WO2010028796A1 - Anticorps hexavalents trispécifiques - Google Patents

Anticorps hexavalents trispécifiques Download PDF

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WO2010028796A1
WO2010028796A1 PCT/EP2009/006498 EP2009006498W WO2010028796A1 WO 2010028796 A1 WO2010028796 A1 WO 2010028796A1 EP 2009006498 W EP2009006498 W EP 2009006498W WO 2010028796 A1 WO2010028796 A1 WO 2010028796A1
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antibody
seq
variable domain
chain variable
ccr5
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PCT/EP2009/006498
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WO2010028796A8 (fr
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Johannes Auer
Michael Brandt
Ulrich Brinkmann
Wilma Dormeyer
Eike Hoffmann
Andreas Jekle
Jun-Ichi Nezu
Juergen Michael Schanzer
Jan Olaf Stancke
Pablo Umana
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F. Hoffmann-La Roche Ag
Roche Glycart Ag
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Publication of WO2010028796A1 publication Critical patent/WO2010028796A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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

Definitions

  • the present invention relates to tri specific hexavalent anti-CCR5/CCR5/CD4 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).
  • 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., Leger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007) 65-74; Wu, C, et al., Nature
  • 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
  • Multivalent Fy-antibody construct having at least four variable domains which are linked with each over via peptide linkers are reported in US 7,129,330.
  • Dimeric and multimeric antigen binding structures are reported in US 2005/0079170.
  • Tri- or tetra-valent 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 6,51 1 ,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 ⁇ thFl)ligOmers7 typically of-the-IgG-elasSj-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.
  • US 2005/0100543 are reported targetable constructs that are multivalent carriers of bi-specific antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specific 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.
  • WO 2008/019817 reports a conjugates of an anti-CCR5 antibody and an antifusogenic peptide.
  • Anti-HIV-1 conjugates for the treatment of HIV disease are reported in WO 2001/043779. Shields, R.L., et al., J. Biol. Chem. report that the lack of fucose in human IgGl N-linked oligosaccharide improve the binding to human FcgammaRIII and antibody-dependent cellular toxicity.
  • a first aspect of the current invention is an antibody comprising six antigen- binding sites each antigen-binding site consisting of a pair of antibody variable domains, whereby every two of said antigen-binding sites bind to the same epitope and the antibody binds to two or three antigens.
  • 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.
  • - four peptidic-linkers, and - four 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
  • four antigen-binding sites are formed by two pairs of single chain antibodies, whereby a) each single chain antibody of a pair binds to the - A -
  • one pair binds to an epitope which is on the same antigen as that of i) and the other pair binds to an epitope on a different antigen
  • 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.
  • Another aspect of the current invention is a method for increasing the activity of a monospecific bivalent antibody, said method comprises the preparation of a trispecific hexavalent variant of said antibody in which the antigen-binding sites bind to two or three different antigens, whereby said monospecific bivalent antibody is modified by the connection to four 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 trispecific hexavalent variant of said parent monospecific bivalent antibody in which to said parent monospecific bivalent antibody two pairs, i.e. four, single chain antibodies are connected via a peptidic- linker, whereby a) each single chain antibody of a pair binds to the same epitope which is different from the epitope of i), b) one pair binds to an epitope which is on the same antigen as that of i) and the other pair binds to an epitope on a different 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) trispecific, and ii) hexavalent, 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, - four peptidic-linkers, and
  • 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
  • two pairs of antigen-binding sites are each formed by one single chain antibody, wherein the antigen-binding sites of each pair bind to the same epitope and wherein one pair of antigen binding sites binds to a non-overlapping epitope on the same antigen as that of the antigen- binding sites of i) and one pair of antigen binding sites binds to an epitope on an antigen different from that of i), iii) said single chain antibodies are both linked to the same kind of terminus of an 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 epitope of an antigen.
  • the terms "bispecific” and “trispecific” antibody as used within the current application denote antibodies that have two or more antigen-binding sites of which in a bispecific antibody a first antigen-binding site binds to a first epitope and a second antigen-binding site binds to a second epitope, or in a trispecific antibody a first antigen-binding site binds to a first epitope, a second antigen-binding site binds to a second epitope and a third antigen-binding site binds to a third epitope, whereby the first, second and third epitopes are different, i.e.
  • Two epitopes are different if a signal reduction of 50% or less, in one embodiment of 25% or less, 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 difference has to be detected at a concentration of 100 nM.
  • SPR surface plasmon resonance
  • a method in which epitope difference of two antibodies binding to the same antigen is determined with the help of a competitive test system.
  • a competitive test system 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 difference 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 difference has to be determined. By detection of the bound labeling there can easily be ascertained the epitope difference.
  • epitope difference is present and both antibodies bind to different epitopes 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.
  • 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 mono-, bi-, or trispecific. That is, e.g., the antibodies may be bispecific even in cases where there are more than two antigen-binding sites (i.e that the antibody is trivalent or multivalent).
  • Multispecific 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.
  • CDR-grafted variant 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
  • 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. After 7 days incubation an 18 hour pulse with 3 H-Thymidine at 1 ⁇ Ci/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.
  • 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 as used within the current application denotes antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • 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 l (GenBank XO 1668), or from germline sequence hVH_3_64 (GenBank M99682) and germline sequence hVK_lD_16 (GenBank KOl 323), or germline sequence hVH_4_59 (GenBank Ll 0088) and germline sequence hVK_l_39
  • 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 one 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 preferably 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
  • the first aspect of the current invention is an antibody comprising six antigen- binding sites whereby said antigen-binding sites are grouped in pairs and the antigen-binding sites of such a pair bind to the different non-overlapping epitopes.
  • 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.
  • 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_heav_y_chainl' ⁇ ndJw ⁇ ' ⁇ fulHength 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).
  • VL antibody light chain variable domain
  • CL antibody light chain constant domain
  • 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:
  • L5 monospecific_bivalent parent antibody and the other pair of single chain antibodies bind to an epitope on a different antigen 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 20 heavy chains and/or to the same kind of terminus (in one embodiment to the
  • either one or no single chain antibody is linked to a chain of the monospecific bivalent antibody, i.e. to each chain of the monospecific bivalent antibody is linked one additional single chain antibody.
  • - 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 pairs of antigen-binding sites are each formed by the heavy and light chain variable domain of one single chain antibody, whereby each pair of antigen-binding sites binds to a different epitope or 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 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.
  • 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: 098, 105, 106, 107, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 141, 142, 143, 144, 145, 146, 14.7,_148,_149,_15p,_lll,_168 ⁇ or_1 ⁇ 9Jsee 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: 115, 116, 121, 122, 168.
  • said peptidic linker comprises the amino acid sequence of SEQ ID NO: 115 or 121.
  • said peptidic-linker consists of the amino acid sequence of SEQ ID NO: 115, 116, 121, 122, 168, in another embodiment of SEQ ID NO: 115 or 121.
  • 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 j;hainTv ⁇ (scFv). J ⁇ singlej ⁇ h ⁇ inj ⁇ nk ⁇ r ⁇ arLC ⁇ rnpi ⁇ s ⁇ ne_o ⁇ more_of the following amino acid sequences and is independently selected for each single chain antibody from SEQ ID NO: 097, 099, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
  • 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: 116,
  • said single-chain-linker comprises the amino acid sequence of SEQ ID NO: 121, 122, 112, in one embodiment of SEQ ID NO: 121. In another embodiment said single-chain-linker consists of the amino acid sequence of SEQ ID NO: 116, 121 , 122, 112. In another embodiment the single- chain-linker is consisting of the amino acid sequence of SEQ ID NO: 121, 122,
  • SEQ ID NO: 112 in one embodiment of SEQ ID NO: 121.
  • Table 2 Single-chain-linker amino acid sequences.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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,
  • 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: 170).
  • 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 K or ⁇ region.
  • constant regions are well known in the state of the art and e.g. described by
  • 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)
  • 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: 093 and 094. hi one embodiment the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 093 with mutations L234A and L235A.
  • the constant heavy chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 094 with mutation S228P.
  • the constant light chain region of the monospecific bivalent parent antibody is of SEQ ID NO: 095:
  • 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.
  • ADCC antibody-dependent cellular cytotoxicity
  • 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.
  • 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.
  • 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.
  • 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
  • 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 Fc ⁇ R 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.
  • 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 and 300.
  • the antibody according to the invention is of human IgGl or IgG3 subclass. In a further embodiment the amount of
  • N-glycolylneuraminic acid 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.
  • 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.
  • NGNA 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).
  • 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 ran 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.
  • 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: 093).
  • 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: 093).
  • 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
  • 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.
  • 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-glycomodif ⁇ ed 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 which are inserted into host cells.
  • 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 there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Expression in NSO cells is described by, e.g., Barnes, L.M., et al., Cytotechnology
  • HEK 293 A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
  • 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.Man ⁇ ctWityjujid ⁇ r ⁇ pi ⁇ i ⁇ on ⁇ wjncj ⁇ p ⁇ rik 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-acetylglucosaminyl transferase I ("GnTI"), the localization domain of marmosidase I, the localization domain of ⁇ (l ,2)-N- acetylglucosaminyl transferase II (“GnTII”), and the localization domain of a- 1,6 core fucosyltransferase.
  • the Golgi localization domain is from mannosidase 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- acetyl glucosaminyl transferase III also known as ⁇ -l,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.144)
  • 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-eell-mediated- cytotoxic activity as reported in US 6,946,292 can be used.
  • 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 e.g. with 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.
  • 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. Tb 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-patienVcomposition, and-mode_of_adminijtration,jwithout being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier 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.
  • ThI ThI responses
  • Th2 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.
  • 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, ex ⁇ cytosis ⁇ oT " 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. In addition 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
  • CCR5 as an HIV-I co-receptor was based on the ability of its ligands, MIP- l ⁇ , MIP- IB 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) 113-1 18; 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., Suppl. A. AIDS 11 (Supp. 093) (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 140 (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: 152 either the amino acid lysine or glutamine can be present, at position 6 of SEQ ID NO: 152 either the amino acid glutamine or glutamic acid can be present, and so on.
  • SEQ ID NO: 152 is the amino acid sequence of the heavy chain variable domain, wherein XOl to X08 denote the variable positions: Gln-Val-Gln-Leu-XOl OO ⁇ -Ser-Gly-Pro-Gly-Leu-Val-XOS-Pro-Ser-Gln-Ser-Leu-
  • 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: 153 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-
  • XlO is either He or Ala
  • Xl 1 is either Phe or Tyr
  • 10 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
  • 15 X 17 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, ⁇ 2tt X22ls ⁇ ithe7Gly ⁇ or Al ⁇
  • X23 is either Asn or Thr
  • X24 is either Leu or VaI
  • One example 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
  • CD4 means human CD4 as described, e.g., in Brady, R.L., and Barclay, A. N., Curr. Top. Microbiol. Immunol. 205 (1996) 1-18 and SwissProt P01730.
  • anti-CD4 antibody denotes an antibody specifically binding to CD4 and 35 preferably inhibiting HIV fusion with a target cell. Binding can be tested in a cell based in vitro ELISA assay (using CD4 expressing CHO cells). Binding is found, if the antibody in question causes an S/N (signal/noise) ratio of 5 or more, preferably 10 or more at an antibody concentration of 100 ng/ml.
  • inhibiting HIV fusion with a target cell refers to inhibiting HIV fusion with a target cell measured in an assay comprising contacting said target cell (e.g. PBMC) with the virus in the presence of the antibody in question in a concentration effective to inhibit membrane fusion between the virus and said cell and measuring, e.g., luciferase reporter gene activity or the HIV p24 antigen concentration.
  • target cell e.g. PBMC
  • membrane fusion refers to fusion between a first cell expressing CD4 polypeptides and a second cell or virus expressing an HIV env protein. Membrane fusion is determined by genetically engineered cells and/or viruses by a reporter gene assay
  • anti-CD4 antibodies are mentioned in e.g. Reimann, K. A., et al., Aids Res. Human Retrovir. 13 (1997) 933- 943, EP 0 512 112, US 5,871,732, EP 0 840 618, EP 0 854 885, EP 1 266 965, US 2006/0051346, WO 1997/46697, WO 2001/43779, US 6,136,310, WO 1991/009966.
  • the anti-CD4 antibody variable domains of the anti-CCR5/CD4 antibody are those reported in US 5,871,732, and Reimann,
  • the anti-CCR5/CD4 antibody is characterized in that it is a non-mimunosuppressive ⁇ rjion-depleting antibody when administered to humans and also does not block binding of HIV gp 120 to human CD4. ⁇
  • Another example is an antibody which is a bispecific tetravalent antibody with antigen-binding sites binding to an epitope of CCR5 and antigen-binding sites binding to an epitope of CD4. It has surprisingly been found that with the admixture of antigen-binding sites to different epitopes on different antigens an improved in vitro biological potency is detectable.
  • a reduction of the IC 5O value from 1077.5 or 987.6 pM (pmol/1) for the monospecific bivalent anti-CCR5 antibodies and from 347.5 pM for the monospecific anti-CD4 antibody to 9.7 pM for the bispecific tetravalent anti-CCR5/CD4 antibody according to the invention can be detected.
  • This reduction was not predictable and, thus, shows a synergistic effect of the pairs of two antigen binding sites binding different epitopes on different antigens located in the same antibody molecule according to the invention.
  • One aspect of the current invention is an antibody which is a trispecific hexavalent antibody with antigen-binding sites binding to two different epitopes on CCR5 and antigen-binding sites binding to an epitope on CD4.
  • the epitopes on CCR5 are either the same or different non-overlapping epitopes. It has surprisingly been found that with the mixture of antigen-binding sites to different epitopes on the same and on different antigens an improved in vitro biological potency is detectable.
  • a reduction of the IC 5O value from 1077.5 or 987.6 pM (pmol/1) for the monospecific bivalent anti-CCR5 antibodies and from 347.5 pM for the monospecific anti-CD4 antibody to 7.0 to 5.5 pM for the trispecific hexavalent anti-CCR5/CD4 antibody according to the invention can be detected.
  • This reduction was not predictable and, thus, shows a synergistic effect of the antigen binding site pairs binding different epitopes on different antigens located in the same antibody molecule according to the invention.
  • This trispecific hexavalent anti-CCR5/CD4 antibody is consisting of:
  • __tp_CCR5 a first pair of two monospecific monovalent single chain antibodies binding __tp_CCR5 each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain- linker
  • CD4 a second pair of two monospecific monovalent single chain antibodies binding to CD4 each consisting of an antibody heavy chain variable domain, an antibody light chain variable domain, and a single-chain- linker.
  • the antigen-binding sites of the trispecific hexavalent anti-CCR5/CD4 antibody according to this aspect of the current invention are characterized by that
  • the first two antigen-binding sites bind to the same epitope of CCR5 as 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, - the second two antigen-binding sites bind to the same epitope of CCR5 as 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: 05
  • the third two antigen-binding sites bind to the same epitope of CD4 as the antigen-binding sites of an antibody with a heavy chain variable domain of SEQ ID NO: 064 and a light chain variable domain of SEQ ID NO: 068, or an antibody with a heavy chain variable domain of SEQ ID NO: 072 and a light chain variable domain of SEQ ID NO: 076, or an antibody with a heavy chain variable domain of SEQ ID NO: 080 and a light chain variable domain of SEQ ID NO: 084, or an antibody with a heavy chain variable domain of SEQ ID NO: 088 and a light chain variable domain of SEQ ID NO: 092.
  • the heavy chain variable domain of said first and second two antigen-binding sites comprise a CDR3 of SEQ ID NO: 001 , 019, 009, 027, 045, or
  • the heavy chain variable domain of said third two antigen-binding sites comprise a CDR3 of SEQ ID NO: 061, 069, 077, or 085.
  • the heavy chain variable domain of said first and second two antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 003, 002, 001, or SEQ ID NO: 021, 020, 019, or SEQ ID NO: 011, 010, 009, or SEQ ID NO: 029, 028, 027, or SEQ ID NO: 047, 046, 045, or SEQ ID NO: 055, 054, 053, and the heavy chain variable domain of said third two antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 063, 062, 061, or SEQ ID NO: 071, 070, 069, or SEQ ID NO: 079, 078, 077, or SEQ ID NO: 087, 086, 085.
  • the antigen-binding sites are characterized that the light chain variable domain of said first and second two antigen-binding sites comprises a CDRl, CDR2, and CDR3 selected from SEQ ID NO: 007, 006, 005, or SEQ ID NO: 025, 024, 023, or SEQ ID NO: 016, 015, 014, or SEQ ID NO: 033, 032, 031, or SEQ ID NO: 051, 050, 049, or SEQ ID NO: 059, 058, 057, and the light chain variable domain of said third two antigen-binding sites comprises a CDRl, CDR2, and
  • the heavy and light chain variable domain of said first and second two antigen-binding sites have an amino acid sequence of SEQ ID NO: 004, 008, or SEQ ID NO: 022, 026, or SEQ ID NO: 012, 017, or SEQ ID NO: 030, 034, or SEQ ID NO: 063, 062, or SEQ ID NO: 061, 067, or are a T-cell epitope depleted, CDR-grafted, or humanized variant thereof
  • the heavy and light chain variable domain of said third two antigen-binding sites have an amino acid sequence of SEQ ID NO: 064, 068, or SEQ ID NO: 072, 076, or SEQ ID NO: 080, 084, or SEQ ID NO: 088, 092, or are a T-cell epitope depleted
  • an aspect of the current invention is a trispecific hexavalent anti-CCR5 antibody with a heavy chain amino acid sequence and a light chain amino acid sequence of SEQ ID NO: 162 and 163.
  • the single chain antibodies optionally in said antibody the single chain antibodies have a disulfide bond between its single chain antibody variable domains.
  • a further aspect of the invention is a trispecific hexavalent anti- CCR5/CD4 antibody with a heavy chain amino acid sequence and a light chain amino acid sequence of SEQ ID NO: 164 and 165, wherein in said antibody the single chain antibodies have a disulfide bond between the single chain antibody variable domains.
  • Another aspect of the current invention is a method for increasing the biological potency of a monospecific bivalent antibody comprising the preparation of a trispecific hexavalent variant of said antibody in which the antigen-binding sites bind to two different antigens, whereby said monospecific bivalent antibody is modified by the connection to four 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.
  • one antigen is CCR5.
  • one antigen is CD4.
  • the epitopes on CCR5 are selected from the epitopes 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.
  • the epitope on CD4 is the epitope to which the antigen-binding sites of an antibody with a heavy chain variable domain of SEQ ID NO: 064 and a light chain variable domain of SEQ ID NO: 068, or an antibody with a heavy chain variable domain of SEQ ID NO: 072 and a light chain variable domain of SEQ ID NO: 076, or an antibody with a heavy chain variable domain of SEQ ID NO: 080 and a light chain variable domain of SEQ ID NO: 084, or an antibody with a heavy chain variable domain of SEQ ID NO: 088 and a light chain variable domain of SEQ ID NO: 092 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.
  • Trispecific hexavalent anti-CCR5/CD4 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.
  • 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.
  • 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).
  • 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 of 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 trispecific hexavalent form of said monospecific bivalent parent antibody in which to said monospecific bivalent parent antibody four monospecific monovalent single chain antibodies are connected via a peptidic-linker, whereby said single chain antibodies are grouped in pairs of which each member binds to the same epitope but to a different epitope from that bound by the antigen-binding sites of the monospecific bivalent parent antibody either of the same antigen or of a different 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>PzO2.1Cl 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:
  • FIG. 1 Structure of an exemplary hexavalent antibody according to the invention.
  • Figure 2 Exemplary size exclusion chromatogram of a bispecific tetravalent antibody a) without disulfide bonds between the variable domains of the single chain antibodies (B AN -4320-
  • Figure 6 Binding epitopes of two anti-CCR5 antibodies combined in a bispecific tetravalent antibody.
  • Figure 7 Comparison of the aggregate formation of bispecific tetravalent antibodies at a concentration of 5 mg/ml and incubation for 7 days at 40°C; squares: BAN-2320-1000 to BAN-2620-1000, triangles: BAN-4320-1000 to BAN-4620-1000.
  • BAN-4320-1000 (filled circles) and BAN-4321-1000 (filled triangles) with mutated CCR5 presented on CHO cells: a) deletion of the N-terminal 8 amino acids, i.e. within the epitope bound by ANN- 1000- 1000, b) Kl 71 A mutation determined by a cell-based ELISA.
  • Figure 9 Analysis of purified proteins a) Structure of exemplary bispecific and trispecific tetravalent and hexavalent antibodies: BHN-2321- 1000, BHA-2321-4321, BHA-2321-5320; b) SDS-PAGE analysis under reducing conditions and staining with Coomassie brilliant blue, not annotated lines are molecular weight marker.
  • Figure 10 A comparison of IC 5O values for different multivalent antibodies determined in PBMC assay: 1 - BNN- 1000- 1000, 2 - HNN- 1000- 1000, 3 - BHN-2321-1000, 4 - BHA-2321-4321, 5 - BHA-2321- 5320.
  • Figure 11 A comparison of IC 50 values for different multivalent antibodies with a resistant HIV-I strain determined in PBMC assay: 1- BNN- 1000- 1000, 2 - HNN- 1000- 1000, 3 - BHN-2321-1000, 4 - BHA-2321-4321. Materials & Methods Recombinant DNA techniques
  • Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
  • 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 1O 7 CCR5 expressing cells (CHO or Ll.2) together with the adjuvant CFA
  • mice immunized according to a are fused with myeloma cells according to Galfre, G., (Methods in Enzymology 73 (1981) 3-46).
  • 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. 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.
  • 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: 093) and human IgG4 (SEQ ID NO: 094) 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: 095) 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 851 10503, a non-immunoglobulin producing mouse myeloma). Transfected cell clones were screened for production of human antibody by ELISA for human IgG
  • 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 ⁇ ijPBMC ⁇ perjwelL_KeyholeJimpgt jiaemocyanin (KLH) was included in the assay as a positive control. After 7 days incubation of cells and peptidic fragments, an 18 hour pulse with 3 H-Thymidine at 1 ⁇ Ci/well was used to assess T-cell proliferation.
  • KLH perjwelL_KeyholeJimpgt jiaemocyanin
  • 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 [Edelman, 008.056., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, 115.093., et al., Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242 (1991)].
  • 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.
  • 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.
  • 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.
  • 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 or an anti-CD4 antibody be constructed whereby both anti-CCR5 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. 13 (1997) 933-943), or SEQ ID NO: 088 and 092
  • 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
  • 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:
  • hygromycin resistance gene as a selection marker, - an origin of replication, oriP, of Epstein-Barr virus (EBV),
  • a beta-lactamase gene which confers ampicillin resistance in E. coli, the immediate early enhancer and promoter from the human cytomegalovirus (HCMV), the human ⁇ l -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-YYY-XXXX-XXX-HC and pUC-Exp- YYY-XXXX-XXXX-LC expression vectors.
  • the string ⁇ YY-XXXX-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,
  • H - anti-CD4 antibody chain obtained from Reimann, K., A., et al., Aids Res. Human Retrovir. 13 (1997) 933-943.
  • 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)
  • H - anti-CD4 antibody domain obtained from Reimann, K., A., et al., Aids Res. Human Retrovir. 13 (1997) 933-943, and N - not present.
  • a - anti-CCR5 antibody domain obtained from DSM ACC 2681,
  • 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 first pair of single chain antibodies) to the full length antibody chain with 1 - no single chain antibodies present
  • the second digit denotes the number of GGGGS units (SEQ ID NO: 1 14) 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 second block of 4 digits denotes the structure of the second pair of single chain antibodies whereby
  • the first digit denotes the position of attachment of the peptidic-linker (to the second pair of single chain antibodies) to the full length antibody chain with
  • 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 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
  • Example 5 The final expression plasmids generated in Example 5 allow the recombinant production of different antibody light chains and different antibody heavy chains. Thus, the in Table 4 listed multivalent multispecific antibodies can be produced. Table 5: Overview of multivalent antibodies.
  • 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 .
  • HEK293-EBNA cells human embryonic kidney cell line 293 expressing Epstein-Barr- Virus nuclear antigen; American type culture collection deposit number ATCC # CRL- 10852
  • 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
  • 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 mM KH 2 PO 4 , 137 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 eluted protein fractions were pooled, concentrated with an Amicon 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).
  • MWCO Amicon Ultra centrifugal filter device
  • GE Superdex200 HiLoad 120 ml 16/60 gel filtration column
  • 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 ( Figure 5 b) and 1Ob)).
  • 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
  • HBS-P (10 mM HEPES, pH 7.4, 150 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 qf_4 ⁇ 0joMj ⁇ nd_injected over 3 minutes at a flow rate of 30 ⁇ l/minute.
  • Running and dilution buffer was HBS-P (1O mM HEPES, pH 7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20; GE Healthcare Biosciences AB, Sweden).
  • the CCR5/CD4 bi- and trispecific antibodies were 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 surface densities of approximately 600 RU for trispecific antibodies and 400 RU for bispecific antibodies.
  • Soluble CD4 (Invitrogen, USA) was diluted with HBS-P to obtain concentrations of 0.09 - 200 nM.
  • binding kinetics of soluble CD4 was measured by injecting each concentration over 3 minutes at a flow rate of 30 ⁇ l/minute with a dissociation time of 5 minutes.
  • the sensor chip was regenerated with 10 mM glycine pH 1.7 for 1 min.
  • the data were fitted using BIAevaluation software (GE Healthcare Biosciences AB, Sweden) determining the rate constants for dissociation (K d ) and association (K a ) kinetics based on the 1 : 1 Langmuir binding model.
  • Table 7 Binding affinity of bispecific and trispecific anti-CCR5/CD4 antibodies.
  • 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
  • Table 8 CCR5 cell ELISA.
  • 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.
  • Table 9 RANTES radioligand binding assay.
  • 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.
  • 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 RPMI 1640 containing 10% FCS (Invitrogen/Gibco), 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM 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. 10 A comparison of IC 50 values for different multivalent antibodies is shown in Figure 10. It can be seen that these bispecific antibodies binding to epitopes on different antigens have an improved in vitro biological potency, e.g. the IC 50 value for BNN- 1000-1000 (1) of 1078 pM or HNN- 1000- 1000 (2) of 348 pM is reduced to 10 pM when employed as tetravalent bispecific antibody (BHN-2321-1000) and even further to 6 to 7 pM when employed as trispecific hexavalent antibody (BHA- 2321-4321 and BHA-2321-5320) according to the invention (3 to 5).
  • Example 15 A comparison of IC 50 values for different multivalent antibodies binding to epitopes on different antigens have an improved in vitro biological potency, e.g. the IC 50 value for BNN- 1000-1000 (1) of 1078 pM or HNN- 1000- 1000 (2) of 348 pM is reduced to 10 pM when employed as tetravalent bispecific
  • the CCR5-tropic HIV-I isolates BAL 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.
  • the full length heavy or light chain of the antibody obtained from a T-cell epitope depleted variant of the antibody obtained from DSM ACC 2683 of SEQ ID NO: 039 and 044 and a scFv nucleic acids with the variable domains of an anti-CD4 antibody of SEQ ID NO: 072 and 076 were connected in a bispecific tetravalent anti-CCR5/CD4 antibody according to the invention using recombinant molecular biology techniques.
  • PBMC peripheral blood mononuclear cells
  • the bispecific anti-CCR5/CD4 antibody showed lower IC 5O values as the corresponding monovalent bispecific full length antibodies.
  • the bispecific anti-CCR5/CD4 antibody has an even lower IC 50 value compared to the parent anti-CD4 antibody although the CCl/85_res virus is resistant to the parent anti-CCR5 antibody showing that by the addition of the further binding sites in the tetravalent bispecific antibody variant BHN-2321-1000 the bindings sites for which the virus has developed a resistance act together with the binding sites to which the virus has developed no resistance thereby increasing the binding affinity of the variant antibody.
  • the trispecific hexavalent variant of the parent antibody BNN- 1000- 1000 showed an even bigger increase of the binding affinity than the tetravalent bispecific variant.
  • Table 12 Antiviral assay with resistant HIV-I strain.

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Abstract

Cette invention concerne un anticorps, caractérisé en ce que ledit anticorps comprend six sites de liaison à l'antigène, constitués chacun par une paire de domaines variables d'anticorps, lesdits sites de liaison à l'antigène se liant deux par deux au même épitope.
PCT/EP2009/006498 2008-09-10 2009-09-08 Anticorps hexavalents trispécifiques WO2010028796A1 (fr)

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