The current invention is related to a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) characterized in that one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of the anti-CCR5 antibody and to the pharmaceutical use of the conjugate.

Description of the Invention

SUMMARY OF THE INVENTION

The invention comprises a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) characterized in that one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody (a number of eight antifusogenic peptides per mAb CCR5 is only possible if the mAb CCR5 comprises eight termini, i.e. is composed e.g. of two heavy chains and two light chains; if the mAb CCR5 comprises a smaller number of C- and N-termini, e.g. as a scFv, the corresponding number of antifusogenic peptides possible at maximum in the conjugate is also reduced, i.e. it is reduced to less than eight).

Preferably the carboxy-terminal amino acid of an anti-CCR5 antibody chain is conjugated to the amino-terminal amino acid of the antifusogenic peptide or the carboxy-terminal amino acid of the antifusogenic peptide is conjugated to the amino-terminal amino acid of the antibody chain, preferably by a peptide bond with or without an intermediate linker.

Preferably the conjugate is characterized by the general formula mAb CCR5-[linker].sub.m-[antifusogenic peptide].sub.n wherein m is independently for each antifusogenic peptide either 0 (i.e. a peptide bond between mAb CCR5 and antifusogenic peptide) or 1 (i.e. a linker between mAb CCR5 and antifusogenic peptide) and n is an integer of from 1 to 8.

A preferred conjugate of a heavy and/or light chain of mAb CCR5 and an antifusogenic peptide ("chain conjugate") is selected from the group consisting of: (1) [antifusogenic peptide]-[linker].sub.m-[heavy chain] (2) [heavy chain]-[linker].sub.m-[antifusogenic peptide] (3) [antifusogenic peptide]-[linker].sub.m-[heavy chain]-[antifusogenic peptide] (4) [antifusogenic peptide]-[linker].sub.m-[light chain] (5) [light chain]-[linker].sub.m-[antifusogenic peptide] (6) [antifusogenic peptide]-[linker].sub.m-[light chain]-[antifusogenic peptide] (7) [antifusogenic peptide]-[linker].sub.m-[heavy chain]-[linker].sub.m-[antifusogenic peptide] (8) [antifusogenic peptide]-[linker].sub.m-[light chain]-[linker].sub.m-[antifusogenic peptide] wherein the linker can be the same or different in (within and between) said chain conjugates, wherein m is an integer of 1 or 0, and m can be independently the same or different in (within and between) said chain conjugates.

("Left side" of the peptide or mAb CCR5 chain means N-terminus, "right side" means C-terminus. In (1) therefore the C-terminus of the antifusogenic peptide is linked by a peptide bond or a linker to the N-terminus of the heavy chain of mAb CCR5).

Preferably the chain conjugates are assembled to conjugates according to the invention comprising a mAb CCR5 (e.g. consisting of two light chains and two heavy chains including the constant Fc domains, a scFv fragment, or a Fab fragment).

Especially preferred chain conjugates are (2), (3), (4), and (7). Especially preferred conjugates according to the invention comprise 2.times.[mAb CCR5 light chain] and 2.times.(2), 2.times.[mAb CCR5 light chain] and 2.times.(3), or 2.times.[mAb CCR5 heavy chain] and 2.times.(4), or 2.times.[mAb CCR5 light chain] and 2.times.(7). The heavy and/or light chain comprises preferably a constant region (Fc).

Preferably the conjugate is characterized in comprising a variable heavy chain domain consisting of an immunoglobulin framework and a CDR3 region selected from the group consisting of the heavy chain CDR3 sequences SEQ ID NO: 16, 17.

Preferably the conjugate is characterized in comprising a variable heavy chain domain consisting of an immunoglobulin framework and a CDR3 region selected from the group consisting of CDR3 sequences SEQ ID NO: 16, 17, a CDR2 region selected from the group consisting of CDR2 sequences SEQ ID NO: 13, 14, 15, and a CDR1 region selected from the group consisting of CDR1 sequences SEQ ID NO: 9, 10, 11, 12.

Preferably the conjugate is characterized in comprising a heavy chain variable domain selected from the group of heavy chain variable domains comprising SEQ ID NO: 1, 3, 5, and 7.

Preferably the conjugate is characterized in comprising a variable light chain domain consisting of an immunoglobulin framework and a CDR1 region selected from SEQ ID NO:18, 19, 20, a CDR2 region selected from SEQ ID NO:21, 22, 23, and a CDR3 region selected from SEQ ID NO:24, 25.

Preferably the conjugate is characterized in comprising as heavy chain CDRs the CDRs of SEQ ID NO:1 and as light chain CDRs the CDRs of SEQ ID NO:2, as heavy chain CDRs the CDRs of SEQ ID NO:3 and as light chain CDRs the CDRs of SEQ ID NO:4, as heavy chain CDRs the CDRs of SEQ ID NO:5 and as light chain CDRs the CDRs of SEQ ID NO:6, or as heavy chain CDRs the CDRs of SEQ ID NO:7 and as light chain CDRs the CDRs of SEQ ID NO:8.

Preferably the conjugate is characterized in comprising a variable heavy and light chain domain independently selected from the group consisting of a) the heavy chain (V.sub.H) variable domain defined by amino acid sequence SEQ ID NO: 1 and the light chain (V.sub.L) variable domain defined by amino acid sequence SEQ ID NO:2; b) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:3 and the light chain variable domain defined by amino acid sequence SEQ ID NO:4; c) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:5 and the light chain variable domain defined by amino acid sequence SEQ ID NO:6; d) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:7 and the light chain variable domain defined by amino acid sequence SEQ ID NO:8.

Preferably the conjugate is characterized in comprising the heavy chain (V.sub.H) variable domain defined by amino acid sequence SEQ ID NO:1 and the light chain (V.sub.L) variable domain defined by amino acid sequence SEQ ID NO:2; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:3 and the light chain variable domain defined by amino acid sequence SEQ ID NO:4; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:5 and the light chain variable domain defined by amino acid sequence SEQ ID NO:6; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:7 and the light chain variable domain defined by amino acid sequence SEQ ID NO:8; a linker selected from the group consisting of the amino acids glycine (G) and asparagine (N), the tripeptide GST, and SEQ ID NO:36-62; and an antifusogenic peptide selected from the group of peptides defined by SEQ ID NO:29 to 35.

Preferably the conjugate is characterized in comprising an antifusogenic peptide selected from the group of peptides comprising C34, T20, T1249, T651, T2635, N36, and DP107.

Preferably the conjugate is characterized in comprising an antifusogenic peptide at each C-terminus of the heavy chains or at each N-terminus of the light chains (two antifusogenic peptides). Preferably the conjugate is characterized in that it comprises an antifusogenic peptide at each C-terminus of the heavy chains and at each N-terminus of the light chains (four antifusogenic peptides).

Preferably the conjugate is characterized in comprising two light chain variable domains of SEQ ID NO:2, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:1, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, in comprising two light chain variable domains of SEQ ID NO:4, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:3, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, in comprising two light chain variable domains of SEQ ID NO:6, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:5, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, or in comprising two light chain variable domains of SEQ ID NO:8, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33.

Preferably the conjugate is characterized in that said anti-CCR5 antibody is of IgG1 subclass. It is also preferred, that said anti-CCR5 antibody is of IgG4 subclass, or of IgG1 or IgG2 subclass, with a mutation in amino acid position S228, L234, L235, and/or D265, and/or contains the PVA236 mutation. Preferably the conjugate is characterized in that said anti-CCR5 antibody of IgG4 subclass has a S228P mutation and said anti-CCR5 antibody of IgG1 subclass has L234A and L235A mutations.

The invention comprises a method for the production of a conjugate according to the invention, characterized in that the method comprises a) cultivating a cell containing one or more plasmids containing one or more nucleic acid molecules encoding a conjugate according to the invention under conditions suitable for the expression of the conjugate, b) recovering the conjugate from the cell or the supernatant.

In one embodiment are the genes encoding the light and heavy chains of mAb CCR5 with or without linked antifusogenic peptide located on the same expression vector or on different expression vectors.

The invention comprises a pharmaceutical composition, containing a conjugate according to the invention, together with a pharmaceutically acceptable excipient or carrier.

The invention comprises the use of a conjugate according to the invention for the manufacture of a medicament for the treatment of viral infections. Preferably the use is characterized in that the viral infection is a HIV infection.

The invention comprises the use of a conjugate according to the invention for the treatment of a patient in need of an antiviral treatment, preferably an anti HIV treatment.

DESCRIPTION OF THE INVENTION

The current invention reports a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) characterized in that one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody. A number of eight antifusogenic peptides per mAb CCR5 is only possible if the mAb CCR5 comprises eight termini, i.e. is composed e.g. of two heavy chains and two light chains. If the mAb CCR5 comprises a smaller number of C- and N-termini, e.g. as a scFv, the corresponding number of antifusogenic peptides possible at maximum in the conjugate is also reduced, i.e. it is reduced to less than eight.

An "antifusogenic peptide" is a peptide which inhibits events associated with membrane fusion or the membrane fusion event itself, including, among other things, the inhibition of infection of uninfected cells by a virus due to membrane fusion. These antifusogenic peptides are preferably linear peptides. For example, they can be derived from the gp41 ectodomain, e.g. such as DP107, DP178. Examples of such peptides can be found in U.S. Pat. No. 5,464,933, U.S. Pat. No. 5,656,480, U.S. Pat. No. 6,013,263, U.S. Pat. No. 6,017,536, U.S. Pat. No. 6,020,459, U.S. Pat. No. 6,093,794, U.S. Pat. No. 6,060,065, U.S. Pat. No. 6,258,782, U.S. Pat. No. 6,348,568, U.S. Pat. No. 6,479,055, U.S. Pat. No. 6,656,906, WO 1996/19495, WO 1996/40191, WO 1999/59615, WO 2000/69902, and WO 2005/067960. For example, the amino acid sequences of such peptides comprise or can be selected from the group of SEQ ID NO: 1 to 10 of U.S. Pat. No. 5,464,933; SEQ ID NO:1 to 15 of U.S. Pat. No. 5,656,480; SEQ ID NO: 1 to 10 and 16 to 83 of U.S. Pat. No. 6,013,263; SEQ ID NO: 1 to 10, 20 to 83 and 139 to 149 of U.S. Pat. No. 6,017,536; SEQ ID NO:1 to 10, 17 to 83 and 210 to 214 of U.S. Pat. No. 6,093,794; SEQ ID NO:1 to 10, 16 to 83 and 210 to 211 of U.S. Pat. No. 6,060,065; SEQ ID NO:1286 and 1310 of U.S. Pat. No. 6,258,782; SEQ ID NO:1129, 1278-1309, 1311 and 1433 of U.S. Pat. No. 6,348,568; SEQ ID NO:1 to 10 and 210 to 238 of U.S. Pat. No. 6,479,055; SEQ ID NO:1 to 171, 173 to 216, 218 to 219, 222 to 228, 231, 233 to 366, 372 to 398, 400 to 456, 458 to 498, 500 to 570, 572 to 620, 622 to 651, 653 to 736, 739 to 785, 787 to 811, 813 to 823, 825, 827 to 863, 865 to 875, 877 to 883, 885, 887 to 890, 892 to 981, 986 to 999, 1001 to 1003, 1006 to 1018, 1022 to 1024, 1026 to 1028, 1030 to 1032, 1037 to 1076, 1078 to 1079, 1082 to 1117, 1120 to 1176, 1179 to 1213, 1218 to 1223, 1227 to 1237, 1244 to 1245, 1256 to 1268, 1271 to 1275, 1277, 1345 to 1348, 1350 to 1362, 1364, 1366, 1368, 1370, 1372, 1374 to 1376, 1378 to 1379, 1381 to 1385, 1412 to 1417, 1421 to 1426, 1428 to 1430, 1432, 1439 to 1542, 1670 to 1682, 1684 to 1709, 1712 to 1719, 1721 to 1753, 1755 to 1757 of U.S. Pat. No. 6,656,906; or SEQ ID NO:5 to 95 of WO 2005/067960. The antifusogenic peptide has an amino acid sequence comprising of from 5 to 100 amino acids, preferably of from 10 to 75 amino acids and more preferred of from 15 to 50 amino acids. Especially preferred antifusogenic peptides are C-34, T-20, T-1249, T-651, T-2635, N-36, (M. J. Root et al., Curr. Pharm. Des. (2004) 10:1805-25) and DP-107 (C. Wild et al., Proc. Natl. Acad. Sci. USA (1994) 91:12676-80). One embodiment comprises the conjugate according to the invention one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein i) said antifusogenic peptides are linear peptides with an amino acid sequence of from 5 to 100 amino acids, and ii) one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody. Another embodiment comprises the conjugate according to the invention having one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein i) said antifusogenic peptides are derived from the gp41 ectodomain, and ii) one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody. The term "gp41 ectodomain" denotes the amino acid sequence starting with amino acid position 561 and ending with amino acid position 620 of HIV-1 gp 160 or starting with amino acid position 50 and ending with amino acid position 109 of HIV-1 gp41 (SEQ ID NO:66) (see also e.g. S. Bar and M. J. Alizon, Virol. (2004) 78:811-20).

The term "antibody" encompasses the various forms of antibody structures including whole antibodies and antibody fragments. The antibody according to the invention is preferably a human antibody, a humanized antibody, a chimeric antibody, a T cell antigen depleted antibody (WO 98/33523, WO 98/52976, and WO 00/34317). Genetic engineering of antibodies is e.g. described in S. L. Morrison et al., Proc. Natl. Acad. Sci. USA (1984) 81:6851-55; U.S. Pat. Nos. 5,202,238 and 5,204,244; L. Riechmann et al., Nature (1988) 332:323-27; M. S. Neuberger et al., Nature (1985) 314:268-70; N. Lonberg, Nat. Biotechnol. (2005) 23:1117-25.

"Antibody fragments" comprise a portion of a full length anti-CCR5 antibody, preferably the variable domains thereof or at least the antigen binding portion thereof. Examples of anti-body fragments are e.g. single-chain antibody molecules (scFv), Fab, F(ab).sub.2 fragments, and the like as long as they retain the characteristics of an anti-CCR5 antibody. ScFv antibodies are, e.g., described in J. S. Huston, Meth. Enzymol. (1991) 203:46-88. Huston also describes linkers and methods for linking of polypeptides useful for the present invention.

"CCR5" means human CCR5 as described, e.g., in M. Oppermann, Cell Signal. (2004) 16:1201-10 and SwissProt P51681. The terms "antibody binding to CCR5", "anti-CCR5 antibody", or "mAb CCR5", which are used interchangeably within this application, mean an antibody specifically binding to CCR5 and preferably inhibiting HIV fusion with a target cell. Binding can be tested in a cell based in vitro ELISA assay (CCR5 expressing CHO cells). Binding is found if the antibody causes an S/N (signal/noise) ratio of 5 or more, preferably 10 or more at an antibody concentration of 100 ng/ml. The term "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 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. The term "membrane fusion" refers to fusion between a first cell coexpressing CCR5 and 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 (e.g. by luciferase reporter gene assay).

Preferred anti-CCR5 antibodies are mentioned in US 2004-0043033, U.S. Pat. No. 6,610,834, US 2003-0228306, US 2003-0195348, US 2003-0166870, US 2003-0166024, US 2003-0165988, US 2003-0152913, US 2003-0100058, US 2003-0099645, US 2003-0049251, US 2003-0044411, US 2003-0003440, U.S. Pat. No. 6,528,625, US 2002-0147147, US 2002-0146415, US 2002-0106374, US 2002-0061834, US 2002-0048786, US 2001/0000241, EP 1 322 332, EP 1 263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, WO 2003/072766, WO 2003/066830, WO 2003/033666, WO 2002/083172, WO 02/22077, WO 01/58916, WO 01/58915, WO 01/43779, WO 01/42308, and WO 2006/103100. Especially preferred anti-CCR5 antibodies are described in WO 2006/103100. An especially preferred anti-CCR5 antibody is characterized in that the antibody comprises a variable heavy chain domain consisting of an immunoglobulin framework and a CDR3 region selected from the group consisting of the heavy chain CDR3 sequences SEQ ID NO:16, 17. A further preferred antibody comprises a variable heavy chain region consisting of an immunoglobulin framework and a CDR3 region selected from the group consisting of CDR3 sequences SEQ ID NO: 16, 17, a CDR2 region selected from the group consisting of CDR2 sequences SEQ ID NO:13, 14, 15, and a CDR1 region selected from the group consisting of CDR1 sequences SEQ ID NO:9, 10, 11, 12. Preferred heavy chain variable domains are shown in SEQ ID NO:1, 3, 5, 7. A preferred anti-CCR5 antibody comprises in addition a variable light chain domain consisting of an immunoglobulin framework and a CDR1 region selected from the group consisting of CDR1 sequences SEQ ID NO:18, 19, 20, a CDR2 region selected from the group consisting of CDR2 sequences SEQ ID NO:21, 22, 23, and a CDR3 region selected from the group of CDR3 sequences SEQ ID NO:24, 25. The anti-CCR5 antibody is preferably characterized in containing as heavy chain CDRs the CDRs of SEQ ID NO: 1 and as light chain CDRs the CDRs of SEQ ID NO:2, as heavy chain CDRs the CDRs of SEQ ID NO:3 and as light chain CDRs the CDRs of SEQ ID NO:4, as heavy chain CDRs the CDRs of SEQ ID NO:5 and as light chain CDRs the CDRs of SEQ ID NO:6, or as heavy chain CDRs the CDRs of SEQ ID NO:7 and as light chain CDRs the CDRs of SEQ ID NO:8.

CDR sequences can be determined according to the standard definition of E. A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). CDRs of SEQ ID NO: 1-8 are shown in SEQ ID NO:9-25.

The anti-CCR5 antibody comprises preferably a variable heavy and light chain domain independently selected from the group consisting of a) the heavy chain (V.sub.H) variable domain defined by amino acid sequence SEQ ID NO: 1 and the light chain (V.sub.L) variable domain defined by SEQ ID NO:2; b) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:3 and the light chain variable domain defined by SEQ ID NO:4; c) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:5 and the light chain variable domain defined by SEQ ID NO:6; d) the heavy chain variable domain defined by amino acid sequence SEQ ID NO:7 and the light chain variable domain defined by SEQ ID NO:8.

The antibody used in the conjugate according to the invention is preferably characterized in that the constant domains are of human origin. Such constant domains are well known in the state of the art and, e.g., described by Kabat (see e.g. G. Johnson and T. T. Wu, Nucleic Acids Res. (2000) 28:214-18). For example, a useful human IgG1 heavy chain constant region (C.sub.H1-Hinge-C.sub.H2-C.sub.H3) comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO:26, 27. For example, a useful human kappa (.kappa.) light chain constant domain comprises an amino acid sequence of a kappa light chain constant domain (.kappa. light chain constant domain, C.sub.L) of SEQ ID NO:28. It is further preferred that the antibody's variable domains are of mouse origin and comprises the antibody variable domain sequence frame of a mouse antibody according to Kabat (see e.g. G. Johnson and T. T. Wu, supra).

A preferred anti-CCR5 antibody shows a binding to the same epitope(s) of CCR5 as does an antibody selected from the group consisting of the antibodies A to E or is inhibited in binding to CCR5 by antibodies A to E due to steric hindrance of binding or competitive binding. Epitope binding is investigated by using alanine scanning according to the method described by W. C. Olson et al. (J. Virol. (1999) 73:4145-55) for epitope mapping. A signal reduction of 75% or more shows that the mutated amino acid(s) contribute to the epitope recognized by said antibody. Binding of the antibody to the same epitope is found, if the amino acids contributing to the epitope are recognized by the investigated antibody and antibody A, B, C, D, or E. Antibody C, which shows lower IC.sub.50 values than antibody 2D7 in HIV assays, binds to an epitope including amino acids on the ECL2 domain of CCR5 (B. Lee et al., J. Biol. Chem. (1999) 274:9617-26) which is different from the epitope recognized by antibody 2D7 (2D7 binds to amino acids K171 and E172 of ECL2A but not to ECL2B amino acids 184-189). Epitope binding for antibody C is found to be 20% for CCR5 mutant K171A or E172A (glu 172 is mutated to ala). 100% epitope binding is defined for wild-type CCR5. A further preferred anti-CCR5 antibody binds to the same epitope as antibody C binds.

The term "epitope" means a protein determinant capable of specific binding to 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. Preferably an antibody according to the invention binds specifically to native but not to denatured CCR5. Such an antibody comprises preferably heavy chain CDR3 of SEQ ID NO: 17, and preferably in addition heavy chain CDRs selected from the group of CDRs of SEQ ID NO:10, 11, 12, 14 and/or 15. Preferably such an antibody is antibody B, C, D, or E, or comprises the variable domains of antibody B, C, D, or E. Preferably an antibody binding to denatured CCR5 is antibody A or comprises the variable domains of antibody A.

The term "variable domain" (variable domain of a light chain (V.sub.L), variable domain of a heavy chain (V.sub.H)) as used herein denotes each domain of the pair of light and heavy chain domains which is involved directly in the binding of the antibody to the antigen. The variable domains of the light and heavy chain have the same general structure, i.e. they possess an "immunoglobulin framework", and each domain comprises four "framework regions" (FR), whose sequences are widely conserved, connected by three "hypervariable regions" (or "complementarity determining regions", CDRs). The framework regions adopt a .beta.-sheet conformation and the CDRs may form loops connecting the .beta.-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.

The terms "antigen-binding portion of an antibody" or "antigen-binding site of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding site of an antibody comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the regions FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (immunoglobulin framework). Especially, the CDR3 region of the heavy chain is the region which contributes most to antigen binding and defines the antibody. Preferably the antibody according to the invention is characterized by comprising in its heavy chain variable domain the CDR3 sequence of SEQ ID NO:16 or SEQ ID NO:17. Complementarity determining (CDR) and framework (FR) regions are determined according to the standard definition of E. A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

The "Fc part" of an anti-CCR5 antibody is not involved directly in binding to CCR5, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. According to the heavy chain constant regions the different classes of immunoglobulins are called .alpha., .delta., .epsilon., .gamma., and .mu., respectively. The antibodies according to the invention are preferably of IgG type. An "Fc part of an antibody" is a term well known to the skilled artisan and defined on basis of papain cleavage of antibodies. The antibodies according to the invention contain as Fc part a human Fc part or an Fc part derived from human origin. In a further embodiment of the invention the Fc part is either an Fc part of a human antibody of the subclass IgG4 or an Fc part of a human antibody of the subclass IgG1, IgG2, or IgG3, which is modified in such a way that no Fc.gamma. receptor (e.g. Fc.gamma.RIIIa) binding and/or no C1q binding as defined below can be detected. Preferably the Fc part is a human Fc part and especially preferred either from human IgG4 subclass or a mutated Fc part from human IgG1 subclass. Further preferred are Fc parts from human IgG1 subclass with mutations L234A and L235A. Further preferred are Fc parts shown in SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 26 with mutations L234A and L235A, SEQ ID NO: 27 with mutation S228P. While IgG4 shows reduced Fc.gamma. receptor (Fc.gamma.RIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which if altered provide also reduced Fc.gamma. receptor binding (R. L. Shields et al., J. Biol. Chem. (2001) 276:6591-604; J. Lund et al., FASEB J. (1995) 9:115-19; A. Morgan et al., Immunol. (1995) 86:319-24; EP 0 307 434). Preferably an antibody according to the invention is in regard to Fc.gamma. receptor binding of IgG4 subclass or of IgG1 or IgG2 subclass, with a mutation in L234, L235, and/or D265, and/or contains the PVA236 mutation. Preferred are the mutations S228P, L234A, L235A, L235E, and/or PVA236 (PVA236 means that the amino acid sequence ELLG (given in one letter amino acid code) from amino acid position 233 to 236 of IgG1 or EFLG of IgG4 is replaced by PVA). Especially preferred are the mutations S228P of IgG4, and L234A and L235A of IgG1. The Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by binding of complement factor C1q to the Fc part of most IgG antibody subclasses. Binding of C1q to an antibody is caused by defined protein-protein interactions at the so called binding site. Such Fc part binding sites are known in the state of the art and described e.g. by T. J. Lukas et al., J. Immunol. (1981) 127:2555-60; R. Brunhouse and J. J. Cebra, Mol. Immunol. (1979) 16: 907-17; D. R. Burton et al., Nature (1980) 288:338-44; J. E. Thommesen et al., Mol. Immunol. (2000) 37:995-1004; E. E. Idusogie et al., J. Immunol. (2000) 164:4178-84; M. Hezareh et al., J. Virol. (2001) 75:12161-68; A. Morgan et al., Immunol. (1995) 86:319-24; and EP 0 307 434. 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 IgG1, IgG2, and IgG3 usually show complement activation including C1q and C3 binding, whereas IgG4 does not activate the complement system and does not bind C1q and C3. An anti-CCR5 antibody which does not bind Fc.gamma. receptor and/or complement factor C1q does not elicit antibody-dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). Preferably, this antibody is characterized in that it binds CCR5, contains an Fc part derived from human origin, and does not bind Fc.gamma. receptors and/or complement factor C1q. More preferably, this antibody is a human, or humanized, or a T-cell antigen depleted antibody. C1q binding can be measured according to Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184. No "C1q binding" is found if in such an assay the optical density (OD) at 492-405 nm is for the test antibody lower than 15% of the value for human C1q binding of the unmodified wild-type antibody Fc part at an antibody concentration of 8 .mu.g/ml. ADCC can be measured as binding of the antibody to human Fc.gamma.RIIIa on human NK cells. Binding is determined at an antibody concentration of 20 .mu.g/ml. "No Fc.gamma. receptor binding" or "no ADCC" means a binding of up to 30% to human Fc.gamma.RIIIa on human NK cells at an antibody concentration of 20 .mu.g/ml compared to the binding of the same antibody as human IgG1 (SEQ ID NO:26).

An antibody used in a conjugate according to the invention include, in addition, such antibodies having "conservative sequence modifications" (variant antibodies), which are amino acid sequence modifications which do not affect or alter the above-mentioned characteristics of the antibody according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-CCR5 antibody can be preferably replaced with another amino acid residue from the same side chain family. A "variant" anti-CCR5 antibody, refers therefore herein to a molecule which differs in amino acid sequence from a "parent" anti-CCR5 antibody's amino acid sequence by up to ten, preferably from about two to about five, additions, deletions, and/or substitutions in one or more of the variable domain regions of the parent antibody outside the heavy chain CDR3 region. Each other heavy chain CDR region comprises at maximum one single amino acid addition, deletion, and/or substitution. The invention comprises a method of modifying the CDR amino acid sequence of a parent antibody binding to CCR5, characterized in selecting a heavy chain variable domain from the group of heavy chain variable domains consisting of SEQ ID NO: 1, 3, 5, 7, and/or a light chain variable domain from the group of light chain variable domains consisting of SEQ ID NO:2, 4, 6, 8, providing a nucleic acid encoding said initial variable domain amino acid sequence, modifying said nucleic acid in that one amino acid is modified in heavy chain CDR1, one amino acid is modified in heavy chain CDR2, 1-3 amino acid are modified in light chain CDR1, 1-3 amino acids are modified in light chain CDR2, and/or 1-3 amino acids are modified in light chain CDR3, expressing and incorporating said modified variable domain(s) amino acid sequence in an antibody structure, measuring whether said antibody binds to CCR5 and selecting said modified variable domain(s)/CDR(s) if the antibody binds to CCR5. Preferably such modifications are conservative sequence modifications. Amino acid sequence modifications can be performed by mutagenesis based on molecular modeling as described by L. Riechmann et al., Nature (1988) 332:323-27, and C. Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86:10029-33.

The term "linker" or "peptidic linker" as used within this application denotes peptide linkers of natural and/or synthetic origin. They are building up of a linear amino acid chain wherein the 20 naturally occurring amino acids are the monomeric building blocks. The chain has a length of from 1 to 50 amino acids, preferred between 1 and 28 amino acids, especially preferred between 3 and 25 amino acids. The linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides, such as polypeptides with a hinge-function. The linker has the function to ensure that a peptide conjugated to an anti-CCR5 antibody can perform its biological activity by allowing the peptide to fold correctly and to be presented properly. Preferably the linker is a "synthetic peptidic linker" that is designated to be rich in glycine, glutamine, and/or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids. This small repetitive unit may be repeated for two to five times to form a multimeric unit. At the amino- and/or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added. Other synthetic peptidic linkers are composed of a single amino acid, that is repeated between 10 to 20 times. At each of the amino- and/or carboxy-terminal end up to six additional arbitrary, naturally occurring amino acids may be present. Preferred linkers are shown in Table 2 (see Original Patent). Especially preferred are linkers [GQ.sub.4].sub.3GNN (SEQ ID NO:40), LSLSPGK (SEQ ID NO:36), LSPNRGEC (SEQ ID NO:37), LSLSGG (SEQ ID NO:61), LSLSPGG (SEQ ID NO:62). All peptidic linkers can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the linkers are themselves peptides, the antifusogenic peptide is connected to the linker via a peptide bond that is formed between two amino acids. The peptidic linker is introduced between the antifusogenic peptide and the anti-CCR5 antibody chain to which the antifusogenic peptide is to be conjugated. Therefore two or three, respectively, possible sequences (in amino- to carboxy-terminal direction) exist: a) antifusogenic peptide-peptidic linker-anti-CCR5 antibody polypeptide chain, or b) anti-CCR5 antibody polypeptide chain-peptidic linker-antifusogenic peptide, or c) antifusogenic peptide-peptidic linker-anti-CCR5 antibody polypeptide chain-peptidic linker-antifusogenic peptide.

In one embodiment of the invention the conjugate is characterized in comprising i) the heavy chain (V.sub.H) variable domain defined by amino acid sequence SEQ ID NO:1 and the light chain (V.sub.L) variable domain defined by SEQ ID NO:2; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:3 and the light chain variable domain defined by SEQ ID NO:4; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:5 and the light chain variable domain defined by SEQ ID NO:6; or the heavy chain variable domain defined by amino acid sequence SEQ ID NO:7 and the light chain variable domain defined by SEQ ID NO:8; ii) a linker selected from the group consisting of the amino acids glycine (G) and asparagine (N), the tripeptide GST, and SEQ ID NO:36-62; and iii) an antifusogenic peptide selected from the group of peptides defined by SEQ ID NO:29 to 35.

A preferred conjugate of a heavy and/or light chain of mAb CCR5 and an antifusogenic peptide(s) ("chain conjugate") is selected from the group consisting of the conjugates (1) [antifusogenic peptide]-[linker].sub.m-[heavy chain], (2) [heavy chain]-[linker].sub.m-[antifusogenic peptide], (3) [antifusogenic peptide]-[linker].sub.m-[heavy chain]-[antifusogenic peptide], (4) [antifusogenic peptide]-[linker].sub.m-[light chain], (5) [light chain]-[linker].sub.m-[antifusogenic peptide], (6) [antifusogenic peptide]-[linker].sub.m-[light chain]-[antifusogenic peptide], (7) [antifusogenic peptide]-[linker].sub.m-[heavy chain]-[linker].sub.m-[antifusogenic peptide], (8) [antifusogenic peptide]-[linker].sub.m-[light chain]-[linker].sub.m-[antifusogenic peptide], wherein the linker can be the same or different both within and between said chain conjugates, wherein m is an integer of 1 or 0, and m can be independently the same or different both within and between said conjugates. For example in a conjugate comprising a chain conjugate (7) and a mAb CCR5 light chain the two linkers in chain conjugate (7) can be the same, i.e. have the same amino acid sequence and length, or can be different, i.e. have different amino acid sequences and/or lengths, or one or both can be absent. For example in a conjugate comprising chain conjugates (2) and (4) the linker contained in chain conjugate (2) and the linker contained in chain conjugate (4) can be the same, i.e. have the same amino acid sequence and length, or can be different, i.e. have different amino acid sequences and/or lengths, or one or both can be absent. In the chain conjugates the linker(s) can be present (m=1) or absent (m=0). Preferred chain conjugates are the chain conjugates (2), (3), (4), and (7). One embodiment of the current invention is a conjugate comprising 2.times.[mAb CCR5 light chain] and 2.times. chain conjugate (2). This conjugate comprises two not conjugate anti-CCR5 antibody light chains and two anti-CCR5 antibody heavy chains conjugated via the C-terminus to the N-terminus of an antifusogenic peptide, optionally with an intermediate linker. Another embodiment of the current invention is a conjugate comprising two mAb CCR5 light chains and two chain conjugates (3). Still another embodiment is a conjugate comprising two mAb CCR5 heavy chains and two chain conjugates (4). A further embodiment of the current invention is a conjugate comprising two mAb CCR5 light chains and two chain conjugates (7). The heavy and/or light chain comprises preferably a constant region (Fc).

The invention further provides a method for the manufacture of a pharmaceutical composition comprising an effective amount of a conjugate according to the invention together with a pharmaceutically acceptable carrier and the use of the conjugate according to the invention for such a method.

The invention further provides the use of a conjugate according to the invention in an effective amount for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from AIDS.

The term "amino acid" as used within this application denotes the group of naturally occurring carboxy .alpha.-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

Methods and techniques known to a person skilled in the art, which are useful for carrying out the current invention, are described e.g. in F. M. Ausubel, ed., Current Protocols in Molecular Biology, Volumes I to III (1997), Wiley and Sons; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

In the conjugates according to the invention the carboxy-terminal amino acid of an anti-CCR5 antibody chain is conjugated via a peptide bond to the amino-terminal amino acid of the antifusogenic peptide or the carboxy-terminal amino acid of the antifusogenic peptide is conjugated via a peptide bond to the amino-terminal amino acid of an anti-CCR5 antibody chain. In one embodiment an intermediate linker is present between the antifusogenic peptide and the anti-CCR5 antibody chain. Thus, the conjugate according to the invention is characterized by the general formula mAb CCR5-[linker].sub.m-[antifusogenic peptide].sub.n wherein m is independently for each antifusogenic peptide either 0 (i.e. a direct peptide bond between mAb CCR5 and the antifusogenic peptide) or 1 (i.e. a linker is present between mAb CCR5 and antifusogenic peptide) and n is an integer of from 1 to 8. In one embodiment n is an integer of from 2 to 8. In another embodiment n is an integer of from 2 to 4. In another embodiment n is an integer of 2 or 4. One embodiment of the invention comprises a conjugate characterized in comprising an antifusogenic peptide at each C-terminus of the heavy chains or at each N-terminus of the light chains of the anti-CCR5 antibody. In this embodiment two antifusogenic peptides are conjugated to one anti-CCR5 antibody. In an other embodiment is the conjugate characterized by comprising an antifusogenic peptide at each C-terminus of the heavy chains and at each N-terminus of the light chains. In this embodiment four antifusogenic peptides are conjugated to one anti-CCR5 antibody.

The antifusogenic peptide which is introduced at a terminus of a mAb CCR5 heavy and/or light chain(s) is small of size compared to the mAb CCR5. For example, the smallest immunoglobulins, immunoglobulins of class G, have a molecular weight of approximately 150 kDa; an antifusogenic peptide has preferably a size (molecular weight) of less than 12.5 kDa, which is equivalent to about 100 amino acids, in general less than 7.5 kDa, which is equivalent to about 60 amino acids. The antifusogenic peptide has an amino acid sequence of from 5 to 100 amino acid residues, preferably of from 10 to 75 amino acid residues, more preferably of from 15 to 50 amino acid residues. The conjugates of the current invention are useful for pharmaceutical, therapeutical, or diagnostical applications. The number of antifusogenic peptides, which can be conjugated to mAb CCR5 heavy and/or light chain(s), is from one to the combined number of amino- and carboxy-termini of the anti-CCR5 antibody polypeptide chains. As the current invention encompasses different anti-CCR5 antibodies the number of antifusogenic peptides can vary. In case of an anti-CCR5 antibody comprising two heavy and two light chains the combined number of amino-termini (N-termini) and carboxy-termini (C-termini) is eight, which is at the same time the totaling maximum number of conjugated antifusogenic peptides; in case e.g. of an anti-CCR5 antibody fragment such as a single chain antibody (scFv) the combined number of termini and therefore the maximum number of conjugatable antifusogenic peptides is two. If a single antifusogenic peptide is conjugated to mAb CCR5, the peptide can occupy any one of the termini of the anti-CCR5 antibody chains. Likewise, if the maximum possible number of peptides is conjugated to mAb CCR5, all termini are occupied by a single peptide. If the number of peptides, which are conjugated to mAb CCR5, is smaller than the maximum possible number, different distributions of the peptides at the termini of the anti-CCR5 antibody chains are possible. For example, if four peptides are conjugated to an immunoglobulin of the G or E class, five different combinations are possible (see Table 1 (see Original Patent)). In two combinations all termini of one kind, i.e. all four amino-termini or all four carboxy-termini of the anti-CCR5 antibody chains, are each conjugated to one single antifusogenic peptide. The other termini are not conjugated. This results in one embodiment in an allocation of the modifications/conjugations in one area of the anti-CCR5 antibody. In the other cases the polypeptides are conjugated to a number of both termini. Within these combinations the conjugated peptides are allocated to different areas of the anti-CCR5 antibody. In either case the sum of conjugated termini is four.

The current invention preferably comprises conjugates in which at least two of the termini are conjugated to an antifusogenic peptide. The amino acid sequences of the antifusogenic peptides can be different, similar or identical. In one embodiment the amino acid sequence identity is in the range of from 90% to less than 100%; these amino acid sequences and the corresponding peptides are defined as similar. In a preferred embodiment the antifusogenic peptides are identical, i.e. have an amino acid identity of 100%.

The present invention comprises a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) wherein one to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody via a peptide bond. In one embodiment the conjugate according to the invention comprises at least two antifusogenic peptides and an anti-CCR5 antibody wherein two to eight antifusogenic peptides are each conjugated to one terminus of the heavy and/or light chains of said anti-CCR5 antibody.

In one embodiment the conjugate according to the invention is characterized i) in comprising two light chain variable domains of SEQ ID NO:2, two chain conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO: 1, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, ii) in comprising two light chain variable domains of SEQ ID NO:4, two chain conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:3, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, iii) in comprising two light chain variable domains of SEQ ID NO:6, two chain conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:5, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33, or iv) in comprising two light chain variable domains of SEQ ID NO:8, two chain conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO:7, a linker of SEQ ID NO:40 and an antifusogenic peptide of SEQ ID NO:33.

The conjugation between the antifusogenic peptide and the anti-CCR5 antibody is performed on the nucleic acid level. Therefore a peptide bond is formed between the antifusogenic peptide and the anti-CCR5 antibody chain with or without an intermediate linker. Thus either the carboxy-terminal amino acid of the antifusogenic peptide is conjugated to the amino-terminal amino acid of an anti-CCR5 antibody chain with or without an intermediate linker, or a carboxy-terminal amino acid of the anti-CCR5 antibody chain is conjugated to the amino-terminal amino acid of the antifusogenic peptide with or without an intermediate linker or both termini of the anti-CCR5 antibody chain are conjugated to an antifusogenic peptide each with or without an intermediate linker. For the recombinant production of the antifusogenic peptide-anti-CCR5 antibody-conjugate according to the invention one or more nucleic acid molecules encoding different polypeptides are required, preferably two to eight nucleic acid molecules are employed. These nucleic acid molecules encode the different anti-CCR5 antibody polypeptide chains of the conjugate and are in the following referred to as structural genes. They can be located on the same expression plasmid (vector) or can alternatively be located on different expression plasmids (vectors). The assembly of the conjugate takes preferably place before secretion of the conjugate and thus within the expressing cells. Therefore the nucleic acid molecules encoding the polypeptide chains of the conjugate are preferably expressed in the same host cell. If after recombinant expression a mixture of conjugates is obtained, the conjugates can be separated and purified by methods known to a person skilled in the art. These methods are well established and widespread used for immunoglobulin purification and are employed either alone or in combination. Such methods are, for example, affinity chromatography using microbial-derived proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange chromatography), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and preparative electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (M. A. Vijayalakshmi, Appl. Biochem. Biotech. (1998) 75:93-102). With recombinant engineering methods known to a person skilled in the art the conjugates can be tailor-made on the nucleic acid/gene level. The nucleic acid sequences encoding immuno-globulins are known and can be obtained for example from genomic databases. Likewise the nucleic acid sequences encoding antifusogenic peptides are known or can easily be deduced from their amino acid sequence. The elements required for the construction of an expression plasmid for the expression of the conjugate of the current invention are, for example, an expression cassette for the anti-CCR5 antibody light chain in its natural and/or modified and/or conjugated version, an expression cassette for the anti-CCR5 antibody heavy chain in its natural and/or modified and/or conjugated version (alternatively the anti-CCR5 antibody light chain and the anti-CCR5 antibody heavy chain can be contained in the same expression cassette, e.g. as bicistronic expression element), a selection marker, and an E. coli replication as well as selection unit. These expression cassettes comprise a promoter, a DNA segment encoding a secretion signal sequence, the structural gene, and a terminator/polyadenylation signal. The elements are assembled in an operatively linked form either on one plasmid encoding all chains of the conjugate, or on two or more plasmids each encoding one or more chains of the conjugate. For the expression of the encoded polypeptides the plasmid(s) is (are) introduced into a suitable host cell. Proteins are preferably produced in mammalian cells such as CHO cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, K562 cells, BHK cells, PER.C6.RTM. cells, and the like. The regulatory elements of the plasmid have to be selected in a way that they are functional in the selected host cell. For the expression the host cell containing the plasmid encoding one or more chains of the conjugate is cultivated under conditions suitable for the expression of the chains. The expressed conjugate chains are functionally assembled. The fully processed antifusogenic peptide-anti-CCR5 antibody-conjugate is secreted into the medium.

An "expression plasmid" is a nucleic acid encoding a polypeptide to be expressed in a host cell. Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, comprising an origin of replication, and a resistance gene, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

One aspect of the current invention is thus a method for the production of a conjugate according to the invention, comprising the following steps a) cultivating a cell containing one or more expression plasmids each comprising one or more nucleic acid molecules encoding a conjugate according to the invention under conditions suitable for the expression of the conjugate, b) recovering the conjugate from the cell or the supernatant.

The term "under conditions suitable for the expression of the conjugate" denotes conditions which are used for the cultivation of a cell expressing a polypeptide and which are known to or can easily be determined by a person skilled in the art. It is known to a person skilled in the art that these conditions may vary depending on the type of cell cultivated and type of polypeptide expressed. In general the cell is cultivated at a temperature, e.g. between 20.degree. C. and 40.degree. C., and for a period of time sufficient to allow effective production of the polypeptide conjugate, e.g. for 4 to 28 days.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption/resorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. In addition to water, the carrier can be, for example, an isotonic buffered saline solution.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, 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 invention preferably comprises the use of a conjugate according to the invention for the treatment of a patient suffering from immunodeficiency syndromes such as AIDS.

Claim 1 of 5 Claims

1. A conjugate comprising an anti-CCR5 antibody (mAb CCR5) comprising two heavy chains and two light chains, having termini comprising an N-terminus and a C-terminus, wherein two or four of said N-terminus or said C-terminus are conjugated with an antifusogenic peptide; wherein said anti-CCR5 antibody comprises a variable heavy chain domain and a variable light chain domain, wherein said variable heavy chain domain consists of an immunoglobulin framework and a CDR3 region selected from the group consisting of heavy chain CDR3 sequences SEQ ID NOS:16 and 17; a CDR2 region selected from the group consisting of heavy chain CDR2 sequences SEQ ID NOS:13, 14, and 15; and a CDR1 region selected from the group consisting of heavy chain CDR1 sequences SEQ ID NOS:9, 10, 11, and 12; and wherein said variable light chain domain consists of an immunoglobulin framework and a CDR1 region selected from SEQ ID NOS:18, 19, and 20; a CDR2 region selected from SEQ ID NOS:21, 22, and 23; and a CDR3 region selected from SEQ ID NOS:24 and 25.

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