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
• • 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
  • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3007—Carcino-embryonic Antigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

• the present invention relates to bispecific antibodies comprising a first antigen binding site specific for a death receptor and a second antigen binding site specific for a second antigen, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
• Monoclonal antibodies are proving to be powerful therapeutic agents in the treatment of cancer owing to the selective targeting of antigens which are differentially expressed on cancer cells.
• the therapeutic strategies of most currently developed monoclonal antibodies include the targeting of tumor-associated antigens to modify tumor-cell biology, inhibition of growth factor receptors, inhibition of angiogenesis, apoptosis induction and cytotoxicity via complement fixa- tion or antibody-dependent cellular cytotoxicity.
• TRAIL target the growth factor receptors that are crucial for cancer cell survival
• trastuzumab Herceptin®
• cetuximab cetuximab
• Targeting of the TRAIL death receptors on cancer cells with agonistic monoclonal antibodies represents a new generation of monoclonal antibody therapy, as they are able to directly induce apoptosis of targeted cells.
• the use of an agonistic monoclonal antibody against the death receptors instead of TRAIL may be advantageous: TRAIL targets multiple receptors including both the death receptors and decoy receptors and, therefore, selectivity is a concern.
• TRAIL has a much shorter blood half-life compared with monoclonal antibodies, a factor which affects dose and schedule parameters. The very short blood half- life of TRAIL would require large and frequent doses compared with monoclonal antibodies. In addition recombinant TRAIL is very difficult and tedious to produce.
• the present invention relates to antibodies combining a death receptor targeting antigen binding site with a second antigen binding site that targets a second antigen. By that the death receptors become cross linked and apoptosis of the target cell is induced.
• the advantage of these bispecific death receptor agonistic antibodies over conventional death receptor targeting antibodies is the specificity of induction of apoptosis only at the site where the second antigen is expressed.
• the present invention relates to a bispecific antibody comprising a first an- tigen binding site specific for a death receptor antigen and a second antigen binding site specific for a second antigen.
• the death receptor is selected from death receptor 4 polypeptide (DR4), death receptor 5 polypeptide (DR5) or FAS polypeptide, preferably human DR4 polypeptide (Seq. Id. No. 1), human DR5 polypeptide (Seq. Id. No. 2) or human FAS polypeptide (Seq. Id. No. 3).
• the second antigen is associated with an oncological disease or rheumatoid arthrithis.
• the second antigen is selected from,carcinoembryonic antigen (CEA) polypeptide, CRIPTO protein, magic roundabout ho- molog 4 (ROB04) polypeptide, melanoma-associated chondroitin sulfate proteoglycan (MCSP) polypeptide, tenascin C polypeptide and fibroblast activation protein (FAP) polypeptide, preferably human CEA polypeptide (Seq. Id. No. 4), human CRIPTO polypeptide (Seq. Id. No. 5), human ROB04 polypeptide (Seq. Id. No. 6), human MCSP polypeptide (Seq. Id. No.
• the bispecific antibody is a dimeric molecule comprising a first antibody comprising the first antigen binding site and a second antibody comprising the second antigen binding site.
• the first and second antibody comprise an Fc part of an antibody heavy chain, wherein the Fc part of the first antibody comprises a first dimerization module and the Fc part of the second antibody comprises a second dimerization module allowing a hetero dimerization of the two antibodies.
• the first dimerization module comprises knobs and the second dimerization module comprises holes according to the knobs into holes strategy (see Carter P.; Ridgway J.B.B.; Presta L.G.: Immunotechnology, Vol- ume 2, Number 1, February 1996 , pp. 73-73(1)).
• the first antibody is an Immunoglobulin (Ig) molecule comprising a light chain and a heavy chain and the second antibody is selected from the group consisting of scFv, scFab, Fab or Fv.
• the bispecific antibody comprises a modified Fc part having a reduced binding affinity for the Fey receptors compared to a wildtpye Fc part e.g. a LALA modification.
• the Ig molecule comprises the first antigen binding site specific for the death receptor and the second antibody comprises the second antigen binding site specific for the second antigen.
• the Ig molecule comprises the second antigen binding site specific for the second antigen and the second antibody comprises the antigen binding site specific for the death receptor.
• the second antibody is fused to the N- or C- terminus of the heavy chain of the Ig molecule.
• the second antibody is fused to the N- or C-terminus of the light chain of the Ig molecule.
• the Ig molecule is an IgG.
• the second molecule is fused to the Ig molecule by a peptide linker, preferably a peptide linker having a length of about 10 - 30 amino acids.
• the second antibody comprises additional cysteine residues to form disulfide bonds.
• the bispecific antibodies according to the invention are at least bivalent and can be triva- lent or multivalent e.g. tetravalent or hexavalent.
• the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the present invention.
• the present invention relates to a bispecific antibody of the present inven- tion for the treatment of cancer or rheumatoid arthritis.
• the present invention relates to a nucleic acid sequence comprising a sequence encoding a heavy chain of a bispecific antibody of the present invention, a nucleic acid sequence comprising a sequence encoding a light chain of a bispecific antibody of the present invention, an expression vector comprising a nucleic acid sequence of the present invention and to a prokaryotic or eukaryotic host cell comprising a vector of the present invention.
• polypeptide is used herein to refer to native amino acid sequences and sequence variants of the polypeptides of the present invention i.e. DR4, DR5, FAS, CEA, CRIPTO, ROB04, MCSP, Tenascin C and FAP from any animal, e.g. mammalian species, including hu- mans.
• “Native polypeptide” refers to a polypeptide having the same amino acid sequence as a polypeptide occurring in nature regardless of its mode of preparation.
• the term “native polypeptide” specifically encompasses naturally occurring truncated or secreted forms, naturally occurring variant forms (e.g. alternatively spliced forms), and naturally occurring allelic variants of the polypeptides of the present invention.
• the amino acid sequences in the Sequence Listing (Seq. Id. No. 1 - 9) refer to native human sequences of the proteins of the present invention.
• polypeptide variant refers to amino acid sequence variants of a native sequence containing one or more amino acid substitution and/or deletion and/or insertion in the native sequence.
• the amino acid sequence variants generally have at least about 75%, preferably at least about 80%), more preferably at least about 85%, even more preferably at least about 90%>, most preferably at least about 95% sequence identity with the amino acid sequence of a native sequence of a polypeptide of the present invention.
• antibody encompasses the various forms of antibody structures including but not being limited to whole antibodies and antibody fragments.
• the antibody according to the in- vention is preferably a fully human antibody, humanized antibody, chimeric antibody, or further genetically engineered antibody as long as the characteristic properties according to the invention are retained.
• Antibody fragments comprise a portion of a full length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof. Examples of antibody fragments in- elude diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.
• chimeric antibody refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other pre- ferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
• FcR Fc receptor
• Chimeric antibodies are also referred to as "class-switched antibodies.”
• Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA seg- ments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions.
• Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244.
• humanized antibody refers to antibodies in which the framework or "comple- mentarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
• CDR framework or “comple- mentarity determining regions”
• a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody.” See e.g. Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268-270.
• Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies.
• humanized antibodies encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.
• human antibody as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).
• Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a se- lection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Bruggemann, M., et al, Year Immunol. 7 (1993) 33-40).
• transgenic animals e.g., mice
• Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581-597).
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Mono- clonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al, J. Immunol. 147 (1991) 86-95).
• human antibody as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by "class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.)
• recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
• recombinant human antibodies have variable and constant regions in a rearranged form.
• the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
• the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
• variable domain denotes each of the pair of light and heavy chain domains which are involved directly in binding the antibody to the antigen.
• the variable light and heavy chain do- mains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions” (or complementary determining regions, CDRs).
• the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
• the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
• the antibody's heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
• antigen-binding site of an antibody when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
• the antigen-binding portion of an antibody comprises amino acid residues from the "complementary determining regions" or "CDRs".
• CDRs complementary determining regions
• FR Framework regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
• CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties.
• CDR and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of Immunological Inter- est, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop".
• Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. "Bispecific antibodies" accord- ing to the invention are antibodies which have two different antigen-binding specificities. Antibodies of the present invention are specific for two different antigens, i.e. death receptor antigen as first antigen and a second antigen.
• the term "monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.
• the term "bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen.
• bispecific antibodies as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule.
• the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule.
• the bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g.”tetravalent” or "hexavalent”).
• Antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).
• Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of full length antibodies to which further antigen-binding sites (e.g., single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) are linked via one or more peptide-linkers.
• the antibodies can be full length from a single species, or be chimerized or humanized.
• a “single chain Fab fragment” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL- CHI -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
• Said single chain Fab fragments a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl and d) VL-CH1 -linker- VH-CL, are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
• these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and posi- tionn 100 in the variable light chain according to Kabat numbering).
• nucleic acid or “nucleic acid molecule”, as used herein, are intended to include DNA molecules and RNA molecules.
• a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
• amino acid denotes the group of naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), argin- ine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
• alanine three letter code: ala, one letter code: A
• argin- ine arg,
• a nucleic acid is "operably linked" when it is placed into a functional relationship with an- other nucleic acid.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• "operably linked” means that the DNA se- quences being linked are colinear, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
• the expressions "cell”, “cell line”, and “cell culture” are used inter- changeably and all such designations include progeny.
• the words “transfectants” and “transfected cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are in- eluded.
• binding refers to the binding of the antibody to an epitope of the antigen in an in-vitro assay, preferably in a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden).
• the affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), kD (dissociation constant), and KD (kD/ka).
• Binding or specifically binding means a binding affinity (KD) of 10-8 mol/1 or less, preferably 10-9 M to 10-13 mol/1.
• Binding of the antibody to the death receptor can be investigated by a BIAcore assay (GE- Healthcare Uppsala, Sweden).
• the affinity of the binding is defined by the terms ka (rate con- stant for the association of the antibody from the antibody/antigen complex), kD (dissociation constant), and KD (kD ka)
• epitope includes any polypeptide determinant capable of specific binding to an antibody.
• epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
• An epitope is a region of an antigen that is bound by an antibody.
• the "Fc part” of an antibody is not involved directly in binding of an antibody to an antigen, but exhibit various effector functions.
• a “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
• antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2.
• the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ respectively.
• the Fc part of an antibody is directly involved in ADCC (antibody- dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding.
• Complement activation (CDC) is initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g.
• Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG4 do not activate the complement system and do not bind Clq and C3.
• the antibodies according to the invention are produced by recombinant means.
• one aspect of the current invention is a nucleic acid encoding the antibody according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention.
• Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity.
• nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods.
• the antibodies according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures.
• the hybridoma cells can serve as a source of such DNA and RNA.
• the DNA may be inserted into expression vectors, which are then trans fected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.
• Amino acid sequence variants (or mutants) of the antibody according to the invention are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
• host cell denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention.
• HEK293 cells and CHO cells are used as host cells.
• the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
• the words “transfectants” and “tansfected cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
• NSO cells Expression in NSO cells is described by, e.g., Barnes, L.M., et al, Cytotechnology 32 (2000) 109-123; Barnes, L.M., et al, Biotech. Bioeng. 73 (2001) 261-270.
• Transient expression is described by, e.g., Durocher, Y., et al, Nucl. Acids. Res. 30 (2002) E9.
• Cloning of variable domains is described by Orlandi, R., et al, Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al, Proc. Natl. Acad. Sci.
• HEK 293 A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
• the regulatory element sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
• Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
• Purification of antibodies is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including al- kaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widespread used for protein purification, such as affinity chromatography with micro- bial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g.
• cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl- sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chro- matography (e.g.
• Ni(II)- and Cu(II)-affinity material size exclusion chromatography
• electrophoretical methods such as gel electrophoresis, capillary electrophoresis
• Gel electrophoresis capillary electrophoresis
• pharmaceutical carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
• a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by cer- tain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
• compositions or dispersions include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• sterile aqueous solutions or dispersions include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the use of such media and agents for pharmaceutically active substances is known in the art.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• cancer refers to proliferative diseases, such as lymphomas, lym- phocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the proliferative diseases
• compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
• Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• the composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
• the carrier preferably is an isotonic buffered saline solution.
• Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as man- nitol or sorbitol, and sodium chloride in the composition.
• transformation refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without daunting cell wall barriers are used as host cells, transfec- tion is carried out e.g. by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 546ff. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transformation is calcium treatment using calcium chloride as described by Cohen, F. N, et al, PNAS. 69 (1972) 7110ff.
• expression refers to the process by which a nucleic acid is transcribed into mR A and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins.
• the transcripts and the encoded polypeptides are collectively referred to as gene product.
• expression in a eukaryotic cell may include splicing of the mRNA.
• a "vector” is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
• the term includes vectors that function primarily for insertion of DNA or R A into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vec- tors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.
• an "expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide.
• An “expression system” usually refers to a suitable host cell comprised of an expression vector that can function to yield a de- sired expression product.
• Figure 1 FACS binding analysis of CEA, DR5 and FAS expression levels on different human cell lines (Lovo, OVCAR-3, AsPC-1, BxPC3, LS174T and MKN-45) using unlabelled, commercially available murine IgGl antibodies (CEA: Abeam # 11330; DR5: R&D # MAB631 ; FAS: BD # 555671) and a common goat anti mouse FITC labelled IgG (Serotec Starl05F) for detection. As controls samples containing only cells or cells and secondary antibody alone were used. Except Lovo cells all tested cell lines express significant amounts of DR5 and FAS on the surface. Compared to that CEA expression was rather low.
• FIG. 1 Analysis of apoptosis induction (DNA fragmentation assay) of different cell lines after 4 firs incubation with commercially available antibodies that are able to induce apoptosis in solution without cross-linking (DR5: R&D # MAB631 ; FAS: Millipore / Upstate: CH11).
• DR5 R&D # MAB631
• FAS Millipore / Upstate: CH11
• Figure 3 Induction of apoptosis (DNA fragmentation assay) of LS174T cells after 4 firs incubation with ApomAb (white bar), ApomAb cross-linked with an anti human Fc antibody (hatched, grey bar), ApomAb_sm3e_A (black bar) and ApomAb_sm3e_Al (stippled grey bar) bispecific molecules.
• CEA binding dependent induction of apoptosis by targeted hyper-cross- linking via the bispecific antibodies can be detected. This effect is in the same range as the apoptosis induced by cross-linking of ApomAb and could be completely abolished by pre-incubation with an excess amount of sm3e IgG. No apoptosis was observed with the controls (cells only or sm3e IgG) and also the ApomAb alone did not induce apoptosis in the used concentration (I ⁇ g / ml).
• Figure 4 Comparison of apoptosis inducing activity of different ApomAb_sm3e bispecific molecules compared to ApomAb (white bar) alone or ApomAb cross-linked with an anti human Fc antibody (hatched, grey bar), in a DNA fragmentation assay with LS 174T cells incubated for 4 firs with apoptosis inducing agents.
• molecules where the sm3e scFv is fused to the C-terminus of the heavy chain of ApomAb (format A, black bar) seem to be more active than constructs in which the sm3e scFv is fused to the C-terminus of the light chain of ApomAb (format B, grey bar).
• disulfide stabilized scFv containing bispecific antibodies (format Al , dotted grey bar and Bl , small grid bar) seem to be slightly inferior to molecules with the wild type scFv.
• FIG. 5 Analysis of apoptosis induction (DNA fragmentation assay) of LS 174T cells after 4 firs of incubation with either Apomab (white bars), ApomAb that was cross-linked with an anti human Fc antibody (hatched, grey bars) or ApomAb PRl A3 A bispecific construct (black bars).
• Apoptosis induction was dependent on the concentration of the antibody used.
• ApomAb alone also induced low levels of apoptosis at high concentrations but this was significantly increased by cross- linking.
• the bispecific ApomAb_PRlA3_A molecule was even more active without secondary cross-linking agent than the cross-linked ApomAb was.
• Figure 6 Analysis of apoptosis induction (DNA fragmentation assay) of Lovo cells after 4 firs of incubation with either Apomab (white bars), ApomAb that was cross-linked with an anti human Fc antibody (grey bars) or ApomAb PRl A3 A bispecific construct (black bars).
• Apoptosis induction was dependent on the concentration of the antibody used.
• ApomAb alone also induced low levels of apoptosis at high concentrations (as described) but this was significantly increased by cross- linking.
• the bispecific ApomAb PRl A3 A molecule was as active on its own as the cross-linked ApomAb was.
• Figure 7 Comparison of DNA fragmentation in LS 174T cells after 4 firs incubation with different apoptosis inducing bispecific antibodies.
• NCCIT cells do not express FAS, only low amounts of CRIPTO but similar amounts of DR5 compared to recombinant HEK293 - CRIPTO cells. The latter cells show low levels of FAS, significant levels of DR5 and rather high levels of CRIPTO expression.
• Figure 10 Apoptosis induction comparison (DNA fragmentation in HEK293-CRIPTO cells) using FAS (HFE7A IgG), FAS (HFE7A IgG) cross-linked via anti human Fc antibody and FAS - CRIPTO bispecific molecules (HFE7A LC020 H3L2D1, in which the wt (A) or disulfide stabilized (Al) CRIPTO scFv is fused to the C-terminus of the heavy chain of HFE7A.
• FIG 11 Apoptosis induction (DNA fragmentation assay) by HFE7A-CRIPTO bispecific molecules in recombinant HEK293 -CRIPTO cells (black bars) compared to recombinant HEK293-FAP (fibroblast activation protein) cells (white bars).
• apoptosis can be induced using an apoptosis inducing commercially available antibody (CH11) and with HFE7A IgG that was cross-linked via a second, Fc specific antibody, whereas HFE7A alone did not induce apoptosis under the used conditions.
• CH11 commercially available antibody
• HFE7A IgG was cross-linked via a second, Fc specific antibody
• Figure 12 FACS binding analysis for determination of surface expression levels of MCSP on different cell lines (MCF7, SkBr3, A431, A549, HCT-116 and U87-MG) using two different antibodies. With both antibodies the same levels of MCSP expression could be detected, indicating that U87-MG showed the highest MCSP expression, HCT-116 with a low MCSP expression whereas all other tested cell lines were MCSP negative (in the range of the negative control such as unstained cells).
• Figure 13 Evaluation of apoptosis capability of U87-MG (A) and HCT-116 (B) cells using soluble and cross-linked ApomAb (black bars) and HFE7A (grey bars) and the relevant control molecules (anti FAS CHl 1, anti DR5 R2 and anti Fc-IgG alone). While in HCT-116 cells apop- to sis could only be induced via the DR5 receptor after four hours and not via FAS, this was different for U87-MG cells. Here, significant apoptosis only could be observed after 24 hours. In contrast to HCT-116 cells in U87-MG apoptosis induction by cross-linked HFE7A was twice as efficient as with cross-linked ApomAb.
• FIG 14 Analysis of apoptosis induction on U87-MG glioma cells after 24 hours incubation with the bispecific HFE7A-MCSP antibody (mAb 9.2.27) in which either the wild type (A format) or disulfide stabilized MCSP scFv (Al format) is fused to the C-terminus of the heavy chain of ApomAb.
• the construct containing the disulfide stabilized scFv demonstrated significantly higher apoptosis that the molecule containing the wild type scFv (although the amount of apoptosis measured by DNA fragmentation was relatively low).
• the induction of apoptosis could be completely abolished by pre-incubation of the cells with an excess of competing MCSP IgG.
• FIG. 15 FACS binding analysis of two different cell lines (SW872 and GM05389) for expression levels of human fibroblast activation protein (FAP) (A).
• FAP human fibroblast activation protein
• A The fluorescence intensity measured with different concentrations of an anti FAP antibody is shown over a range of three magnitudes (black, grey and hatched bars). Negative control reactions as secondary antibody and cells only ate shown as stippled and white bars, respectively.
• the GM05389 cells demonstrate expression of FAP over all tested antibody concentrations that was above background, with the SW872 cells FAP expression only could be detected with the highest antibody concen- tration used (10 ⁇ g / ml), indicating that these cells are not suitable for FAP based binding / apoptosis induction experiments.
• Apoptosis induction is compared to ApomAb (+ / - cross-linking with 10 fold excess of anti-human-Fc- antibody) and negative controls. All constructs were used at concentrations of 0.1 and 1.0 ⁇ g / ml. Under the used assay conditions the bispecific ApomAb_PRlA3_scFab construct (black bars) clearly shows a concentration dependent induction of apoptosis which is in the same range as observed with hyper-cross-linked ApomAb (grey bars) and which is significantly higher as with ApomAb alone (hatched bars).
• Figure 18 Analysis of apoptosis induction of LS 174T cells by ApomAb (alone, hatched bars or hyper-cross-linked, grey bars) compared to bispecific trivalent constructs (Apo- mAb_sm3e_scFab; 2x1 valency, black bars) and negative controls.
• the assay was performed for 4 firs using the constructs in concentrations of 0.1 and 1.0 ⁇ g / ml.
• the bispecific Apo- mAb_sm3e_scFab construct is able to induce apoptosis in a concentration dependent manner in the same range as hyper-cross-linked ApomAb does.
• Figure 19 Analysis of in-vivo efficacy of ApomAb and the bispecific DR5 agonistic anti- body ApomAb_sm3e_Al compared to the vehicle control in an intrasplenic metastatic model using the human colon carcinoma cell line LS174T. Random groups of ten mice each were treated either with PBS (black line), with ApomAb (black circles) or the ApomAb-sm3e_Al bispecific antibody (black squares). The percentage of survival is plotted against the time course of the experiment. Examples
• Example 1 Design of bispecific antibodies recognizing human death receptor 5 and human CEA
• bispecific antibodies comprising a full length antibody binding to a first antigen (human death receptor, DR5) combined with two single chain Fv fragments binding to a second antigen (human carcino embryonic antigen, CEA) fused via a peptide linker either to the C-terminus of the heavy or light chain of the full length antibody are described.
• the antibody domains and the linker in said single chain Fvs have the following orientation: VH - linker - VL.
• ApomAb antibody described by Adams in US2007 / 0031414 Al were used.
• CEA antigen binding scFvs the sequences of the variable light and heavy chains of PR1A3 (Bodmer et al, 1999; US5965710) and sm3e (Begent et al, 2003; US7232888 B2) were used.
• PR1A3 Bodmer et al, 1999; US5965710
• sm3e Begent et al, 2003; US7232888 B2
• variants containing cysteine residues at Kabat position 44 in the variable heavy chain and Kabat position 100 in the variable light chain were produced to generate interchain disulfide bridges between VH and VL. This had the aim to stabilize the scFv molecule to minimize potential aggregation tendency.
• a summary of the different bispecific death receptor agonistic antibody molecules consisting of ApomAb - CEA combinations is given in table 1.
• the description of the design of the different molecules can be concluded from the molecule names, where the first part characterizes the death receptor targeting IgG (e.g. ApomAb), the second name describes the source of the CEA targeting scFv (e.g. PR1A3 or sm3e) and the letter and number combination describes the fusion position and disulfide stabilization property of the scFv.
• Table 1 Description of the different bispecific death receptor agonistic antibodies targeting human DR5 and human CEA with their relevant characteristics.
• Example 2 Expression and purification of bispecific death receptor agonistic anti- bodies
• All molecules could be produced and purified in sufficient amounts and with appropriate quality for further characterization and testing.
• the yields after purification were in the range of about 5 mg / L with some deviations for some molecules.
• the yield for ApomAb- sm3e-Bl was significantly lower (2.19 mg / L) while of the corresponding construct, ApomAb- PR1A3 B1 even more than 11 mg / L could be purified.
• Example 3 Induction of apoptosis by death receptor bispecific DR5 - CEA antibody molecules: The human DR5 death receptor agonistic antibody ApomAb induces apoptosis of DR5 expressing tumor cells, such as the colon cancer cell lines LSI 80 or Colo-205. In- vitro, ApomAb on its own mediates significant apoptosis which can be dramatically increased by cross-linking of the ApomAb-bound DR5 with antibodies binding to the human Fc region of ApomAb.
• Apoptosis also translates into in-vivo where it could be shown for different tumor models that ApomAb exhibits significant efficacy (Jin et al, 2008; Adams et al, 2008), most probably by cross-linking events via the human Fc-receptors.
• DR5 - CEA bispecific antibodies for tumor site targeted cross-linking of DR5 with subsequent indue- tion of apoptosis the activity of ApomAb - CEA bispecific molecules in terms of apoptosis mediation was analyzed in- vitro.
• DR5 - CEA bispecific antibody molecules are able to induce tumor antigen binding dependent apoptosis of target cells DNA fragmentation in tumor cells after incubation with death receptor agonistic bispecific antibodies as a measure of apoptosis was analyzed using a cell death detection ELISA assay.
• Cell dissociation buffer Gibco - Invitrogen # 13151-014
• Cells were harvested by centrifugation (4 min, 400 x g), washed with FACS buffer (PBS / 0.1 % BSA) and the cell num- ber was adjusted to 1.111 X 106 cells / ml in FACS buffer. 180 ⁇ of this cell suspension was used per well of a 96 well round bottom plate, resulting in 2 x 105 cell per well. The cells were incubated for 30 min at 4 °C with the first antibody in appropriate dilution.
• the cells were harvested by centrifugation (4 min, 400 x g), supernatant was completely removed and cells were washed once with 150 ⁇ of FACS buffer.
• the cells were resuspended in 150 ⁇ FACS buffer and incubated with the secondary antibody (in case unlabelled first antibody was used) for 30 min at 4 °C in the dark.
• the secondary antibody in case unlabelled first antibody was used
• FACS buffer cells were resuspended in 200 ⁇ of FACS buffer and analyzed in a HTS FACSCanto II (BD, Software FACS Diva).
• the cells could be fixed with of 200 ⁇ of 2 % PFA (paraformaldehyde) in FACS buffer for 20 min at 4 °C and analyzed later. All assays were performed in triplicates.
• FIG 1 the results of FACS binding analysis of different tumor cell lines with three specific antibodies recognizing CEA, DR5 or FAS are shown. Except the Lovo cells all other tested cell lines express the tested antigens at different levels. CEA expression was highest in MKN-45 cells and more or less similar in OVCAR-3, AsPC-1, BxPC-3 and LS174T. In terms of DR5 expression the AsPC-1 and BxPC.3 cells express most of the receptor compared to the other cell lines followed by OVCAR-3 and MKN-45 whereas LS174T has the lowest DR5 expression level. Regarding FAS expression the cell lines were different but all showing significant FAS expression.
• the bispecific antibodies were used in a final concentration of 0.01 - 10 ⁇ g / ml; control antibodies were used at 0.5 ⁇ g / ml and cross-linking antibodies were used at 100 ⁇ g / ml. Competing antibodies were used at a 100 fold excess.
• the cells were incubated for 4 - 24 firs at 37 °C, 5 % C0 2 to allow induction of apoptosis.
• the cells were harvested by centrifugation (10 min, 200 x g) and incubated for 1 h at room temperature in 200 ⁇ of lysis buffer (supplied by the kit). Intact cells were sedimented by centrifugation (10 min, 200 x g) and 20 ⁇ of the supernatant was analyzed according to the manufacturer's recommendations for induction of apoptosis. A set of cell lines also was analyzed for the ability to undergo apoptosis by incubation with commercially available antibodies against DR5 or FAS which are known to cross-link the death receptors already in solution (figure 2).
• ASPC-1 cells are very resis- tant to apoptosis induction via death receptor cross-linking. Based on these results the two cell lines Lovo and LS174T were chosen to analyze apoptosis induction by tumor antigen targeted cross-linking of DR5.
• the two bispecific molecules showed very similar activity, demonstrating that the stabili- zation of the molecule by insertion of interchain disulfides does not affect biological activity.
• the cells were pre-incubated with an excess of sm3e IgG (100-fold higher concentration compared to the bispecific constructs) no apoptosis can be induced anymore, indicating that the sm3e IgG blocks all CEA antigen on the cell surface and prevents additional binding of the bis- pecific death receptor agonistic molecule.
• the ApomAb - sm3e constructs described above worked very well in terms of antigen dependent specific induction of apoptosis as shown in figures 3 and 4.
• This CEA antibody, sm3e exhibits a very high affinity towards its antigen (low pico molar range).
• additional constructs, analogous to the former ones, were generated.
• the CEA targeting scFv was engineered using the sequence of the CEA antibody PR1A3 which has a rather low affinity to CEA which is in the micromolar range.
• FIG 5 the induction of apoptosis on LS174T cells by ApomAb, cross-linked ApomAb and ApomAb_PRlA3 bispecific antibody (wild type PR1A3 scFv fused to C-terminus of the ApomAb heavy chain) is shown over a concentration range from 0.01 to 10.0 ⁇ g / ml.
• ApomAb on its own exhibits a certain degree of concentration dependent apoptosis induction which could be significantly increased by cross-linking of ApomAb with an anti human Fc antibody.
• the bis- pecific ApomAb-PRlA3 molecule also demonstrated concentration dependent induction of apoptosis, which at a concentration of 10.0 ⁇ g / ml, was even higher as with the cross-linked ApomAb at concentration at the same concentration indicating that it is not absolutely necessary to use the highest affine tumor antigen binders in this bispecific death receptor agonistic antibody format to achieve good in- vitro efficacy in terms of apoptosis induction.
• the ApomAb PRl A3 bispecific antibody in the absence of any cross-linking molecule, showed the same induction of DNA fragmentation as the hyper-cross-linked ApomAb antibody demonstrating that the apoptosis inducing effect using death receptor agonistic antibodies is a general phenomenon that can be applied to all apoptosis competent cell lines.
• ApomAb - PR1 A3 shows only about one third of the apoptosis induction in LS174T cells compared to ApomAb - sm3e. Furthermore there seem to exist intrinsic differences in the different molecules which also are reflected in the capability of induction of apoptosis. In the cases in which the PR1 A3 scFv is fused to ApomAb there is no difference in activity between molecules where the scFv is fused to either the C-terminus of the heavy or light chain. Both molecules show the same induction of apoptosis. In contrast to this, constructs containing the sm3e scFv behave different.
• Example 4 Generation of bispecific death receptor agonistic antibodies targeting FAS (CD95) and CRIPTO as the tumor antigen and evaluation of these molecules in-vitro:
• CRIPTO is a GPI-anchored growth factor that is reported to be over-expressed in cancer cells, but low or absent in normal cells. CRIPTO is found to be up-regulated in colon tumors and liver metastasis. As a member of the EGF family, it is considered to be an autocrine growth factor that plays a role in proliferation, metastasis, and/or survival of tumor cells. This growth factor activates a number of signaling pathways through several potential receptors or co-receptors.
• bispecific antibodies targeting FAS as the death receptor and CRIPTO as the tumor antigen were generated. These molecules consist of a full length IgGl antibody (recognizing FAS) to which CRIPTO targeting scFvs are fused to the C-terminus of the heavy chain.
• the sequences of the HFE7A antibody was used (Haruyama et al, 2002), which is a human / mouse cross- reactive antibody against CD95.
• the CRIPTO scFv was generated from sequences of a humanized anti-CRIPTO antibody that was generated by immunization (LC020 H3L2D1).
• the scFv was generated using standard recombinant DNA techniques and fused by a short peptide linker to the C-terminus of the FAS IgGl heavy chain.
• the order of the single domains in the scFv is VH - (G4S)4 linker VL.
• FIG. 10 summarizes the results of in- vitro experiments for induction of apoptosis on HEK-CRIPTO cells using either HFE7A, cross-linked HFE7A or the HFE7A - CRIPTO bispeci- fic constructs. There is no significant apoptosis induction with HFE7A or CRIPTO (LC020) alone.
• HFE7A LC020 A wild type CRIPTO scFv
• HFE7A LC020 A1 disulfide stabilized scFv
• Example 5 Generation of FAS - MCSP bispecific death receptor agonistic antibodies and evaluation of their apoptosis induction potential.
• antigens that are directly expressed and displayed on the tumor cell surface also other antigens are being considered for targeted cross-linking of death receptors to induce apoptosis.
• antigens from the stroma or neo vasculature are antigens from the stroma or neo vasculature.
• MCSP melanoma associated chondroitin sulfate proteoglycan
• MCSP is expressed on the majority of melanoma cells but also on glioma cells and on neo vasculature.
• monoclonal antibodies targeting human MCSP have been described but none of them was suitable to be used in cancer therapy due to missing efficacy (e.g. lack of ADCC). Therefore MCSP antibodies might gain value if used in a bispecific format that is able to mediate tumor site targeted apoptosis.
• bispecific death receptor agonistic antibodies were generated in which a MCSP specific scFv (wild type or disulfide stabilized) is fused to the C-terminus of the anti FAS antibody HFE7A. These scFvs are fused via a short peptide linker to HFE7A.
• the sequences of the variable light and heavy chains to generate the MCSP targeting scFv were taken from the MCSP antibody 9.2.27 (Beavers et al, 1996; US5580774).
• U-87MG cells apoptosis could be induced by both, anti FAS and anti DR5 antibodies (figure 13 A) while this was different for HCT-116 cells.
• apoptosis only could be induced with anti DR5 antibodies (figure 13 B). Therefore U-87MG cells were chosen to be used as target cells for future apoptosis induction experiments.
• Figure 14 shows the results obtained from apoptosis induction experiments with the glioma cell line U-87MG after treatment with FAS agonistic bispecific antibodies (in a concentration of 1 ⁇ g / ml) consisting of FAS targeting HFE7A IgG which is combined with a MCSP binding scFv (9.2.27).
• Example 6 A DR5 - FAP death receptor agonistic bispecific antibody is able to me- diate apoptosis of one cell line via cross-linking by a second cell line.
• Another approach of induction of apoptosis by cross-linking of death receptors as DR5 is targeting the stroma surrounding the tumor. In that case the targeted antigen is not displayed directly by the tumor cells but by a second, different cell type.
• FAP fibro- blast activation protein
• This protein is expressed on activated fibroblast as they are found in the tumor stroma.
• bispecific molecules were generated that consist of an IgGl part that recognizes DR5 and a FAP binding scFv that is fused to the C-terminus of the heavy chain of the antibody.
• the sequence of the DR5 targeting IgG was taken from the ApomAb sequence as described in US2007 / 0031414 Al .
• the sequence of variable heavy and light chain of the FAP binding scFv was taken from a Fab anti FAP molecule isolated by phage display as shown in sequence # 1 and 2.
• the FAP scFv is fused by a (G4S)2 connector to the C-terminus of the anti DR5 IgG heavy chain.
• a (G4S)2 connector to the C-terminus of the anti DR5 IgG heavy chain.
• the second cell line (the effector cell line) has to be apoptosis negative (either by apoptosis resistance or by not expressing DR5) but needs to express FAP on the surface.
• One possible effector cell line that fulfils the desired criteria is the human fibroblast cell line GM05389.
• this cell line expresses significant levels of FAP compared to the cell line SW872 which only showed FAP expression with the highest tested antibody concentration (10 ⁇ g / ml) but does not undergo apoptosis by non-cross-linked ApomAb as seen in figure 15 B. Therefore this cell line seems to be a potential effector cell line in an apop- tosis assay where DNA fragmentation of a target cell line is induced by cross-linking via an antigen expressed on a second cell line.
• the human breast-adenocarcinoma cell line MDA-MB-231 was used that expresses low levels of DR5 and is sensitive to DR5 mediated apoptosis induction.
• figure 16 the results of induction of DNA fragmentation of GM05389 cells and MDA-MB-231 cells compared to the combination of both cell lines by tumor targeted cross-linking of DR5 via FAP is summarized.
• Example 7 Fusion of CEA single chain Fab molecules (scFab) to ApomAb for the generation of DR5 - CEA bispecific agonistic antibodies.
• scFab single chain Fab's
• the orientation of the different domains of the scFab's is as follows: VL - CL - VH - CHI .
• the C-terminus of the constant light chain (CL) is connected to the N-terminus of the variable heavy chain (VH) via a 34mer peptide linker. Fusion of the scFab occurs by a G4S connector (either 2mer or 4mer).
• Single chain Fab containing bispecific antibodies were generated in two basically different formats: in one format two scFab's are fused to the C-terminus of the heavy or light chain of ApomAb (bispecific, tetravalent homodimeric molecules).
• a bispecific mole- cule was constructed in which only one scFab is fused to the C-terminus of only one ApomAb heavy chain (bispecific, trivalent heterodimeric molecule). This heterodimerization was achieved by using the so-called knob into holes technology which uses Fc mutations that only allow formation of heterodimeric IgG molecules.
• FIG 17 the results of apoptosis induction experiments in which Apo- mAb PRl A3_scFab is compared to ApomAb or hyper-cross-linked ApomAb are shown.
• the gastric cancer cell line MKN-45 was used and apoptosis was measured after 24 firs using a DNA fragmentation assay.
• the bispecific construct exhibits apoptosis induction activity that is in the same range as can be observed with ApomAb that was cross-linked via an anti Fc antibody, and which is significantly higher as with the ApomAb alone.
• apoptosis induction with ApomAb on its own is rather high, which most probably is due to the elongated incubation time of 24 firs which is necessary to demonstrate maximum apoptosis induction on the used MKN-45 cell line (in contrast to e.g. LS174T cells with which the assay is only run for four firs).
• trivalent DR5 agonistic antibodies monovalent for the tumor target, CEA, and bivalent for DR5
• a molecule was generated in which a CEA scFab (sm3e specificity) was fused to the C-terminus of the ApomAb heavy chain (containing the knob mutation).
• Example 8 DR5 - CEA bispecific agonistic antibody with superior in-vivo efficacy compared to ApomAb
• SCID beige mice were treated with intras- plenic injection of 3 x 106 tumor cells.
• a scout animal was tested for tumor en- graftment as a criterion to start with the antibody treatment one day later.
• the treatment con- sisted of a series of three injections (each 10 mg / kg, i.v. in intervals of seven days). Each day the animals were analyzed for demonstrating termination criteria.
• Figure 19 summarizes the results obtained in this in-vivo experiment.
• the survival duration of three groups of mice is compared. While the control group (PBS, black line) was completely terminated 37 days post tumor injection the group treated with ApomAb (filled circles) showed a prolonged survival (maximum of 44 days).
• the group treated with the bispecific antibody (Apo- mAb_sm3e_Al, black squares) even showed longer survival (52 days) than the group that had obtained ApomAb alone.
• the cells were grown in standard DMEM medium (Invitrogen) containing 10 % FCS (Gibco, # 16000) at 37 °C in humidified incubators with 5 % C02 atmosphere. 48 firs prior to transfection 3 x 107 cells were inoculated in 200 ml DMEM / 10 % FCS in roller bottles (Falcon # 353069, 1400 cm 2 ) and were incubated at 37 °C in a roller bottle incubator (0.3 rpm). For transfection 880 ⁇ g total DNA (440 ⁇ g for each, heavy and light chain vector) + 4.4 ml CaCl 2 were filled up with H 2 0 to a total volume of 8.8 ml. The solution was mixed shortly.
• DMEM medium Invitrogen
• FCS Gibco, # 16000
• the proteins were produced by transient expression in HEK293 EBNA cells. All bispecific molecules described here were purified in two steps using standard procedures, such as protein A affinity purification (Akta Explorer) and size exclusion chromatography.
• Akta Explorer protein A affinity purification
• size exclusion chromatography size exclusion chromatography
• buffer A 50 mM sodiumphosphate, pH 7.0, 250 mM NaCl
• All used target cell lines were analyzed for relative expression levels of tumor-related anti- gens and FAS or DR5 death receptors before apoptosis assays were performed.
• Cell dissociation buffer Gibco - Invitrogen # 13151-014
• Cells were harvested by centrifugation (4 min, 400 x g), washed with FACS buffer (PBS / 0.1 % BSA) and the cell number was adjusted to 1.1 1 1 X 106 cells / ml in FACS buffer. 180 ⁇ of this cell suspension was used per well of a 96 well round bottom plate, resulting in 2 x 105 cell per well. The cells were incubated for 30 min at 4 °C with the first antibody in appropriate dilution.
• the cells were harvested by centrifugation (4 min, 400 x g), supernatant was completely removed and cells were washed once with 150 ⁇ of FACS buffer.
• the cells were resuspended in 150 ⁇ FACS buffer and incubated with the secondary antibody (in case unlabelled first antibody was used) for 30 min at 4 °C in the dark.
• the secondary antibody in case unlabelled first antibody was used
• FACS buffer cells were resuspended in 200 ⁇ of FACS buffer and analyzed in a HTS FACSCanto II (BD, Software FACS Diva).
• the cells could be fixed with of 200 ⁇ of 2 % PFA (paraformaldehyde) in FACS buffer for 20 min at 4 °C and analyzed later. All assays were performed in triplicates.
• the Cell Death Detection ELISA PLUS kit from Roche was used for determination of induced apoptosis.
• 104 cells per well of a 96-well plate (after detaching, and determination of cell number and viability) were seeded in 200 ⁇ appropriate medium and were incubated over night at 37 °C in a 5 % C0 2 atmosphere. The next day the medium was replaced by fresh medium containing the apoptosis inducing antibodies, control antibodies and other controls in appropriate concentrations:
• the bispecific antibodies were used in a final concentration of 0.01 - 10 ⁇ g / ml; control antibodies were used at 0.5 ⁇ g / ml and cross-linking antibodies were used at 100 ⁇ g / ml. Com- peting antibodies were used at a 100 fold excess.
• the cells were incubated for 4 - 24 firs at 37 °C, 5 % C0 2 to allow induction of apoptosis.
• the cells were harvested by centrifugation (10 min, 200 x g) and incubated for 1 h at room temperature in 200 ⁇ of lysis buffer (supplied by the kit). Intact cells were sedimented by centrifugation (10 min, 200 x g) and 20 ⁇ of the supernatant was analyzed according to the manufac- turer's recommendations for induction of apoptosis.

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