patient experiencing this type of adverse 5 reaction was not allowed to receive additional monoclonal antibody.
  • the study medication was given as a single intravenous infusion.
  • the compUance was verified with pharmacokinetic assessments and with radioimmunoscintigraphic images.
  • GTT Gamma Glutaryl Transpeptidase
  • bilirubin urea
  • uric-acid thyroid stimulating hormone (TSH), haemoglobin (Hb), haematocrit (Ht), mean corpuscular volume (MCV), reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the screening visit or on day 1 pre-infusion at 21, 48 and 144 hrs p.i. and at six weeks post infusion (p.i.)-
  • HAHA was assessed in a serum sample obtained at the screening visit, after one week (144 hrs) and after six weeks
  • the SPECT scan and planar scan was done once 21 hrs p.i.
  • the pre-infusion assessment had to occur no more than 3 weeks prior to the actual infusion.
  • Blood samples were tested for: Glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, sGOT, sGPT, Alkaline Phosphatase, GGT, bilirubin, urea, uric-acid, TSH, Hb, Ht, MCV, reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the screening visit or on day 1 pre-infusion, at 21 hrs p.i., at 48 hrs p.i. and at 144 hrs p.i.. During weeks 2-6 p.i. safety blood samples were obtained at least weekly.
  • HAHA was assessed in a serum sample obtained at the screening visit, at 144 hrs and six weeks post infusion.
  • planar scan was done at 21, 48 (optional), 72, 144 hrs p.i. and optionally at two weeks p.i.
  • the SPECT scan was done at 72 hrs p.i.
  • Soluble CD44v6 was measured from a serum sample obtained pre-infusion, at 21, 48 and 144 hrs p.i and six weeks p.i. x : Disease assessment was to be repeated every 6 weeks until disease progression or loss to follow-up. CT thorax was done at baseline and repeated at follow-up if there were abnormalities. 3.5.1.1 Radioimmunoscintigraphy and dosimetry
  • RIS radioimmunoscintigraphy
  • the initial activity of the caUbration source had to be 100-200 MBq 186 Re and this source had to be used during all whole body imaging studies.
  • the energy-window and peak settings, the scanspeed, the scanlength, the scanning date, the time of starting the scan and the scan duration had to be reported.
  • the anterior-posterior thickness of the neck and of the abdomen had to be measured, while the patient was in supine position on the scanning table.
  • SPECT images were obtained using a double headed rotating gamma camera equipped with a low energy coUimator. Acquisition required at least thirty minutes. Twenty percent symmetric windows were centred at the 137 keV photon peaks.
  • Planar imaging included the following minimal requirements: matrix 128x128 (detail) or 256 x 256 (whole body) and a minimum of 400000 counts with a maximum acquisition time of 10 minutes for detail and 60 minutes for whole body.
  • SPECT imaging included the following minimal requirements: 64 images, matrix size 64 x 64, 360 degree circular orbit, 60 second acquisitions per angle.
  • ROI's regions of interest
  • irregular ROI's around the organs which accumulated 186 Re (e.g. Uver, spleen and left kidney) and around the tumour had to be drawn.
  • One or more representative background regions had to be drawn. These regions had to be mirrored to the posterior images.
  • the s number of pixels and the counts per pixel in each region had to be reported.
  • the number of counts in the regions for aU imaging time points had to be recorded digitally in a spreadsheet.
  • the amount of activity in the organs, tumour and the total body was estimated from the geometric mean counts in the ROI's of the anterior and posterior views. Background and o attenuation correction were applied when indicated.
  • the activity in the urine was not used to estimate the absorbed dose in the bladder as originaUy planned. Instead the dynamic bladder model was used. The residence times in the organs and the rest of the body were calculated and imported in the MIRDOSE3 program.
  • a filtered back projection algorithm was used for tomographic image reconstruction using a ramp filter.
  • CD44v6 antigen expression was assessed by immunohistochemistry using cryostate sections, which were first incubated with mMAb BIWA 1, foUowed by anti-mouse Immunoglobulin G (IgG) secondary reagent. The surgical specimen and the biopsies were investigated histo/cytopathologically.
  • IgG Immunoglobulin G
  • Biopsies of primary tumour, suspect lymph node, and if possible from normal tissues in s the surgical specimen like normal mucosa, normal lymph node, fat and muscle were taken.
  • AU biopsies were weighed and the amount of 9 m Tc was measured.
  • AU data were converted to percentages injected dose/kilogram tissue. Specific uptake of radioactivity into tumour was evaluated by comparing %ID/kg tumour with %ID/kg normal tissue.
  • a specimen radiograph was made to show the exact size and location of the lymph nodes involved. This radiograph was made while the specimen was being immersed in ethanol 96%, which has the same X-ray absorption as fat. 5 6. AU the nodes visualised with the X-ray were indicated on the Polaroid and specimen radiograph.
  • tumour response was assessed with tumour measurements as assessed clinically and/or with CT, MRI or bone scintigraphy investigations. Evaluation was done according to response criteria of the World Health Organisation (WHO). See Appendix VI of the protocol, (R96-0941). o 3.5.1.4 Physical Examination
  • CT computed tomography
  • MRI magnetic resonance imaging
  • bone scintigraphy For tumour involvement of the head and neck region MRI was generally preferred. In case there were bone lesions, CT was preferred.
  • CT was preferred.
  • 5 Part B all radiological disease assessment parameters obtained at baseline were repeated six weeks after the infusion and every six weeks until progression or lost to follow-up.
  • a CT thorax was obtained at baseline, and was repeated at follow-up if there were tumour lesions in the thorax. Ultrasound might have been employed as additional technique of tumour imaging. Guidelines for the investigations are given below: 0 Primary tumour / loco-regional recurrence
  • Part B For patients participating in Part A of the study and patients with a loco-regional recurrence (Part B) CT scan and/or MRI of the head and neck region had to be performed.
  • Computed tomography of the Head and Neck region was preferred over spiral CT. However, spiral CT might have been helpful in patients who were unable to cooperate.
  • the patient had to be examined in supine position, the neck slightly hyperextended, the head immobilised and the shoulders relaxed and pushed downwards. The patient had to breath quietly with use of abdominal rather than chest muscles.
  • the area to be scanned was determined from the initial overview made in the lateral projection. The plane of the scan had to be parallel to that of the vocal cords. Contiguous three millimetre-slices had to be used routinely. Imaging had to be performed from skull base to upper mediastinum.
  • Spiral CT-scans were obtained. Patients were examined in supine position and had to raise their arms above their heads. Patients had to breath quietly with use of abdominal rather than chest muscles. The patient was scanned from just above the lungs to the level of the adrenal glands. Images had to be photographed in mediastinal and lung setting.
  • Spiral scans were obtained. Patients were examined in supine position and had to raise their arms above their heads. Patients had to breath quietly. Oral contrast was used in all patients. The area to be scanned ran from just above the diaphragm to the symphysis. Images had to be photographed in abdominal and liver setting.
  • Soluble CD44v6 had to be measured in serum. Concentrations were determined by means of a validated enzyme-linked immuno-sorbent assay (ELISA) that was based on a commercially available test kit and conducted in accordance with current international guidelines at the Boehringer Ingelheim Department of Pharmacokinetics and Drug MetaboUsm, Biberach, Germany. Blood samples (to be processed to serum) of 5 mL were obtained pre- infusion, at 21, 48 and 144 hrs p.i. and six weeks after the infusion. Samples were aUowed to clot and centrifuged to prepare serum.
  • ELISA enzyme-linked immuno-sorbent assay
  • the sample for ELISA measurement was put in cryotubes, labeUed carefuUy to enable unique identification, stored at -20°C until radioactivity has decreased ( 186 Re: 4 weeks, 9 m Tc: 3 days) and sent to Bl Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany in batches every four weeks.
  • the serum samples were sent on dry ice.
  • Blood samples were collected for glucose, sodium, potassium, calcium, chloride, creatinine, total protein, albumin, serum glutamic oxalacetic transaminase (sGOT), serum glutamic pyruvic transaminase (sGPT), alkaline phosphatase (AP), gamma glutaryl transpeptidase (GGT), bilirubin, urea, uric-acid, thyroid stimulating hormone (TSH), haemoglobin (Hb), haematocrit (Ht), median corpuscular volume (MCV), reticulocytes, leucocytes, neutrophils, bands, lymphocytes, basophils, eosinophils, monocytes and platelets at the following time points: • At the screening visit or on day one pre-infusion and • At 21, 48 and 144 hrs post infusion.
  • sGOT serum glutamic oxalacetic transaminase
  • sGPT serum glutamic pyruvic
  • Baseline laboratory assessments pre-infusion could have been obtained either at the screening visit or on day one of the study, provided the samples were obtained less then 21 days before the infusion of radiolabelled hMAb BIWA 4 and all required assessments were done. All required laboratory test results had to be reviewed and checked for eligibility by the responsible physician prior to the antibody infusion. The same also applied to a second hMAb BIWA 4 administration in Part B of the study.
  • Urine samples were collected for standard hospital screening (protein, blood, and glucose) at the time points:
  • HAHA The presence and/or the development of HAHA was evaluated in serum samples. Therefore o blood samples (to be processed to serum) of 5 mL were taken at the screening visit, at one week (144 hrs p.i.) and at six weeks after infusion of the antibody. For patients receiving two BIWA 4 administrations in Part B of the study an additional HAHA sample was collected before the second administration. Serum level was evaluated by means of validated ELISA methods. s Samples were allowed to clot and centrifuged to prepare serum.
  • Murine and chimeric MAb U36 (anti-CD44v6-epitope with defined overlapping epitope specificity) was so far safe in HNSCC patients; no toxicities except for myelotoxicity (caused by 186 Re-labels) were seen in a Phase I dose-escalation RIT study with cMAb U36. It was theoreticaUy possible that hypersensitivity reactions to radiolabelled hMAb BIWA 4 might occur. Monoclonal antibodies have been administered to several thousands of patients for diagnostic applications.
  • BIWA 4 potential reactions to intravenously administered BIWA 4 might include hypotension, transient fever and chills, skin rashes, dyspnoea, itching, nausea, and anaphylaxis.
  • Vital signs blood pressure, temperature, pulse rate and respiratory rate were therefore recorded at the screening visit and at the following time points: pre-infusion and 10, 60, 120 minutes post-infusion and after 6 weeks. Additionally vital signs were recorded at 240 minutes post-infusion for patients participating in Part B only.
  • radionucUdes were used.
  • the radiation burden associated with the gamma emitting radionuclide 99m Tc was similar to that encountered in many routine nuclear medicine procedures, and was known to be small. To minimise radiation exposure to the bladder and kidneys the patients were well hydrated and asked to void at frequent intervals during the first 48 hours (96 hours after infusion with 186 Re-labelled antibody).
  • the primary efficacy variables determined in Part A of the trial were the biopsy distribution and the radioimmunoscintigraphy. Description of the methods how to obtain the data is given in section 3.5.1.1 and 3.5.1.2. 5
  • the uptake in the normal tissue and tumour was measured in biopsies of the surgical specimen and the uptake expressed as percent of the injected dose per kg (% ID/kg).
  • the time course of uptake in the tumour and other tissues was evaluated and compared concerning the different doses of BIWA 4.
  • AU sample tubes were labelled with the following information: Trial number, patient number, sample identification (i.e., serum, plasma, or blood), time relative to infusion, actual time, o actual date and isotope (i.e., 99m Tc or 186 Re).
  • sample identification i.e., serum, plasma, or blood
  • time relative to infusion actual time
  • o actual date and isotope i.e., 99m Tc or 186 Re
  • the volume of each urine sample was measured s and recorded and all urine samples were labelled with the following information: Trial number, patient number, total sample volume, collection interval, actual time, date and isotope (i.e., 99m Tc or 186 Re).
  • Plasma samples were measured by validated ELISA methods at the Boehringer Ingelheim Department of Pharmacokinetics and Drug Metabolism, Biberach, Germany. Radioactivity counting of the samples were performed in full blood, serum and urine by the investigator.
  • the primary pharmacokinetic parameters are the time point at which the maximum drug concentration is observed (T max ), maximum drug concentration observed (C max ), area under the 5 concentration-time curve (AUQo ⁇ ⁇ , terminal elimination half-life (t 2 ), volume of distribution (terminal phase and predicted steady state), total body clearance (CL), and mean residence
  • WinNonlin® software package was used to perform non-compartmental pharmacokinetic s analysis on the plasma concentration vs. time profiles of immunoreactive hMAb BIWA 4 and of total blood radioactivity levels, as a function of time, following intravenous administration.
  • a compartmental pharmacokinetic model was initially intended to describe the distribution and metabolism of 186 Re-labelled BIWA 4 in humans. It was intended to combine data from BIWA 4 plasma levels, full blood and serum radioactivity measurements, radioactivity from whole body images and specific regions of interest, the administered dose 186 Re-labelled o BIWA 4, the dose of unlabelled BIWA 4, soluble CD44v6 levels and the assessment of the radiolabelled antibody prior to infusion.
  • DLT was defined as drug-related CTC grade 3 non-haematologic toxicity or drug-related CTC grade 4 haematologic toxicity, excluding nausea and vomiting without adequate antiemetic o treatment.
  • the MTD was defined as the dose level at which less than two out of six patients developed drug-related DLT.
  • Netherlands was contacting and/or visiting the study site to monitor the progress of the study. There were frequent contacts with the investigator and onsite visits for the purpose of data 0 audits, including the comparison of source documents with Case Report Forms and drug accountability checks. The investigator or his/her designee had to be avaUable to the Boehringer Ingelheim The Netherlands representative during these onsite visits.
  • Part A of this phase I trial was an uncontroUed, rising dose sequential group study to determine the safety, tolerabiUty, biodistribution and pharmacokinetics of a single infusion of 99m Tc- labelled hMAb BIWA 4 in patients with advanced squamous cell carcinoma of the head and neck.
  • Three hMAb BIWA 4 dose levels were used in this part of the study with three patients planned at each dose level.
  • AU patients had a proven tumour of the head and neck and were destined for surgery.
  • Part B of this phase I trial was an open uncontroUed dose escalation study to determine the 0 safety, tolerabiUty, MTD, pharmacokinetics and preliminary therapeutic effects of a single infusion of I86 Re-labelled hMAb BIWA 4 in patients with advanced squamous cell carcinoma of the head and neck for whom no curative treatment options were available.
  • the foUowing pharmacokinetic parameters were determined from plasma or serum, urine levels and imaging data as described below:
  • a compartmental pharmacokinetic model was planned to be developed to define the distribution and metabolism of 186 Re-labelled hMAb BIWA 4 in humans.
  • the model collated s data from serum and urine radioactivity measurements, radioactivity from whole body images and specific regions of interest, the administered dose 186 Re-labeUed hMAb BIWA 4, the cold- loading dose of hMAb BIWA 4, soluble CD44v6 levels and the assessment of the radiolabelled antibody prior to infusion.
  • Tumour response was the parameter for therapeutic efficacy. Tumour response was assessed according to the WHO guidelines. The sum of the products of the largest perpendicular diameters of all measured tumour lesions were the primary parameter for tumour response. 5 There were separate criteria for the assessment of response of bone lesions.
  • the intention to treat subset consists of all patients who received an infusion of radiolabelled hMAb BIWA 4 and for whom data after baseline were available.
  • S The per protocol subset consists of evaluable patients. A patient was evaluable if the following criteria were met: s Part A:
  • the immunoreactive fraction in at least one of both performed assays was larger than 60%. o 4. Adequate biopsies and scintigraphic images were available to assess the biodistribution.
  • Non evaluable patients were replaced.
  • the evaluable subset was planned to consist of 9 patients and the intent to treat subset consisted of at least 9 patients.
  • the number of patients depended on the toxicity encountered. Patients who were evaluable for 5 toxicity but not for response were not replaced.
  • the primary objectives of pharmacokinetic analysis were: 0 1. To determine and compare the disposition of total radioactivity and immunoreactive hMAb BIWA 4 following administration of single doses of 99m Tc-labeUed hMAb BIWA 4 and 186 Re-labeUed hMAb BIWA 4.
  • the specific pharmacokinetic parameters that were generated include: T max , C max , AUC (0- infinite time), terminal elimination half-life, volume of distribution (terminal phase and predicted s steady state), total body clearance, and mean residence time (0- infinite time). Cumulative urinary excretion of radioactivity over time were determined from total urine output.
  • the secondary analyses were performed for the intention to treat subset of patients participating in Part B of the study.
  • the secondary analyses were restricted to key endpoints: e.g.: Response rate, duration of response, time to progression. 5 Interim Analysis
  • Part A three patients per dose level provide some information on safety of the hMAb BIWA 4 and on the anticipated correct dose of 50 mg unlabelled hMAb BIWA 4.
  • MAG3 will be replaced by MAG2GABA-TFP in the future development of the BIWA 4 project.
  • Results from Part A confirmed a dose of 50 mg BIWA 4 as the optimal dose for treatment.
  • Data from Part B indicate that patients may clinicaUy benefit from 186 Re-BIWA 4 therapy at a radiation dose below that dose at which DLT was observed.
  • Concentrations of BIWA 4 measured were dose-proportional and a moderate amount of the o dose administered was excreted via the kidneys.
  • the mean age of all patients included was 56 years (range 37 to 78). Nineteen patients were males and eleven were females (TABLE 5.2: 1).
  • Stage of the disease was local operable for all patients included in Part A while patients from Part B had recurrent disease (15 patients) and/or metastases (3 patients). One patient had local inoperable disease. In one patient the information is missing.
  • Concomitant diseases or relevant medical history were reported in 28 patients (TABLE 5.2: 1). Concomitant therapy was required by all patients. The most often used medications were analgesics, sedatives, lactulose, H 2 -blockers, anti-emetics and antimicrobial agents.
  • BIWA 4 BIWA 4 BIWA 4 30 40 50 60 n 3 4 3 2 4 3 6 5 30 age [mean] 45.7 58.5 58.7 57.0 61.0 53.0 57.0 60.4 56.4 male/female 2/1 2/2 1/2 1/1 3/1 2/1 5/1 3/2 19/11 cone, disease 3 3 2 2 4 3 6 5 28 concom.
  • Lesion sizes (including metastases) of the tumours ranged from 14 mm 2 to 10304 mm 2 .
  • the primary tumour site was moderately differentiated in the majority of the patients. Lymph nodes were affected in 13 patients (TABLE 5.2: 2).
  • the dose of 50 mg BIWA 4 was confirmed in Part A of the trial to be the optimal dose concerning blood concentrations and selective tumour uptake .
  • the plasma concentrations of BIWA 4 were dose-proportional in the range of 25 mg to 100 mg BIWA 4 in Part A and peaked at 0.9 hours with a terminal elimination half-life of 54 - 74 hours and 94 hours for Part A and Part B, respectively.
  • Intent-to-treat (ITT) subset The relative biodistribution of 99m Tc-BIWA 4 was highest in the tumour in all three dosing groups except for patient 1 (25 mg BIWA 4), patient 5 (50 mg BIWA 4, no tumour cells) and patient 9 (100 mg BIWA 4), respectively.
  • the uptake in tumour ranged from 6 to 17 % ID/kg, 5 to 28 % ID/kg and 13 to 17 % ID/kg for the dose group 25 mg, 50 mg and 100 mg, respectively.
  • the calculated mean ratio of tumour uptake versus uptake in bone marrow was 1.7, 2.6 and 2 in the 25 g, 50 mg and 100 mg dose group, respectively, (TABLES 7.2: 1 and 7.2: 2).
  • Per-protocol (PP) subset The relative biodistribution of 99m Tc-BIWA 4 was highest in the tumour in all three dosing groups except for patient 1 (25 mg BIWA 4).
  • the uptake in tumour ranged from 6 to 17 % ID/kg, 23 to 28 % ID/kg and 16 % ID/kg for the dose group 25 mg, 50 mg and 100 mg, respectively.
  • the calculated mean ratio of tumour uptake versus uptake in bone marrow was 1.7, 3.2 and 2.5 in the 25 mg, 50 mg and 100 mg dose group, respectively, (TABLES 7.2: 1 and 7.2: 3).
  • Radioiummunoscintigraphic examinations were done immediately after infusion and 21, 48, 72, 144 and 336 hours after infusion. Hardly any uptake in tumour was observed directly after the infusion. Relative uptake in tumour appeared to be dose-dependent and increased over time reaching medium and high uptake after 72 to 144 hours with a decline after 336 hours. o Biodistribution of radioactivity was similar in bone marrow, lung, liver, kidney and intestine and did not reveal the same dose dependent effect (except for the intestine). Moreover, relative uptake of radioactivity appeared to be constant or modestly decreased over time and was similar for all doses of radioactivity. Uptake in bone marrow was lowest for most treatment groups. s 5.4.1.2 Secondary endpoint(s)
  • CD44v6 expression in tumour in Part A Data of determination of CD44v6 antigen expression is avaUable for seven patients. CD44v6 antigen expression was observed in more than 90% and 80% of the tumour cells in five and two patients, respectively, while CD44v6 antigen expression was detected in more than 90% of the cells of lymph node metastases in the four patients for which lymph node was available for evaluation. Homogenous CD44v6 antigen expression was observed in all patients in the 5 mucosa.
  • Soluble CD44v6 was determined in aU patients treated with BIWA 4 in the present trial.
  • VaUdated assays were used for analysis of plasma samples. Analysis of quality control samples s produced results of high accuracy and precision.
  • the signal linearity of the scintillation crystal in the gamma counter was not tested. It can be assumed based on the type of instrument at least in the energy range that was used in the current study. Calibration samples showed typically 40000-200000 counts per minute vs less 0 than 50-100 counts per minute in background samples. The precision of the seven measurements per calibration standard was regularly within 2%. The same applies for the precision of the individual triplicate measurements in blood, serum and urine. Blood and serum samples showed typicaUy activities between 2000 and 100000 counts per minute.
  • Part A consisted of 10 patients receiving a single BIWA 4 dose ranging from 25 to 100 mg with a constant radiolabelled dose of 20 mCi 99m Tc.
  • the pharmacokinetic parameters in plasma for BIWA 4 measured by ELISA following a short intravenous infusion are given in TABLE 5.4.2.2: 1.
  • the geometric mean half-life for BIWA 4 measured by ELISA in plasma foUowing intravenous infusion ranged from 54 to 73.8 hours for the three treatment groups studied in Part A.
  • the geometric mean half-life for the 99m Tc-radiolabeled BIWA 4 for the same samples was shorter at 39.4 hours in serum and 46.4 hours in blood. The discrepancy between the two mean estimates was attributed to longer sampling times possible with BIWA 4 due to radioactivity assay restrictions.
  • Part B consisted of 20 patients receiving BIWA 4 doses of 50 mg with varying radiolabelled doses of 18fr Re-BTWA 4 ranging from 20-60 mCi m 2 . Three patients received a second intravenous infusion while the other 17 patients received only a single course of therapy.
  • Results from Part A confirmed a dose of 50 mg BIWA 4 as the optimal dose for treatment based on blood concentrations and tissue uptake level.
  • the distribution as assessed by radio scintigraphy and biopsy measurements was in almost aU cases highest in the tumour as 0 compared to other tissues. Uptake of radioactivity increased in the tumour over time.
  • CD44v6 expression was present in more than 80 % and 90% of the cells of primary tumour and lymph node metastases, respectively, and in aU mucosa specimens obtained.
  • the amount of soluble CD44v6 detected tended to increase for the 186 Re-BIWA 4 treated patients.
  • the plasma concentrations of BIWA 4 peaked at 0.92 hours and the antibody was eliminated with a geometric mean half-life of 94 hours for BIWA 4 determined by ELISA.
  • C m a x and AUC values were similar to those obtained in Part A of the trial for the 50 mg BIWA 4 dose group.
  • Dose limiting toxicity occurred at a dose of 60 mCi/m 2 while a dose of 50 mCi/m 2 186 Re-BIWA 4 turned out to be the maximum tolerated dose in the present trial.
  • the dose limiting toxicity were adverse events from the bone marrow i.e. thrombocytopenia and leucopenia.
  • AU ten patients treated in Part A of the trial were administered one single dose of 99m Tc-BIWA 4.
  • Doses of BIWA 4 were either 25 mg, 50 mg or 100 mg while 99m Tc was 20 mCi.
  • Part B 20 patients were administered one single dose of 186 Re-BIWA 4. Three patients received a second dose of 50 mCi/m 2 due to stable disease (no change). The dose of BIWA 4 was kept stable at 50 mg while the radioactivity of 186 Re was increased in 10 mCi/m 2 body surface increments.
  • the dose was calculated according to body surface area (TABLE 6.1: 1).
  • the radiochemical purity of the drug was more than 95 % and the immunoreactive fraction was higher than 80 %.
  • Non-haematological dose-limiting toxicity defined as dmg-related CTC grade 3 or 4 non- haematological toxicity occurred in 4 patients (at 30 mCi/m 2 one patient experienced rash and Quincke's oedema, two patients experienced fever at 60 mCi m 2 and one patient treated with 0 60 mCi/m 2 experienced fatigue.
  • Part A s
  • the following adverse events sorted by SOC, preferred term and the three dose groups of BIWA 4 were reported in more than one patient in Part A of the trial (TABLE 6.2.2: 1). More details as well as the preferred terms can be found in section 7.3.1.
  • Haemorrhage (not specified) 1 (33.3) 1 (25.0) 0 (0) 2 (20.0) given are SOCs and preferred terms in case they were reported in more than one patient; figure in brackets denote percentage of patients treated
  • Dmg-related adverse events were reported in 75 % of the patients.
  • Dmg-related leucopenia was reported in 50 % and thrombocytopenia in 55 % of the patients, respectively.
  • Adverse events considered dmg-related are provided in TABLE 6.2.2: 4 graded according to the CTC.
  • CTC grade 1 1 0 2 6 5 14 face oedema 1 n.a. 0 0 0 1 leucopenia 0 n.a. 1 2 0 3 moniliasis 0 n.a. 0 0 1 1 mucositis 0 n.a. 0 0 1 1 platelets abnormal 0 n.a. 0 1 0 1 purpura 0 n.a. 0 0 1 1 stomatitis 0 n.a. 0 1 0 1 thrombocytopenia 0 n.a. 1 2 2 5
  • CTC grade 2 0 1 0 7 7 15 anaemia n.a. 0 n.a. 1 1 2 gout n.a. 0 n.a. 1 0 1 leucopenia n.a. 0 n.a. 1 2 3 mucositis n.a. 0 n.a. 2 3 5 stomatitis n.a. 0 n.a. 0 1 1 taste loss n.a. 0 n.a. 1 0 1 thrombocytopenia n.a. 1 n.a. 1 0 2
  • CTC grade 4 0 0 0 3 5 8 anaemia n.a. n.a. n.a. 0 1 1 granulocytopenia n.a. n.a. n.a. 1 2 3 leucopenia n.a. n.a. n.a. 1 2 3 thrombocytopenia n.a. n.a. n.a. 1 0 1 given is the number of adverse events; n.a. not applicable
  • Thrombocytopenia and leucopenia were dose-dependent (TABLES 6.2.2: 3 and 6.2.2: 4) and dose-limiting. Time course is provided in section 6.4.2.1. causing dose-Umiting toxicity (TABLES 6.2.2: 3 and 6.2.2: 4).
  • HAHAs were detected in two patients (see section 12.4.3).

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US7947496B2 (en) 1999-10-08 2011-05-24 Hoffmann-La Roche Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US8048416B2 (en) 1999-10-08 2011-11-01 Hoffmann-La Roche Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US7189397B2 (en) 1999-10-08 2007-03-13 Arius Research Inc. Cytotoxicity mediation of cells evidencing surface expression of CD44
US7084257B2 (en) 2001-10-05 2006-08-01 Amgen Inc. Fully human antibody Fab fragments with human interferon-gamma neutralizing activity
AU2005214331B2 (en) 2004-02-12 2011-09-15 Eisai, Inc. Monoclonal antibodies that specifically bind to folate receptor alpha
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AU2006241099B2 (en) 2005-04-22 2012-04-19 Eisai, Inc. Antibodies with immune effector activity and that internalize in folate receptor alpha-positive cells
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AU2011212485B2 (en) 2010-02-04 2014-04-17 University Of Miami A monoclonal antibody to CD44 for use in the treatment of head and neck squamous cell carcinoma
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