WO2010028798A1 - Multivalent antibodies - Google Patents

Multivalent antibodies Download PDF

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Publication number
WO2010028798A1
WO2010028798A1 PCT/EP2009/006500 EP2009006500W WO2010028798A1 WO 2010028798 A1 WO2010028798 A1 WO 2010028798A1 EP 2009006500 W EP2009006500 W EP 2009006500W WO 2010028798 A1 WO2010028798 A1 WO 2010028798A1
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WO
WIPO (PCT)
Prior art keywords
antibody
monospecific
seq
ccr5
antigen
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PCT/EP2009/006500
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French (fr)
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WO2010028798A8 (en
Inventor
Johannes Auer
Michael Brandt
Ulrich Brinkmann
Wilma Dormeyer
Eike Hoffmann
Andreas Jekle
Jun-Ichi Nezu
Juergen Michael Schanzer
Jan Olaf Stracke
Pablo Umana
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F. Hoffmann-La Roche Ag
Roche Glycart Ag
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Application filed by F. Hoffmann-La Roche Ag, Roche Glycart Ag filed Critical F. Hoffmann-La Roche Ag
Publication of WO2010028798A1 publication Critical patent/WO2010028798A1/en
Publication of WO2010028798A8 publication Critical patent/WO2010028798A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16023—Virus like particles [VLP]

Definitions

  • the present invention relates to monospecific tetravalent anti-CCR5 antibodies, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
  • Engineered proteins such as bi- or multispecific antibodies capable of binding two or more antigens are known in the art.
  • Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
  • a wide variety of recombinant antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S.L., Nature Biotech 25 (2007) 1233- 1234).
  • All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., and Leger, O., Pathobiology 74 (2007) 3-14). It has to be kept in mind that one may want to retain effector functions, such as e.g. complement- dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.
  • CDC complement- dependent cytotoxicity
  • ADCC antibody dependent cellular cytotoxicity
  • WO 2007/024715 are reported dual variable domain immunoglobulins as engineered multivalent and multispecific binding proteins.
  • a process for the preparation of biologically active antibody dimers is reported in US 6,897,044.
  • Multivalent Fy-antibody construct having at least four variable domains which are linked with each over via peptide linkers are reported in US 7,097,330.
  • Dimeric and multimeric antigen binding structures are reported in US 2005/0079170.
  • Tri- or tetravalent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a connecting structure, which protein is not a natural immunoglobulin are reported in US 6,511,663.
  • tetravalent bispecific antibodies are reported that can be efficiently expressed in prokaryotic and eukaryotic cells, and are useful in therapeutic and diagnostic methods.
  • a method of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782.
  • Bispecific tetravalent receptors are reported in US 5,959,083.
  • Engineered antibodies with three or more functional antigen antigen-binding sites are reported in WO 2001/077342.
  • Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580.
  • WO 1992/004053 reports homoconjugates typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant are covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 whereby the oligomers, typically of the IgG class, are secreted having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules. Sheep-derived antibodies and engineered antibody constructs are reported in US 6,350,860 which can be used to treat diseases wherein interferon gamma activity is pathogenic.
  • targetable constructs that are multivalent carriers of bi-specif ⁇ c antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specif ⁇ c antibodies.
  • Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917.
  • stabilized binding molecules that consist of or comprise a stabilized scFv are reported.
  • Mantis, N.J., et al. J. Immunol. 179 (2007) 3144-3152
  • Tetravalent bispecific receptors the preparation and use thereof are reported in US 5,959,083.
  • novel tetravalent bispecific antibodies are reported in US 2007/0122405.
  • US 2007/0122405 reports human G-protein chemokine receptor (CCR5) HDGNRlO. Diagnostics and therapeutics for diseases associated with C-C chemokine receptor 5 (CCR5) are reported in WO 2005/106489.
  • a first aspect of the current invention is an antibody comprising four antigen- binding sites each antigen-binding site consisting of a pair of antibody variable domains, whereby every antigen-binding site binds to the same or an overlapping epitope.
  • the antibody is consisting of four polypeptides which are linked via inter-polypeptide disulfide bonds.
  • the antigen-binding sites of the antibody are each consisting of an antibody heavy chain variable domain and an antibody light chain variable domain.
  • the antibody comprises two full length antibody light chains and two full length antibody heavy chains.
  • two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker connecting the antibody heavy chain variable domain and the antibody light chain variable domain of the single chain antibody.
  • two antigen-binding sites are each formed by a pair of heavy and light chain variable domains of the monospecific bivalent antibody and both bind to the same epitope, ii) two antigen-binding sites are each formed by one single chain antibody and both bind to the same or an overlapping epitope as in i),
  • said single chain antibodies are each linked to one heavy chain or to one light chain via said peptidic-linker, whereby each antibody chain is linked only to one single chain antibody.
  • the single chain antibodies have a disulfide bond between the heavy and light chain variable domain.
  • the monospecific bivalent antibody is of human IgGl or IgG4 subclass.
  • the monospecific bivalent antibody is of IgG4 subclass with the additional mutation S228P, or the antibody is of human IgGl subclass with the mutations L234A and L235A.
  • the monospecific bivalent antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65% or lower (numbering according to Kabat).
  • nucleic acid molecule encoding an antibody according to the invention and a cell comprising the nucleic acid according to the invention.
  • the invention also comprises a method for increasing the activity of a monospecific bivalent antibody, whereby the method comprises the preparation of a monospecific tetravalent variant of said antibody in which all antigen-binding sites bind to the same antigen, whereby said monospecific bivalent antibody is modified by the connection to monospecific monovalent single chain antibodies each connected via a peptidic-linker to a single C- or N-terminus of the antibody chains of said monospecific bivalent antibody.
  • a pharmaceutical composition comprising an antibody according to the invention is comprised. Additionally a method for the production of a medicament for the treatment of immunosuppression comprising an antibody according to the invention as well as a method for the production of a medicament for the treatment of allograft rejection, or COPD, or rheumatoid arthritis comprising an antibody according to the invention is reported.
  • a further aspect of the current invention is a method for the production of a medicament for the treatment of an HIV infection comprising a variant of a parent monospecific bivalent antibody to which said HIV strain has developed a resistance which variant is a monospecific tetravalent variant of said parent monospecific bivalent antibody in which to said parent monospecific bivalent antibody two single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies bind to the same epitope either the same or an overlapping epitope from that bound by the antigen-binding sites of the parent monospecific bivalent antibody on the same antigen .
  • the invention further encompasses a method for the production of an antibody according to the invention comprising the following steps:
  • the current invention reports an antibody which is i) monospecific
  • iii) consisting of: - a monospecific bivalent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain, - two peptidic-linkers, and two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, with a single-chain-linker in between,
  • two antigen-binding sites are each formed by a pair of heavy and light chain variable domains of the monospecific bivalent antibody and both bind to the same epitope, ii) two antigen-binding sites are each formed by one single chain antibody and both bind to the same or an overlapping epitope as the antigen-binding sites of i),
  • said single chain antibodies are both linked to the same kind of terminus of a heavy chain or a light chain of the monospecific bivalent antibody via said peptidic-linker, whereby each antibody chain of the monospecific bivalent antibody which is linked to a single chain antibody is linked only to one single chain antibody.
  • the term "monoclonal” as used herein refers to a preparation of antibody molecules of a single amino acid composition, preferably produced by a single cell and/or its progeny. Nevertheless may these antibody molecules vary in introduced post-translational modifications e.g. in the glycostructure.
  • the term "monospecific" antibody as used within the current application denotes an antibody that has one or more antigen-binding sites each of which bind to the same or an overlapping epitope of one antigen. Two epitopes are identical or overlapping if a signal reduction of 50% or more, in one embodiment of 75% or more, is detected by a surface plasmon resonance (SPR) assay using the immobilized antibody and antigen with the epitope in question at a concentration of 20-50 nM and the antibody for which the epitope identity or overlap has to be detected at a concentration of 100 nM.
  • SPR surface plasmon resonance
  • ELISA cell-based enzyme immunoassay
  • the antibody for which the epitope identity or overlap has to be detected competes with the antibody for the binding to immobilized antigen.
  • the immobilized antigen is incubated with the antibody in labeled form and an excess of the antibody for which the epitope identity or overlap has to be determined. By detection of the bound labeling there can easily be ascertained the epitope identity or overlap.
  • epitope identity or overlap is present and both antibodies bind to the same or an overlapping epitope on the same antigen.
  • epitope denotes a protein determinant capable of specific binding to the antigen-binding site of an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the term “valent” as used within the current application denotes the presence of a specified number of antigen-binding sites in an antibody molecule. As such, the terms “bivalent”, and “tetravalent” denote the presence of two antigen-binding site and four antigen-binding sites, respectively, in an antibody molecule.
  • each of the antigen-binding sites comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL).
  • each of the antigen-binding sites is formed by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • Antibodies of the present invention have more than two antigen-binding sites and are monospecific.
  • Monospecific 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 term "CDR-grafted variant” as used within the current application denotes a variant of an antibody variable domain comprising complementary determining regions (CDRs or hypervariable regions) from one source or species and framework regions (FRs) from a different source or species, usually prepared by recombinant DNA techniques. CDR-grafted variants of variable domains comprising murine, rabbit or canine CDRs and human FRs are preferred.
  • T-cell epitope depleted variant denotes a variant of a variable domain of an antibody which was modified to remove or reduce immunogenicity by removing potential human T-cell epitopes (peptide sequences with the capacity to bind to MHC Class II molecules).
  • the removal of potential T-cell stimulating epitopes can be evaluated in an in vitro T-cell proliferation assay using peripheral blood mononuclear cells (PBMC) from healthy donors to provide both T-cells and antigen presenting cells (APC).
  • PBMC peripheral blood mononuclear cells
  • APC antigen presenting cells
  • Overlapping peptidic fractions (9 to 15 amino acids in length) of the variable domain and PBMC are set up in cultures in 96 well plates with 5 ⁇ M peptidic fraction and 2 x 10 5 PBMC per well.
  • a potential T-cell epitope is defined as a peptidic fraction giving a stimulation index (SI) greater than 2 in at least 2 independent PBMC populations, although SIs which are just under 2 may also be included in the analysis.
  • SI stimulation index
  • humanized variant denotes a variant of a variable domain of an antibody, which is reconstituted from the complementarity determining regions (CDRs) of non-human origin, e.g. from a non-human species, and from the framework regions (FRs) of human origin, and which has been further modified by amino acid addition and/or deletion and/or mutation in order to also reconstitute or improve the binding affinity and specifity and optionally to reduce the immunogenicity of the original non-human variable domain.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Such humanized variants are usually prepared by recombinant DNA techniques. The reconstitution of the affinity and specifity of the parent non-human variable domain is the critical step, for which different methods are currently used.
  • the suited positions for such mutations can be identified e.g. by sequence or homology analysis, by choosing the human framework (fixed frameworks approach; homology matching; best-fit), by using consensus sequences, by selecting FRs from several different human mAbs, or by replacing non-human residues on the three dimensional surface with the most common residues found in human mAbs ("resurfacing” or "veneering").
  • human antibody denotes antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the variable heavy chain and light chain regions are in one embodiment derived from germline sequence hVH_3_33 (GenBank L06618) and germline sequence hVK_3_l 1 (GenBank XO 1668), or from germline sequence hVH_3_64 (GenBank M99682) and germline sequence hVK lD l ⁇ (GenBank K01323), or germline sequence hVH_4_59 (GenBank L10088) and germline sequence hVK_l_39 (GenBank X59315), or from germline sequence hVH_2_26 (GenBank M99648) and germline sequence hVK_l_9 (GenBank Z00013), or from germline sequence hVH_4_30_4 (GenBank Z14238) and germline sequence hVK_l_27 (GenBank X63398).
  • the constant regions of the antibody are in one embodiment constant regions of human IgGl or IgG4 type. Such regions can be allotypic and are described by, e.g., Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218, and the databases referenced therein.
  • binding refers to the binding of the antibody to an epitope of an antigen in an in vitro assay, in one embodiment in a cell-based ELISA with CHO cells expressing wild-type antigen. Binding means in one embodiment a binding affinity (K D ) of 10 "8 M or less, in another embodiment 10 "13 M to 10 "9 M.
  • nucleic acid molecule as used within the current application is intended to include DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but in one embodiment is double-stranded DNA.
  • variable domain denotes the part of an antibody heavy and light chain which is directly involved in binding of an antibody to its antigen, generally this is the N-terminal part of the antibody heavy and light chain of a monospecific bivalent antibody.
  • Variable domains have a general structure. Each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three “hypervariable regions” or “complementarity determining regions”.
  • FR framework
  • hypervariable regions or “complementarity determining regions”.
  • CDR complementary determining region
  • HVR hypervariable region
  • the term "framework" region (FR) denotes those variable domain amino acid residues other than those of the hypervariable regions. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the regions FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4.
  • 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".
  • CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody.
  • CDRs and FRs are determined according to the standard definition of Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • the first aspect of the current invention is an antibody comprising four antigen- binding sites whereby said antigen-binding sites are grouped in pairs and the antigen-binding site bind to the same or an overlapping epitope.
  • the architecture of the antibody according to the invention allows for multiple different formats to be generated, such as monospecific tetravalent antibodies, bispecific tetravalent antibodies, and trispecific hexavalent antibodies.
  • the antibody according to the invention always has an even number of antigen-binding sites in order to allow the grouping of said antigen-binding sites in pairs, whereby each antigen-binding site of such a pair binds to the same epitope of the same antigen.
  • the antibodies according to the invention always have at least two antigen-binding sites binding to the same, i.e. identical, epitope.
  • the antibody according to the invention comprises a full length parent antibody as scaffold.
  • full length antibody denotes an antibody consisting of two “full length antibody heavy chain” and two “full length antibody light chain”.
  • a “full length antibody heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody hinge region, an antibody constant domain 2 (CH2), an antibody constant domain 3 (CH3), and optionally an antibody constant domain 4 (CH4) in case of an antibody of the subclass IgE.
  • a “full length antibody light chain” is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL). The full length antibody chains a linked together via inter-polypeptide disulfide bonds between the CL-domain and the CHl domain and between the hinge regions of the full length antibody heavy chains.
  • the antigen-binding sites in the antibody according to the invention are each formed by a pair of two variable domains, i.e. of one heavy chain variable domain and one light chain variable domain.
  • the minimal antigen-binding site determinant in an antibody is the heavy chain CDR3 region.
  • One embodiment of the current invention is an antibody which has the following characteristics: - it is a monospecific antibody, it is tetravalent antibody, and it is consisting of: a monospecific bivalent parent antibody consisting of two full length antibody heavy chains and two full length antibody light chains whereby each chain is comprising only one variable domain,
  • two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker between said two antibody variable domains of the single chain antibodies, whereby said single chain antibodies bind to the same or an overlapping epitope as the antigen-binding sites of the monospecific bivalent parent antibody.
  • said single chain antibodies are linked pair wise to the same kind of terminus (C- and/or N-terminus) of the monospecific bivalent antibody heavy chains or, alternatively to the same kind of terminus (in one embodiment to the C-terminus) of the monospecific bivalent antibody light chains of the parent antibody.
  • either one or no single chain antibody is linked to a chain of the monospecific bivalent antibody, i.e. to two chains of the monospecific bivalent antibody is linked at most one additional single chain antibody.
  • the antibody is characterized by - two antigen-binding sites are each formed by the two pairs of heavy and light chain variable domains of the monospecific bivalent parent antibody and both bind to the same epitope, the additional two antigen-binding sites are each formed by the heavy and light chain variable domain of one single chain antibody and both bind to the same or an overlapping epitope on the same antigen as the antigen-binding sites of the parent antibody, the single chain antibodies are each linked to one heavy chain or to one light chain via a peptidic-linker, whereby each antibody chain terminus is linked only to one single chain antibody.
  • peptidic-linker as used within the invention denotes a peptide, which is in one embodiment of synthetic origin.
  • peptidic-linkers are used to link the different antigen-binding sites and/or antibody fragments eventually comprising the different antigen-binding sites (e.g. single chain Fv, full length antibodies, a VH domain and/or a VL domain, Fab, (Fab)2, Fc part) together to form an antibody according to the invention
  • the peptidic-linkers can comprise one or more of the following amino acid sequences or are in another embodiment independently selected from SEQ ID NO: 066, 073, 074, 075, 076, 077, 078, 079, 080, 081, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 094, 095, 096, 097, 099, 101, 102, 103, 104, 105, 109, 1 10, 1 1 1, 112, 1 13, 1 14, 115, 116, 117, 118, 119, 126 or 127 (see Table 1) as well as further arbitrarily selected amino acids.
  • said peptidic-linkers are peptides with an amino acid sequence with a length of from at least 10 amino acids up to 30 amino acids.
  • said peptidic-linker comprises the amino acid sequence of SEQ ID NO: 083, 084, 089, 090, 126.
  • said peptidic linker comprises the amino acid sequence of SEQ ID NO: 083.
  • said peptidic-linker consists of the amino acid sequence of SEQ ID NO: 083, 084, 089, 090, 126, in another embodiment of SEQ ID NO: 083.
  • Table 1 Peptidic-linker amino acid sequences.
  • single-chain-linker denotes a peptide, which is in one embodiment of synthetic origin. These single-chain-linkers according to invention are used to link a VH and a VL domain to form a single chain Fv (scFv).
  • the single-chain-linker can comprise one or more of the following amino acid sequences and is independently selected for each single chain antibody from SEQ ID NO: 065, 067, 068, 069, 070, 071, 072, 073, 074, 075, 076, 077, 078, 079, 080, 081, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 094, 095, 096, 098, 099, 100, 101, 102, 103, 104, 106, 107, 108, 109, 110, 111, 112, 1 13, 114, 115, 116, 117, 118 (see Table 2) as well as further arbitrarily selected amino acids.
  • said single-chain-linker is a peptide with an amino acid sequence with a length of at least 15 amino acids, in another embodiment with a length of from at least 20 amino acids up to 30 amino acids.
  • said single-chain-linker comprises the amino acid sequence of SEQ ID NO: 084, 089, 090, 080.
  • said single-chain-linker comprises the amino acid sequence of SEQ ID NO: 089, 090, 080, in one embodiment of SEQ ID NO: 089.
  • said single-chain-linker consists of the amino acid sequence of SEQ ID NO: 084, 089, 090, 080.
  • the single- chain-linker is consisting of the amino acid sequence of SEQ ID NO: 089, 090, 080, in one embodiment of SEQ ID NO: 089.
  • the multivalent antibodies according to the current invention have improved characteristics compared to the respective bivalent parent antibodies. They show an increased in vitro biological potency and may provide benefits such as increased activity/efficacy, reduced dose and/or frequency of administration and concomitantly cost savings compared to the application of two or more individual antibodies in combination. It has further been found that not all position combinations of antigen-binding sites result in a multivalent antibody that can be recombinantly produced. Thus, it has surprisingly been found that the selection of the antigen-binding site(s) and therewith the variable domains has to be made carefully.
  • the downstream processing of antibodies is very complicated. For example, not only for formulated drugs but also for intermediates in downstream processing (DSP) concentrated solutions are required to achieve low volumes for economic handling and application storage. But with increasing concentration of the antibody a tendency to form aggregates can be observed. These aggregated antibodies have impaired characteristics compared to the isolated antibody. It has now been found that aggregation of the antibodies according to the invention can be reduced by the introduction of disulfide bonds between the heavy and light chain variable domains of the single chain antibodies connected to the monospecific bivalent parent antibody ( Figure 2). This improved stability is not only useful during the production process but also for the storage of the antibodies ( Figure 3). In one embodiment the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is independently for each single chain antibody selected from:
  • heavy chain variable domain position 44 to light chain variable domain position 100
  • heavy chain variable domain position 105 to light chain variable domain position 43
  • heavy chain variable domain position 101 to light chain variable domain position 46.
  • the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100.
  • the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 105 and light chain variable domain position 43. It has further been found that with increased single-chain-linker length the formation of aggregates is reduced.
  • the number of GGGGS units in the linker is from 3 to 6. In a preferred embodiment the number of GGGGS units is 5 or 6.
  • constant region denotes the sum of the domains of an antibody other than the variable region.
  • the constant region is not involved directly in antigen-binding, but exhibits various effector functions.
  • antibodies are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2.
  • the heavy chain constant regions that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ ,.. respectively.
  • the light chain constant regions which can be found in all five antibody classes are called K (kappa) and ⁇ (lambda).
  • the antibody according to the invention has a light chain constant region of the kappa class (SEQ ID NO: 063) or the lambda class (SEQ ID NO: 128).
  • constant region derived from human origin denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain ⁇ or ⁇ region.
  • constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).
  • an antibody according to the invention has a reduced FcR binding compared to an IgGl antibody and the monospecific bivalent parent antibody is in regard to FcR binding of IgG4 subclass or of IgGl or IgG2 subclass with a mutation in S228, L234, L235 and/or D265, and/ or contains the PVA236 mutation.
  • the mutations in the monospecific bivalent parent antibody are S228P, L234A, L235A, L235E and/or PVA236.
  • the mutations in the monospecific bivalent parent antibody are in IgG4 S228P and in IgGl L234A and L235A. Constant heavy chain regions shown in SEQ ID NO: 061 and 062. In one embodiment the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 061 with mutations L234A and L235A. In another embodiment the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 062 with mutation S228P. In another embodiment the constant light chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 063.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CIq complement factor CIq
  • IgG antibody subclasses IgG antibody subclasses
  • complement-binding site Such constant region binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al., J. Immunol. 127 (1981) 2555- 2560; Brunhouse, R. and Cebra, J. J., MoI. Immunol.
  • Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
  • ADCC antibody-dependent cellular cytotoxicity
  • complement-dependent cytotoxicity denotes a process initiated by binding of complement factor CIq to the Fc part of most IgG antibody subclasses. Binding of CIq to an antibody is caused by defined protein-protein interactions at the so called complement-binding site.
  • Fc part binding sites are known in the state of the art (see above). Such Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
  • Antibodies of subclass IgGl, IgG2, and IgG3 usually show complement activation including CIq and C3 binding, whereas IgG4 does not activate the complement system and does not bind CIq and/or C3.
  • Cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and US 6,602,684.
  • IgGl type antibodies the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain.
  • ADCC antibody dependent cellular cytotoxicity
  • the monospecific bivalent parent antibody glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65% or lower (Numbering according to Kabat). In another embodiment is the amount of fucose within said sugar chain is between 5% and 65%, in another embodiment between 20% and 40%.
  • “Asn297” according to the invention means amino acid asparagine located at about position 297 in the Fc region (numbering according to Kabat). Based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than ⁇ 3 amino acids) upstream or downstream of position 297, i.e. between position 294 and 300.
  • the antibody according to the invention is of human IgGl or IgG3 subclass.
  • the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha- 1,3 -galactose is 1% or less within said sugar chain.
  • the sugar chain show preferably the characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell.
  • the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell
  • NGNA as used within this application denotes the sugar residue N-glycolylneuraminic acid.
  • a constant region of IgGl subclass is employed in the monospecific bivalent parent antibody
  • a constant region of IgG4 subclass, or modified/mutated IgGl subclass is employed in the monospecific bivalent parent antibody.
  • the present invention refers in one embodiment to an antibody with a monospecific bivalent parent antibody that has a reduced binding to or does not bind Fc ⁇ receptor and/or complement factor CIq. Such an antibody show reduced or no triggering of antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
  • such a monospecific bivalent parent antibody is characterized in that it contains a constant region derived from human origin, and does not bind or shows a reduced binding of Fc receptors and/or complement factor CIq. No "CIq binding" is found if in such an assay the optical density (OD) at 492 nm reduced by the optical density at the reference wavelength of 405 nm is for the test antibody lower than 15% of the value for human CIq binding of the unmodified wild-type monospecific bivalent parent antibody constant region at an antibody concentration of 8 ⁇ g/ml. Reduced "CIq binding" is in the range of from 15% to 30% of the value for human CIq binding of the unmodified wild-type monospecific bivalent parent antibody constant region at the same conditions.
  • ADCC can be measured as binding of the antibody to human Fc ⁇ RIIIa on human NK cells. Binding is determined at an antibody concentration of 20 ⁇ g/ml. "No Fc ⁇ receptor binding” or “no ADCC” means a binding of up to 30% to human Fc ⁇ RIIIa on human NK cells at an antibody concentration of 20 ⁇ g/ml compared to the binding of the same antibody as human IgGl (SEQ ID NO: 061). "Reduced Fc ⁇ receptor binding” or “reduced ADCC” means a binding of from 30% up to 60% to human Fc ⁇ RIIIa on human NK cells compared to the binding of the same antibody as human IgGl (SEQ ID NO: 061).
  • the monospecific bivalent parent antibody is an antibody that does bind Fc ⁇ receptor and/or complement factor CIq.
  • Such an antibody which does bind Fc receptor and/or complement factor CIq does elicit antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • this antibody is characterized in that the monospecific bivalent parent antibody contains an Fc part derived from human origin, and does also bind Fc receptors and/or complement factor CIq.
  • CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F. H., Glycoconjugate J. 14 (1997) 201-207.
  • Antibodies which are recombinantly expressed in non-glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85%.
  • the modified oligosaccharides of the monospecific bivalent parent antibody may be hybrid or complex. In one embodiment the bisected, reduced/not-fucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/not-fucosylated oligosaccharides are complex.
  • amount of fucose means the amount of said sugar within the sugar chain at Asn297, related to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry and calculated as average value.
  • the relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures identified in an N-Glycosidase F treated sample (e.g. complex, hybrid and oligo- and high-mannose structures, respectively) by MALDI-TOF.
  • the antibody according to the invention is 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 and said expression vectors are transfected into a host cell.
  • 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 “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom 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.
  • control 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.
  • a nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein 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 sequences being linked are contiguous, 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, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • An antibody according to the invention with a reduced amount of fucose can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity and a polypeptide having ManII activity in an amount to fucosylate according to the invention the oligosaccharides in the Fc region.
  • the polypeptide having GnTIII activity is a fusion polypeptide.
  • a ⁇ ,6-fucosyltransferase activity of the host cell can be decreased or eliminated according to US 6,946,292 to generate glycomodified host cells.
  • the amount of antibody fucosylation can be predetermined e.g. either by fermentation conditions or by combination of at least two antibodies with different fucosylation amount.
  • the antibody according to the invention with a reduced amount of fucose can be produced in a host cell by a method comprising: (a) culturing a host cell engineered to express at least one polynucleotide encoding a fusion polypeptide having GnTIII activity and/or ManII activity under conditions which permit the production of said antibody and which permit fucosylation of the oligosaccharides present on the Fc region of said antibody in an amount according to the invention; and (b) isolating said antibody.
  • the polypeptide having GnTIII activity is a fusion polypeptide, in one embodiment comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi resident polypeptide selected from the group consisting of the localization domain of mannosidase II, the localization domain of ⁇ (l,2)-N-acetylglucosaminyltransferase I ("GnTI"), the localization domain of marmosidase I, the localization domain of ⁇ (l,2)-N- acetylglucosaminyltransferase II (“GnTII”), and the localization domain of a- 1 ,6 core fucosyltransferase.
  • the Golgi localization domain is from marmosidase II or GnTI.
  • polypeptide having GnTIII activity refers to polypeptides that are able to catalyze the addition of an N-acetylglucosamine (GIcNAc) residue in ⁇ -1,4 linkages to the ⁇ - linked mannoside of the trimannosyl core of N-linked oligosaccharides.
  • GIcNAc N-acetylglucosamine
  • ⁇ -l,4-N- acetylglucosaminyltransferase III also known as ⁇ -l,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.112)
  • NC-IUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
  • Golgi localization domain refers to the amino acid sequence of a Golgi resident polypeptide which is responsible for anchoring the polypeptide in location within the Golgi complex. Generally, localization domains comprise amino terminal "tails" of an enzyme.
  • a host cell that is able and engineered to allow the production of an antibody with modified glycoforms can be used.
  • a host cell has been further manipulated to express increased levels of one or more polypeptides having GnTIII activity.
  • CHO cells are preferred as such host cells.
  • cells producing antibody compositions having high antibody-dependent cell-mediated cytotoxic activity as reported in US 6,946,292 can be used.
  • 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 alkaline/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 microbial 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 chromatography (e.g.
  • Ni(II)- and Cu(II)-affinity material size exclusion chromatography
  • electrophoretical methods such as gel electrophoresis, capillary electrophoresis
  • Gel electrophoresis capillary electrophoresis
  • One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention.
  • Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition.
  • a further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention.
  • the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
  • 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 certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • 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.
  • Pharmaceutical carriers 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, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. These 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.
  • composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier is in one embodiment 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 mannitol or sorbitol, and sodium chloride in the composition.
  • CCR5 is a human chemokine receptor (see e.g. Swiss Prot P51681 and Mueller, A., and Strange, P.G., Int. J. Biochem. Cell Biol. 36 (2004) 35-38) within the "cluster" chemokines that are produced primarily during inflammatory responses and control the recruitment of neutrophils (CXC chemokines) and macrophages and subsets of T-cells. (T-helper ThI and Th2 cells). ThI responses are typically those involving cell-mediated immunity effective against viruses and tumors, for example, whereas Th2 responses are believed to be pivotal in allergies. Therefore, inhibitors of these chemokine receptors may be useful as immunomodulators.
  • ThI responses overactive responses are dampened, for example, in autoimmunity including rheumatoid arthritis or, for Th2 responses, to lessen asthma attacks or allergic responses including atopic dermatitis (see e.g. Schols, D., Curr. Top. Med. Chem. 4 (2004) 883-893; Mueller, A., and Strange, P.G., Int. J. Biochem. Cell Biol. 36 (2004) 35-38; Kazmierski, W.M., et al., Curr. Drug Targets Infect. Disord. 2 (2002) 265-278; Lehner, T., Trends Immunol. 23 (2002) 347-351).
  • the antibodies inhibit one or more functions of human CCR5, such as ligand binding to CCR5, signaling activity (e.g. activation of a mammalian T-protein, induction of a rapid and transient increase in the concentration of cytosolic free Ca 2+ , and/or stimulation of a cellular response (e.g. stimulation of chemotaxis, exocytosis or inflammatory mediator release by leukocytes, integrin activation).
  • the antibodies inhibit binding of RANTES, MIP-I alpha, and/or MIP-I beta, to human CCR5 and/or inhibit functions mediated by human CCR5, like leukocyte trafficking, T-cell activation, inflammatory mediator release, and/or leukocyte degranulation.
  • An antibody according to the invention in one embodiment does not inhibit chemokine binding in a binding assay to CCRl, CCR2, CCR3, CCR4, CCR6, and CXCR4 in an antibody concentration up to 100 ⁇ g/ml.
  • Chemokines and their receptors are known to participate in allograft rejection by mediating leukocyte trafficking.
  • Panzer, U., et al. Transplantation 78 (2004) 1341-50
  • Luckow, B., et al. (Eur. J. Immunol. 34 (2004) 2568-78) observed decreased intragraft levels of metalloproteinases and arteriosclerosis in CCR5- deficient animals.
  • Gao, W., et al. Transplantation 72 (2001) 1199-1205) demonstrated prolonged allograft survival in mice treated with CCR5-specific monoclonal antibody and in CCR5-deficient mice.
  • WO 01/78707 refers to a method of inhibiting graft rejection comprising administering an antagonist of CCR5 function.
  • CCR5 is also used by most HIV-I primary isolates and is critical for the establishment and maintenance of infection.
  • CCR5 function is dispensable for human health, probably because CCR5 is part of a highly redundant chemokine network as receptor for the ⁇ chemokines MIP l ⁇ , MIP- l ⁇ and RANTES, which share many overlapping functions, and most of which have alternative receptors (Rossi, D., and Zlotnik, A., Annu. Rev. Immunol. 18 (2000) 217-242).
  • CCR5 as an HIV-I co-receptor was based on the ability of its ligands, MIP- l ⁇ , MIP- l ⁇ and RANTES, to block infection by R5 but not R5X4 or X4 isolates (Cocchi, F., et al., Science 270 (1995) 1811-1815). HIV-I infection is initiated by interactions between the viral envelope glycoprotein (Env) and a cellular receptor complex comprised of CD4 plus a chemokine receptor (Pierson, T.C., and Doms, R. W., Immuno. Lett. 85 (2003) 113-118; and Kilby, J.M., and Eron, J.N., Engl. J. Med.
  • the chemokine receptor CCR5 is a co-receptor for macrophage-tropic (R5) strains and plays a crucial role in the sexual transmission of HIV-I (Berger, E. A., AIDS 1 1 (Supp. 061) (1997) S3-S16; Bieniasz, P.D., and Cullen, B.R., Frontiers in Bioscience 3 (1998) D44-D58; Littman, D.R., Cell 93 (1998) 677-680).
  • Antibodies against CCR5 are e.g. PRO 108 (Olson, W.C., et al., J. Virol. 73 (1999) 4145-4155) and 2D7 (Samson, M., et al., J. Biol. Chem. 272 (1997) 24934-24941).
  • variable domains of the anti-CCR5 antibody some positions are variable within the given boundaries, e.g. at position 5 of SEQ ID NO: 120 either the amino acid lysine or glutamine can be present, at position 6 of SEQ ID NO: 120 either the amino acid glutamine or glutamic acid can be present, and so on.
  • SEQ ID NO: 120 is the amino acid sequence of the heavy chain variable domain, wherein XOl to X08 denote the variable positions:
  • XOl is either Lys or GIn
  • X02 is either GIn or GIu
  • X03 is either Arg or Lys
  • X04 is either Leu or Pro
  • X05 is either Met or Lys
  • X06 is either He or Thr
  • X07 is either Ser or Thr
  • X08 is either He or Thr (SEQ ID NO: 120).
  • SEQ ID NO: 121 is the amino acid sequence of the variable light chain domain, wherein XlO to X24 denote the variable positions:
  • Xl 1 is either Phe or Tyr
  • Xl 2 is either GIn or Pro
  • Xl 3 is either Ser or Ala
  • Xl 4 is either GIn or Lys
  • Xl 5 is either VaI or He
  • Xl 6 is either GIn or Asp
  • Xl 7 is either Ser or Thr
  • Xl 8 is either Leu or Ala
  • Xl 9 is either Lys or Thr
  • X20 is either Asn or Ser
  • X21 is either Leu or Ala
  • X22 is either GIy or Ala
  • X23 is either Asn or Thr
  • X24 is either Leu or VaI (SEQ ID NO: 121).
  • One aspect of the invention is an antibody which is a monospecific tetravalent antibody with antigen-binding sites binding to a single epitope of CCR5. It has surprisingly been found that with the doubling of the antigen-binding sites an improved in vitro biological potency is detectable. For example in a PBMC antiviral assay according to Example 14 a reduction of the IC 50 value from 1077.5 pM (pmol/1) to 87.8 pM can be detected. This provides for a synergistic effect of the four antigen-binding sites in this antibody according to the invention.
  • This monospecific tetravalent anti-CCR5 antibody is consisting of:
  • - a monospecific bivalent anti-CCR5 parent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain, - two peptidic-linkers, and two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker.
  • the four antigen-binding sites of the monospecific tetravalent anti-CCR5 antibody according to this aspect of the current invention bind to the same epitope or an overlapping epitope as an antibody obtained from DSM ACC 2681 or DSM ACC 2683.
  • the heavy chain variable domain of said antigen-binding sites comprises a CDR3 of SEQ ID NO: 001, or 009, or 035.
  • the heavy chain variable domain of said antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 003, 002, 001, or SEQ ID NO: 011, 010, 009, or SEQ ID NO: 037, 036, 035.
  • the antigen- binding sites are characterized that the light chain variable domain of said antigen- binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 007, 006, 005, or SEQ ID NO: 016, 015, 014, or SEQ ID NO: 042, 041, 040.
  • the heavy and light chain variable domain of said antigen-binding sites have an amino acid sequence of SEQ ID NO: 004, 008, or SEQ ID NO: 012, 017, or SEQ ID NO: 039, 044 or are a T-cell epitope depleted, CDR-grafted, or humanized variant thereof.
  • One aspect of the invention is therefore a monospecific tetravalent anti-CCR5 antibody with a heavy chain amino acid sequence of SEQ ID NO: 122 and a light chain amino acid sequence of SEQ ID NO: 123.
  • This antibody binds to the same epitope as an antibody obtained from DSM ACC 2681.
  • the single chain antibodies have a disulfide bond between its single chain antibody variable domains.
  • Another aspect of the invention is a monospecific tetravalent anti-CCR5 antibody with a heavy chain amino acid sequence of SEQ ID NO: 124 and a light chain amino acid sequence of SEQ ID NO: 125, wherein in said antibody the single chain antibodies have a disulfide bond between the single chain antibody variable domains. This antibody binds to the same epitope as an antibody obtained from DSM ACC 2683.
  • Another aspect of the current invention is a method for increasing the biological potency of a monospecific bivalent parent antibody comprising the preparation of a monospecific tetravalent variant of said antibody in which all antigen-binding sites bind to the same or an overlapping epitope on the same antigen, whereby said monospecific bivalent antibody is modified by the connection to two monospecific monovalent single chain antibodies each connected via a peptidic-linker each to a single C- or N-terminus of the antibody chains of said monospecific bivalent antibody.
  • said method is the antigen CCR5.
  • all antigen-binding sites bind to the same or an overlapping epitope of the same antigen.
  • said epitope is that to which the antigen-binding sites of an antibody obtained from DSM ACC 2681, or DSM ACC 2682, or DSM ACC 2683, or DSM ACC 2684, or an antibody with a heavy chain variable domain of SEQ ID NO: 048 and a light chain variable domain of SEQ ID NO: 052, or an antibody with a heavy chain variable domain of SEQ ID NO: 056 and a light chain variable domain of SEQ ID NO: 060 bind.
  • Another aspect of the current invention is a pharmaceutical composition comprising an antibody according to the invention.
  • One embodiment of this aspect is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of immunosuppression.
  • Another embodiment is the use of an antibody according to the invention for the treatment of immunosuppression.
  • a further embodiment is a method for the manufacture of a medicament for the treatment of immunosuppression comprising an antibody according to the invention.
  • Another embodiment is a method of treatment of immunosuppression by administering an antibody according to the invention to a patient.
  • the immunosuppression is HIV infection.
  • Monospecific tetravalent anti-CCR5 antibodies which have a reduced amount of fucose and are non-depleting are useful for the manufacture of a medicament for the treatment of acute or chronic allograft rejection, or COPD, or rheumatoid arthritis.
  • one embodiment is the use of an antibody according to the invention with a reduced amount of fucose for the manufacture of a medicament for the treatment of allograft rejection, or COPD, or rheumatoid arthritis.
  • a further embodiment is the use of an antibody according to the invention with a reduced amount of fucose for the treatment of allograft rejection, or COPD, or rheumatoid arthritis.
  • Transplantation is performed according to the state of the art with numerous cell types, tissue types and organ types, e.g. pancreatic islets, corneal, bone marrow, stem cells, skin graft, skeletal muscle, aortic and aortic valves, and organs as heart, lung, kidney, liver, and pancreas.
  • the invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from GvHD or HvGD (e.g. after transplantation).
  • the invention comprises also a method for the treatment of a patient suffering from such GvHD and HvGD.
  • the invention also provides the use of an antibody according to the invention in an effective amount for the manufacture of a pharmaceutical agent, in one embodiment together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from inflammatory mediator release mediated by CCR5.
  • graft rejection denotes the response of the human immune system to transplanted tissue. If tissue is transplanted from a donor to a host the human leukocyte antigen genes of the donor's tissue are likely to be different from those of the host's tissue. Thus, the host's immune system recognized the transplanted tissue as foreign and effects an immune response called graft rejection. This graft rejection reaction is called "graft versus host disease" (GvHD).
  • GvHD graft versus host disease
  • the current invention provides a method of treating or preventing acute and chronic organ transplant rejection in a mammal, including a human, characterized in administering to said mammal an antibody according to the invention. Also is provided an antibody according to the invention for the treatment or prevention of acute and chronic organ transplant rejection in a mammal, including a human.
  • Another aspect of the current invention is the use of a variant of a monospecific bivalent parent antibody to which a HIV strain has developed a resistance for the manufacture of medicament for the treatment an HIV infection by said HIV strain said medicament comprising a variant of said monospecific bivalent parent antibody to which said HIV strain has developed a resistance which is a bispecific or trispecif ⁇ c tetravalent or hexavalent form of said monospecific bivalent parent antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic- linker, whereby said single chain antibodies are grouped in pairs in which each member of a pair binds to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen.
  • the yield of the monospecific tetravalent antibody according to the invention is depending on the one hand on the terminus to which the single chain antibodies are connected, on the length of the linkers and on the additional stabilization of the single chain antibodies by additional disulfide bonds.
  • Table 3 Yield of antibodies according to the invention.
  • the single chain antibodies connected to the C-terminus of the full length heavy or light antibody chain.
  • the single chain antibody comprises the single chain antibody a disulfide bond between residue 100 of the light chain variable domain and residue 44 of the heavy chain domain.
  • the peptidic linker consisting of two G4S units.
  • hybridoma cell lines used in the invention m ⁇ CCR5>Pz01.063, m ⁇ CCR5>Pz02.1Cl l, m ⁇ CCR5>Pz03.1C5 and m ⁇ CCR5>PzO4.1F6, were deposited, under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany:
  • Figure 1 Structures of an exemplary tetravalent antibody according to the invention.
  • Figure 2 Exemplary size exclusion chromatogram of an antibody a) without disulfide bonds between the variable domains of the single chain antibodies (BA-4320), b) with disulfide bonds between the variable domains of the single chain antibodies (BA-
  • Figure 3 Exemplary comparison of the storage dependent formation of aggregates of exemplary antibodies according to the invention for storage at 4 °C for 5 days: a) without disulfide bonds between the variable domains of the single chain antibodies, b) with disulfide bonds between the variable domains of the single chain antibodies.
  • Figure 4 Binding epitopes of two anti-CCR5 antibodies.
  • Figure 5 A comparison of IC 50 values for different multivalent antibodies determined by PBMC assay: 1 - BN- 1000, 2 - AN- 1000, 3- AN-
  • DNA sequencing DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
  • Desired nucleic acids were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis.
  • the nucleic acids which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids.
  • the plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned nucleic acids was confirmed by DNA sequencing.
  • mice Female Balb/c mice are given a primary intraperitoneal immunization with 10 7 CCR5 expressing cells (CHO or Ll .2) together with the adjuvant CFA (complete Freund's adjuvant). This is followed by one further intraperitoneal immunization after 4-6 weeks with again 10 7 CCR5 expressing cells together with IFA (incomplete Freund's adjuvant). Thereafter each mouse is administered with again 10 7 CCR5 expressing cells (CHO or Ll.2) in PBS at 4-6 weeks intervals. Subsequently the last immunizations are carried out intraperitoneally with again 10 7 CCR5 expressing cells or intravenously using 2x10 6 CCR5 expressing cells on the 3rd or 4th day before fusion.
  • CFA complete Freund's adjuvant
  • mice immunized according to a) are fused with myeloma cells according to Galfre, G., Methods in Enzymology 73 (1981) 3-46.
  • About 1x10 8 spleen cells of the immunized mouse are mixed with about the same number of myeloma cells (P3x63-Ag8.653, ATCC CRL 1580), fused and cultivated subsequently in HAZ medium (100 mmol/1 hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640 + 10% FCS). After ca. 10 days the primary cultures are tested for specific antibody production.
  • Vectors for the expression of chimeric human-mouse antibodies have been constructed as follows.
  • a heavy chain expression vector was constructed by linking a heavy chain variable domain to human IgGl (SEQ ID NO: 061) and human IgG4 (SEQ ID NO: 062) constant region in the expression vector pSVgpt.
  • a light chain expression vector was constructed by linking a light chain variable domain to human Kappa light chain constant region (SEQ ID NO: 063) in the expression vector pSVhyg. 5' flanking sequence including the leader signal peptide, leader intron and the murine immunoglobulin promoter, and 3' flanking sequence including the splice site and intron sequence was introduced using the vectors VH-PCRl and VK-PCRl as templates.
  • the heavy and light chain expression vectors were co-transfected into NSO cells (ECACC No 85110503, a non- immunoglobulin producing mouse myeloma). Transfected cell clones were screened for production of human antibody by ELISA for human IgG.
  • NSO cells ECACC No 85110503, a non- immunoglobulin producing mouse myeloma.
  • PBMC Peripheral blood mononuclear cells
  • APC antigen presenting cells
  • Forty donors were selected for screening in T-cell assays based on human HLA-DR typing. This enables the screening of peptides in the T-cell assay against greater than 85% of DR alleles expressed in the world population.
  • Peptidic fragments of 15 amino acids length were designed to cover the variable regions of the antibody, each overlapping by 12 residues. The sequence was extended at the end of the variable regions to include the first few residues of the human constant regions to which they will be joined in the final engineered antibodies.
  • Peptidic fragments and PBMC were set up in sextuplicate cultures in 96 well plates with 5 ⁇ M peptide and 2 x 10 5 PBMC per well.
  • Keyhole limpet haemocyanin (KLH) was included in the assay as a positive control.
  • an 18 hour pulse with 3 H-Thymidine at 1 ⁇ Ci/well was used to assess T-cell proliferation.
  • a potential T-cell epitope is defined as a peptide giving a stimulation index (SI) greater than 2 in at least 2 independent donors, although SIs which are just under 2 may also be included in the analysis. All donors responded to KLH with SIs ranging from 2.2 to 32.9.
  • SI stimulation index
  • Expression plasmids encoding variant anti-CCR5 antibodies of IgGl and IgG4 subclass can be obtained by site-directed mutagenesis of the wild type expression plasmids using the QuickChangeTM Site-Directed mutagenesis Kit (Stratagene) and are described in Table 4. Amino acids are numbered according to EU numbering
  • S228P denotes that the amino acid serine at Kabat amino acid position 228 is changed to proline
  • L234A and L235A denotes that the amino acid Leucine at Kabat amino acid position 234 and 235 is each changed to the amino acid Alanine.
  • nucleic acids for the full length antibody heavy and light chains of any anti-CCR5 antibody conjugated to single chain antibodies comprising the variable domains of a different or the same anti-CCR5 antibody be constructed whereby both antibodies bind to the same or different, non-overlapping epitopes on CCR5.
  • nucleic acids for the full length antibody heavy and light chains of an anti- CCR5 antibody obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from the antibodies with variable domain amino acid sequence of SEQ ID NO: 039 and 044, or of SEQ ID NO: 048 and 052, or of SEQ ID NO: 056 and 060 conjugated to single chain antibodies comprising the variable domains of a an anti-CCR5 antibody obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from SEQ ID NO: 039 and 044, or SEQ ID NO: 048 and 052, or SEQ ID NO: 056 and 060 can be obtained whereby either only variable domains are combined which bind to the same epitope on CCR5 or only variable domains are combined which bind to different, non-overlapping epitopes on CCR5.
  • nucleic acids encoding full length heavy and light antibody chains connected to single chain antibodies were obtained in which the single chain antibody binds to the same or a different antigen than the full length antibody, e.g. one antigen is the CCR5 and one antigen is the CD4.
  • Such antibodies comprise variable domains binding to CD4 of SEQ ID NO: 064 and 068 (US 5,871,732), or SEQ ID NO: 072 and 076, or SEQ ID NO: 080 and 084 (Reimann, K.A., et al., Aids Res. Human Retrovir.
  • variable domains binding to CCR5 obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from SEQ ID NO: 039 and 044, or SEQ ID NO: 048 and 052, or SEQ ID NO: 056 and 060.
  • the basis vector used for the construction of all heavy and light chain scFv fusion protein encoding expression plasmids was composed of the following elements: a hygromycin resistance gene as a selection marker, an origin of replication, oriP, of Epstein-Barr virus (EBV),
  • HCMV human cytomegalovirus
  • poly A human ⁇ l -immunoglobulin polyadenylation
  • the nucleic acids comprising the heavy or light chain and a scFv nucleic acids in combination with optional peptidic-linker- variation, single-chain-linker- variation and cysteine modifications, which have been constructed as outlined in Example 4, were cloned into the plasmid pGA18 (ampR).
  • the pGA18 (ampR) plasmids carrying the heavy and light chain encoding nucleic acids as well as the basis vector were digested with BamHI and Xbal restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis.
  • Purified heavy and light chain encoding nucleic acids were ligated to the isolated basis vector BamHI/Xbal fragment resulting in the final pUC-Exp- YY-XXXX-HC and pUC- Exp- YY-XXXX-LC expression vectors.
  • the string 'YY-XXXX' in the expression vector denotes the structure of the encoded antibody, whereby X is a digit and Y is a character.
  • a - anti-CCR5 antibody chain obtained from DSM ACC 2681 B - anti-CCR5 antibody chain obtained from DSM ACC 2683,
  • F - anti-CCR5 antibody domain of SEQ ID NO: 048 (heavy chain variable domain) or SEQ ID NO: 052 (light chain variable domain)
  • G - anti-CCR5 antibody domain of SEQ ID NO: 056 (heavy chain variable domain) or SEQ ID NO: 060 (light chain variable domain)
  • the first block of 4 digits denotes the structure of the first pair of single chain antibodies whereby
  • the first digit denotes the position of attachment of the peptidic-linker (to the pair of single chain antibodies) to the full length antibody chain with
  • the second digit denotes the number of GGGGS units (SEQ ID NO: 082) of which the single-chain-linker is consisting,
  • the third digit denotes the number of GGGGS units of which the peptidic- linker is consisting
  • the fourth digit denotes the position of the disulfide bond in the single chain antibody with
  • the final expression vectors were transformed into E. coli cells, expression plasmid DNA was isolated (Miniprep) and subjected to restriction enzyme analysis and DNA sequencing. Correct clones were grown in 118 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep) and sequence integrity confirmed by DNA sequencing.
  • the final expression plasmids generated in Example 5 allow the recombinant production of different antibody light chains and different antibody heavy chains.
  • the antibodies according to the invention were generated by transient transfection of suspension culture HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr- Virus nuclear antigen; American type culture collection deposit number ATCC # CRL- 10852) cultivated in FreeStyleTM 293 Expression medium containing 250 ⁇ g/ml G418 (Roche Molecular Biochemicals, Germany) at 37°C/5% CO 2 .
  • FuGENETM 6 Transfection Reagent (Roche Molecular Biochemicals, Germany) was used in a ratio of reagent ( ⁇ l) to DNA ( ⁇ g) ranging from 3:1 to 6:1.
  • the light and heavy chains were expressed from two different plasmids using a molar ratio of light chain to heavy chain encoding plasmid from 1 :2 to 2:1.
  • Antibody containing cell culture supernatants were harvested at day 7 after transfection by centrifugation at 1000 x g for 10 minutes followed by filtration through a sterile filter (0.22 ⁇ m).
  • the antibodies were also expressed by transient transfection of human embryonic kidney 293 -F cells using the FreeStyleTM 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyleTM 293-F cells were cultivated in FreeStyleTM 293 Expression medium at 37°C/8% CO 2 .
  • the cells were seeded in fresh medium at a density of 1-2 x 10 6 viable cells/ml on the day of transfection.
  • the DNA-293fectinTM complexes were prepared in Opti-MEM ® I medium (Invitrogen, USA) using 325 ⁇ l of 293fectinTM (Invitrogen, Germany) and 250 ⁇ g of heavy and light chain encoding plasmid DNA in a 1 :1 molar ratio for a 250 ml final transfection volume.
  • the antibody containing cell culture supernatants were clarified 7 days after transfection by centrifugation at 14,000 x g for 30 minutes and filtration through a sterile filter (0.22 ⁇ m). Supernatants were stored at -20°C until purification.
  • the expressed and secreted antibodies were purified in two steps by affinity chromatography using Protein A-SepharoseTM (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the antibody containing clarified culture supernatants were applied on a HiTrap Protein A HP (5 ml) column equilibrated with PBS buffer (10 mM Na 2 HPO 4 , 1 raM KH 2 PO 4 , 105 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed out with equilibration buffer. The antibodies were eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing fractions were neutralized with 0.1 ml 1 M TRIS, pH 8.5.
  • the protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.
  • the purity and the molecular weight of the antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1 ,4-dithiotreitol) and staining with Coomassie brilliant blue).
  • the NuP AGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-20% TRIS-Glycine gels).
  • the aggregate content of antibody samples was analyzed by high-performance SEC using a Superdex 200 analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM KH 2 PO 4 , 250 mM KCl, pH 7.0 running buffer at 25°C. 25 ⁇ g protein were injected on the column at a flow rate of 0.5 ml/min and eluted isocratic over 50 minutes.
  • concentrations of 0.1 mg/ml, 1 mg/ml and 5 mg/ml of purified proteins were prepared and incubated at 4°C, 37°C and 40°C, respectively, for 7 or 28 days and then evaluated by high-performance SEC (Figure 2).
  • HBS-P (10 mM HEPES, pH 7.4, 118 mM NaCl, 0.005% Surfactant P20; GE Healthcare Biosciences AB, Sweden) was used as running buffer during immobilization.
  • the antibodies in question were diluted with PBS, 0.005% (v/v) Tween20, pH 6.0 to a concentration of 450 nM and injected over 3 minutes at a flow rate of 30 ⁇ l/minute. Afterwards the sensor chip was regenerated for 1 minute with PBS, pH 8.0, 0.005% (v/v) Tween20. Data analysis was performed with the BIAevaluation software (BIAcore, Sweden).
  • CHO-CCR5, CHO-CCR5-dN8 or CHO-CCR5-K171A (K) or CHO-CCR5-K171A/E172A (KE) cells per well were seeded into 96-well tissue culture plates and incubated overnight at 37°C. Thereafter, the cell culture medium was aspirated, 50 ⁇ l new medium containing serially diluted bispecific or control antibodies was added and plates were incubated for 2 hours at 4°C. Then, cells were fixed in PBS containing 0.05% (v/v) glutaraldehyde for 10 min.
  • HRP horseradish peroxidase
  • IgG immunoglobulin G
  • 125I-RANTES (regulated on activation normal T-cell expressed and secreted) was purchased from PerkinElmer Life Sciences Inc. (USA). Binding assays were performed on CHO cells expressing recombinant human CCR5 receptor with 1251- labeled RANTES. Cells were plated in 96-well culture plates at 1.5 x 10 5 cells/well in ice cold binding buffer (phenol red- free Fl 2 medium supplemented with freshly made 0.1% BSA and 0.1% NaN 3 ). Serially diluted CCR5 multivalent and control antibodies were added to the cells, followed by addition of 100 pM 1251-labeled RANTES.
  • plasmid pNL4-3 ⁇ env HAV pNL4-3 genomic construct with a deletion within the env gene
  • pCDNA3.1/NL-BAL env pcDNA3.1 plasmid containing NL-BaI env gene (obtained from NIBSC Centralized Facility for AIDS Reagents)] were co- transfected into the HEK 293FT cell line (Invitrogen, USA), cultured in Dulbecco's' modified minimum medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/mL Penicillin, 100 ⁇ g/mL Streptomycin, 2 mM L-glutamine and 0.5 mg/mL geniticin (all media from Invitrogen/Gibco).
  • DMEM Dulbecco's' modified minimum medium
  • FCS fetal calf serum
  • the supernatants containing pseudotyped viruses were harvested two days following transfection, and cellular debris was removed by filtration through a 0.45 ⁇ m pore size PES (polyethersulfone) filter (Nalgene) and stored at -80°C in aliquots.
  • virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells yielding approximately 1.5 x 10 5 RLU (relative light units) per well.
  • Multivalent and control antibodies were serially diluted in 96-well plates. The assay was carried out in quadruplicates. Each plate contained cell control and virus control wells.
  • PBMC peripheral blood mononuclear cells
  • Human PBMC were isolated from buffy-coats (obtained from the Stanford Blood Center) by a Ficoll-Paque (Amersham, USA) density gradient centrifugation according to manufacturer's protocol. Briefly, blood was transferred from the buffy coats in 50 ml conical tubes and diluted with sterile Dulbecco's phosphate buffered saline (Invitrogen/Gibco) to a final volume of 50 ml. Twenty- five ml of the diluted blood was transferred to two 50 ml conical tubes, carefully underlayerd with 12.5 ml of Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at room temperature for 20 min. at 450 x g without braking.
  • Ficoll-Paque Analogen/Gibco
  • the white cell layer was carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove remaining red blood cells, cells were incubated for 5 min. at room temperature with ACK lysis buffer (Biosource) and washed one more time with PBS. PBMC were counted and incubated at a concentration of 2 to 4 x 10 6 cells/ml in RPMIl 640 containing 10% FCS (Invitrogen/Gibco), 1% penicillin/streptomycin, 2 raM L-glutamine, 1 raM sodium-pyruvate, and 2 ⁇ g/ml Phytohemagglutinin (Invitrogen) for 24 h at 37°C. Cells were incubated with 5 Units/ml human IL-2 (Roche Molecular Biochemicals) for a minimum of 48 h prior to the assay.
  • FIG. 5 A comparison of IC 50 values for different multivalent antibodies is shown in Figure 5. It can be seen that the tetravalent antibodies have an improved in vitro biological potency even better than the combination of the individual isolated parent antibodies, e.g. it can be seen that the doubling of the antigen-binding sites for the same epitope in case of AA-2320 provides for a reduction of the IC 50 from 988 pM to 88 pM.
  • the CCR5-tropic HIV-I isolates BaI and CC 1/85 were passaged in vitro in the presence of increasing concentrations of the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044.
  • CD8-depleted PBMC and genetically diverse, high titer viruses were used to facilitate fast resistance development.
  • the sensitivity of resistant and no drug control viruses (NDC) to the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044 and other anti-CCR5 antibodies was measured in PBMC and single-cycle assays.

Abstract

Herein is reported an antibody, characterized in that said antibody comprises four antigen-binding sites each consisting of a pair of antibody variable domains, whereby each of said antigen-binding sites bind to the same epitope.

Description

Multivalent antibodies
The present invention relates to monospecific tetravalent anti-CCR5 antibodies, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
Background of the Invention Engineered proteins, such as bi- or multispecific antibodies capable of binding two or more antigens are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
A wide variety of recombinant antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S.L., Nature Biotech 25 (2007) 1233- 1234).
Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al., Nature Biotech 23 (2005)
1126-1136; Fischer N., and Leger O., Pathobiology 74 (2007) 3-14; Shen, J., et al.,
Journal of Immunological Methods 318 (2007) 65-74; Wu, C, et al., Nature Biotech. 25 (2007) 1290- 1297).
All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., and Leger, O., Pathobiology 74 (2007) 3-14). It has to be kept in mind that one may want to retain effector functions, such as e.g. complement- dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Fc receptor binding, by maintaining a high degree of similarity to naturally occurring antibodies.
In WO 2007/024715 are reported dual variable domain immunoglobulins as engineered multivalent and multispecific binding proteins. A process for the preparation of biologically active antibody dimers is reported in US 6,897,044. Multivalent Fy-antibody construct having at least four variable domains which are linked with each over via peptide linkers are reported in US 7,097,330. Dimeric and multimeric antigen binding structures are reported in US 2005/0079170. Tri- or tetravalent monospecific antigen-binding protein comprising three or four Fab fragments bound to each other covalently by a connecting structure, which protein is not a natural immunoglobulin are reported in US 6,511,663. In WO 2006/020258 tetravalent bispecific antibodies are reported that can be efficiently expressed in prokaryotic and eukaryotic cells, and are useful in therapeutic and diagnostic methods. A method of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers is reported in US 2005/0163782. Bispecific tetravalent receptors are reported in US 5,959,083. Engineered antibodies with three or more functional antigen antigen-binding sites are reported in WO 2001/077342. Multispecific and multivalent antigen-binding polypeptides are reported in WO 1997/001580. WO 1992/004053 reports homoconjugates typically prepared from monoclonal antibodies of the IgG class which bind to the same antigenic determinant are covalently linked by synthetic cross-linking. Oligomeric monoclonal antibodies with high avidity for antigen are reported in WO 1991/06305 whereby the oligomers, typically of the IgG class, are secreted having two or more immunoglobulin monomers associated together to form tetravalent or hexavalent IgG molecules. Sheep-derived antibodies and engineered antibody constructs are reported in US 6,350,860 which can be used to treat diseases wherein interferon gamma activity is pathogenic. In US 2005/0100543 are reported targetable constructs that are multivalent carriers of bi-specifϊc antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specifϊc antibodies. Genetically engineered bispecific tetravalent antibodies are reported in WO 1995/009917. In WO 2007/109254 stabilized binding molecules that consist of or comprise a stabilized scFv are reported. Mantis, N.J., et al. (J. Immunol. 179 (2007) 3144-3152) report inhibition of HIV-I infectivity and epithelial cell transfer by human monoclonal IgG and IgA antibodies carrying the bl2 V region. Tetravalent bispecific receptors, the preparation and use thereof are reported in US 5,959,083. In WO 2006/020258 novel tetravalent bispecific antibodies are reported. US 2007/0122405 reports human G-protein chemokine receptor (CCR5) HDGNRlO. Diagnostics and therapeutics for diseases associated with C-C chemokine receptor 5 (CCR5) are reported in WO 2005/106489.
Summary of the Invention
A first aspect of the current invention is an antibody comprising four antigen- binding sites each antigen-binding site consisting of a pair of antibody variable domains, whereby every antigen-binding site binds to the same or an overlapping epitope.
In one embodiment the antibody is consisting of four polypeptides which are linked via inter-polypeptide disulfide bonds. In another embodiment the antigen-binding sites of the antibody are each consisting of an antibody heavy chain variable domain and an antibody light chain variable domain. In still a further embodiment the antibody comprises two full length antibody light chains and two full length antibody heavy chains.
In one embodiment the antibody according to the invention is
i) monospecific, and
ii) tetravalent, and
iii) consisting of:
- a monospecific bivalent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain,
- two peptidic-linkers, and
- two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker connecting the antibody heavy chain variable domain and the antibody light chain variable domain of the single chain antibody.
In another embodiment of this aspect of the invention
i) two antigen-binding sites are each formed by a pair of heavy and light chain variable domains of the monospecific bivalent antibody and both bind to the same epitope, ii) two antigen-binding sites are each formed by one single chain antibody and both bind to the same or an overlapping epitope as in i),
iii) said single chain antibodies are each linked to one heavy chain or to one light chain via said peptidic-linker, whereby each antibody chain is linked only to one single chain antibody.
In still a further embodiment the single chain antibodies have a disulfide bond between the heavy and light chain variable domain. In one embodiment the monospecific bivalent antibody is of human IgGl or IgG4 subclass. In still a further embodiment the monospecific bivalent antibody is of IgG4 subclass with the additional mutation S228P, or the antibody is of human IgGl subclass with the mutations L234A and L235A. In another embodiment the monospecific bivalent antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65% or lower (numbering according to Kabat).
Further aspects of the invention are a nucleic acid molecule encoding an antibody according to the invention and a cell comprising the nucleic acid according to the invention.
The invention also comprises a method for increasing the activity of a monospecific bivalent antibody, whereby the method comprises the preparation of a monospecific tetravalent variant of said antibody in which all antigen-binding sites bind to the same antigen, whereby said monospecific bivalent antibody is modified by the connection to monospecific monovalent single chain antibodies each connected via a peptidic-linker to a single C- or N-terminus of the antibody chains of said monospecific bivalent antibody.
Also a pharmaceutical composition comprising an antibody according to the invention is comprised. Additionally a method for the production of a medicament for the treatment of immunosuppression comprising an antibody according to the invention as well as a method for the production of a medicament for the treatment of allograft rejection, or COPD, or rheumatoid arthritis comprising an antibody according to the invention is reported.
A further aspect of the current invention is a method for the production of a medicament for the treatment of an HIV infection comprising a variant of a parent monospecific bivalent antibody to which said HIV strain has developed a resistance which variant is a monospecific tetravalent variant of said parent monospecific bivalent antibody in which to said parent monospecific bivalent antibody two single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies bind to the same epitope either the same or an overlapping epitope from that bound by the antigen-binding sites of the parent monospecific bivalent antibody on the same antigen .
The invention further encompasses a method for the production of an antibody according to the invention comprising the following steps:
a) cultivating a cell comprising a nucleic acid encoding an antibody according to the invention under conditions suitable for the expression of said antibody, b) recovering said antibody from the cultivation medium or from the cells, c) purifying said antibody in order to produce an antibody according to the invention.
Detailed Description of the Invention
The current invention reports an antibody which is i) monospecific, and
ii) tetravalent, and
iii) consisting of: - a monospecific bivalent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain, - two peptidic-linkers, and two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, with a single-chain-linker in between,
whereby
i) two antigen-binding sites are each formed by a pair of heavy and light chain variable domains of the monospecific bivalent antibody and both bind to the same epitope, ii) two antigen-binding sites are each formed by one single chain antibody and both bind to the same or an overlapping epitope as the antigen-binding sites of i),
iii) said single chain antibodies are both linked to the same kind of terminus of a heavy chain or a light chain of the monospecific bivalent antibody via said peptidic-linker, whereby each antibody chain of the monospecific bivalent antibody which is linked to a single chain antibody is linked only to one single chain antibody.
Methods and techniques known to a person skilled in the art, which are useful for carrying out the current invention, are described e.g. in Ausubel, F.M., ed., Current Protocols in Molecular Biology, Volumes I to III (1997), Wiley and Sons; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
The term "monoclonal" as used herein refers to a preparation of antibody molecules of a single amino acid composition, preferably produced by a single cell and/or its progeny. Nevertheless may these antibody molecules vary in introduced post-translational modifications e.g. in the glycostructure.
The term "monospecific" antibody as used within the current application denotes an antibody that has one or more antigen-binding sites each of which bind to the same or an overlapping epitope of one antigen. Two epitopes are identical or overlapping if a signal reduction of 50% or more, in one embodiment of 75% or more, is detected by a surface plasmon resonance (SPR) assay using the immobilized antibody and antigen with the epitope in question at a concentration of 20-50 nM and the antibody for which the epitope identity or overlap has to be detected at a concentration of 100 nM. Alternatively a method can be used in which epitope identity or overlap of two antibodies binding to the same antigen is determined with the help of a competitive test system. For this purpose, for example with the help of a cell-based enzyme immunoassay (ELISA) employing cells expressing recombinant antigen epitopes, it is tested if the antibody for which the epitope identity or overlap has to be detected competes with the antibody for the binding to immobilized antigen. For this purpose, the immobilized antigen is incubated with the antibody in labeled form and an excess of the antibody for which the epitope identity or overlap has to be determined. By detection of the bound labeling there can easily be ascertained the epitope identity or overlap. If there is a signal reduction of more than 70%, in one embodiment of more than 80%, at the same concentration, or a displacement of more than 80%, in one embodiment of more than 90%, at higher concentrations, preferably in the case of 103-105-fold excess of the antibody for which epitope identity or overlap has to be determined, referred to the known antibody, then epitope identity or overlap is present and both antibodies bind to the same or an overlapping epitope on the same antigen.
The term "epitope" as used within the current application denotes a protein determinant capable of specific binding to the antigen-binding site of an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The term "valent" as used within the current application denotes the presence of a specified number of antigen-binding sites in an antibody molecule. As such, the terms "bivalent", and "tetravalent" denote the presence of two antigen-binding site and four antigen-binding sites, respectively, in an antibody molecule.
As used herein, the term "antibody" refers to a binding protein that comprises antigen-binding sites. The term "antigen-binding site" as used within the current application denotes the region(s) of an antibody molecule to which a ligand actually binds. In one embodiment of the current invention each of the antigen- binding sites comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL). In another embodiment each of the antigen-binding sites is formed by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
Antibodies of the present invention have more than two antigen-binding sites and are monospecific. Monospecific 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 term "CDR-grafted variant" as used within the current application denotes a variant of an antibody variable domain comprising complementary determining regions (CDRs or hypervariable regions) from one source or species and framework regions (FRs) from a different source or species, usually prepared by recombinant DNA techniques. CDR-grafted variants of variable domains comprising murine, rabbit or canine CDRs and human FRs are preferred.
The term "T-cell epitope depleted variant" as used within the current application denotes a variant of a variable domain of an antibody which was modified to remove or reduce immunogenicity by removing potential human T-cell epitopes (peptide sequences with the capacity to bind to MHC Class II molecules). The removal of potential T-cell stimulating epitopes can be evaluated in an in vitro T-cell proliferation assay using peripheral blood mononuclear cells (PBMC) from healthy donors to provide both T-cells and antigen presenting cells (APC). Overlapping peptidic fractions (9 to 15 amino acids in length) of the variable domain and PBMC are set up in cultures in 96 well plates with 5 μM peptidic fraction and 2 x 105 PBMC per well. After 7 days incubation an 18 hour pulse with 3H-Thymidine at 1 μCi/well is used to assess T-cell proliferation. A potential T-cell epitope is defined as a peptidic fraction giving a stimulation index (SI) greater than 2 in at least 2 independent PBMC populations, although SIs which are just under 2 may also be included in the analysis. By this method interaction between amino acid side chains of the variable domain and specific binding pockets with the MHC class II binding groove are identified. The identified immunogenic regions are mutated to reduce or eliminate immunogenicity. Such methods are described in general in, e.g., WO 98/52976.
The term "humanized variant" as used within the current application denotes a variant of a variable domain of an antibody, which is reconstituted from the complementarity determining regions (CDRs) of non-human origin, e.g. from a non-human species, and from the framework regions (FRs) of human origin, and which has been further modified by amino acid addition and/or deletion and/or mutation in order to also reconstitute or improve the binding affinity and specifity and optionally to reduce the immunogenicity of the original non-human variable domain. Such humanized variants are usually prepared by recombinant DNA techniques. The reconstitution of the affinity and specifity of the parent non-human variable domain is the critical step, for which different methods are currently used. In one method it is determined whether it is beneficial to introduce mutations, so called back- and/or forward-mutations, in the non-human CDRs as well as in the human FRs. The suited positions for such mutations can be identified e.g. by sequence or homology analysis, by choosing the human framework (fixed frameworks approach; homology matching; best-fit), by using consensus sequences, by selecting FRs from several different human mAbs, or by replacing non-human residues on the three dimensional surface with the most common residues found in human mAbs ("resurfacing" or "veneering").
The term "human antibody" as used within the current application denotes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The variable heavy chain and light chain regions are in one embodiment derived from germline sequence hVH_3_33 (GenBank L06618) and germline sequence hVK_3_l 1 (GenBank XO 1668), or from germline sequence hVH_3_64 (GenBank M99682) and germline sequence hVK lD lό (GenBank K01323), or germline sequence hVH_4_59 (GenBank L10088) and germline sequence hVK_l_39 (GenBank X59315), or from germline sequence hVH_2_26 (GenBank M99648) and germline sequence hVK_l_9 (GenBank Z00013), or from germline sequence hVH_4_30_4 (GenBank Z14238) and germline sequence hVK_l_27 (GenBank X63398). The constant regions of the antibody are in one embodiment constant regions of human IgGl or IgG4 type. Such regions can be allotypic and are described by, e.g., Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218, and the databases referenced therein.
As used herein, the term "binding" refers to the binding of the antibody to an epitope of an antigen in an in vitro assay, in one embodiment in a cell-based ELISA with CHO cells expressing wild-type antigen. Binding means in one embodiment a binding affinity (KD) of 10"8 M or less, in another embodiment 10"13 M to 10"9 M.
The term "nucleic acid molecule" as used within the current application is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but in one embodiment is double-stranded DNA.
The term "variable domain" as used within the current application denotes the part of an antibody heavy and light chain which is directly involved in binding of an antibody to its antigen, generally this is the N-terminal part of the antibody heavy and light chain of a monospecific bivalent antibody. Variable domains have a general structure. Each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" or "complementarity determining regions". The terms "complementary determining region" (CDR) or "hypervariable region" (HVR), which are used interchangeably within the current application, denote the amino acid residues of an antibody which are responsible for antigen-binding. The term "framework" region (FR) denotes those variable domain amino acid residues other than those of the hypervariable regions. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the regions FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-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". Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody. CDRs and FRs are determined according to the standard definition of Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).
The first aspect of the current invention is an antibody comprising four antigen- binding sites whereby said antigen-binding sites are grouped in pairs and the antigen-binding site bind to the same or an overlapping epitope.
Thus, with antibodies according to the invention it is now possible to
i) provide antibodies having multiple antigen-binding sites,
ii) retain the possibility in such antibodies to have/modify CDC and/or ADCC and/or FcRn-binding, and
iii) provide antibodies with an improved expression yield.
The architecture of the antibody according to the invention allows for multiple different formats to be generated, such as monospecific tetravalent antibodies, bispecific tetravalent antibodies, and trispecific hexavalent antibodies. Thus, the antibody according to the invention always has an even number of antigen-binding sites in order to allow the grouping of said antigen-binding sites in pairs, whereby each antigen-binding site of such a pair binds to the same epitope of the same antigen. With this architecture the antibodies according to the invention always have at least two antigen-binding sites binding to the same, i.e. identical, epitope. In one embodiment the antibody according to the invention comprises a full length parent antibody as scaffold. The term "full length antibody" denotes an antibody consisting of two "full length antibody heavy chain" and two "full length antibody light chain". A "full length antibody heavy chain" is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHl), an antibody hinge region, an antibody constant domain 2 (CH2), an antibody constant domain 3 (CH3), and optionally an antibody constant domain 4 (CH4) in case of an antibody of the subclass IgE. A "full length antibody light chain" is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL). The full length antibody chains a linked together via inter-polypeptide disulfide bonds between the CL-domain and the CHl domain and between the hinge regions of the full length antibody heavy chains.
The antigen-binding sites in the antibody according to the invention are each formed by a pair of two variable domains, i.e. of one heavy chain variable domain and one light chain variable domain. The minimal antigen-binding site determinant in an antibody is the heavy chain CDR3 region.
One embodiment of the current invention is an antibody which has the following characteristics: - it is a monospecific antibody, it is tetravalent antibody, and it is consisting of: a monospecific bivalent parent antibody consisting of two full length antibody heavy chains and two full length antibody light chains whereby each chain is comprising only one variable domain,
- two peptidic-linkers, two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker between said two antibody variable domains of the single chain antibodies, whereby said single chain antibodies bind to the same or an overlapping epitope as the antigen-binding sites of the monospecific bivalent parent antibody. In one embodiment said single chain antibodies are linked pair wise to the same kind of terminus (C- and/or N-terminus) of the monospecific bivalent antibody heavy chains or, alternatively to the same kind of terminus (in one embodiment to the C-terminus) of the monospecific bivalent antibody light chains of the parent antibody. In one embodiment either one or no single chain antibody is linked to a chain of the monospecific bivalent antibody, i.e. to two chains of the monospecific bivalent antibody is linked at most one additional single chain antibody.
This structure is exemplified in Figure 1.
In a further embodiment the antibody is characterized by - two antigen-binding sites are each formed by the two pairs of heavy and light chain variable domains of the monospecific bivalent parent antibody and both bind to the same epitope, the additional two antigen-binding sites are each formed by the heavy and light chain variable domain of one single chain antibody and both bind to the same or an overlapping epitope on the same antigen as the antigen-binding sites of the parent antibody, the single chain antibodies are each linked to one heavy chain or to one light chain via a peptidic-linker, whereby each antibody chain terminus is linked only to one single chain antibody. The term "peptidic-linker" as used within the invention denotes a peptide, which is in one embodiment of synthetic origin. These peptidic-linkers according to invention are used to link the different antigen-binding sites and/or antibody fragments eventually comprising the different antigen-binding sites (e.g. single chain Fv, full length antibodies, a VH domain and/or a VL domain, Fab, (Fab)2, Fc part) together to form an antibody according to the invention The peptidic-linkers can comprise one or more of the following amino acid sequences or are in another embodiment independently selected from SEQ ID NO: 066, 073, 074, 075, 076, 077, 078, 079, 080, 081, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 094, 095, 096, 097, 099, 101, 102, 103, 104, 105, 109, 1 10, 1 1 1, 112, 1 13, 1 14, 115, 116, 117, 118, 119, 126 or 127 (see Table 1) as well as further arbitrarily selected amino acids.
In one embodiment said peptidic-linkers are peptides with an amino acid sequence with a length of from at least 10 amino acids up to 30 amino acids. In one embodiment said peptidic-linker comprises the amino acid sequence of SEQ ID NO: 083, 084, 089, 090, 126. In another embodiment said peptidic linker comprises the amino acid sequence of SEQ ID NO: 083. In another embodiment said peptidic-linker consists of the amino acid sequence of SEQ ID NO: 083, 084, 089, 090, 126, in another embodiment of SEQ ID NO: 083.
Table 1: Peptidic-linker amino acid sequences.
Figure imgf000015_0001
Figure imgf000016_0001
The term "single-chain-linker" as used within the invention denotes a peptide, which is in one embodiment of synthetic origin. These single-chain-linkers according to invention are used to link a VH and a VL domain to form a single chain Fv (scFv). The single-chain-linker can comprise one or more of the following amino acid sequences and is independently selected for each single chain antibody from SEQ ID NO: 065, 067, 068, 069, 070, 071, 072, 073, 074, 075, 076, 077, 078, 079, 080, 081, 083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 094, 095, 096, 098, 099, 100, 101, 102, 103, 104, 106, 107, 108, 109, 110, 111, 112, 1 13, 114, 115, 116, 117, 118 (see Table 2) as well as further arbitrarily selected amino acids. In one embodiment said single-chain-linker is a peptide with an amino acid sequence with a length of at least 15 amino acids, in another embodiment with a length of from at least 20 amino acids up to 30 amino acids. In one embodiment said single-chain-linker comprises the amino acid sequence of SEQ ID NO: 084, 089, 090, 080. In another embodiment said single-chain-linker comprises the amino acid sequence of SEQ ID NO: 089, 090, 080, in one embodiment of SEQ ID NO: 089. In another embodiment said single-chain-linker consists of the amino acid sequence of SEQ ID NO: 084, 089, 090, 080. In another embodiment the single- chain-linker is consisting of the amino acid sequence of SEQ ID NO: 089, 090, 080, in one embodiment of SEQ ID NO: 089.
Table 2: Single-chain-linker amino acid sequences.
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
It has now been found that the multivalent antibodies according to the current invention have improved characteristics compared to the respective bivalent parent antibodies. They show an increased in vitro biological potency and may provide benefits such as increased activity/efficacy, reduced dose and/or frequency of administration and concomitantly cost savings compared to the application of two or more individual antibodies in combination. It has further been found that not all position combinations of antigen-binding sites result in a multivalent antibody that can be recombinantly produced. Thus, it has surprisingly been found that the selection of the antigen-binding site(s) and therewith the variable domains has to be made carefully.
Due to their chemical and physical properties, such as molecular weight and domain architecture including secondary modifications, the downstream processing of antibodies is very complicated. For example, not only for formulated drugs but also for intermediates in downstream processing (DSP) concentrated solutions are required to achieve low volumes for economic handling and application storage. But with increasing concentration of the antibody a tendency to form aggregates can be observed. These aggregated antibodies have impaired characteristics compared to the isolated antibody. It has now been found that aggregation of the antibodies according to the invention can be reduced by the introduction of disulfide bonds between the heavy and light chain variable domains of the single chain antibodies connected to the monospecific bivalent parent antibody (Figure 2). This improved stability is not only useful during the production process but also for the storage of the antibodies (Figure 3). In one embodiment the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is independently for each single chain antibody selected from:
i) heavy chain variable domain position 44 to light chain variable domain position 100, ii) heavy chain variable domain position 105 to light chain variable domain position 43, or iii) heavy chain variable domain position 101 to light chain variable domain position 46.
In one embodiment the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100.
In one embodiment the disulfide bond between the variable domains of the single chain antibodies comprised in the antibody according to the invention is between heavy chain variable domain position 105 and light chain variable domain position 43. It has further been found that with increased single-chain-linker length the formation of aggregates is reduced. Thus, in one embodiment the number of GGGGS units in the linker is from 3 to 6. In a preferred embodiment the number of GGGGS units is 5 or 6.
It has been found that with the introduction of the single disulfide bonds only in the single chain antibodies and with the selection of the linker length an increase in the expression yield and also in the long-term stability in solution of the antibody according to the invention can be achieved.
The term "constant region" as used within the current applications denotes the sum of the domains of an antibody other than the variable region. The constant region is not involved directly in antigen-binding, but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, such as IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The heavy chain constant regions that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ,.. respectively. The light chain constant regions which can be found in all five antibody classes are called K (kappa) and λ (lambda). In one embodiment the antibody according to the invention has a light chain constant region of the kappa class (SEQ ID NO: 063) or the lambda class (SEQ ID NO: 128).
The term "constant region derived from human origin" as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGl, IgG2, IgG3, or IgG4 and/or a constant light chain κ or λ region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., (see e.g. Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). While antibodies of the IgG4 subclass show reduced Fc receptor (FcγRIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which, if altered, provide also reduced Fc receptor binding (Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-9; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434). In one embodiment an antibody according to the invention has a reduced FcR binding compared to an IgGl antibody and the monospecific bivalent parent antibody is in regard to FcR binding of IgG4 subclass or of IgGl or IgG2 subclass with a mutation in S228, L234, L235 and/or D265, and/ or contains the PVA236 mutation. In one embodiment the mutations in the monospecific bivalent parent antibody are S228P, L234A, L235A, L235E and/or PVA236. In another embodiment the mutations in the monospecific bivalent parent antibody are in IgG4 S228P and in IgGl L234A and L235A. Constant heavy chain regions shown in SEQ ID NO: 061 and 062. In one embodiment the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 061 with mutations L234A and L235A. In another embodiment the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 062 with mutation S228P. In another embodiment the constant light chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 063.
The constant region of an antibody is directly involved in ADCC (antibody- dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by binding of complement factor CIq to the constant region of most IgG antibody subclasses. Binding of CIq to an antibody is caused by defined protein-protein interactions at the so called complement-binding site. Such constant region binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al., J. Immunol. 127 (1981) 2555- 2560; Brunhouse, R. and Cebra, J. J., MoI. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., MoI. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434. Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is measured in one embodiment by the treatment of a preparation of CCR5 expressing cells with an antibody according to the invention in the presence of effector cells such as freshly isolated PBMC or purified effector cells from buffy coats, like monocytes or natural killer (NK) cells or a permanently growing NK cell line.
The term "complement-dependent cytotoxicity (CDC)" denotes a process initiated by binding of complement factor CIq to the Fc part of most IgG antibody subclasses. Binding of CIq to an antibody is caused by defined protein-protein interactions at the so called complement-binding site. Such Fc part binding sites are known in the state of the art (see above). Such Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclass IgGl, IgG2, and IgG3 usually show complement activation including CIq and C3 binding, whereas IgG4 does not activate the complement system and does not bind CIq and/or C3. Cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide component as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and US 6,602,684. IgGl type antibodies, the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright, A. and Morrison, S.L., Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al. Nature Biotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpression in Chinese hamster ovary (CHO) cells of β(l,4)-N-acetylglucosaminyl transferase III ("GnTIII"), a glycosyltransferase catalyzing the formation of bisected oligosaccharides, significantly increases the in vitro ADCC activity of antibodies. Alterations in the composition of the Asn297 carbohydrate or its elimination affect also binding to FcγR and CIq (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al., Biotechnol. Bioeng. 74 (2001) 288-294; Mimura, Y., et al., J. Biol. Chem. 276 (2001) 45539-45547; Radaev, S., et al., J. Biol. Chem. 276 (2001) 16478-16483; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R.L., et al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L.C., et al., J. Immunol. Methods 263 (2002) 133-147).
Methods to enhance cell-mediated effector functions of monoclonal antibodies are reported e.g. in WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2004/065540, WO 2005/011735, WO 2005/027966, WO 1997/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739.
Therefore in one embodiment of the current invention is the monospecific bivalent parent antibody glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65% or lower (Numbering according to Kabat). In another embodiment is the amount of fucose within said sugar chain is between 5% and 65%, in another embodiment between 20% and 40%. "Asn297" according to the invention means amino acid asparagine located at about position 297 in the Fc region (numbering according to Kabat). Based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than ±3 amino acids) upstream or downstream of position 297, i.e. between position 294 and 300. In one embodiment the antibody according to the invention is of human IgGl or IgG3 subclass. In a further embodiment the amount of N-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount of N-terminal alpha- 1,3 -galactose is 1% or less within said sugar chain. The sugar chain show preferably the characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell.
The term "the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell" denotes that the sugar chain at Asn297 of the monospecific bivalent parent antibody according to the invention has the same structure and sugar residue sequence except for the fucose residue as those of the same antibody expressed in unmodified CHO cells, e.g. as those reported in WO 2006/103100.
The term "NGNA" as used within this application denotes the sugar residue N-glycolylneuraminic acid.
In an embodiment in which ADCC and/or CDC is/are required, a constant region of IgGl subclass is employed in the monospecific bivalent parent antibody, in embodiments in which reduced or no ADCC and/or CDC is/are required, a constant region of IgG4 subclass, or modified/mutated IgGl subclass is employed in the monospecific bivalent parent antibody. The present invention refers in one embodiment to an antibody with a monospecific bivalent parent antibody that has a reduced binding to or does not bind Fcγ receptor and/or complement factor CIq. Such an antibody show reduced or no triggering of antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). In one embodiment such a monospecific bivalent parent antibody is characterized in that it contains a constant region derived from human origin, and does not bind or shows a reduced binding of Fc receptors and/or complement factor CIq. No "CIq binding" is found if in such an assay the optical density (OD) at 492 nm reduced by the optical density at the reference wavelength of 405 nm is for the test antibody lower than 15% of the value for human CIq binding of the unmodified wild-type monospecific bivalent parent antibody constant region at an antibody concentration of 8 μg/ml. Reduced "CIq binding" is in the range of from 15% to 30% of the value for human CIq binding of the unmodified wild-type monospecific bivalent parent antibody constant region at the same conditions. ADCC can be measured as binding of the antibody to human FcγRIIIa on human NK cells. Binding is determined at an antibody concentration of 20 μg/ml. "No Fcγ receptor binding" or "no ADCC" means a binding of up to 30% to human FcγRIIIa on human NK cells at an antibody concentration of 20 μg/ml compared to the binding of the same antibody as human IgGl (SEQ ID NO: 061). "Reduced Fcγ receptor binding" or "reduced ADCC" means a binding of from 30% up to 60% to human FcγRIIIa on human NK cells compared to the binding of the same antibody as human IgGl (SEQ ID NO: 061). In another embodiment of the current invention the monospecific bivalent parent antibody is an antibody that does bind Fcγ receptor and/or complement factor CIq. Such an antibody which does bind Fc receptor and/or complement factor CIq does elicit antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). In one embodiment this antibody is characterized in that the monospecific bivalent parent antibody contains an Fc part derived from human origin, and does also bind Fc receptors and/or complement factor CIq.
Glycosylation of human IgGl or IgG3 occurs at Asn297 as core fucosylated biantennary complex oligosaccharide terminated with up to two Gal residues. Human constant heavy chain regions of the IgGl or IgG3 subclass are reported in detail by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brueggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T.W., et al., Methods Enzymol. 178 (1989) 515-527. These structures are designated as GO, Gl (θf-1,6- or α-1,3-), or G2 glycan residues, depending from the amount of terminal Gal residues (Raju, T.S., Bioprocess Int., April 2003, 44-53). CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F. H., Glycoconjugate J. 14 (1997) 201-207. Antibodies which are recombinantly expressed in non-glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85%. The modified oligosaccharides of the monospecific bivalent parent antibody may be hybrid or complex. In one embodiment the bisected, reduced/not-fucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/not-fucosylated oligosaccharides are complex.
According to the invention "amount of fucose" means the amount of said sugar within the sugar chain at Asn297, related to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry and calculated as average value. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures identified in an N-Glycosidase F treated sample (e.g. complex, hybrid and oligo- and high-mannose structures, respectively) by MALDI-TOF.
The antibody according to the invention is produced by recombinant means. Thus, 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. For the expression of the antibodies as aforementioned in a host cell, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods and said expression vectors are transfected into a host cell. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). General methods for recombinant production of antibodies are well- known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., MoI. Biotechnol. 16 (2000) 151- 160; Werner, R.G., Drug Res. 48 (1998) 870-880.
The term "host cell" as used in the current application denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention. In one embodiment HEK293 cells and CHO cells are used as host cells. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom 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.
Expression in NSO cells is described by, e.g., Barnes, L.M., et al., Cytotechnology 32 (2000) 077-091 ; 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. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. 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 control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein 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. Generally, "operably linked" means that the DNA sequences being linked are contiguous, 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, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
An antibody according to the invention with a reduced amount of fucose can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity and a polypeptide having ManII activity in an amount to fucosylate according to the invention the oligosaccharides in the Fc region. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively a\ ,6-fucosyltransferase activity of the host cell can be decreased or eliminated according to US 6,946,292 to generate glycomodified host cells. The amount of antibody fucosylation can be predetermined e.g. either by fermentation conditions or by combination of at least two antibodies with different fucosylation amount.
The antibody according to the invention with a reduced amount of fucose can be produced in a host cell by a method comprising: (a) culturing a host cell engineered to express at least one polynucleotide encoding a fusion polypeptide having GnTIII activity and/or ManII activity under conditions which permit the production of said antibody and which permit fucosylation of the oligosaccharides present on the Fc region of said antibody in an amount according to the invention; and (b) isolating said antibody. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide, in one embodiment comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi resident polypeptide selected from the group consisting of the localization domain of mannosidase II, the localization domain of β(l,2)-N-acetylglucosaminyltransferase I ("GnTI"), the localization domain of marmosidase I, the localization domain of β(l,2)-N- acetylglucosaminyltransferase II ("GnTII"), and the localization domain of a- 1 ,6 core fucosyltransferase. In one embodiment the Golgi localization domain is from marmosidase II or GnTI.
As used herein, a "polypeptide having GnTIII activity" refers to polypeptides that are able to catalyze the addition of an N-acetylglucosamine (GIcNAc) residue in β-1,4 linkages to the β- linked mannoside of the trimannosyl core of N-linked oligosaccharides. This includes fusion polypeptides exhibiting enzymatic activity similar to, but not necessarily identical to, an activity of β-l,4-N- acetylglucosaminyltransferase III, also known as β-l,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.112), according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of GnTIII, but rather substantially similar to the dose- dependence in a given activity as compared to the GnTIII (i.e. the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, in one embodiment, not more than about tenfold less activity, and in a further embodiment, not more than about three-fold less activity relative to the GnTIII). As used herein, the term "Golgi localization domain" refers to the amino acid sequence of a Golgi resident polypeptide which is responsible for anchoring the polypeptide in location within the Golgi complex. Generally, localization domains comprise amino terminal "tails" of an enzyme.
For the production of antibodies according to the invention with a reduce amount of fucose likewise a host cell that is able and engineered to allow the production of an antibody with modified glycoforms can be used. Such a host cell has been further manipulated to express increased levels of one or more polypeptides having GnTIII activity. CHO cells are preferred as such host cells. Likewise cells producing antibody compositions having high antibody-dependent cell-mediated cytotoxic activity as reported in US 6,946,292 can be used.
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 alkaline/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 microbial 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 chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102).
One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
As used herein, "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. In one embodiment 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 certain 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. Pharmaceutical carriers 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.
The phrases "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, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. These 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.
Regardless of the route of administration selected, 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. In addition to water, the carrier is in one embodiment 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. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
CCR5 is a human chemokine receptor (see e.g. Swiss Prot P51681 and Mueller, A., and Strange, P.G., Int. J. Biochem. Cell Biol. 36 (2004) 35-38) within the "cluster" chemokines that are produced primarily during inflammatory responses and control the recruitment of neutrophils (CXC chemokines) and macrophages and subsets of T-cells. (T-helper ThI and Th2 cells). ThI responses are typically those involving cell-mediated immunity effective against viruses and tumors, for example, whereas Th2 responses are believed to be pivotal in allergies. Therefore, inhibitors of these chemokine receptors may be useful as immunomodulators. For ThI responses, overactive responses are dampened, for example, in autoimmunity including rheumatoid arthritis or, for Th2 responses, to lessen asthma attacks or allergic responses including atopic dermatitis (see e.g. Schols, D., Curr. Top. Med. Chem. 4 (2004) 883-893; Mueller, A., and Strange, P.G., Int. J. Biochem. Cell Biol. 36 (2004) 35-38; Kazmierski, W.M., et al., Curr. Drug Targets Infect. Disord. 2 (2002) 265-278; Lehner, T., Trends Immunol. 23 (2002) 347-351).
The antibodies inhibit one or more functions of human CCR5, such as ligand binding to CCR5, signaling activity (e.g. activation of a mammalian T-protein, induction of a rapid and transient increase in the concentration of cytosolic free Ca2+, and/or stimulation of a cellular response (e.g. stimulation of chemotaxis, exocytosis or inflammatory mediator release by leukocytes, integrin activation). The antibodies inhibit binding of RANTES, MIP-I alpha, and/or MIP-I beta, to human CCR5 and/or inhibit functions mediated by human CCR5, like leukocyte trafficking, T-cell activation, inflammatory mediator release, and/or leukocyte degranulation. An antibody according to the invention in one embodiment does not inhibit chemokine binding in a binding assay to CCRl, CCR2, CCR3, CCR4, CCR6, and CXCR4 in an antibody concentration up to 100 μg/ml.
Chemokines and their receptors are known to participate in allograft rejection by mediating leukocyte trafficking. Panzer, U., et al. (Transplantation 78 (2004) 1341-50) reported CCR5-positive T cell recruitment in acute human allograft rejection, and Luckow, B., et al., (Eur. J. Immunol. 34 (2004) 2568-78) observed decreased intragraft levels of metalloproteinases and arteriosclerosis in CCR5- deficient animals. Further, Gao, W., et al. (Transplantation 72 (2001) 1199-1205) demonstrated prolonged allograft survival in mice treated with CCR5-specific monoclonal antibody and in CCR5-deficient mice. Schroeder, C, et al., J. Immunol. 179 (2007) 2289-2299, explored the effects of a CCR5 antagonist in a cynomolgus monkey cardiac allograft model for investigation of CCR5 modulation during inflammation and alloimmunity. Moreover, a retrospective study in human transplant recipient cohorts uncovered that CCR5-deficient patients (delta 32) showed prolonged allograft survival (Fischereder, M., et al., Lancet 357 (2001) 1758-1761).
In experimental models, due to the redundancy of receptor-ligand interaction, the deficiency or blockade of a single chemokine does not protect the allograft from acute rejection (Fischereder, M., et al., Lancet 357 (2001) 1758; Gao, W., et al., Transplantation 72 (2001) 1199-1205; Hancock, W. W., et al., Curr. Opin. Immunol. 12 (2000) 51 1-516; Hancock, W.W., et al., Curr. Opin. Immunol. 15 (2003) 479-486).
WO 01/78707 refers to a method of inhibiting graft rejection comprising administering an antagonist of CCR5 function. CCR5 is also used by most HIV-I primary isolates and is critical for the establishment and maintenance of infection. In addition CCR5 function is dispensable for human health, probably because CCR5 is part of a highly redundant chemokine network as receptor for the α chemokines MIP lα, MIP- lβ and RANTES, which share many overlapping functions, and most of which have alternative receptors (Rossi, D., and Zlotnik, A., Annu. Rev. Immunol. 18 (2000) 217-242). The identification of CCR5 as an HIV-I co-receptor was based on the ability of its ligands, MIP- lα, MIP- lβ and RANTES, to block infection by R5 but not R5X4 or X4 isolates (Cocchi, F., et al., Science 270 (1995) 1811-1815). HIV-I infection is initiated by interactions between the viral envelope glycoprotein (Env) and a cellular receptor complex comprised of CD4 plus a chemokine receptor (Pierson, T.C., and Doms, R. W., Immuno. Lett. 85 (2003) 113-118; and Kilby, J.M., and Eron, J.N., Engl. J. Med. 348 (2003) 2228-2238). The chemokine receptor CCR5 is a co-receptor for macrophage-tropic (R5) strains and plays a crucial role in the sexual transmission of HIV-I (Berger, E. A., AIDS 1 1 (Supp. 061) (1997) S3-S16; Bieniasz, P.D., and Cullen, B.R., Frontiers in Bioscience 3 (1998) D44-D58; Littman, D.R., Cell 93 (1998) 677-680).
Antibodies against CCR5 are e.g. PRO 108 (Olson, W.C., et al., J. Virol. 73 (1999) 4145-4155) and 2D7 (Samson, M., et al., J. Biol. Chem. 272 (1997) 24934-24941).
Additional antibodies are mentioned in US 2004/0043033, US 6,610,834,
US 2003/0228306, US 2003/0195348, US 2003/0166870, US 2003/0166024,
US 2003/0165988, US 2003/0152913, US 2003/0100058, US 2003/0099645,
US 2003/0049251, US 2003/0044411, US 2003/0003440, US 6,528,625, US 2002/0147147, US 2002/0146415, US 2002/0106374, US 2002/0061834,
US 2002/0048786, US 2001/0000241, EP 1 322 332, EP 1 263 791, EP 1 207 202,
EP 1 161 456, EP 1 112 006, WO 2003/072766, WO 2003/066830,
WO 2003/033666, WO 2002/083172, WO 2002/22077, WO 2001/58916,
WO 2001/58915, WO 2001/43779, and WO 2001/42308. Novel CCR5 monoclonal antibodies with potent and broad-spectrum anti-HIV activities, whereby synergistical antiviral effects were found are reported by Ji, C, et al., Antivir. Res.
74 (2007) 125-137.
In the variable domains of the anti-CCR5 antibody some positions are variable within the given boundaries, e.g. at position 5 of SEQ ID NO: 120 either the amino acid lysine or glutamine can be present, at position 6 of SEQ ID NO: 120 either the amino acid glutamine or glutamic acid can be present, and so on. SEQ ID NO: 120 is the amino acid sequence of the heavy chain variable domain, wherein XOl to X08 denote the variable positions:
Gln-Val-Gln-Leu-X01-X02-Ser-Gly-Pro-Gly-Leu-Val-X03-Pro-Ser-Gln-Ser-Leu- Ser-Ile-Thr-Cys-Thr-Val-Ser-Gly-Phe-Pro-Leu-Gly-Ala-Phe-Gly-Val-His-Trp-Val- Arg-Gln-Ser-Pro-Gly-Lys-Gly-X04-Glu-Tφ-Leu-Gly-Val-Ile-Tφ-Lys-Gly-Gly- Asn-Thr-Asp-Tyr-Asn-Ala-Ala-Phe-XOS-Ser-Arg-Leu-Arg-Ile-Thr-Lys-Asp-Asn- Ser-Lys-Ser-Gln-Val-Phe-Phe-Arg-Met-Asn-Ser-Leu-Gln-Thr-Asp-Asp-Thr-Ala- X06-Tyr-Tyr-Cys- Ala- Lys-Val-Asn-Leu- Ala- Asp- Ala-Met- Asp-Tyr-Trp-Gly-Gln- Gly-Thr-X07-Val-X08-Val-Ser-Ser, wherein
XOl is either Lys or GIn, X02 is either GIn or GIu, X03 is either Arg or Lys, X04 is either Leu or Pro, X05 is either Met or Lys, X06 is either He or Thr, X07 is either Ser or Thr, X08 is either He or Thr (SEQ ID NO: 120).
Likewise denoted SEQ ID NO: 121 is the amino acid sequence of the variable light chain domain, wherein XlO to X24 denote the variable positions:
Asp-Ile-Gln-Met-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ser-Ala-Ser-Val-Gly-Glu-Thr-Val-
Thr-Ile-Thr-Cys-Arg-Ala-Ser-Gly-Asn-XlO-His-Gly-Tyr-Leu-Ala-Tφ-Xl l-Gln- Gln-Lys-X^-Gly-Lys-XB-Pro-XM-Leu-Leu-XlS-Tyr-Asn-Thr-Lys-Thr-Leu-
Ala-Glu-Gly-Val-Pro-Ser-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Xlό-Phe-Xπ-
X18-X19-Ile-X20-Ser-X21-Gln-Pro-Glu-Asp-Phe-X22-X23-Tyr-Tyr-Cys-Gln-
His-His-Tyr-Asp-Leu-Pro-Arg-Thr-Phe-Gly-Gly-Gly-Thr-Lys-X24-Glu-Ile-Lys, wherein Xl 0 is either He or Ala,
Xl 1 is either Phe or Tyr,
Xl 2 is either GIn or Pro,
Xl 3 is either Ser or Ala,
Xl 4 is either GIn or Lys, Xl 5 is either VaI or He,
Xl 6 is either GIn or Asp,
Xl 7 is either Ser or Thr,
Xl 8 is either Leu or Ala,
Xl 9 is either Lys or Thr, X20 is either Asn or Ser,
X21 is either Leu or Ala,
X22 is either GIy or Ala,
X23 is either Asn or Thr,
X24 is either Leu or VaI (SEQ ID NO: 121). One aspect of the invention is an antibody which is a monospecific tetravalent antibody with antigen-binding sites binding to a single epitope of CCR5. It has surprisingly been found that with the doubling of the antigen-binding sites an improved in vitro biological potency is detectable. For example in a PBMC antiviral assay according to Example 14 a reduction of the IC50 value from 1077.5 pM (pmol/1) to 87.8 pM can be detected. This provides for a synergistic effect of the four antigen-binding sites in this antibody according to the invention.
This monospecific tetravalent anti-CCR5 antibody is consisting of:
- a monospecific bivalent anti-CCR5 parent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain, - two peptidic-linkers, and two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker.
The four antigen-binding sites of the monospecific tetravalent anti-CCR5 antibody according to this aspect of the current invention bind to the same epitope or an overlapping epitope as an antibody obtained from DSM ACC 2681 or DSM ACC 2683.
In one embodiment the heavy chain variable domain of said antigen-binding sites comprises a CDR3 of SEQ ID NO: 001, or 009, or 035. In another embodiment the heavy chain variable domain of said antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 003, 002, 001, or SEQ ID NO: 011, 010, 009, or SEQ ID NO: 037, 036, 035. In a further embodiment the antigen- binding sites are characterized that the light chain variable domain of said antigen- binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 007, 006, 005, or SEQ ID NO: 016, 015, 014, or SEQ ID NO: 042, 041, 040. In a final embodiment of this aspect the heavy and light chain variable domain of said antigen-binding sites have an amino acid sequence of SEQ ID NO: 004, 008, or SEQ ID NO: 012, 017, or SEQ ID NO: 039, 044 or are a T-cell epitope depleted, CDR-grafted, or humanized variant thereof. One aspect of the invention is therefore a monospecific tetravalent anti-CCR5 antibody with a heavy chain amino acid sequence of SEQ ID NO: 122 and a light chain amino acid sequence of SEQ ID NO: 123. This antibody binds to the same epitope as an antibody obtained from DSM ACC 2681. Optionally in said antibody the single chain antibodies have a disulfide bond between its single chain antibody variable domains. Another aspect of the invention is a monospecific tetravalent anti-CCR5 antibody with a heavy chain amino acid sequence of SEQ ID NO: 124 and a light chain amino acid sequence of SEQ ID NO: 125, wherein in said antibody the single chain antibodies have a disulfide bond between the single chain antibody variable domains. This antibody binds to the same epitope as an antibody obtained from DSM ACC 2683.
Another aspect of the current invention is a method for increasing the biological potency of a monospecific bivalent parent antibody comprising the preparation of a monospecific tetravalent variant of said antibody in which all antigen-binding sites bind to the same or an overlapping epitope on the same antigen, whereby said monospecific bivalent antibody is modified by the connection to two monospecific monovalent single chain antibodies each connected via a peptidic-linker each to a single C- or N-terminus of the antibody chains of said monospecific bivalent antibody. In one embodiment of said method is the antigen CCR5. In a further embodiment all antigen-binding sites bind to the same or an overlapping epitope of the same antigen. In a further embodiment said epitope is that to which the antigen-binding sites of an antibody obtained from DSM ACC 2681, or DSM ACC 2682, or DSM ACC 2683, or DSM ACC 2684, or an antibody with a heavy chain variable domain of SEQ ID NO: 048 and a light chain variable domain of SEQ ID NO: 052, or an antibody with a heavy chain variable domain of SEQ ID NO: 056 and a light chain variable domain of SEQ ID NO: 060 bind.
Another aspect of the current invention is a pharmaceutical composition comprising an antibody according to the invention. One embodiment of this aspect is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of immunosuppression. Another embodiment is the use of an antibody according to the invention for the treatment of immunosuppression. A further embodiment is a method for the manufacture of a medicament for the treatment of immunosuppression comprising an antibody according to the invention. Another embodiment is a method of treatment of immunosuppression by administering an antibody according to the invention to a patient. In one embodiment the immunosuppression is HIV infection.
Monospecific tetravalent anti-CCR5 antibodies which have a reduced amount of fucose and are non-depleting are useful for the manufacture of a medicament for the treatment of acute or chronic allograft rejection, or COPD, or rheumatoid arthritis. Thus, one embodiment is the use of an antibody according to the invention with a reduced amount of fucose for the manufacture of a medicament for the treatment of allograft rejection, or COPD, or rheumatoid arthritis. A further embodiment is the use of an antibody according to the invention with a reduced amount of fucose for the treatment of allograft rejection, or COPD, or rheumatoid arthritis. Still another embodiment is a method for the manufacture of a medicament for the treatment of allograft rejection, or COPD, or rheumatoid arthritis comprising an antibody according to the invention with a reduced amount of fucose. Still a further embodiment is a method of treatment of allograft rejection, or COPD, or rheumatoid arthritis by administering an antibody according to the invention with a reduced amount of fucose to a patient.
Transplantation is performed according to the state of the art with numerous cell types, tissue types and organ types, e.g. pancreatic islets, corneal, bone marrow, stem cells, skin graft, skeletal muscle, aortic and aortic valves, and organs as heart, lung, kidney, liver, and pancreas. The invention comprises the use of the antibodies according to the invention for the treatment of a patient suffering from GvHD or HvGD (e.g. after transplantation). The invention comprises also a method for the treatment of a patient suffering from such GvHD and HvGD. The invention also provides the use of an antibody according to the invention in an effective amount for the manufacture of a pharmaceutical agent, in one embodiment together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from inflammatory mediator release mediated by CCR5.
The term "graft rejection" as used within this application denotes the response of the human immune system to transplanted tissue. If tissue is transplanted from a donor to a host the human leukocyte antigen genes of the donor's tissue are likely to be different from those of the host's tissue. Thus, the host's immune system recognized the transplanted tissue as foreign and effects an immune response called graft rejection. This graft rejection reaction is called "graft versus host disease" (GvHD). Thus, the current invention provides a method of treating or preventing acute and chronic organ transplant rejection in a mammal, including a human, characterized in administering to said mammal an antibody according to the invention. Also is provided an antibody according to the invention for the treatment or prevention of acute and chronic organ transplant rejection in a mammal, including a human.
Another aspect of the current invention is the use of a variant of a monospecific bivalent parent antibody to which a HIV strain has developed a resistance for the manufacture of medicament for the treatment an HIV infection by said HIV strain said medicament comprising a variant of said monospecific bivalent parent antibody to which said HIV strain has developed a resistance which is a bispecific or trispecifϊc tetravalent or hexavalent form of said monospecific bivalent parent antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic- linker, whereby said single chain antibodies are grouped in pairs in which each member of a pair binds to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen. Also encompassed in the current invention are i) the use of a variant antibody of a monospecific bivalent parent antibody to which an HIV strain has developed a resistance which is a bispecific or trispecifϊc tetravalent or hexavalent form of said monospecific bivalent parent antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies are grouped in pairs in which each member binds to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen for the treatment of HIV infection; ii) a method for the manufacture of a medicament for the treatment of an HIV infection comprising a variant of a parent monospecific bivalent antibody to which said HIV strain has developed a resistance which variant is a bispecific or trispecific tetravalent or hexavalent variant of said parent monospecific bivalent antibody in which to said parent monospecific bivalent antibody two or four single chain antibodies are connected via a peptidic- linker, whereby every two of said single chain antibodies bind to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the parent monospecific bivalent antibody of the same antigen; and iii) a method of treatment of an HIV infection by administering a variant antibody of a monospecific bivalent parent antibody to which an HIV strain has developed a resistance which is a bispecific or trispecific tetravalent or hexavalent form of said monospecific bivalent parent antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies are grouped in pairs in which each member binds to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen to a patient. In one embodiment each of the heavy chains or the light chains of said parent antibody is connected either to one or to no single chain antibody via a peptidic linker.
It has now been surprisingly found that not all combinations of the parent antibody and the two or four single chain antibodies can be produced in good yield or even can be produced at all. The yield of the monospecific tetravalent antibody according to the invention is depending on the one hand on the terminus to which the single chain antibodies are connected, on the length of the linkers and on the additional stabilization of the single chain antibodies by additional disulfide bonds.
In the following Table 3 the yield of different multivalent antibodies is shown.
Table 3: Yield of antibodies according to the invention.
Figure imgf000039_0001
Figure imgf000040_0001
n.d. = not detectable
It can seen from Table 3 that conjugates in which the single chain antibodies are connected to the C-terminus of the heavy or light chain expressed better than for example antibodies with the single chain antibodies conjugated to the N-terminus of the heavy and light chain. Additionally it can be seen that only the expression of antibodies in which the single chain antibodies have a disulfide bond between residue 44 of the heavy chain variable domain and residue 100 of the light chain variable domain yield product. Additionally can be seen that a peptidic linker of two G4S units provides for the best expression results.
Thus, in one embodiment of the invention are the single chain antibodies connected to the C-terminus of the full length heavy or light antibody chain. In a further embodiment comprises the single chain antibody a disulfide bond between residue 100 of the light chain variable domain and residue 44 of the heavy chain domain. In still afurther embodiment of the invention is the peptidic linker consisting of two G4S units.
The hybridoma cell lines used in the invention, m<CCR5>Pz01.063, m<CCR5>Pz02.1Cl l, m<CCR5>Pz03.1C5 and m<CCR5>PzO4.1F6, were deposited, under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany:
Figure imgf000041_0001
The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Description of the Figures
Figure 1 Structures of an exemplary tetravalent antibody according to the invention. Figure 2 Exemplary size exclusion chromatogram of an antibody a) without disulfide bonds between the variable domains of the single chain antibodies (BA-4320), b) with disulfide bonds between the variable domains of the single chain antibodies (BA-
4321).
Figure 3 Exemplary comparison of the storage dependent formation of aggregates of exemplary antibodies according to the invention for storage at 4 °C for 5 days: a) without disulfide bonds between the variable domains of the single chain antibodies, b) with disulfide bonds between the variable domains of the single chain antibodies. Figure 4 Binding epitopes of two anti-CCR5 antibodies. Figure 5 A comparison of IC50 values for different multivalent antibodies determined by PBMC assay: 1 - BN- 1000, 2 - AN- 1000, 3- AN-
1000 + BN- 1000, 4 - AA-2320.
Materials & Methods
Recombinant DNA techniques
Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, CoId Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.
DNA and protein sequence analysis and sequence data management
General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242. Amino acids of antibody chains are numbered according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242). The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NTI Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
DNA sequencing DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
Nucleic acid synthesis
Desired nucleic acids were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The nucleic acids which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned nucleic acids was confirmed by DNA sequencing.
Example 1 Production of monoclonal antibodies to human CCR5
a) Immunization of mice
Female Balb/c mice are given a primary intraperitoneal immunization with 107 CCR5 expressing cells (CHO or Ll .2) together with the adjuvant CFA (complete Freund's adjuvant). This is followed by one further intraperitoneal immunization after 4-6 weeks with again 107 CCR5 expressing cells together with IFA (incomplete Freund's adjuvant). Thereafter each mouse is administered with again 107 CCR5 expressing cells (CHO or Ll.2) in PBS at 4-6 weeks intervals. Subsequently the last immunizations are carried out intraperitoneally with again 107 CCR5 expressing cells or intravenously using 2x106 CCR5 expressing cells on the 3rd or 4th day before fusion.
b) Fusion and cloning
The spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., Methods in Enzymology 73 (1981) 3-46. About 1x108 spleen cells of the immunized mouse are mixed with about the same number of myeloma cells (P3x63-Ag8.653, ATCC CRL 1580), fused and cultivated subsequently in HAZ medium (100 mmol/1 hypoxanthine, 1 μg/ml azaserine in RPMI 1640 + 10% FCS). After ca. 10 days the primary cultures are tested for specific antibody production. Primary cultures which exhibit a positive reaction with CCR5 in cell ELISA and no cross-reaction with non-transfected parental cells are cloned in 96-well cell culture plates by means of limiting dilution or a fluorescence activated cell sorter. The cell lines deposited were obtained in this manner.
c) Recombinant production of antibodies
Vectors for the expression of chimeric human-mouse antibodies have been constructed as follows. A heavy chain expression vector was constructed by linking a heavy chain variable domain to human IgGl (SEQ ID NO: 061) and human IgG4 (SEQ ID NO: 062) constant region in the expression vector pSVgpt. A light chain expression vector was constructed by linking a light chain variable domain to human Kappa light chain constant region (SEQ ID NO: 063) in the expression vector pSVhyg. 5' flanking sequence including the leader signal peptide, leader intron and the murine immunoglobulin promoter, and 3' flanking sequence including the splice site and intron sequence was introduced using the vectors VH-PCRl and VK-PCRl as templates. The heavy and light chain expression vectors were co-transfected into NSO cells (ECACC No 85110503, a non- immunoglobulin producing mouse myeloma). Transfected cell clones were screened for production of human antibody by ELISA for human IgG. Example 2
Identification of potential T-cell epitopes
Peripheral blood mononuclear cells (PBMC) from healthy donors are used to provide both T-cells and antigen presenting cells (APC). Forty donors were selected for screening in T-cell assays based on human HLA-DR typing. This enables the screening of peptides in the T-cell assay against greater than 85% of DR alleles expressed in the world population. Peptidic fragments of 15 amino acids length were designed to cover the variable regions of the antibody, each overlapping by 12 residues. The sequence was extended at the end of the variable regions to include the first few residues of the human constant regions to which they will be joined in the final engineered antibodies. Peptidic fragments and PBMC were set up in sextuplicate cultures in 96 well plates with 5 μM peptide and 2 x 105 PBMC per well. Keyhole limpet haemocyanin (KLH) was included in the assay as a positive control. After 7 days incubation of cells and peptidic fragments, an 18 hour pulse with 3H-Thymidine at 1 μCi/well was used to assess T-cell proliferation.
In the present assay, a potential T-cell epitope is defined as a peptide giving a stimulation index (SI) greater than 2 in at least 2 independent donors, although SIs which are just under 2 may also be included in the analysis. All donors responded to KLH with SIs ranging from 2.2 to 32.9.
Example 3
Construction of constant region variant anti-CCR5 antibodies of the IgGl and
IgG4 subclass
Expression plasmids encoding variant anti-CCR5 antibodies of IgGl and IgG4 subclass can be obtained by site-directed mutagenesis of the wild type expression plasmids using the QuickChange™ Site-Directed mutagenesis Kit (Stratagene) and are described in Table 4. Amino acids are numbered according to EU numbering
[Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242 (1991)] Table 4: Constant region variants.
Figure imgf000045_0001
Explanation of mutations: S228P denotes that the amino acid serine at Kabat amino acid position 228 is changed to proline; L234A and L235A denotes that the amino acid Leucine at Kabat amino acid position 234 and 235 is each changed to the amino acid Alanine.
Example 4
Construction of encoding nucleic acids
Nucleic acids encoding the anti-CCR5 antibody heavy chain of SEQ ID NO: 039 with an additional C-terminal peptidic-linker of the amino acid sequence GGGGSGGGGS (SEQ ID NO: 083) connecting the heavy chain to a single chain antibody with the variable domains of the antibody obtained from DSM ACC 2681 of SEQ ID NO: 004 and 008 joined by a single-chain-linker of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 084), were prepared by gene synthesis with 5'- and 3 '-terminal flanking BamHI and Xbal restriction sites. In a similar manner, nucleic acids encoding for the antibody heavy chain of SEQ ID NO: 039 with N-terminal connected single chain antibody and unmodified heavy chain of SEQ ID NO: 039 were prepared.
Nucleic acid sequences encoding antibody heavy chains with connected single chain antibodies (scFv) with
i) cysteine residues for disulfide stabilization in the heavy chain variable domain (Kabat position 44, 101 or 105) of the connected scFv, and/or ii) variable single-chain-linker length, and/or iii) variable peptidic-linker length,
were prepared by nucleic acid synthesis with flanking BamHI/EcoNI (N-terminal scFv) or EcoNI/Xbal (C-terminal scFv) restriction sites and the corresponding overlapping region of the antibody heavy chain of SEQ ID NO: 039. Nucleic acids encoding the anti-CCR5 antibody light chain of SEQ ID NO: 044 with an additional C-terminal peptidic-linker of the amino acid sequence GGGGSGGGGS (SEQ ID NO: 083) connecting the light chain to a single chain antibody with the variable domains of the antibody obtained from DSM ACC 2681 of SEQ ID NO: 004 and 008 joined by a single-chain-linker of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 084), were synthesized with 5'- and 3 '-terminal flanking BamHI and Xbal restriction sites hi a similar manner, nucleic acids encoding for the antibody light chain of SEQ ID NO: 044 with N-terminal connected single chain antibody and unmodified light chain of SEQ ID NO: 044 were prepared.
Nucleic acid sequences encoding antibody light chains with connected single chain antibodies (scFv) with
i) cysteine residues for disulfide stabilization in the light chain variable domain (Kabat position 43, 46, or 100) of the connected scFv, and/or ii) variable single-chain-linker length, and/or iii) variable peptidic-linker length,
were prepared by nucleic acid synthesis with flanking BamHI/Bsgl (N-terminal scFv) or Bsgl/Xbal (C-terminal scFv) restriction sites and the corresponding overlapping region of the antibody light chain of SEQ ID NO: 044.
All constructs were designed with a 5 '-end DNA sequence coding for a leader peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 064), which targets proteins for secretion in eukaryotic cells.
Likewise can nucleic acids for the full length antibody heavy and light chains of any anti-CCR5 antibody conjugated to single chain antibodies comprising the variable domains of a different or the same anti-CCR5 antibody be constructed whereby both antibodies bind to the same or different, non-overlapping epitopes on CCR5.
Thus, nucleic acids for the full length antibody heavy and light chains of an anti- CCR5 antibody obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from the antibodies with variable domain amino acid sequence of SEQ ID NO: 039 and 044, or of SEQ ID NO: 048 and 052, or of SEQ ID NO: 056 and 060 conjugated to single chain antibodies comprising the variable domains of a an anti-CCR5 antibody obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from SEQ ID NO: 039 and 044, or SEQ ID NO: 048 and 052, or SEQ ID NO: 056 and 060 can be obtained whereby either only variable domains are combined which bind to the same epitope on CCR5 or only variable domains are combined which bind to different, non-overlapping epitopes on CCR5.
Finally, nucleic acids encoding full length heavy and light antibody chains connected to single chain antibodies were obtained in which the single chain antibody binds to the same or a different antigen than the full length antibody, e.g. one antigen is the CCR5 and one antigen is the CD4. Such antibodies comprise variable domains binding to CD4 of SEQ ID NO: 064 and 068 (US 5,871,732), or SEQ ID NO: 072 and 076, or SEQ ID NO: 080 and 084 (Reimann, K.A., et al., Aids Res. Human Retrovir. 13 (1997) 933-943), or SEQ ID NO: 088 and 092 (WO 1991/009966), and variable domains binding to CCR5 obtained from DSM ACC 2681, DSM ACC 2682, DSM ACC 2683, and DSM ACC 2684 or selected from SEQ ID NO: 039 and 044, or SEQ ID NO: 048 and 052, or SEQ ID NO: 056 and 060.
Example 5
Construction of the expression plasmids The basis vector used for the construction of all heavy and light chain scFv fusion protein encoding expression plasmids was composed of the following elements: a hygromycin resistance gene as a selection marker, an origin of replication, oriP, of Epstein-Barr virus (EBV),
- an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli a beta-lactamase gene which confers ampicillin resistance in E. coli,
- the immediate early enhancer and promoter from the human cytomegalovirus (HCMV),
- the human γl -immunoglobulin polyadenylation ("poly A") signal sequence, and
- unique BamHI, and/or Xbal, and/or Bsgl, and/or EcoNI restriction sites.
The nucleic acids comprising the heavy or light chain and a scFv nucleic acids in combination with optional peptidic-linker- variation, single-chain-linker- variation and cysteine modifications, which have been constructed as outlined in Example 4, were cloned into the plasmid pGA18 (ampR). The pGA18 (ampR) plasmids carrying the heavy and light chain encoding nucleic acids as well as the basis vector were digested with BamHI and Xbal restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis. Purified heavy and light chain encoding nucleic acids were ligated to the isolated basis vector BamHI/Xbal fragment resulting in the final pUC-Exp- YY-XXXX-HC and pUC- Exp- YY-XXXX-LC expression vectors.
The string 'YY-XXXX' in the expression vector denotes the structure of the encoded antibody, whereby X is a digit and Y is a character.
The two characters denote the composition of the basic construct:
The first character denotes the origin and specificity of the full length antibody chains with
A - anti-CCR5 antibody chain obtained from DSM ACC 2681, B - anti-CCR5 antibody chain obtained from DSM ACC 2683,
C - anti-CCR5 antibody chain obtained from DSM ACC 2682, D - anti-CCR5 antibody chain obtained from DSM ACC 2684, E - anti-CCR5 antibody chain of SEQ ID NO: 039 (heavy chain) or SEQ ID NO: 044 (light chain), F - anti-CCR5 antibody chain of SEQ ID NO: 048 (heavy chain variable domain) or SEQ ID NO: 052 (light chain variable domain), G - anti-CCR5 antibody chain of SEQ ID NO: 056 (heavy chain variable domain) or SEQ ID NO: 060 (light chain variable domain).
The second character denotes the origin and specificity of the pair of single chain antibodies with
A - anti-CCR5 antibody domain obtained from DSM ACC 2681, B - anti-CCR5 antibody domain obtained from DSM ACC 2683, C - anti-CCR5 antibody domain obtained from DSM ACC 2682, D - anti-CCR5 antibody domain obtained from DSM ACC 2684, E - anti-CCR5 antibody domain of SEQ ID NO: 039 (heavy chain) or SEQ
ID NO: 044 (light chain),
F - anti-CCR5 antibody domain of SEQ ID NO: 048 (heavy chain variable domain) or SEQ ID NO: 052 (light chain variable domain), G - anti-CCR5 antibody domain of SEQ ID NO: 056 (heavy chain variable domain) or SEQ ID NO: 060 (light chain variable domain), and N - not present.
The first block of 4 digits denotes the structure of the first pair of single chain antibodies whereby
- the first digit denotes the position of attachment of the peptidic-linker (to the pair of single chain antibodies) to the full length antibody chain with
1 - no single chain antibodies present,
2 - a single chain antibody is connected to each of the C-termini of the full length heavy chain,
3 - a single chain antibody is connected to each of the N-termini of the full length heavy chain,
4- a single chain antibody is connected to each of the C-termini of the full length light chain, and 5 - a single chain antibody is connected to each of the N-termini of the full length light chain.
- the second digit denotes the number of GGGGS units (SEQ ID NO: 082) of which the single-chain-linker is consisting,
- the third digit denotes the number of GGGGS units of which the peptidic- linker is consisting, and
- the fourth digit denotes the position of the disulfide bond in the single chain antibody with
1 - disulfide bond between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain, 2 - disulfide bond between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain, and 3 - disulfide bond between position 101 of the heavy chain variable domain and position 46 of the light chain variable domain.
All other expression plasmids for heavy and light chain fusion protein constructs with N- and C-terminal scFv attachments were prepared in a similar manner after the synthesis of the corresponding nucleic acids. For the cysteine-, peptidic-linker- and single-chain-linker modified constructs, the newly synthesized nucleic acids were digested with either BamHI/EcoNI, BamHI/Bsgl, EcoNI/Xbal or Bsgl/Xbal restriction enzymes and ligated to the analogously treated basis vector fragment.
The final expression vectors were transformed into E. coli cells, expression plasmid DNA was isolated (Miniprep) and subjected to restriction enzyme analysis and DNA sequencing. Correct clones were grown in 118 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep) and sequence integrity confirmed by DNA sequencing.
Example 6
Transient expression in HEK293 EBNA and HEK293 cells
The final expression plasmids generated in Example 5 allow the recombinant production of different antibody light chains and different antibody heavy chains. The antibodies according to the invention were generated by transient transfection of suspension culture HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr- Virus nuclear antigen; American type culture collection deposit number ATCC # CRL- 10852) cultivated in FreeStyle™ 293 Expression medium containing 250 μg/ml G418 (Roche Molecular Biochemicals, Germany) at 37°C/5% CO2. For transfection FuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals, Germany) was used in a ratio of reagent (μl) to DNA (μg) ranging from 3:1 to 6:1. The light and heavy chains were expressed from two different plasmids using a molar ratio of light chain to heavy chain encoding plasmid from 1 :2 to 2:1. Antibody containing cell culture supernatants were harvested at day 7 after transfection by centrifugation at 1000 x g for 10 minutes followed by filtration through a sterile filter (0.22 μm). The antibodies were also expressed by transient transfection of human embryonic kidney 293 -F cells using the FreeStyle™ 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyle™ 293-F cells were cultivated in FreeStyle™ 293 Expression medium at 37°C/8% CO2. The cells were seeded in fresh medium at a density of 1-2 x 106 viable cells/ml on the day of transfection. The DNA-293fectin™ complexes were prepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of 293fectin™ (Invitrogen, Germany) and 250 μg of heavy and light chain encoding plasmid DNA in a 1 :1 molar ratio for a 250 ml final transfection volume. The antibody containing cell culture supernatants were clarified 7 days after transfection by centrifugation at 14,000 x g for 30 minutes and filtration through a sterile filter (0.22 μm). Supernatants were stored at -20°C until purification.
Example 7
Purification of the antibodies
The expressed and secreted antibodies were purified in two steps by affinity chromatography using Protein A-Sepharose™ (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, the antibody containing clarified culture supernatants were applied on a HiTrap Protein A HP (5 ml) column equilibrated with PBS buffer (10 mM Na2HPO4, 1 raM KH2PO4, 105 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed out with equilibration buffer. The antibodies were eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing fractions were neutralized with 0.1 ml 1 M TRIS, pH 8.5. Then, the eluted protein fractions were pooled, concentrated with an Ami con Ultra centrifugal filter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded on a Superdex200 HiLoad 120 ml 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM Histidin, 108 mM NaCl, pH 6.0. Fractions containing purified bispecific antibodies with less than 5% high molecular weight aggregates were pooled and stored in 1.0 mg/ml and 0.1 mg/ml aliquots at -80°C. Example 8
Analysis of purified proteins
The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. The purity and the molecular weight of the antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1 ,4-dithiotreitol) and staining with Coomassie brilliant blue). The NuP AGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-20% TRIS-Glycine gels). The aggregate content of antibody samples was analyzed by high-performance SEC using a Superdex 200 analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM KH2PO4, 250 mM KCl, pH 7.0 running buffer at 25°C. 25 μg protein were injected on the column at a flow rate of 0.5 ml/min and eluted isocratic over 50 minutes. For stability analysis, concentrations of 0.1 mg/ml, 1 mg/ml and 5 mg/ml of purified proteins were prepared and incubated at 4°C, 37°C and 40°C, respectively, for 7 or 28 days and then evaluated by high-performance SEC (Figure 2). The integrity of the amino acid backbone of reduced antibody light and heavy chains was verified by nano-electrospray Q-TOF mass spectrometry after removal of N-glycans by enzymatic treatment with Peptide-N-Glycosidase F (Roche Molecular Biochemicals).
Example 9
BIAcore analysis of interaction of CCR5 monospecific antibodies with FcRn
All surface plasmon resonance measurements were performed on a BIAcore 3000 instrument (GE Healthcare Biosciences AB, Sweden) at 25°C. Running and dilution buffer was PBS (1 mM KH2PO4, 1O mM Na2HPO4, 105 mM NaCl, 2.7 mM KCl), pH 6.0, 0.005% (v/v) Tween20. The soluble human FcRn was diluted in 10 mM sodium-acetate, pH 5.0 and immobilized on a CM5 biosensor chip using the standard amine coupling kit (GE Healthcare Biosciences AB, Sweden) to obtain FcRn surface densities of approximately 1000 RU. HBS-P (10 mM HEPES, pH 7.4, 118 mM NaCl, 0.005% Surfactant P20; GE Healthcare Biosciences AB, Sweden) was used as running buffer during immobilization. The antibodies in question were diluted with PBS, 0.005% (v/v) Tween20, pH 6.0 to a concentration of 450 nM and injected over 3 minutes at a flow rate of 30 μl/minute. Afterwards the sensor chip was regenerated for 1 minute with PBS, pH 8.0, 0.005% (v/v) Tween20. Data analysis was performed with the BIAevaluation software (BIAcore, Sweden).
Table 5: FcRn binding analysis with BIAcore.
Figure imgf000052_0001
n.d. = not determined
Example 10 CCR5 CeU ELISA
Twenty thousand CHO-CCR5, CHO-CCR5-dN8 or CHO-CCR5-K171A (K) or CHO-CCR5-K171A/E172A (KE) cells per well were seeded into 96-well tissue culture plates and incubated overnight at 37°C. Thereafter, the cell culture medium was aspirated, 50 μl new medium containing serially diluted bispecific or control antibodies was added and plates were incubated for 2 hours at 4°C. Then, cells were fixed in PBS containing 0.05% (v/v) glutaraldehyde for 10 min. After washing three times with assay medium, 50 μl per well of horseradish peroxidase (HRP)-conjugated sheep anti-mouse immunoglobulin G (IgG) antibody (GE Healthcare, USA) diluted 1 :2,000 was added to the plates. The plates were incubated at room temperature for 2 h. After extensive washes with PBS, 50 μl per well tetramethylbenzidine substrate (Roche Applied Science, Germany) was added for color development. The reactions were stopped by adding 25 μl of 1 M sulfuric acid to the reaction mixture. Plates were read at 450 to 620 nm on an Envision plate reader (Perkin-Elmer, Shelton, CT, USA).
Table 6: CCR5 cell ELISA.
Figure imgf000053_0001
n.d. =not determined
Example 11
RANTES radioligand binding assay
125I-RANTES (regulated on activation normal T-cell expressed and secreted) was purchased from PerkinElmer Life Sciences Inc. (USA). Binding assays were performed on CHO cells expressing recombinant human CCR5 receptor with 1251- labeled RANTES. Cells were plated in 96-well culture plates at 1.5 x 105 cells/well in ice cold binding buffer (phenol red- free Fl 2 medium supplemented with freshly made 0.1% BSA and 0.1% NaN3). Serially diluted CCR5 multivalent and control antibodies were added to the cells, followed by addition of 100 pM 1251-labeled RANTES. After 2 h of incubation at room temperature with gentle shaking, cells were harvested onto GF/C UniFilter plates (PerkinElmer Life Sciences Inc.) using a cell harvester. UniFilter plates were pretreated with 0.3% PEI/0.2% BSA for 30 min prior to harvest. Filter plates were washed five times with 25 mM pH 7.1 HEPES buffer containing 500 mM NaCl, 1 mM CaCl2 and 5 mM MgCl2. Plates were dried in 70°C oven for 20 min. Afterwards 40 μl scintillation fluid was added and radioactivity was measured using TopCount NXT (PerkinElmer). In all experiments, each data point was assayed in duplicate. Data are presented as the percentage of counts obtained in absence of cold competing ligand. Curve fitting and subsequent data analysis were carried out using GraphPad PRISM software (Intuitive Software for Science, San Diego, CA, USA) and IC50 values were calculated using non-linear regression analysis.
i Table 7: RANTES radioligand binding assay.
Figure imgf000054_0001
n.d. =not determined
Example 12
Single-cycle antiviral assay
For the production of pseudotyped NL-BaI viruses, plasmid pNL4-3Δenv (HIV pNL4-3 genomic construct with a deletion within the env gene) and pCDNA3.1/NL-BAL env [pcDNA3.1 plasmid containing NL-BaI env gene (obtained from NIBSC Centralized Facility for AIDS Reagents)] were co- transfected into the HEK 293FT cell line (Invitrogen, USA), cultured in Dulbecco's' modified minimum medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/mL Penicillin, 100 μg/mL Streptomycin, 2 mM L-glutamine and 0.5 mg/mL geniticin (all media from Invitrogen/Gibco). The supernatants containing pseudotyped viruses were harvested two days following transfection, and cellular debris was removed by filtration through a 0.45 μm pore size PES (polyethersulfone) filter (Nalgene) and stored at -80°C in aliquots. For normalization in assay performance, virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells yielding approximately 1.5 x 105 RLU (relative light units) per well. Multivalent and control antibodies were serially diluted in 96-well plates. The assay was carried out in quadruplicates. Each plate contained cell control and virus control wells. The equivalent of 1.5 x 105 RLU of virus stocks were added to each well, then 2.5 x 104 JC53-BL cells were added to each well, with a final assay volume of 200 μl per well. After 3 day incubation at 37°C, 90% Relative Humidity, and 5% CO2, media were aspirated and 50 μl of Steady-Glow® Luciferase Assay System (Promega) was added to each well. The assay plates were read on a Luminometer (Luminoskan, Thermo Electron Coφoration) after 10 minutes of incubation at room temperature. Percent inhibition of luciferase activity was calculated for each dose point after subtracting the background, and ICso-values were determined by using XLfit curve fitting software for Excel (version 3.0.5 Buildl2; Microsoft).
Table 8: Single-cycle antiviral assay
Figure imgf000055_0001
n.d. =not determined
Example 13
Antiviral assay in peripheral blood mononuclear cells (PBMC)
Human PBMC were isolated from buffy-coats (obtained from the Stanford Blood Center) by a Ficoll-Paque (Amersham, USA) density gradient centrifugation according to manufacturer's protocol. Briefly, blood was transferred from the buffy coats in 50 ml conical tubes and diluted with sterile Dulbecco's phosphate buffered saline (Invitrogen/Gibco) to a final volume of 50 ml. Twenty- five ml of the diluted blood was transferred to two 50 ml conical tubes, carefully underlayerd with 12.5 ml of Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at room temperature for 20 min. at 450 x g without braking. The white cell layer was carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove remaining red blood cells, cells were incubated for 5 min. at room temperature with ACK lysis buffer (Biosource) and washed one more time with PBS. PBMC were counted and incubated at a concentration of 2 to 4 x 106 cells/ml in RPMIl 640 containing 10% FCS (Invitrogen/Gibco), 1% penicillin/streptomycin, 2 raM L-glutamine, 1 raM sodium-pyruvate, and 2 μg/ml Phytohemagglutinin (Invitrogen) for 24 h at 37°C. Cells were incubated with 5 Units/ml human IL-2 (Roche Molecular Biochemicals) for a minimum of 48 h prior to the assay.
In a 96 well round bottom plate, 1 x 105 PBMC were infected with HIV-I NLBaI (NL4.3 strain (Adachi, A., et al., J. Virol. 59 (1986) 284-291) with the env of BaL (gpl20)) in the presence of serially diluted multivalent and control antibodies. The amount of virus used was equivalent to 1.2 ng HIV-I p24 antigen/well. Infections were set up in quadruplicates. Plates were incubated for 6 days at 37°C. Virus production was measured at the end of infection by using a p24 ELISA (Perkin- Elmer, Shelton, CT) according to the manufacturer's instructions. For all antiviral assays, the 50% inhibitory concentration (IC50) was calculated by using the sigmoid dose-response model with one antigen-binding site in Microsoft XLfit.
Table 9: Antiviral assay.
Figure imgf000056_0001
n.d. =not determined
A comparison of IC50 values for different multivalent antibodies is shown in Figure 5. It can be seen that the tetravalent antibodies have an improved in vitro biological potency even better than the combination of the individual isolated parent antibodies, e.g. it can be seen that the doubling of the antigen-binding sites for the same epitope in case of AA-2320 provides for a reduction of the IC50 from 988 pM to 88 pM.
Example 14
Generation of HIV-I strains with resistance to the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044
The CCR5-tropic HIV-I isolates BaI and CC 1/85 were passaged in vitro in the presence of increasing concentrations of the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044. CD8-depleted PBMC and genetically diverse, high titer viruses were used to facilitate fast resistance development. The sensitivity of resistant and no drug control viruses (NDC) to the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044 and other anti-CCR5 antibodies was measured in PBMC and single-cycle assays.
Passaging of BaI and CCl/85 in the presence of increasing concentrations of the antibody resulted in the selection of two highly resistant viruses. The NDC viruses remained sensitive to the antibody. In the presence of a second anti-CCR5 antibody recognizing an epitope at the N-terminus of CCR5, the phenotype was reversed; both resistant virus (Bal res and CCl/85_res) were more sensitive to said second antibody compared to their respective NDC virus, suggesting a shift in the binding ability of these Envs.
Sequence analysis revealed that Bal res differed from BaI NDC in only two amino acid positions (K163N and S531A). K163N is located in the V2 region, whereas S531A is located in gp41. S531A was also detected in CCl/85_res, which had additional mutations throughout gpl20. Furthermore, pseudotyped virus containing Env genes of CCl/85_res and Bal_res, but not of BaI NDC and CC1/85 NDC, were also resistant to the anti-CCR5 antibody comprising variable domains of SEQ ID NO: 039 and 044 and the anti-CCR5 monoclonal antibody 2D7.

Claims

Patent Claims
1. A monospecific tetravalent anti-CCR5 antibody, characterized in that said antibody is consisting of:
a monospecific bivalent anti-CCR5 parent antibody consisting of two full length antibody light chains and two full length antibody heavy chains each chain comprising only one variable domain,
- two peptidic-linkers, and two monospecific monovalent single chain antibodies each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain-linker.
2. Monospecific tetravalent anti-CCR5 antibody according to claim 1, characterized in that said four antigen-binding sites bind to the same epitope or an overlapping epitope as an antibody obtained from DSM ACC 2681 or DSM ACC 2683.
3. Monospecific tetravalent anti-CCR5 antibody according to claim 1, characterized in that the heavy chain variable domain of said antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 003, 002, 001, or SEQ ID NO: 01 1, 010, 009, or SEQ ID NO: 037, 036, 035.
4. Monospecific tetravalent anti-CCR5 antibody according to claim 3, characterized in that the light chain variable domain of said antigen-binding site comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 007, 006, 005, or SEQ ID NO: 016, 015, 014, or SEQ ID NO: 042, 041, 040.
5. Monospecific tetravalent anti-CCR5 antibody according to any one of claims 3 or 4, characterized in that the heavy and light chain variable domain of said antigen-binding sites have an amino acid sequence of SEQ ID NO: 004, 008, or SEQ ID NO: 012, 017, or SEQ ID NO: 039, 044 or are a T-cell epitope depleted, CDR-grafted, or humanized variant thereof.
6. Monospecific tetravalent anti-CCR5 antibody according to any one of claims 1 and 3 to 5, characterized in that said heavy chain has an amino acid sequence of SEQ ID NO: 122 and said light chain has an amino acid sequence of SEQ ID NO: 123.
7. Monospecific tetravalent anti-CCR5 antibody according to any one of claims 1 and 3 to 6, characterized in that said the single chain antibodies have a disulfide bond between the single chain antibody variable domains.
8. Monospecific tetravalent anti-CCR5 antibody according to any one of claims 1 or 3 to 5, characterized in that said heavy chain has an amino acid sequence of SEQ ID NO: 124 and said light chain has an amino acid sequence of SEQ ID NO: 125.
9. Monospecific tetravalent anti-CCR5 antibody according to any one of claims 1 and 3 to 6 and 8, characterized in that said antibody the single chain antibodies have a disulfide bond between the single chain antibody variable domains.
10. Antibody according to any one of the preceding claims, characterized in that said antibody is glycosylated with a sugar chain at Asn297 whereby the amount of fucose within said sugar chain is 65% or lower
11. Method for increasing the biological potency of a monospecific bivalent anti- CCR5 parent antibody comprising the preparation of a monospecific tetravalent or monospecific hexavalent variant of said antibody in which all antigen-binding sites bind to the same or an overlapping epitope on the same antigen, whereby said monospecific bivalent antibody is modified by the connection to two or four monospecific monovalent single chain antibodies each connected via a peptidic-linker each to a single C- or N-terminus of the antibody chains of said monospecific bivalent parent antibody whereby to each light or heavy chain of the parent antibody either no or one single chain antibody is connected.
12. Method according to claim 11, characterized in that said epitope is that to which the antigen-binding sites of an antibody obtained from DSM ACC 2681, or DSM ACC 2682, or DSM ACC 2683, or DSM ACC 2684, or an antibody with a heavy chain variable domain of SEQ ID NO: 048 and a light chain variable domain of SEQ ID NO: 052, or an antibody with a heavy chain variable domain of SEQ ID NO: 056 and a light chain variable domain of SEQ ID NO: 060 binds.
13. Pharmaceutical composition comprising an antibody according to any one of claims 1 to 10.
14. Use of an antibody according any one of claims 1 to 9 for the manufacture of a medicament for the treatment of immunosuppression.
15. Use of an antibody according to claim 10 for the manufacture of a medicament for the treatment of acute or chronic allograft rejection, or COPD, or rheumatoid arthritis.
16. Pharmaceutical composition comprising a variant of a monospecific bivalent parent antibody to which a HIV strain has developed a resistance, characterized in that said variant of said monospecific bivalent anti-CCR5 parent antibody is a monospecific tetravalent or a monospecific hexavalent anti-CCR5 antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies all bind to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen.
17. Use of a variant of a monospecific bivalent parent antibody to which a HIV strain has developed a resistance for the manufacture of medicament for the treatment an HIV infection by said HIV strain, characterized in that said variant of said monospecific bivalent anti-CCR5 parent antibody is a monospecific tetravalent or a monospecific hexavalent anti-CCR5 antibody in which to said monospecific bivalent parent antibody two or four monospecific monovalent single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies all bind to the same epitope which is the same or an overlapping epitope as that bound by the antigen-binding sites of the monospecific bivalent parent antibody of the same antigen.
18. Method for the production of an antibody according to any one of claims 1 to 10 comprising the following steps:
a) cultivating a cell comprising a nucleic acid encoding an antibody according to any one of claims 1 to 10 under conditions suitable for the expression of said antibody, b) recovering said antibody from the cultivation medium or from the cells, c) purifying said antibody in order to produce an antibody according to any one of claims 1 to 10.
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