BRPI0715794A2 - ccr5 antibody conjugate and antifusogenic peptide - Google Patents

Abstract

CCR5 ANTIBODY AND ANTIFUSOGENIC PEPTIDE ASSEMBLY. The present invention relates to a conjugate comprising one or more anti-genomic peptides and an antibody to an HIV gp120-binding cell surface receptor, characterized in that one to eight antifusogenic peptides are each conjugated to one or more antifusogenic peptides. terminating the heavy and / or light bitches of said antibody against a cell surface receptor that binds HIV gp12O to the pharmaceutical use of said conjugate.

Description

Descriptive Report of the Invention Patent for "CCR5 ANTIBODY AND ANTIFUSOGENIC PEPTIDE ASSEMBLY".

The present invention relates to a conjugate of a CCR5 antibody and an antifusogenic peptide wherein one to eight antifusogenic peptides are each conjugated to a heavy and / or light chain termination of an anti-CCR5 antibody. Antifusogenic peptides may be different, similar or identical at the amino acid level. Background of the Invention

Human immunodeficiency virus (HIV) infection of cells is effected by a process in which the cell membrane to be infected and the viral membrane are fused. A general scheme for this process is proposed: The viral envelope glycoprotein complex (gp120 / gp41) interacts with a cell surface receptor located on the membrane of the cell to be infected. Binding of gp120, for example, to the CD4 receptor in combination with a corrector such as CCR-5 or CX-CR-4 causes a change in the conformation of the gp120 / gp41 complex. As a result of this conformational change the gp41 protein is able to insert into the target cell membrane. This insertion is the beginning of the membrane fusion process. It is well known that the amino acid sequence of gp41 protein varies in different strains of HIV because of naturally occurring polymorphisms. But the same domain architecture can be recognized, more precisely, a fusion signal, two repeated hepta domains (HR1, HR2) and a transmembrane domain (in the N- to C-terminal direction). The fusion (or fusogenic) domain is suggested to participate in the insertion and disintegration of the cell membrane. HR regions are constructed from multiple strands comprising seven "heptad" amino acids (see, for example, Shu, W., et al., Biochemistry 38 (1999) 5378-5385). In addition to the "heptads" one or more motifs similar to Ieucine zippers are present. This composition is responsible for forming a coiled-coil structure of the gp41 proteins and also peptides derived from these domains. Super-propellers are generally oligomers that consist of two or more interacting propellers. Peptides with deduced amino acid sequences from the HR1 or HR2 domain of gp41 are efficient in vitro and in vivo inhibitors of HIV entry into cells (for examples of peptides see, e.g., US 5,464,933, US 5,656,480, US 6,258 .782, US 6,348,568, or US 6,656,906). For example, T20 (also known as DP178, Fuzeon®, an HR2 peptide), T651 (US 6,479,055), and T2635 (WO 2004/029074) are very potent inhibitors of HIV infection. Attempts have been made to increase the efficacy of HR2-derived peptides with, for example, amino acid substitutions or chemical crosslinking (Sia, SK, et al., Proc. Natl. Acad. Sci. USA 99 (2002) 14664-14669; Otaka, A., et al., Angew. Chem. Int. Ed. 41 (2002) 2937-2940).

Conjugation of peptides to certain molecules may alter their pharmacokinetic properties, for example, the serum half-life of such peptide conjugates may be increased. Conjugations are reported, for example, for polyethylene glycol (PEG) and interleukin-6 (EP 0 442 724), for PEG and erythropoietin (WO 01/02017), for chimeric molecules comprising Endostatin and immunoglobulins (US 2005/008649 ), for secreted antibody-based fusion proteins (US 2002/147311), for fusion polypeptides comprising albumin (US 2005/0100991; human serum albumin US 5,876,969), for PEGylated polypeptides (US 2005/0114037 ), and for interferon fusions. Immunotoxins comprising Gelonin and an antibody (WO 94/26910), modified transferrin-antibody fusion proteins (US 2003/0226155), antibody-cytokine fusion proteins (US 2003/0049227) are also described in the art. ), and fusion proteins consisting of a peptide with immunostimulatory, membrane transport or homophilic activity and an antibody (US 2003/0103984). WO 2004/085505 Long acting biologically active conjugates consisting of biologically active compounds chemically linked to macromolecules are reported.

The CCR5 correceptor is used by most HIV-1 isolates and is critical for the establishment and maintenance of infection. In addition, the CCR5 function is unnecessary for human health. A mutant CCR5 allele, "CCR5 Δ32", encodes a non-functional truncated protein (Samson, M., et al., Nature 382 (1996) 722-725; Dean, M., et al., Science 273 ( 1996) 1856-1862). Homozygous individuals for the mutation have no CCR5 expression and are strongly protected from HIV-1 infection. They show no observable phenotypic consequence and are highly resistant to M-tropic HIV infection, whereas heterozygous individuals show delayed disease progression (Schwarz, MK and Wells, TN, Nat. Rev. Drug Discov. 1 (2002) 347 - 358). Lack of CCR5 has no apparent adverse consequences, probably because CCR5 is part of a highly redundant chemokine network as a receptor for ΜΙΡ-Ια, MIP-1 β, and RANTES α-chemokines, which share many common functions. , and most of which have alternative receptors (Rossi, D. and Zlotnik, A., Ann. Rev. Immunol. 18 (2000) 217-242). Identification of CCR5 as an HIV-1 correceptor was based on the ability of its ligands, ΜΙΡ-Ια, ΜΙΡ-1β, and RANTES, to block infection by R5 isolates, but not by R5X4 or X4 isolates (Cocchi, F ., et al., Science 270 (1995) 1811-1815). CCR5 is also a receptor for "aggregated" chemokines, which are produced primarily during inflammatory responses and control neutrophil recruitment (CXC chemokines), macrophages, and a subset of T cells (Th1 and Th2 helper T cells). Th1 responses are typically those involving effective cell-mediated immunity against viruses and tumors, proinflammatory responses responsible for the death of intracellular parasites, and the perpetuation of autoimmune responses, for example, while Th2 responses are. believed to be the leading allergy sufferers. Therefore, inhibitors of these chemokine receptors may be useful as immunomodulators. For Th1 responses, overactive responses are reduced, for example, in autoimmunity including rheumatoid arthritis, or for Th2 responses, asthma attacks or allergic responses including allergic responses including atopic dermatitis (see for example Schols , D., Curr. Top, Med. Chem. 4 (2004) 883-893; Mueller, A., and Strange, PG, Int. J. Biochem. Cell Biol. 36 (2004) 35-38; Kazmierski, WM , et al., Curr. Drug Targets Infect Disorders 2 (2002) 265-278; Lehner, T., Trends Immunol. 23 (2002) 347-351). Human CCR5 antibodies are, for example, PRO 140 (Olson, WC, et al., J. Virol. 73 (1999) 4145-4155), and / or 2D7 (Samson, M., et al., J. Biol. Chem. 272 (1997) 24934-24941). Additional antibodies are mentioned in US 2004/0043033, US 6,610,834, US 2003/0228306, US 2003/0195348, US 2003/0166870, US 2003/0166024, US 2003/0165988, US 2003/0152913, US 2003 / 0100058, US 2003/0099645, US 2003/0049251, US 2003/0044411, US 2003/0003440, US 6.528.625, US 2002/0147147, US 2002/0146415, US 2002/0106374, US 2002/0061834, US 2002 / 0048786, US 2001/0000241, EP 1 322 332, EP 1 263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, 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 EP 05007138.0.

CCR5 antibody polyethylene glycol conjugates are known from US 2003/0228306. US 2003/0215421 refers to chemicin-toxin conjugates. WO 01/43779 relates to anti-CD4 antibody and anti-CCR5 antibody conjugates and anti-CD4 antibody and HIV-1 fusion inhibitor peptide conjugates. CCR5 antibody conjugates and toxins are mentioned in EP 1 346 731. Summary of the Invention The present invention relates to a conjugate comprising one to

eight antifusogenic peptides and an antibody against an HIV g120 binding cell surface receptor, characterized in that one to eight, preferably two to four antifusogenic peptides, each combine to a heavy and / or light chain termination. of said antibody against an HIV gp120 binding cell surface receptor (a number of eight antifusogenic peptides per antibody is possible only if the antibody comprises eight terminations, i.e. is composed of, for example, two heavy chains and two light chains, if the antibody comprises a smaller number of C- and -N-termini, for example as an scFv, the corresponding number of possible maximum antifusogenic peptides in the conjugate will also be reduced, i.e. (it is reduced to less than eight).

The invention further comprises a conjugate comprising one or more antifusogenic peptides and an anti-CCR5 antibody (mAb C-CR5) characterized in that at most one to eight antifusogenic peptides are each conjugated to a heavy and / or light chain termination. of said anti-CCR5 antibody (a number of eight antifusogenic peptides per mAb CCR5 is only possible if the mAb CCR5 comprises eight terminations, i.e. is composed of, for example, two heavy chains and two light chains; if the mAb CCR5 comprises fewer number of C- and -N-terminations, for example as scFv, the corresponding number of maximum possible antifungal peptides in the conjugate will also be reduced (i.e. 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 ligand.

Preferably the conjugate is characterized by the general formula

mAb CCR5 - [Ligating] - [antifusogenic peptide] n where m is independently for each antifusogenic peptide 0 (i.e., a peptide bond between mAb CCR5 and the antifusogenic peptide) or 1 (this is, a ligand between mAb CCR5 and antifusogenic peptide) and η is an integer 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] - [ligand] m- [heavy chain]

(2) [heavy chain] - [ligand] m- [antifusogenic peptide]

(3) [antifusogenic peptide] - [ligand] m- [heavy chain] - [antifusogenic peptide]

(4) [antifusogenic peptide] - [ligand] m- [light chain]

(5) [light chain] - [ligand] m- [antifusogenic peptide]

(6) [antifusogenic peptide] - [ligand] m- [light chain] - [antifusogenic peptide]

(7) [antifusogenic peptide] - [ligand] m- [heavy chain] - [ligand] m- [antifusogenic peptide]

(8) [antifusogenic peptide] - [ligand] m- [light chain] - [ligand] m- [antifusogenic peptide]

wherein the Ligand may be the same or different in (within or between) said chain conjugates, where m is an integer of 1 or 0, and may independently be the same or different in (within or between) said conjugate chains. jail. (The left side of the CCR5 peptide or mAb chain means

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

Preferably the chain conjugates are grouped to conjugates according to the invention comprising the CCR5 mAb (e.g. consisting of two light chains and two heavy chains including the Fc1 constant 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 2x [CCR5 mAb light chain] and 2x (2), 2 χ [CCR5 mAb light chain] and 2 χ (3), or 2 χ [CCR5 mAb heavy chain] ] and 2 χ (4), or 2 χ [light chain of mAb CCR5] and 2 χ (7). The heavy chain and / or light chain preferably comprises a constant region (Fc). Preferably the conjugate is characterized in that it comprises

a heavy chain variable domain consisting of an immunoglobulin backbone and a CDR3 region selected from the group consisting of heavy chain CDR3 sequences SEQ ID NO: 16, 17.

Preferably, the conjugate is characterized by comprising a heavy chain variable domain consisting of an immunoglobulin structure 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 that it comprises a heavy chain variable domain selected from the group of chain variable domains comprising SEQ ID NO: 1, 3, 5, and 7.

Preferably the conjugate is characterized in that it comprises a light chain variable domain consisting of an immunoglobulin structure 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 as heavy chain CDRs as CDRs of SEQ ID NO: 1 and as light chain CDRs as SEQ ID NO: 2, as heavy chain CDRs as 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 SEQ ID NO: 6, or as heavy chain CDRs the CDRs of SEQ ID NO: 7 and as light chain CDRs are the CDRs of SEQ ID NO: 8.

Preferably the conjugate is characterized by comprising a heavy and light chain variable domain independently selected from the group consisting of

a) the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the light chain variable domain (V1) defined by the amino acid sequence SEQ ID NO: 2;

b) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 4;

c) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by the amino acid sequence SEQ

ID NO: 6; d) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 8.

Preferably the conjugate is characterized by comprising the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the light chain variable domain (V1) defined by the amino acid sequence SEQ ID NO: 2; or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 4; or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 6; or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 8; a ligand selected from the group consisting of the amino acids glycine (G) and asparagine, a tripeptide GST and SEQ ID NO: 36 to 62 and SEQ ID NO: 67 to 70; and an antifusogenic peptide selected from the group of peptides defined by SEQ ID NO: 29 to 35 and SEQ ID NO: 73.

Preferably the conjugate is characterized in that it comprises an antifusogenic peptide selected from the group of peptides comprising C34, T20, T1249, T651, T2635, N36, DP107, and afp-1.

Preferably the conjugate is characterized in that it comprises an antifusogenic peptide at each heavy chain C-terminus or light chain N-terminus (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 that it comprises 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 ligand of SEQ ID NO. : 40 and an antifusogenic peptide of SEQ ID NO: 33, 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 ligand of SEQ ID NO: 40 and an antifusogenic peptide of SEQ ID NO: 33, comprising two light chain variable domains of SEQ ID NO: 6, two conjugates of type (2) each comprising one domain heavy chain variable of SEQ ID NO: 5, a ligand of SEQ ID NO: 40 and an antifusogenic peptide of SEQ ID NO: 33, or comprising two light chain variable domains of SEQ ID NO: 8, two conjugates of the type (2) each comprising a heavy chain variable domain of SEQ ID NO: 7, an ligand 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 the IgGI subclass. It is also preferred that said anti-CCR5 antibody is from the IgG4 subclass, or IgGI or IgG2 subclass, with an amino acid position mutation S228, L234, L235, and / or D265, and / or contains the PVA236 mutation. Preferably the conjugate is characterized in that the antibody said anti-CCR5 antibody of subclass IgG4 has an S228P mutation and said anti-CCR5 antibody of IgGI subclass has mutations L234A and L235A.

The invention further comprises a conjugate comprising two or more antifusogenic peptides and an anti-CD4 antibody (mAb CD4) characterized in that two to eight antifusogenic peptides, preferably two or four, are each conjugated to a heavy chain termination. and / or light weight of said anti-CD4 antibody (a number of eight antifungal peptides per mAb CD4 is only possible if the mAb CD4 antibody comprises eight terminations, ie it is composed of, for example, two heavy chains and two light chains, if the CD4 mAb comprises a lower number of C- and -N-terminations, for example as an scFv, the corresponding number of possible antifusogenic peptides in the conjugate is also reduced, ie it is reduced. to less than eight). Preferably the carboxy terminal amino acid of a chain

of antibody is conjugated to the amino-terminal amino acid of the antifungal 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 ligand.

Preferably the conjugate is characterized by the general formula

antibody - [ligand] m- [antifusogenic peptide] n where m is independently for each antifusogenic peptide

0 (i.e., a ligand between antibody and antifusogenic peptide) or 1 (i.e., ligand between antibody and antifusogenic peptide) and η is an integer from 2 to 8, preferably an even integer from 2 at 8, more preferably 2 or 4. Preferably said antibody is CD4 mAb.

A preferred conjugate of a heavy and / or light chain of a

The antibody and an antifusogenic peptide ("chain conjugate") is selected from the group consisting of (in the N-terminal to C-terminal direction):

(1) [antifusogenic peptide] - [ligand] m- [heavy chain]

(2) [heavy chain] - [ligand] m- [antifusogenic peptide]

(3) [antifusogenic peptide] - [ligand] m- [heavy chain] - [peptide]

antifusogenic deo]

(4) [antifusogenic peptide] - [ligand] m- [light chain]

(5) [light chain] - [ligand] m- [antifusogenic peptide]

(6) [antifusogenic peptide] - [ligand] m- [light chain] - [peptide

antifusogenic]

(7) [antifusogenic peptide] - [ligand] m- [heavy chain] - [ligand] m- [antifusogenic peptide]

(8) [antifusogenic peptide] - [ligand] m- [light chain] - [ligand] m- [antifusogenic peptide]

where the ligand may be the same or different in (within and between)

said chain conjugates, wherein m is an integer of 1 or 0, and m may be independently the same or different in (within and between) said conjugates.

(The left side of the peptide or antibody chain means

N-termination, the right side means C-termination. In (1) therefore, the C-terminus of the antifusogenic peptide is bound by a peptide bond or a ligand to the N-terminus of the antibody heavy chain). Preferably the conjugates are grouped to conjugates according to the invention comprising a CD4 mAb (e.g., consisting of two light chains and two heavy chains including constant Fc domains, an scFv fragment, or a Fab fragment).

Especially preferred chain conjugates are (2), (3), (4),

and (7). Especially preferred conjugates according to the invention comprise 2x [CD4 mAb light chain] and 2x (2), 2 χ [CD4 mAb light chain] and 2 χ (3), or 2 χ [CD4 mAb heavy chain]. ] and 2 χ (4), or 2 χ [CD4 mAb light chain] and 2 χ (7). The heavy and / or light chain preferably comprises a constant region.

Preferably the conjugate is characterized by comprising a heavy chain variable domain consisting of an immunoglobulin structure and a CDR3 region selected from the heavy chain CDR3 sequences of SEQ ID NO: 103, 104, or 105. Preferably the conjugate is characterized by understand

a heavy chain variable domain of an immunoglobulin structure and a CDR3 region selected from the CDR3 sequences of SEQ ID NO: 103, 104, or 105, a CDR2 region selected from the CDR2 sequences of SEQ ID NO : 100, 101, or 102, and a CDR1 region selected from the CDR1 sequences of SEQ ID NO: 97, 98, or 99.

Preferably the conjugate is characterized in that it comprises a heavy chain variable domain wherein the heavy chain variable domain comprises SEQ ID NO: 82, or 83.

Preferably the conjugate is characterized in that it comprises a light chain variable domain consisting of an immunoglobulin structure and a CDR1 region selected from SEQ ID NO: 87, 88, 89, or 90, a CDR2 region selected from SEQ ID NO: 91, 92, or 93, and a CDR3 region selected from SEQ ID NO: 94, 95, or 96.

Preferably the conjugate is characterized by comprising as heavy chain CDRs the CDRs of SEQ ID NO: 81, and as light chain CDRs the CDRs of SEQ ID NO: 75, as heavy chain CDRs the CDRs of SEQ ID NO: 82, and as light chain CDRs the CDRs of SEQ ID NO: 76, as heavy chain CDRs the CDRs of SEQ ID NO: 84, and as light chain CDRs the CDRs of SEQ ID NO: 78, as heavy chain CDRs as CDRs of SEQ ID NO: 85, and as light chain CDRs the CDRs of SEQ ID NO: 79, or as heavy chain CDRs the SEQ ID NO: 86, and as light chain CDRs the SEQ ID NO. : 80.

Preferably the conjugate is characterized by comprising a heavy and light chain variable domain independently selected from:

a) the heavy chain variable domain defined by the sequence

of amino acid SEQ ID NO: 81, and the defined light chain variable domain

by amino acid sequence SEQ ID NO: 75;

b) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 82, and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 76;

c) the heavy chain variable domain defined by the sequence

amino acid sequence SEQ ID NO: 84, and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 78;

d) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 85, and the light chain variable domain defined

by the amino acid sequence SEQ ID NO: 79,

e) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 86, and the light chain variable domain defined by the amino acid sequence

SEQ ID NO: 80.

Preferably the conjugate is characterized in that it comprises the

heavy chain variable domain defined by amino acid sequence SEQ ID NO: 81, and light chain variable domain defined by amino acid sequence SEQ ID NO: 75; or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 82, and the heavy chain variable domain

light chain defined by amino acid sequence SEQ ID NO: 76; or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 84, and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 78; the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 85, and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 79, or the heavy chain variable domain defined by the sequence of amino acid SEQ ID NO: 86, and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 80; a ligand selected from the amino acids glycine (G) and asparagine (Ν), the tripeptide GST, at SEQ ID NO: 36 to 62 and SEQ ID NO: 67 to 70; and an antifusogenic peptide selected from the antifusogenic peptides of SEQ ID NO: 29 to 35 or SEQ ID NO: 73. Preferably the conjugate is characterized in that it comprises

an antifusogenic peptide selected from antifusogenic peptides C34, T20, T1249, T651, T2635, N36, DP107, or afp-1.

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

Preferably the conjugate is characterized in that it comprises two light chain variable domains of SEQ ID NO: 75, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO: 81, a ligand of SEQ ID NO. : 69, and an antifusogenic peptide of SEQ ID NO: 73, comprising two light chain variable domains of SEQ ID NO: 76, two conjugates of type (2) each comprising a heavy chain variable domain of SEQ ID NO : 82, a ligand of SEQ ID NO: 69, and an antifusogenic peptide of SEQ ID NO: 73, comprising two light chain variable domains of SEQ ID NO: 78, two conjugates of type (2) each comprising one heavy chain variable domain of SEQ ID NO: 84, a ligand of SEQ ID NO: 69, and an antifusogenic peptide of SEQ ID NO: 73, comprising two light chain variable domains of SEQ ID NO: 79, two conjugates of the type (2) each comprising a heavy chain variable domain of SEQ ID N O: 85, a ligand of SEQ ID NO: 69, and an antifusogenic peptide of SEQ ID NO: 73, or by comprising two light chain variable domains of SEQ ID NO: 80, two conjugates of the type (2 ) each comprising a heavy chain variable domain of SEQ ID NO: 86, a ligand of SEQ ID NO: 69, and an antifungal peptide of SEQ ID NO: 73. Preferably the conjugate is characterized in that the said anti-

body is either subclass IgGI or subclass lgG4. It is also preferred that said antibody is of the IgG4 or IgGI subclass or of the IgG2 subclass, with an amino acid position mutation S228, L234, L235, and / or D265, and / or contains the PVA236 mutation. Preferably said conjugate is wherein said lgG4 subclass antibody has an S228P mutation and said IgGI subclass antibody has mutations L234A and L235A.

Especially preferred is said antibody either an anti-CD4 antibody or an anti-CCR5 antibody.

The invention comprises a method for producing a conjugate according to the invention, wherein the method comprises

a) culturing 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 expression of the

conjugate,

b) recovering the conjugate from the cell or supernatant.

In one embodiment are genes encoding the light and heavy chains of mAb CCR5 or mAb CD4 with or without linked antifungal peptide located on the same or 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 an HIV infection.

The invention comprises the use of a conjugate according to the invention for the treatment of a patient in need of antiviral treatment, preferably anti-HIV treatment. Description of the Invention One aspect of the present invention is a conjugate comprising

It comprises one or more antifusogenic peptides and an anti-CCR5 antibody (mAb CCR5) characterized in that one to eight antifusogenic peptides are each coupled to a heavy and / or light chain termination of said anti-CCR5 antibody. A number of eight antifusogenic peptides per mAb CCR5 is only possible if the mAb CCR5 comprises eight terminations, that is, it consists of two heavy chains and two light chains. If the CCR5 mAb comprises fewer C-and -N-terminations, for example as an scFv, the corresponding maximum number of possible antifusogenic peptides in the conjugate is also reduced, i.e. it is reduced to less than eight .

Another aspect of the present invention is a conjugate comprising two or more antifusogenic peptides and an anti-CD4 antibody (mAb CD4) characterized in that two to eight antifusogenic peptides are each conjugated to a heavy and / or light chain termination. said anti-CD4 antibody.

When reference is made below to the antibody in general or to the CCR5 mAb or CD4 mAb, reference is also made, where appropriate, to the other mAb and corresponding sequences.

An "antifusogenic peptide" is a peptide that inhibits events associated with membrane fusion or the membrane fusion event itself, including, among other things, inhibiting infection of cells not infected with a virus due to membrane fusion. These anti-fusogenic peptides are preferably linear peptides. For example, they may be derived from the gp41 ectodomain, for example, such as DP107, DP178. Examples of such peptides can be found in US 5,464,933, US 5,656,480, US 6,013,263, US 6,017,536, US 6,020,459, US 6,093,794, US 6,060,065, US 6,258,782, US 6,348 .568, US 6,479,055, US 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 may be selected from the group of SEQ ID NO: 1 to 10 of US 5,464,933; SEQ ID NO: 1 to 15 of US 5,656,480; SEQ ID NO: 1 to 10 and 16 to 83 of US 6,013,263; SEQ ID NO: 1 to 10, 20 to 83, and 139 to 149 of US 6,017,536; SEQ ID NO: 1 to 10, 17 to 83 and 210 to 214 of US 6,093,794; SEQ ID NO: 1 to 10, 16 to 83 and 210 to 211 of US 6,060,065; SEQ ID NO: 1286 and 1310 of US 6,258,782; SEQ ID NO: 1129, 1278 to 1309, 1311 and 1433 of US 6,348,568; SEQ ID NO: 1 to 10 and 210 to 238 of US 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, 885 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 to US 6,656,906; or SEQ ID NO: 5 to 95 of WO 2005/067960. The antifusogenic peptide has an amino acid sequence comprising from 5 to 100 amino acids, preferably from 10 to 75 amino acids and more preferably from 15 to 50 amino acids. Especially preferred antifusogenic peptides are C-34, T-20, T-1249, T-651, T-2635, N-36 (Root, MJ, et al., Curr. Pharm. Des. 10 (2004) 1805-1825 ) and DP-107 (Wild, C, et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12676-12680) and afp-1 (SEQ ID NO: 29 to 35 and SEQ ID NO: 73) . In one embodiment the conjugate according to the invention comprises one or more antifusogenic peptides and an antibody wherein i) the antifusogenic peptides are linear peptides with an amino acid sequence of 5 to 100 amino acids, and ii) one to eight antifusogenic peptides. each is coupled with a termination of the heavy and / or light chains of said antibody. In another embodiment the conjugate according to the invention comprises one or more antifusogenic peptides and an antibody, for example an anti-CCR5 antibody (mAb CCR5), wherein i) antifusogenic peptides are derived from the gp41 ectodomain and (ii) one to eight antifungal peptides are each conjugated to a heavy and / or light chain termination of said antibody. The term "gp41 ectodomain" denotes the amino acid sequence beginning with amino acid position 561 and ending with amino acid position 620 of HIV-1 gp160 or starting with amino acid position 50 and ending with amino acid position 50. HIV-I gp41 amino acid 109 (SEQ ID NO: 66) (see also, for example, Bar, S. and Alizon, MJ Virol. 78 (2004) 811-820).

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 manipulation of antibodies is described, for example, in Morrison, S.L., et al., Proc. Natl. Acad I know. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244; Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger, M.S., et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23 (2005) 1117-1125.

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

"CCR5" means human CCR5 as described, for example, in Oppermann, M., Cell Signal. 16 (2004) 1201-1210 and SwissProt P51681. The terms "CCR5 antibody binding", "anti-CCR5 antibody", or "CCR5 mAb", which are used interchangeably within this application, mean an antibody that specifically binds CCR5 and preferably inhibits fusion. of HIV with a target cell. Binding can be tested in an in vitro cell-based ELISA assay (CCR5-expressing CHO cells). Binding is found if the antibody causes an S / R (signal / noise) ratio of 5 or more, preferably 10 or more at an antibody concentration of 100 ng / ml. The term "inhibition of HIV fusion with a target cell" refers to the inhibition of 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 target cell. antibody at a concentration effective to inhibit membrane fusion between the virus and said cell and measure, for example, Iuciferase reporter gene activity or HIV p24 antigen concentration. The term "membrane fusion" refers to the fusion between a first cell that co-expresses CCR5 and CD4 polypeptides and a second cell or virus that expresses 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, US 6,610,834, US 2003/0228306, US 2003/0195348, US 2003/0166870, US 2003/0166024, US 2003/0165988, US 2003/0152913, US 2003 / 0100058, US 2003/0099645, US 2003/0049251, US 2003/0044411, US 2003/0003440, US 6.528.625, US 2002/0147147, US 2002/0146415, US 2002/0106374, US 2002/0061834, US 2002 / 0048786, US 2001/0000241, EP 1 322 332, EP 1 263 791, EP 1 207 202, EP 1 161 456, EP 1 144 006, 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 structure and a CDR3 region selected from the group consisting of heavy chain CDR3 sequences SEQ ID NO : 16, 17. An additionally preferred antibody comprises a heavy chain variable region of an immunoglobulin backbone and a CDR3 region selected from the group consisting of the CDR3 sequences SEQ ID NO: 16, 17, a region CDR2 selected from the group consisting of the sequences of SEQ ID NO: 13, 14, 15, and a CDR1 region selected from the group consisting of the sequences of CDR1 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 to a light chain variable domain consisting of an immunoglobulin structure and a reg CDR1 ion selected from the group consisting of the CDR1 sequences SEQ ID NO: 18, 19, 20, a CDR2 region selected from the group consisting of the CDR2 sequences SEQ ID NO: 21, 22, 23, and a CDR3 region selected from the CDR3 sequence group SEQ ID NO: 24, 25. The anti-CCR5 antibody is preferably characterized as containing heavy chain CDRs as SEQ ID NO: 1 and as light chain CDRs as 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 Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Behesda, MD ( 1991). The CDRs of SEQ ID NO: 1 to 8 are shown in SEQ ID NOs: 9 to 25. The anti-CCR5 antibody preferably comprises a heavy and light chain variable domain independently selected from the group consisting of:

a) the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the light chain variable domain

(V1) defined by SEQ ID NO: 2;

b) the heavy chain variable domain defined by the 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 the sequence

of amino acid SEQ ID NO: 5 and the light chain variable domain defined by SEQ ID NO: 6;

d) the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by SEQ ID NO: 8.

"CD4" means human CD4, as described, for example, in Brady, R.L. and Barclay, A.N., Curr. Top. Microbiol. Immunol. 205 (1996) 1-18 and SwissProt P01730. The terms "CD4 binding antibody", "anti-CD4 antibody", or "CD4 mAb", which are used interchangeably within this application, denote an antibody that specifically binds CD4 and preferably inhibits the fusion of HIV with a target cell. Binding can be tested in a cell-based in vitro ELISA (CHO cells expressing CD4). 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. The term "inhibiting fusion of HIV with a target cell" refers to inhibiting fusion of HIV with a target cell as measured in an assay comprising contacting said target cell (e.g. PBMC) with the virus in the presence of the target cell. antibody in question at a concentration effective to inhibit membrane fusion between the virus and said cell and to measure, for example, the activity of the Iuciferase reporter gene or the concentration of HIV p24 antigen. The term "membrane fusion" refers to the fusion between a first cell expressing CD4 polypeptides and a second cell or virus expressing an HIV env protein. Membrane fusion is determined by cells and / or viruses genetically engineered by a reporter gene assay (e.g., by a luciferase reporter gene assay). Exemplary anti-CD4 antibodies are mentioned, for example, in 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 97/46697, WO 01/43779, US 6,136 310, WO 91/009966.

"CXCR4" means human CXCR4, as described, for example, in Feng, Y., et al., Science 272 (1996) 809-810, or Tamamura, H. and Fujii, N., Expert Opinion on Therapeutic Targets. 9 (2005) 1267-1282. The terms "CXCR4 binding antibody", "anti-CXCR4 antibody" or "CXCR4 mAb", which are used interchangeably within this application, denotes an antibody that specifically binds CXCR4 and preferably inhibits the fusion of HIV with a target cell. Binding can be tested in a cell-based in vitro ELISA assay (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. The term "inhibiting fusion of HIV with a target cell" refers to inhibiting fusion of HIV with a target cell as measured in an assay comprising contacting said target cell (e.g. PBMC) with the virus in the presence of the antibody in question at a concentration effective to inhibit membrane fusion between the virus and said cell and to measure, for example, luciferase reporter gene activity or HIV p24 antigen concentration. The term "membrane fusion" refers to the fusion between a first cell expressing CXCR4 polypeptides and a second cell or virus expressing an HIV env protein. Membrane fusion is determined by cells and / or viruses genetically engineered by a reporter gene assay (for example, a luciferase reporter gene assay). Exemplary anti-CXCR4 antibodies are mentioned, for example, Strizki, J.M., et al., J. Virol. 71 (1997) 5678-5683 (Ab 12G5), US 7,138,496. An "antibody to an HIV gp120 binding cell surface receptor" denotes within the present invention an antibody that is binding to a cell surface receptor which is used by the HIV gp120 antibody to mediate. binding the virus to the cell surface of a cell, thereby preventing gp120 binding to said receptor and also binding of HIV to a cell surface. HIV gp120 is derived from the proteolytic cleavage of the HIV gp160 precursor (see, for example, Brenneman, D.E., etal., Int. Rev. Neurobiol. 32 (1990) 305-353). The second fragment of this cleavage is HIV gp41. These two fragments non-covalently associate and mediate cell binding and fusion of HIV and a cell. Exemplary cell surface receptors are the CD4 receptor, or chemokine receptors such as the CCR5 receptor, CXCR4 receptor or CXCR5 receptor. Therefore, in one embodiment the antibody against an HIV gp120 binding cell surface receptor is an anti-CD4 antibody, or an anti-CCR5 antibody, or an anti-CXCR4 antibody, or an anti-CXCR5 antibody.

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 art, and, for example, described by Kabat (see, for example, Johnson, G., and Wu1 T.T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful human IgGI heavy chain constant region (CH1-hinge-CH2-CH3) comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 26, 27. For example, a chain constant domain Useful kappa (κ) heavy chain comprises an amino acid sequence of a kappa light chain constant domain (light chain constant domain κ, CL) of SEQ ID NO: 28. Further, it is preferred that the antibody variable domains originate from mice and which comprise the antibody variable domain sequence structure of a mouse antibody according to Kabat (see, for example, Johnson, G., and Wu1 TT, NucIeicAcids Res. 28 (2000)). ) 214-218).

A preferred anti-CCR5 antibody shows binding to the same CCR5 epitope (s) than an antibody selected from the group consisting of antibodies A to E or is inhibited on CCR5 binding by AaE antibodies. due to steric hindrance of binding or competitive binding. Epitope binding is investigated by the use of alanine selection according to the method described by Olson, W.C., et al. (J. Virol. 73 (1999) 4145-4155) for epitope mapping. A signal reduction of 75% or more shows that the mutated amino acid (s) contributes to the epitope recognized by said antibody. Antibody binding to the same epitope is found if amino acids contributing to the epitope are recognized by the investigated antibody and antibody A, B, C, D, or Ε. Antibody C, which shows lower IC 50 values than antibody 2D7 in HIV1 assays, binds to an epitope that includes amino acids in the CCR5 ECL2 domain (Lee, B., et al., J. Biol. Chem. 274 (1999) 9617-9626) which is different from the epitope recognized by the 2D7 antibody (2D7 binds to ECL2A amino acids K171 and E172 but not to ECL2B amino acids 184 to 189). Epitope binding for antibody C is seen to be 20% for mutant CCR5 K171A or E172A (glu 172 is mutated for ala). 100% epitope binding is defined for wild type CCR5. An additionally preferred anti-CCR5 antibody binds to the same epitope as antibody C. The term "epitope" means a protein determinant capable of

to specifically bind to an antibody. Epitopes generally consist of groups of chemically active surface 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 nonconformational epitopes are distinct in that the binding to the former but not to the latter is lost in the presence of denaturing solvents. Preferably an antibody according to the invention specifically binds to native CCR5, but not denatured CCR5. Such antibody preferably comprises heavy chain CDR3 of SEQ ID NO: 17, and preferably additionally heavy chain CDRs selected from the group of CDRs of SEQ ID NO: 10, 11, 12, 14 and / or 15. Preferably such antibody is antibody B, C, D, or E, or comprises antibody variable domains B, C, D, or E. Preferably an antibody that binds to CCR5 is antibody A or comprises antibody variable A domains.

The term "variable domain" (light chain variable domain (VL), heavy chain variable domain (Vh)) as used herein denotes each domain of the light and heavy chain pair of domains that is directly involved in binding. from antibody to antigen. The light and heavy chain variable domains have the same general structure, that is, they have an "immunoglobulin structure" and each domain comprises four "framework regions" (FR) 1 whose sequences are widely conserved, connected. by three "hypervariable regions" (or "complementarity determining regions", CDRs). The framework regions adopt a β-leaf conformation and the CDRs may form loops that connect the β-leaf structure. The CDRs in each chain are maintained in their three-dimensional structure by the framework regions and together with the CDRs of the other chain form the antigen binding site. The heavy and light chain CDR3 regions of the antibody play a particularly important role in the binding specificity / affinity of the antibodies according to the invention and therefore provide an additional object of the invention. The terms "antigen binding portion of an antibody" or

"antibody antigen binding site" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen binding site of an antibody comprises the amino acid residues of the "complementarity determining regions" or "CDRs". "Backbone" or "FR" regions are those whose variable domain regions other than the hypervariable region residues of an antibody comprise from the N-terminus to -C the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 regions. (immunoglobulin structure). Especially, the heavy chain CDR3 region is the region that most contributes to antigen binding and defines the antibody. Preferably the anti-CCR5 antibody according to the invention is characterized in that it comprises in its heavy chain variable domain the CDR3 sequence of SEQ ID NO: 16 or SEQ ID NO: 17. Complementarity Determination Regions (CDR) and structure (FR) are determined according to the standard definition of Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed Public Health Service, National Institutes of Health, Bethesda, MD (1991).

The "Fc portion" of an anti-CCR5 antibody is not directly involved in CCR5 binding, but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into classes: IgA, IgD, IgE, IgG, and IgM, and several of these may further be divided into subclasses (isotypes), for example, IgGI, IgG2, IgG3, and IgG4, IgAI and IgA2. According to the heavy chain constant regions the different classes of immunoglobulins are called α, δ, ε, γ, and μχ respectively. The antibodies according to the invention are preferably of the IgG type. An "Fc portion of an antibody" is a term well known in the art and defined based on papain cleavage of antibodies. Antibodies according to the invention contain as a Fc moiety a human Fc moiety, or a human derived Fc moiety. In a further embodiment of the invention the Fc moiety is an Fc moiety of a human IgG4 subclass antibody or an Fc moiety of a human IgGI subclass, IgG2, or IgG3 antibody, which is modified such that no ligand is present. Reception to the Fcy receptor (eg FcyRIIIa) and / or to C1q as defined below can be detected. Preferably the Fc portion is an especially preferred human Fc portion of the IgG4 subclass or a mutated Fc portion of the IgGI subclass. Additionally preferred are the Fc moieties of the IgGI subclass with mutations L234A and L235A. Additionally preferred are the Fc moieties 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. Although IgG4 shows reduced binding to Fcy receptor (FcyRIIIa), antibodies from other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (Fe carbohydrate loss), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Thrys7, Gln311, Asn434, and His435 are also reduced altered residues. Fcy receptor (Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9 (1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0 307 434). Preferably an antibody according to the invention is, and with respect to Fcy receptor binding, IgG4 subclass or IgGI subclass with a mutation in L234, L235, and / or D265, and / or contains the PVA236 mutation. Preferred are mutations S228P, L234A, L235A, L235E, and / or PVA236 (PVA236 means that the amino acid sequence ELLG (given in one letter amino acid code) of the IgGI or EFLG amino acid position 233 to 236 is substituted by PVA). Especially preferred are the IgG4 S228P mutations, and IgGI L234A and L235A. Fc moiety of an antibody is directly involved in ADCC (antibody dependent cell mediated cytotoxicity) and CDC (complement dependent cytotoxicity). Complement activation (CDC) is initiated by ligation of complement factor C1q to the Fc portion of most IgG antibody subclasses. C1q binding to an antibody is caused by protein-protein interactions defined at the so-called binding site. Such Fc pore linkage sites are known in the art and described, for example, by Lukas1 T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. 'et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434. Such Fc pore linkage sites are characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to (Kabat Index Ε.U.). Antibodies of the subclasses lgG1, lgG2, and lgG3 generally show complement activation including C1q and C3 binding, while lgG4 does not activate the complement system and is not bound to C1q and C3. An anti-CCR5 antibody that does not bind to Fcy receptor and / or complement factor C1q does not elicit cellular cytotoxicity depending on

antibody tooth (ADCC) and / or complement-dependent cytotoxicity (CDC). Preferably, this antibody is characterized in that it binds to CCR5, contains a human derived Fc moiety, and does not bind to Fcy receptors and / or complement factor C1q. More preferably, this antibody is a human, or humanized antibody or a T cell antigen-depleted antibody. C1q binding may be measured according to ldusogie, 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 at 405 nm for the test antibody is less than 15% of the human C1q binding value of the wild-type Fc moiety. unmodified at an antibody concentration of 8 pg / ml. ADCC can be measured as antibody binding to human FcyRIIIa in human NK cells. Binding is determined at an antibody concentration of 20 pg / ml. "No Fey receptor binding" or "no ADCC" means binding of up to 30% to human FcyRIIIa in human NK cells at an antibody concentration of 20 pg / ml compared to the same antibody binding as human IgGI (SEQ ID NO: 26).

An antibody used in a conjugate according to the invention further includes such antibodies which have "conservative sequence modifications" (variant antibodies), which are amino acid sequence modifications that do not affect or alter the characteristics mentioned above of the antibody according to the invention.

Modifications may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR mediated mutagenesis. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. Families of amino acid residues that have 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), chains

nonpolar laterals (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched laterals (eg threonine, valine, isoleucine) and aromatic laterals (eg tyrosine) , phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a human anti-CCR5 antibody may preferably be substituted for another amino acid residue of the same chain family. A "variant" anti-CCR5 antibody therefore refers to a molecule that differs in amino acid sequence from an amino acid sequence of the "parent" anti-CCR5 antibody by up to ten, preferably from about two to about five. additions, deletions, and / or substitutions in one or more of the parental antibody variable domain regions outside the heavy chain CDR3 region. Each other heavy chain CDR region comprises at most one Amino Acid Adhesion, Delegation and / or Substitution Lineage. The invention comprises a method of modifying the CDR amino acid sequence of a CCR5-binding parent antibody, characterized in that it selects a heavy chain variable domain from the group of heavy chain variable domains consisting of SEQ ID NO: 1 ， 3, 5, 7 '82，83 and / or an Ieve chain variable domain from the Ieve chain variable domain group consisting of SEQ ID NO: 2，4, 6, 8，76，77 provide an acid encoding said initial variable domain sequence, modifying said nucleic acid wherein one amino acid is modified in heavy chain C-DR1, one amino acid is modified in heavy chain CDR2, 1 to 3 amino acids are modified in chain CDR1 1 to 3 amino acids are modified in the light chain CDR2 and / or 1 to 3 amino acids are modified in the light chain CDR3, expressing and incorporating said amino acid sequence of the variable domain (s) modified (s) into an antibody structure, measure if said antibody if Ii select CCR5 and select said modified variable domain (s) / CDR (s) if antibody binds to CCR5. Preferably, such modifications are conservative sequence modifications. Amino acid sequence modifications can be made by molecular modeling-based mutagenesis as described by Riechmann1 L. 'et al., Nature 332 (1988) 323-327, and Queen, C, et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033. The term "binder" or "peptide binder" as used within this application denotes peptide binders of natural and / or synthetic origin. They are formed of a linear amino acid chain in which the 20 naturally occurring amino acids are the monomeric building blocks. The chain has a length of 1 to 50 amino acids, preferred between 1 and 28 amino acids, especially preferred between 3 and 25 amino acids. The binder may contain repetitive amino acid sequences or naturally occurring polypeptide sequences, such as polypeptides with a hinge function. The binder has the function of ensuring that a peptide conjugated to an anti-CCR5 antibody can perform its biological activity by allowing the peptide to fold properly and to display properly. Preferably, it is the binder and a "synthetic peptide binder" that is designed to be rich in glycine, glutamine, and / or serine residues. These residues are arranged, for example, into small repetitive units of up to five amino acids, such as GGGGS, QQQQG, or SSSSG. This small repetitive unit may be repeated 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 peptide binders are composed of an amino acid kinetic, which is repeated 10 to 20 times, such as, for example, serine in the SSSSSSSSSSSSS binder. At each of the terminal amino- and / or carboxy termini up to six naturally occurring arbitrary amino acids may be present. Preferred binders are shown in Table 2. Especially preferred are Binders [GQ4] 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), G3 [SG4] 2SG (SEQ ID NO: 69). All peptide ligands may be encoded by a nucleic acid molecule and therefore may be recombinantly expressed. Since the ligands are peptides themselves, the antifusogenic peptide is connected to the ligand by a peptide bridge that is formed between two amino acids. The peptide binder is introduced between ο

antifusogenic peptide and the Anti-CCRS antibody chain to which antifusogenic peptide should be conjugated. Therefore, two or three, respectively, possible sequences (in the amino to carboxy terminal direction) exist: a) antifusogenic peptide - peptide ligand - anti-CCR5 antibody polypeptide chain, or b) polypeptide chain anti-CCR5 antibody antibody - Peptide ligand - antifusogenic peptide, or c) antifusogenic peptide - Peptide ligand - anti-CCR5 antibody polypeptide chain - Peptide ligand - antifusogenic peptide.

In one embodiment of the invention is the mAb CCR5 conjugate and comprises i) the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the eve variable chain domain (VL1) defined by the SEQ ID NO: 2; or ο heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and ο heavy chain variable Ieve defined by SEQ ID NO: 4; or ο heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and ο heavy chain variable Ieve defined by SEQ ID NO: 6; or ο heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and ο heavy chain variable Ieve defined by SEQ ID NO: 8; ii) a ligand selected from the group consisting of the amino acids glycine (G) and asparagine (N) 1 ο tripeptide GST, and SEQ ID NO: 36 to 62, and SEQ ID NO: 67 to 70; and iii) an antifusogenic peptide selected from the group of peptides defined by SEQ ID NO: 29 to 35, or 73.

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] - [ binder] m- [heavy chain], (2) [heavy chain] - [binder] m- [anti-fusogenic peptide], (3) [antifusogenic peptide] - [binder] m- [heavy chain] - [peptide antifusogenic deo], (4) [antifusogenic peptide] - [ligand] m- [light chain], (5) [light chain] - [ligand] m- [antifusogenic peptide], (6) [antifusogenic peptide] - [binder] m- [light chain] - [antifusogenic peptide], (7) [antifusogenic peptide] - [binder] m- [heavy chain] - [binder] m- [antifusogenic peptide], (8) [peptide antifusogenic] - [ligand] m- [light chain] - [ligand] m- [antifusogenic peptide], wherein the ligand may be the same or different, both within and between said chain conjugates, where me is a number 1 or O, and may be independently the same or different, both within and between the the conjugates. For example, in a conjugate comprising a chain conjugate (7) and a mAb CCR5 Ieve chain the two linkers in the chain conjugate (7) may be the same, that is, have the same amino acid sequence, or they may be different, that is, have different amino acid sequences and / or lengths, or one or both may be absent. For example, in a conjugate comprising chain conjugates (2) and (4) ο ligand contained in the chain conjugate (2) and ο ligand contained in the chain conjugate (4) may be the same, i.e. the same amino acid sequence and length, or may be different, that is, have different amino acid sequences and / or lengths, or one or both may be absent. In chain conjugates the linker (s) may be present (m = 1) or absent (m = 0). Preferred chain conjugates are the chain conjugates (2), (3), (4), and (7). One embodiment of the present invention is a conjugate comprising 2 χ [Ive mAb CCR5 chain] and 2 χ chain conjugate (2). This conjugate comprises two light chains of unconjugated anti-CCR5 antibody and two heavy chains of conjugated anti-CCR5 antibody through the C-terminus to N-termination of an antifusogenic peptide, optionally with an intermediate ligand. Another embodiment of the present invention is a conjugate comprising two CCR5 mAb Ieve 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 present invention is a conjugate comprising two CCR5 mAb Ieve chains and two Ieve chain conjugates (7). The heavy and / or light chain preferably comprises a constant region (Fe).

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 purposes.

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

The term "amino acid" as used within this application shall

note ο naturally occurring carboxy α-amino acid group comprising alanine (three letter code: ala, one Ietra code, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp , D), cysteine (cis, C), glutamine (gin, Q), gutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), yeucine (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).

Known methods and techniques of one of skill in the art, which are useful for carrying out the present invention, are described, for example, Ausubel, F.M., ed., Current Protocols in Molecular Biology, Volumes I to III (1997), Wiley and Sons; Sambrook et al., Molecular Cloning: A Manual Laboratory, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

In conjugates according to the invention the carboxy-terminal amino acid of an antibody chain is conjugated via a peptide linker to the antifusogenic peptide peptide or the carboxy-terminal amino acid of the antifusogenic peptide and conjugated via a peptide linkage to the amino acid -terminal of an antibody chain. In one embodiment an intermediate ligand is present between the antifusogenic peptide and the antibody chain. Thus, it is conjugated according to the invention and characterized by the general formula

antibody - [ligand] m- [antifusogenic peptide] wherein m is independently for each antifusogenic peptide or O (i.e., a direct peptide linkage between ο antibody and antifusionogenic peptide) or 1 (i.e., ο ligand is present between ο antibody and antifusogenic peptide) and η and an integer from 1 to 8. In one embodiment, η is an integer from 2 to 8. In another embodiment, η is an integer from 2 to 4. In another embodiment , η is an integer of 2 or 4. One embodiment of the invention comprises a conjugate comprising an antifusogenic peptide at the C-terminus of the heavy chains or at each N-terminus of the antibody I-chains. In this embodiment two antifusogenic peptides are conjugated to an antibody. In another embodiment, it is conjugated and characterized in that it comprises an anti-fusogenic peptide at each heavy chain C-terminus and at each light chain N-terminus. In this embodiment four antifusogenic peptides are conjugated to an antibody. In one embodiment said antibody is mAb CCR5 or mAb CD4.

The antifusogenic peptide that is introduced at a terminus of the heavy and / or light chain (s) of a CCR5 mAb is small in size compared to CCR5 mAb. For example, the smallest immunoglobulins, class G immunoglobulins, have a molecular weight of approximately 150 kDa; an antifusogenic peptide preferably has a size (molecular weight) of less than 12.5 kDa, which is equivalent to about 100 amino acids, generally less than 7.5 kDa, which is equivalent to about 60 amino acids. The antifusogenic peptide has an amino acid sequence of 5 to 100 amino acid residues, preferably 10 to 75 amino acids, more preferably 15 to 50 amino acid residues. The conjugates of the present invention are useful for pharmaceutical, therapeutic or diagnostic applications. The number of antifusogenic peptides, which may be conjugated to mAb CCR5 heavy and / or heavy chain (s), and one to the combined number of amino and carboxy chain terminates of anti-CCR5 antibody polypeptide. Since the present invention encompasses different anti-CCR5 antibodies, the number of antifusogenic peptides may vary. In the case of an anti-CCR5 antibody comprising two light and two heavy chains, the combined number of amino termini (N-terminus) and carboxy terminates (C-terminus) and eight, which is at the same time the total maximum peptide number conjugated antifungal agents; in the case, for example, of an anti-CCR5 antibody fragment such as a kinetic chain antibody (scFv) is the combined number of terminations and thus the maximum number of conjugated antifusogenic peptides and two. If an antifusogenic peptide kinetic is conjugated to the CCR5 mAb, the peptide may occupy any of the anti-CCR5 antibody chain terminations. Similarly, if the maximum possible number of peptides is conjugated to mAb CCR5, all terminations will be occupied by a peptide kinetic. If the number of peptides which are conjugated to mAb CCR5 is less than the maximum possible number, different distributions of the peptides at the anti-CCR5 antibody chain terminations are possible. For example, if four peptides are conjugated to a class G or E immunoglobulin, five different combinations will be possible (see Table 1). In two combinations, all terminations of the type, that is, all four amino termini or all four carboxy termini of the anti-CCR5 antibody chains, are conjugated to an antifusogenic peptide kinetic. The other terminations are not combined. This results in a modality in an allocation of modifications / conjugations in an area of the anti-CCR5 antibody. In other cases, the polypeptides are conjugated to a number of both terminations. Within these combinations, conjugated peptides are allocated to different areas of the anti-CCR5 antibody. In either case, the sum of the conjugate terminations is four.

Table 1: Possible combination for the conjugation of four peptides as

terminations of an anti-CCR5 antibody composed of four polypeptide chains.

A Busy amino terminus number Busy carboxy terminus number Total occupied number of terminations 4 O 4 3 1 4 2 2 4 1 3 4 O 4 4

The present invention preferably comprises conjugates

wherein at least two of the terminations are conjugated to an antifusogenic peptide. The amino acid sequences of antifusogenic peptides may be different, similar or identical. In one embodiment, the amino acid sequence identity is in the range of 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, that is, they have a 100% amino acid identity.

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 conjugated to a heavy and / or light chain termination of said anti-CCR5 antibody. through a peptide ligation. 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 coupled to a heavy and / or short chain termination thereof. anti-CCR5 antibody.

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

SEQ ID NO: 40 is an antifusogenic peptide of SEQ ID NO: 33. Conjugation between antifusogenic peptide and anti-CCR5 antibody is performed at the nucleic acid level. Therefore, a peptide ligation is formed between the antifusogenic peptide and the anti-CCR5 antibody chain with or without an intermediate ligand. 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. and is conjugated to the amino-terminal amino acid of the antifusogenic peptide with or without an intermediate ligand or both anti-CCR5 antibody chain terminations are conjugated to an antifusogenic peptide each with or without an intermediate ligand. For 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. Such nucleic acid molecules encode the different conjugate anti-CCR5 antibody polypeptide chains and are referred to below as structural genes. They may be located on the same expression plasmid (vector) or alternatively they may be located on different expression plasmids (vectors). The conjugation of the conjugate preferably occurs prior to conjugate secretion and thus within the same expressing cells. Therefore, nucleic acid molecules encoding the conjugate polypeptide chains are preferably expressed in the same host cell. If, after recombinant expression, a conjugate mixture is obtained, the conjugates may be separated and purified by methods known to the person skilled in the art. These methods are well established and widely used for immunoglobulin purification and are employed alone or in combination. Such methods are, for example, affinity chromatography using microbiologically derived proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins)). , exchange

anion (aminoethyl resins) and mixed mode exchange chromatography), thiophilic adsorption (for example with beta-mercaptoethanol and other SH ligands), hydrophobic interaction chromatography or aromatic adsorption (for example with phenyl sepharose, resins aza-arenophilic or aminophenylboronic acid), metal chelate affinity chromatography (eg Ni (II) - and Cu (II) affinity material), size exclusion chromatography and preparative electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102). With recombinant manipulation methods known to one of skill in the art, conjugates can be produced at the nucleic acid / gene level. Nucleic acid sequences encoding immunoglobulins are known and can be obtained, for example, from genomic databases. Similarly, nucleic acid sequences encoding antifusogenic peptides are known or can be readily deduced from their amino acid sequences. The elements necessary for constructing an expression plasmid for expression of a conjugate of the present invention are, for example, an expression cassette for the anti-CCR5 antibody Ieve chain in its natural and / or modified and / or modified version. conjugated, an anti-CCR5 antibody heavy chain expression cassette in its natural and / or modified and / or conjugated version (alternatively, the anti-CCR5 antibody light chain and anti-CCR5 antibody heavy chain may contained in the same expression case, for example as a bicistronic expression element), a selection marker and an E. coli replication unit as well as a selection unit. Such expression cassettes comprise a promoter, a DNA segment encoding a secretion signal sequence, the structural gene and a termination / polyadenylation signal. The elements are assembled in an operably linked form in both a plasmid encoding all conjugate chains and two or more plasmids each encoding one or more conjugate chains. For expression of the encoded polypeptides, the plasmid (s) and (s) are introduced into a suitable host cell. Proteins are preferred

produced in mammalian cells such as CHO1 cells, NSO1 cells, Sp2 / 0 cells, COS cells, HEK cells, K562 cells, BHK1 cells PER.C6® cells and the like. Regulatory elements should be selected such that they are functional in the selected host cell. For expression, the host cell containing the plasmid encoding one or more chains of the conjugate is cultured under conditions suitable for expression of the chains. The expressed conjugate chains are functionally joined. 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, for example, for E. coli, which comprises a replication origin and a resistance gene, a eukaryotic selection marker and one or more expression cassettes for expression of the structural gene (s) of interest comprising a promoter, a structural gene and a transcription terminator that includes a polyadenylation signal. Gene expression is generally placed against a promoter and such a structural gene is said to be "operably linked" to the promoter. Similarly, a regulatory element and a central promoter are operatively linked if the regulatory element modulates the activity of the central promoter.

One aspect of the present invention is, therefore, a method for producing a conjugate according to the invention, comprising the following steps:

a) culturing 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 expression of the conjugate,

(b) recover the cell or supernatant conjugate.

The term "under conditions suitable for expression of the conjugate" denotes conditions which are used for the purpose of a cell expressing a polypeptide and which are known or can be readily determined by one of skill in the art. It is well known to one skilled in the art that such conditions may vary depending on the type of cultured cell and the type of expressed polypeptide. In general, the cell is grown at a temperature, for example, between 20 ° C and 40 ° C, and for a period of time sufficient to allow effective production of the polypeptide conjugate, for example, 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 adsorptive retarding agents and the like, which are compatible physiologicamerite. Preferably, the vehicle 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 dispersions. The use of such media and agents for a pharmaceutically active substance is known in the art. In addition to water, the vehicle may 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 in pharmaceutically acceptable dosage forms by conventional methods known to the art. those skilled in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be altered to provide an amount of the active ingredient that is effective in obtaining the desired therapeutic response for a particular patient, composition and method. of administration without being toxic to the patient. The dosage level selected 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 to be employed, other drugs, compounds and / or materials used in combination with the particular composition employed, age, sex, weight, general health condition and previous medical history.

of the patient to be treated and similar 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.

The following examples, Sequence List, Figures, and Deposition

These are provided to assist in understanding the present invention, the true scope of which is described in the appended claims. It is understood that modifications may be made to the described procedures without departing from the spirit of the invention. Description of the Figures:

Figure 1: Ieve κ chain plasmid map of mAb CCR5

Figure 2: Plasmid map of mAb CCR5 γ1 heavy chain. Figure 3: Plasmid map of the

mAb CCR5 γ1 heavy chain conjugate Figure 4: Plasmid map of the CD4 mAb Ieve κ chain.

Figure 5: Plasmid map of CD4 mAb γ1 heavy chain.

Figure 6: Plasmid map of CD4 mAb γ1 heavy chain conjugate. Anti-CCR5 Antibody Deposit

Preferred mAb CCR5 expressing hybridoma cell lines Useful in the conjugates according to the invention were deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany.

Deposit Cell Line No. Deposit Date m <CCR5> Pz01.F3 DSM ACC 2681 18.08.2004 m <CCR5> Pz02.1C11 DSM ACC 2682 18.08.2004 m <CCR5> DSM ACC 2683 18.08.2004 m <CCR5 > Pz04.1F6 DSM ACC 2684 18.08.2004

expression vector 4900 of expression vector 4901 of expression vector 4995 of expression vector 6310 of expression vector 6309 of expression vector of antibody Nomenclature 6303 of antibody

<CCR5 »Pz011.F3: Antibody A SEQ ID NO: 1 '2 <CCR5> Pz02.1CII: Antibody B SEQ ID NO: 3' 4 <CCR5> Pz03.1C5: Antibody C SEQ ID NO: 5, 6 <CCR5 > F3.1H12.2E5: Antibody D SEQ ID NO: 7, 8

<CCR5> Pz04.1F6: Antibody E

The CD4 mAb variable domain 1 is reported in SEQ ID NO: 10, 15, 45 'and 56 of US 5,871,732.

Variable domains 2 and 4 of mAb CD4 are reported in Remannmann, K.A., et al., Aids Res. Human Retrovir. 13 (1997) 933-943.

The variable domain 3 of mAb CD4 is reported in Figures 3, 4 '12, and 13 of WO 91/009966.

Antibody Sequences. antifusogenic peptide sequences and peptide linker sequences

SEQ ID NO 1 heavy chain, <CCR5> variable domain Pz01.F3 SEQ ID NO 2 heavy chain, <CCR5> variable domain Pz01 .F3 SEQ ID NO 3 heavy chain, <CCR5> variable domain Pz02.1C11 SEQ ID NO 4 light chain, <CCR5> variable domain Pz02.1C11 SEQ ID NO 5 heavy chain, <CCR5> Pz03.1C5 variable domain SEQ ID NO 6 light chain, <CCR5> Pz03.1C5 variable domain SEQ ID NO 7 chain heavy, <CCR5> F3.1H12.2E5 variable domain SEQ ID NO 8 light chain, <CCR5> F3.1H12.2E5 variable domain SEQ IDNO 9 mAb heavy chain CDR1 SER IDNO 10 mAb heavy chain CDR1 CCR5 SEQ IDNO 11 mAb heavy chain CDR1 CCR5 SEQ IDNO 12 mAb heavy chain CDR1 CCR5 SEQ IDNO 13 mAb heavy chain CDR2 CCR5 SEQ ID NO 14 CCR5 mAb heavy chain CDR2 SEQ IDNO 15 mAb CCR5 SEQ ID NO 16 mAb heavy chain CDR3 mAb CCR5 SEQ ID NO 17 mAb CCR5 heavy chain CDR3 SEQ ID NO 18 mAb CCR5 light chain CDR1 10

15

20

25

30

SEQ ID NO: 19 mAb CCR5 Ieve Chain CDR1 SEQ ID NO: 20 mAb CCR5 Ieve Chain CDR1 SEQ ID NO: 21 mAb CCR5 Ieve Chain CDR2 SEQ ID NO: 22 NO: 23 mAb Ic-chain CDR2 CCR5 SEQ ID NO: 24 mAb Ic-chain CDR3 CCR5 SEQ ID NO: 25 mAb Ic-chain CDR3 CCR5 SEQ ID NO: 26 γ1 heavy chain constant region SEQ ID NO: 27 heavy chain constant region γ4 SEQ ID NO: 28 Ieve K chain constant domain SEQ ID NO: 29 C34 SEQ ID NO: 30 T20 SEQ ID NO: 31 T1249 SEQ ID NO: 32 T651 SEQ ID NO: 33 T2635 SEQ ID NO : 34 N36 SEQ ID NO: 35 DP107 SEQ ID NO: 36 to 62, 67 to 70 Binding Peptides SEQ ID NO: 63 Chain I Amino Acid Sequence

SEQ ID NO: 64

SEQ ID NO: 65

SEQ SEQ

ID ID

NINTH:

66 71

SEQ ID NO: 72

SEQ SEQ

ID NO: ID NO:

73

74

i κ of mature CCR5 mAb

Mature CCR5 mAb γ1 heavy chain amino acid sequence

Heavy chain amino acid sequence of HIV-1 mature CCR5 mAb conjugate gp41

Ieve κ chain amino acid sequence of mature CD4 mAb

MAb γΙ heavy chain amino acid sequence

Mature CD4

afp-1

Conjugate Heavy Chain Amino Acid Sequence

mature CD4 mAb SEQ ID NO 75 murine CD4 mAb Ive 1 variable domain SEQ ID NO 76 chimeric mAb CD4 Ieve 1 stranded domain SEQ ID NO 77 chimeric mAb CD4 Ieve 1 strand IQ 1 chain chimeric CD4 mAb Ieve 2 chain variable domain SEQ ID NO 79 chimeric CD4 mAb Ieve chain variable 3 domain SEQ ID NO 80 chimeric CD4 mAb Ieve chain variable 4 domain SEQ ID NO 81 mAb heavy chain 1 variable domain 1 Mu-Ι-ΪΠΛ CD4 SEQ ID NO: 82 ΓΙΠΟ mAb chimeric CD4 heavy chain 1 variable domain SEQ ID NO: 83 chimeric mAb CD4 heavy chain 1 variable SEQ ID NO: 84 mAb CD4 chimericx heavy chain 2 variable domain ) SEQ ID NO: 85 chimeric mAb CD4 heavy chain 3 variable domain SEQ ID NO: 86 mAb CD4 chimericx heavy chain 4 variable domain) SEQ ID NO: 87 mAb CD4 Ieve chain light chain CDR1 SEQ ID NO: 88 CDR1 CD4 mAb Ieve chain strand SEQ ID NO: 89 CD4 mAb Ieve chain strand SEQ ID NO: 90 CDR1 CD4 mAb Ieve chain ID SEQ: NO: 91 CD4 mAb Ieve chain IDR SEQ ID NO: 92 CD4 mAb Ieve chain IDR SEQ ID NO: 93 CDA mAb Ieve chain CDR2 SEQ ID NO: 94 chain CDR3 CD4 mAb eve SEQ ID NO: 95 CD4 mAb eve CD4 mAb eve CD4 mAb eve CDR3 SEQ ID NO: 97 CD4 mAb heavy chain CDR1 SEQ ID NO: 98 mAb CD4 SEQ ID NO: 99 mAb heavy chain CDR1 CD4 SEQ ID NO: 100 mAb heavy chain CDR2 CD4 SEQ ID NO: 101 mAb CD4 heavy chain CDR2 SEQ ID NO: 102 mAb CD4 heavy chain CDR2 SEQ ID NO: 103 CD4 mAb heavy chain CDR3 SEQ ID NO: 104 CD4 mAb heavy chain CDR3 SEQ ID NO: 105 CD4 mAb heavy chain CDR3 Table 2: Binder

N0 Binding Peptides SEQ ID NO: 1 lslspgk 36 2 lspnrgec 37 3 (GQ4) 3 38 4 [gq4] 3g 39 [gq4i3gnn 40 6 GGGfSG4J2SGG 41 7 ggg [sg4] 2sgn 42 8 [sg4j3 43 9 [sg4i3g 44] 3t 45 11 [sg4i3gg 46 12 [sg4j3ggt 47 13 [sg4j3ggn 48 14 [sg4j3ggn 49 [SG4J5 50 16 [SG4J5G 51 17 (sg4j5gg 52 18 [sg] 15g 54 g (s) 15gas 55 21 g - 22 n - 23 gst - 24 f (G) 4Sl3GAS 56 [(g) 4sl3g 57 26 i (g) 4s] 5g 58 27 I (G) 4Sl3GG 59 28 i (g) 4sl5gg 60 29 lslsgg 61 lslspgg 62 31 [G3Sl5 67 32 [G3Sl5GGG 68 33 gjsgd 2sg 69 34 gjsgj7sg1 70 Examples

Materials and Methods

General information regarding the nucleotide sequences of the human immunoglobulin heavy and Ieve chains is given in: Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered according to EU number (Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, EA, et al., Sequences of Proteins from Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

Recombinant DNA Techniques:

Usual methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. Gene Synthesis:

Desired gene segments were prepared from oligonucleotides made by chimeric synthesis. Segments 100 to 600 bp in length, which are flanked by Linnose1 restriction endonuclease cleavage sites, were joined by oligonucleotide annealing and ligation, including PCR amplification, and subsequently cloned into the pCR2 cloning vector. 1-TOPO-TA (Invitrogen Corp., USA) through A-projections ("Α-overhangs"). The DNA sequence of subcloned gene fragments was confirmed by DNA sequencing. Protein Determination:

Protein concentration of the conjugate was determined by determining the optical density (OD) at 280 nm using the coefficient of

molar extinction calculated based on amino acid sequence. Example 1 ： Production of Anti-CCR5 Antibody Expression Plasmids

The genetic segments encoding the anti-CCR5 antibody (I) antibody Ieve chain variable domain and the human Iappa (Ic) constant chain domain were joined as if they were gene segments for the antibody heavy chain variable domain anti-CCR5 and the human γ1 heavy chain constant domains (CHl-hinge-9a-CH2-CH3).

In the case of mAB CCR5 of SEQ ID NO: 63/64, the heavy and light chain variable domains are derived from a mouse antibody and the heavy and light chain constant domains are derived from a human antibody (C -kappa and IgGI).

Subsequently1 the genomic segment encoding a complete Ieve chain of anti-CCR5 antibody was ligated at the N-terminus and / or -C termination with a nucleic acid encoding an antifusogenic peptide including a linker sequence and / or genomic segment encoding a chain. A complete heavy weight of anti-CCR5 antibody was ligated at the N- and / or -C-terminus with a nucleic acid encoding an antifusogenic peptide including a ligand binding sequence. a) Vector 4900

Vector 4900 is an expression plasmid for transient expression of an IAB CCR5 Ieve strand (genomically organized expression cassette; exon-introri organization) in HEK293 cells.

In addition to the mE CCR5 Ieve κ chain expression cassette, this vector contains:

-a hygromycin resistance gene as a selectable marker

questionable,

an Epstein-Barr virus (EBV) origin of replication, oriP, a pUC18 vector replication origin that allows replication of this plasmid into E. coli cells, and

a β-lactamase gene that confers ampicillin resistance

in E. coli.

The transcription unit of the IAB κ chain gene of mAB CCR5 is composed of the following elements:

-the immediate early enhancer and promoter of human cytomegalovirus,

-a synthetic untranslated 5 'region, - an immunoglobulin heavy chain signal sequence

murine including a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-intron-L2]),

-the Ieve chain is a mature variable anti-murine CCR5 antibody encoding the segment arranged with a unique 5 'end Bsml restriction site (L2 signal sequence), and a splice donor site and a Notl Cinical restriction site at the 3 'end,

-a human / mouse Ieve κ chain intron 2, -ο human κ-light gene constant domain, -human immunoglobulin ("poly A") signal sequence, and,

The Ascl and Fsel Linearic restriction sites at the 5 'and 3' ends respectively.

Plasmid map of mAB CCR5 IEve κ chain expression vector 4900 is shown in Figure 1. The amino acid sequence of mAB CCR5 mature κ-light chain (without signal sequence) is shown in SEQ ID NO : 63. b) Vector 4991

Vector 4991 is an expression plasmid for transient expression of a mRNA γ1 heavy chain CCR5 (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

In addition to the mAB CCR5 γ1-heavy chain expression cassette this vector contains:

-a hygromycin resistance gene as a selectable marker

questionable,

- an origin of replication, oriP, from Epstein-Barr virus (EBV) 1

- an origin of replication of the pUC18 vector that allows replication

cation of this plasmid in E. coli is a β-lactamase gene that confers ampicillin resistance

in E. coli.

The mRNA CCR5 γΐ-heavy chain gene transcription unit is composed of the following elements: - It is an early cytoplasmic enhancer and promoter.

human megalovirus,

-a synthetic 5 'untranslated region,

-a murine immunoglobulin heavy chain signal sequence including a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-intron-L2]),

-the mature variable heavy chain of murine anti-CCR5 antibody encoding the segment arranged with a 5 'end Bsml restriction site kinetic (L2 signal sequence), and a splice donor site and site. Notl Lineage restriction at the 3 'end, - a human / mouse heavy chain hybrid intron 2

including the mouse heavy chain enhancer element (poron JH3, JH4) (Neuberger, M.S., EMBO J. 2 (1993) 1373-1378)

-the genomic constant domains of the human heavy γ1 gene

manna,

- the γ1-polyadenylation ("poly A") signal sequence

human immunoglobulin and,

- the Ascl and SgrAI ionic restriction sites at the 5 'and 3' ends respectively.

Plasmid map of mAB CCR5 γ1-heavy chain expression vector 4901 is shown in Figure 2. The amino acid sequence of mAB CCR5 mature γ1-heavy chain (without signal sequence) is shown in SEQ ID NO: 64. c) Vector 4995

Vector 4995 is an expression plasmid for transient expression of an anti-CCR5 antibody chimeric heavy chain peptide conjugate (genomically organized expression cassette;

exon-intron zagation) in HEK293 cells. The vector 4995 is derived from plasmid 4991 by the fact that the γΐ-heavy chain of mAB CCR5 is ligated at the C-terminus with a nucleic acid encoding the antifusogenic peptide T-2635 (SEQ ID NO: 33) and the ligand peptide sequence. [GQ4] 3GNN (SEQ ID NO: 40).

In addition to the expression peptide conjugate cassette

γΙ-heavy chain of anti-CCR5 antibody this vector contains:

-a hygromycin resistance gene as a selectable marker

questionable,

- an Epi-Barr virus (EBV) 1 oriP replication origin 1 - a pUC18 vector replication origin that allows replication

cation of this plasmid in E. coli, and

a β-lactamase gene that confers ampicillin resistance

in E. coli.

The transcription unit of the anti-CCR5 antibody chimeric peptide conjugate is γ e-heavy chain and is composed of the following elements:

early-enhancer and promoter of human cytomegalovirus,

- a synthetic untranslated 5 'region, - an immunoglobulin heavy chain signal sequence

murine including a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-intron-L2]),

-the mature variable heavy chain of the murine anti-CCR5 antibody encoding the segment arranged with a 5 'end Bsml restriction site kinetic (L2 signal sequence), and a splice donor site and site. Notl restriction restriction at the 3 'end,

- a human / mouse heavy chain hybrid intron 2, including the mouse heavy chain enhancer (poron JH3, JH4) (Neuberger, MS, EMBO J. 2 (1993) 1373-1378) - the domains genomic constants of the gene / 1 -human weight-

at the,

the antifusogenic peptide T-2635, the ligand peptide sequence [GQ4] 3GNN, the human γ1-immunoglobulin polyadenylation ("poly A") signal sequence, and

- Ascl and SgrAI kinetic restriction sites at the 5 'and 3' ends respectively.

Plasmid map of mAB CCR5 γΙ-heavy chain peptide conjugate expression vector 4995 is shown in Figure 3. The conjugate mature heavy chain amino acid sequence (without signal sequence) is shown in SEQ ID NO: 65. Example 2:

Production of the final anti-CCR5 antibody expression plasmids

Fusion genes (antibody heavy and / or ve chain fusion genes) comprising a CCR5 mAB gene segment, an optional ligand gene segment, and an antifungal gene peptide segment have been joined by methods and techniques of fusion. recombination processes by connecting the concordant nucleic acid segments. Nucleic acid sequences encoding peptide ligands and antifusogenic polypeptides were synthesized by chimeric synthesis and then ligated into an E. coli plasmid for amplification. Subcloned nucleic acid sequences were verified by DNA sequencing. Example 3:

Transient Expression of Immunoglobulins and Immunoglobulin Variants in EBNA HEK293 Cells

Recombinant anti-CCR5 antibodies and anti-CCR5 antibody variants were generated by transient transfection of adherent culture HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr virus nuclear antigen; American type culture collection ATCC deposition number # CRL-10852) grown in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) supplemented with 10% FCS ultra-low IgG (fetal bovine serum, Gibco), 2 mM Glutamine (Gibco), 1% volume non-essential amino acids by volume Gibco) and 250 mg / ml of

G418 (Roche Molecular Biochemicals). For transfection FuGE-NΕ® 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a reagent ratio (μΙ) to DNA (pg) ranging from 3: 1 to 6: 1. The Ieve and heavy chains, including the Ieve and heavy chains of the anti-CCF5 antifusogenic peptide-antibody peptide conjugate were expressed from two different plasmids using a heavy chain Ieve-chain molar ratio encoding 1: 2 plasmid at 2: 1, respectively. Antifusogenic peptide-anti-CCR5 antibody conjugates containing cell length supernatants were collected on day 4 to 11 after transfection. General information regarding recombinant expression of human immunoglobulins, for example in HEK293 cells, is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203. Example 4

Expression analysis using SDS PAGE, Western blotting, and detection with immunoglobulin-specific antibody conditons. The expressed and secreted antifusogenic peptide conjugates

anti-CCR5 antibody were processed by sodium polyacrylamide dodecyl sulfate (SDS) gel electrophoresis (SDS-PAGE) and the separate chains of anti-CCR5 antibody and anti-CCR5 antibody-antifusogenic peptide conjugate They were transferred from the gel to a membrane and subsequently detected by an immunological method. SDS-PAGE:

LDS Sample Buffer, 4x Concentrate (4x): 4g glycerol, 0.682g TRIS-base, 0.666g TRIS-hydrochloride, 0，8g LDS (lithium dodecyl sulfate), 0.006g EDTA (ethylenediaminetetraacetic acid), 0.75 ml of a 1 wt% (w / w) solution of Serva Blue G250 in water, 0.75 ml of a 1 wt% (w / w) phenol red solution and water to make a total volume of 10 ml.

The culture broth containing antifusogenic peptide-anti-CCR5 antibody secreted conjugate was centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was mixed with 1/4 volume (v / v) 4x LDS sample buffer and 1/10 volume (v / v) 1.4-

0.5 M dithiothreitol (DTT). The samples were then incubated for 10 min at 70 ° C and the protein separated by SDS-PAGE. The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast Gels (pH 6.4) and a NuPAGE® MOPS Running Buffer were used.

Western blot:

Transfer Buffer: 39 mM glycine, 48 mM TRIS hydrochloride, 0.04 wt% SDS (w / w) and 20 wt% ethanol (v / v).

After SDS-PAGE, the separate strands of antifusogenic peptide-anti-CCR5 antibody conjugate were electrophoretically transferred to a nitrocellulose filter membrane (pore size: 0.45 μιτι) according to "Semi Blotting Method". Burnette (Burette, WN, Anal. Biochem. 112 (1981) 195-203). Immunological Detection:

TBS Buffer: 50 mM TRIS hydrochloride, 150 mM NaCl, pH adjusted to 7.5.

Blocking Solution: 1% (w / v) Western Blocking Reagent (Molecular Molecular Biochemicals) in TBS buffer.

TBST Buffer: 1x TBS buffer with 0.05% Tween-20 by volume (v / v).

For immunological detection western blotting membranes

they were incubated with shaking at room temperature twice for 5 minutes in TBS buffer and once for 90 minutes in blocking solution. Detection of peptide and immunoglobulin conjugate chains:

Heavy Chain: For detection of the heavy chain of the antifusogenic peptide-anti-CCR5 antibody conjugate, a purified peroxidase conjugated rabbit anti-human IgG antibody (DAKO, Code No. P.0214) was used.

Light chain: The Ieve chain of the anti-fusion-gene peptide-anti-CCR5 antibody conjugate was detected with one with a purified peroxidase-conjugated human anti-Ieve kappa chain antibody (DAKO, Code No. P01212).

For visualization of antibody Ieve and heavy chains, Iavated and blocked Western blot membranes were first incubated in the case of a heavy chain with a purified peroxidase-conjugated rabbit anti-human IgG antibody or It is an Ieve chain with a purified peroxidase-conjugated human anti-Ieve kappa rabbit antibody in a 1: 10,000 dilution in 10 ml blocking solution at 4 ° C with overnight stirring. After washing the membranes three times with TBST buffer and once with TBS buffer for 10 min at room temperature, the Western blot membranes were treated with a chemiluminescent Luminol / peroxide solution (Lumi- Lightpujs Western Blotting Substrate, Roche Molecular). Biochemicals). Then the membranes were incubated in 10 ml Luminol / peroxide solution for 10 seconds to 5 minutes and the emitted Light was subsequently detected with a LUMI-Imager FL Analyzer (Roche Molecular Biochemicals) and / or recorded with a film. x-ray. The intensity of the markings was quantified with the LumiAnaIyst Program (Version 3.1). Multiple-Immunoblot Labeling:

Secondary peroxidase labeled antibody conjugate used for detection can be removed from the labeled blot by incubating the membrane for one hour at 70 ° C in 1M TRIS-hydrochloride buffer (pH 6.7) containing 100 mM beta-mercaptoethanol and SDS at 20% (w / v). After this treatment the blot may be labeled with a different secondary antibody a second time. Prior to the second detection, the blot is washed three times at room temperature with shaking in TBS buffer for 10 minutes each time.

Example 5

Affinity Purification. dialysis and concentration of peptide and immunoglobulin compounds

Expressed and secreted antifusogenic peptide-anti-CCR5 antibody conjugates were purified by affinity chromatography using Protein A-Sepharose® CL-4B (former GE Healthcare Amersham Bioscience, Sweden) according to known methods. Briefly,

After centrifugation (10,000 g for 10 minutes) and filtration through a 0.45 μιτι filter, clarified culture supernatants containing the peptide-immunoglobulin conjugate were applied to a Protein A-Sepharose® CL column. -4B equilibrated with PBS buffer (10 mM Na 2 HPO 4, 1 mM KH 2 PO 4, 137 mM NaCl and 2.7 mM KCI, pH 7.4). Unlinked proteins were removed with PBS equilibration buffer and 0.1 M citrate buffer, pH 5.5. Antifusogenic peptide-anti-CCR5 antibody conjugates were eluted with 0.1 M citrate buffer, pH 3.0 and fragments containing the conjugate were neutralized with 1M TRIS-Base. Then, antifusogenic peptide-anti-CCR5 antibody conjugates were intensively dialyzed against PBS buffer at 4 ° C, concentrated with an Ultrafree®-CL Centrifugal Filter Unit equipped with a Biomax-SK membrane (Millipore Corp., USA) and stored in an ice-water bath at 0 ° C. The integrity of the conjugates was analyzed by SDS-PAGE in the presence and absence of a reducing agent and stained with bright Coomassie blue as described in Example 4. Aggregation of antifusogenic peptide-anti-CCR5 antibody conjugates was analyzed. by size exclusion analytical chromatography. Example 6

Deglycosylation of peptide and immunoglobulin conjugates Anti-CCR5 antibody N-linked carbohydrates and conjugates

of antifusogenic peptide-anti-CCR5 antibody were cleaved by enzymatic treatment with Peptide-N-Glycosidase F (PNGaseF, Roche Molecular Biochemicals, Mannheim, Germany or Prozyme, San Leandro1 CA). Next, anti-CCR5 antibodies and antifusogenic peptide-anti-CCR5 antibody conjugates were incubated at 37 ° C for 12 to 24 h using PNG-seF 50 mU per mg N-glycosylated protein in PBS buffer with a concentration of about 2 mg / ml protein. Then, Peptide-N-Glycosidase F was separated by preparative gel filtration according to known methods. Briefly, anti-CCR5 antibodies and PNGaseF-treated antifusogenic peptide-anti-CCR5 antibody conjugates were applied to a Superose® 12 10/300 GL column (GE Healthcare former Amer-

Bioscience, Sweden) equilibrated with PBS buffer (10 mM Na 2 HPO 4, 1 mM KH 2 PO 4, 137 mM NaCl and 2.7 mM KCI, pH 7.4) and then eluted with equilibration buffer at a flow rate of 0.5 to 1.0 ml / min using the Amersham Bioscience Akta Exploratory Chromatography System (former GE Healthcare Amersham Bioscience, Sweden).

Example 7

Kinetic cycle antiviral activity assay

For the production of pseudotyped NL-Bal viruses, the plasmid pNL4-3Aenv (HIV pNL4-3 genomic construct with a deletion within the env gene) and pCDNA3.1 / NL-BAL env [pcDNA3.1 plasmid containing the NL env gene -BaI (obtained from NIBSC Centralized Facility for AIDS Reagents)] were co-transfected into HEK 293FT cell line (Invitrogen) grown in Dulbecco's minimal modified medium (DMEM) containing 10% fetal bovine serum (FCS), 100 U / ml Penicillin, 100 Mg / ml Streptomycin, 2 mM L-Glutamine and 0.5 mg / ml Geniticin (all Invitrogen / Gibco media). Supernatants containing pseudotyped viruses were collected two days after transfection and cell debris was removed by filtration through a 0.45 µm PES (polyethersulfone) filter (NaIgene) and stored at -80 ° C in aliquots. . For normalization for assay performance, virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells generating approximately 1.5 χ 105 RLU (relative light units) per well. Test antifusogenic peptide-anti-CCR5 antibody conjugates, reference antibodies, and reference antifusogenic peptides (T-20, T-1249, T-651, and T-2635) were serially diluted in 96-well plates. The assay was performed in quadruplicates. Each plate contained control cell and control virus wells. The equivalent of 1.5 x 105 RLU of virus stocks was added to each well, then 2.5 x 104 JC53-BL cells were added to each well, with a final assay volume of 200 μΙ per well. After 3 days incubation at 37 ° C, 90% Relative Humidity and 5% CO2, the media was aspirated and 50 μΙ Steady-Glo® Luciferase Assay System (Promega) was added to each well. The test plates

They were read on a Luminometer (Luminoskan, Thermo Electron Corporation) after 10 min incubation at room temperature. Percent inhibition of Iuciferase activity was calculated for each dose after background subtraction, and IC50 and IC90 values were determined using the Excel XLfit curve fitting program (version 3.0.5 Buildl2; Microsoft ).

The results are shown in Table 3.

Table 3: Antiviral activity of antifusogenic polypeptides, antibodies and

antifusogenic peptide-anti-CCR5 antibody conjugates

Compound Antiviral activity IC50 (ng / ml) IC90 (ng / ml) Inactive inactive reference 1 antibody (inert) Inactive inactive reference antibody 2 (inert) T-20 206 3955 T-1249 11 90 T-651 11 139 T- 2635 14 161 mAb CCR5 (4901/4900) 114 2387 Peptide-mAb CCR5 chimeric conjugate (4995/4900) 7 45

Example 8

Cell-Cell Fusion Assay On day 1, HeLa cells expressing gp160 (2 x 104 cells / 50

μΙ / well) are seeded in a white 96 microtiter plate in DMEM medium supplemented with 10% FCS and 2 Mg / ml doxicillin. On the day 2 ’1 μ | Sample of supernatant or well control antibody is added to a clean 96 microtiter plate. Then 100 μΙ containing 8x10 4 CEM-NKr-Luc cells suspended in medium are added and incubated for 30 min at 37 ° C. HeLa cell cuvette media is aspirated from the 96-well plate, 100 μΙ of the 200 μΙ antibody / CEM-NKr-Luc mixture is added and incubated overnight at 37 ° C. On day 3, 100 μΙ / well Bright-Glo® Luciferase Test Substrate (1,4-dithiothreitol and sodium dithionite; Promega Corp., USA) are added and the Luminescence

and measured after a minimum of 15 min incubation at RT. Materials:

HeLa-R5-16 cells (cell line to express HIV gp160 after doxicillin induction) are grown in DMEM medium containing nutrients and 10% FCS with 400 Mg / ml G418 and 200 pg / ml Hygromycin B. CEM NKR-CCR5-LUC (Catalog No. 5198, a T-cell line available from NIH AIDS Research & Reference Reagent Program McKesson BioServices Corporation Germantown1 MD 20874, USA). Cell Type: CEM.NKR-CCR5 (Cat. # 4376) and transfected (electroporation) to express the uciferase gene under the transcriptional control of HIV-2 LTR and propagated in RPMI 1640 containing 10% fetal bovine serum, glu- 4 mM tamine, penicillin / streptomycin (100 U / ml Penicillin, 100 pg / ml Streptomycin), and 0.8 mg / ml geniticin sufate (G418). Growth Characteristics: Round lymphoid cells, morphology not very variable. The cells grow in suspension as single cells, which can form small clumps. Raise 1:10 twice a week. Special Features: Expresses Iuciferase activity after HIV LTR transactivation. Suitable for infection with primary HIV-2 isolates, neutralization and drug sensitivity assays (Spenlehauer, C, et al. 'Virology 280 (2001) 292-300; Trkola1 A., et al., J. Virol. 73 (1999) 8966-8974). The cell line was obtained from the NIH AIDS Research and Reference Reagent Program, NIAID, NIH by Drs. John Moore and Catherine Spenlehauer. Bright-Glo® Luciferase Assay Buffer (Promega Corp. USA, Part No. E2264B), Bright-Glo® Luciferase Assay Substrate (Promega Corp. USA, part No. EE26B). Example 9

Antiviral activity assay in peripheral blood mononuclear cells (PBMC)

Human PBMCs are isolated from the buffy-coats layer (obtained from Stanford Blood Center) by Ficoll-Paque density gradient centrifugation (Amersham, Piscataway, New Jersey, USA) according to the protocol. from the manufacturer. Briefly, the blood is transferred from the white cell layer into 50 ml conical tubes and diluted with Dulbecco's phosphate buffered saline (Invitrogen / Gibco) to a final volume of 50 ml. Twenty-five ml of diluted blood is transferred to two 50 ml conical tubes, carefully covered with 12.5 ml Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at room temperature for 20 min at 450 χ g with slow stop. The white cell layer is carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove the remaining red blood cells, the cells are incubated for 5 min at room temperature with ACK Lysis Buffer (Biosource) and washed again with PBS. PBMC are counted and incubated in a concentration of 2 to 4 χ 106 cells / ml in RPMI1649 containing 10% FCS (Invitrogen / Gibco), 1% penicillin / streptomycin, 2 mM L-glutamine, 1 mM sodium and Phytohemagglutinin 2 Mg / ml (Invitrogen) for 24 h at 37 ° C. Cells are incubated with 5 Units / ml human IL-2 (Roche Molecular Biochemicals) for a minimum of 48 h before assay. In a 96 well round bottom plate 1 x 105 PBMC are infected with the HIV-I JR-CSF virus (Koyanagi, Y., et al., Science 236 (1987) 819-822) in the presence of conjugates of Serially diluted peptide-immunoglobulin, reference immunoglobulin and reference peptide (T-20, T-1249, T-651 and T-2635) tests. The amount of virus used is equivalent to 1.2 ng of HIV-1 p24 antigen / well. Infections are done in quadruplicate. The plates are incubated for 6 days at 37 ° C. Virus production is measured at the end of infection using p24 ELISA (HIV-I p24 ELISA # NEK050B, Perkin Elmer / NEN) using a dose-response format sigmoid model with a Microsoft Excel Fit site (version 3.0.5 Build 12; equation 205; Microsoft). Example 10

Production of anti-CD4 antibody expression plasmids

Gene segments encoding the anti-human CD4 antibody variable Ive (V1) domain and the Ieve kappa (Cl) constant domain were ligated as if they were gene segments for

It is the variable domain (Vh) of the anti-CD4 antibody heavy chain and the human γΐ-heavy chain (CH1 -dobradiga-CH2-CH3) constant domains.

In the case of CD4 mAb of SEQ ID NO: 71/72 'the heavy and light chain variable domains are derived from a mouse antibody that has been humanized as described, for example, by Reimann1 KA, et al. , Aids Res. Human Retrovir. 13 (1997) 933-943 or US 5,871,732, and the heavy and light chain constant domains are derived from a human antibody (C-kappa and IgGI).

Subsequently, the gene segment encoding an anti-CD4 antibody complete Ieve chain was ligated at the N and / or -C termination with a nucleic acid encoding an antifusogenic peptide including a linker sequence and / or the gene segment encoding a complete anti-CD4 antibody heavy chain was ligated at the N- and / or -C-terminus with a nucleic acid encoding an antifusogenic peptide including a ligand binding sequence. a) Vector 6310

Vector 6310 is an expression plasmid, for example, for transient expression of the CD4 mAb Ieve chain (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

In addition to the mAb CD4 κ-light chain expression cassette, this vector contains:

-a neomycin resistance gene as a selectable marker

questionable,

an origin of replication of the pUC18 vector that allows replication of this plasmid into E. coli cells, and an β-lactamase gene that confers ampicillin resistance.

in E. coli.

The transcription unit of the κ-light chain gene of mAb CD4 is composed of the following elements:

-the immediate early enhancer and promoter of human cytomegalovirus,

-a 5 'region not translated from a germline of

human antibody, -a murine immunoglobulin heavy chain signal sequence including a signal sequence intron (signal sequence 1, intra η, signal sequence 2 [L1-intron-L2]),

-the mature variable eve chain κ of anti-human CD4 antibody encoding the segment arranged with a splice donor site and a CirH-BamHI restriction site at the 3 'end,

-a human κ-light truncated intron 2, -ο human κ-light gene constant domain, -the bovine growth hormone ("poly A") signal sequence (bGH), and,

-The Ascl and SgrAI Linearic Restriction Sites at the 3 'end.

The plasmid map of the CD4 mAb κ-Ieve chain expression vector 6310 is shown in Figure 4. The amino acid sequence of the CD4 mAb mature κ-light chain (without signal sequence) is shown in SEQ ID NO: 71 b) Vector 6309

Vector 6309 is an expression plasmid, for example, for transient expression of the CD4 mAb γ1-heavy chain (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

In addition to the mAb CD4 γ heavy chain expression cassette, this vector contains:

-an origin of replication of the pUC18 vector that allows the replication of this plasmid into E. coli cells, and

a β-lactamase gene that confers ampicillin resistance

in E. coli.

The transcription unit of the mAb CD4 y1-heavy chain gene is composed of the following elements: - It is the immediately early enhancer and promoter of

human megalovirus,

-a 5 'untranslated region of a human antibody germline,

-a murine immunoglobulin heavy chain signal sequence including a signal sequence intron (signal sequence 1 ， intra η, signal sequence 2 [L1-intron-L2]), - the mature variable antibody heavy chain -CD4 human-

wherein it encodes the segment arranged with a splice donor site and a 3 'end Xhol restriction site Kinetic,

-a mouse / human heavy chain hybrid intron 2,

- the human genomic constant domains of the γΙ gene are

output containing the L234A and L235A mutation,

-the bovine growth hormone ("poly A") polyadenylation (bGH) signal sequence, and,

-the SgrAI kinetic restriction site at the 3 'end.

Plasmid map of γ1-chain expression vector 6309

mAb heavy chain and shown in Figure 3. The amino acid sequence of the mature mAb CD4 / 1-heavy chain (no signal sequence) is shown in SEQ ID NO: 72. c) Vector 6303

Vector 6303 is an expression plasmid, for example, for

transient expression of an anti-CD4 antibody chimeric yl-chain peptide chimeric peptide (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

Vector 6303 is derived from vector 6309 such that γΙ-

CD4-linked and thymidine mAb heavy chain, but a C-terminal amino acid Kinetics, that is, the residue of the C-terminal heavy-chain lysine is removed with a nucleic acid encoding the afp-1 antifusogenic peptide (SEQ ID NO: 73) and ο glycine-serine peptide linker of SEQ ID NO: 69.

In addition to the peptide chimeric conjugate expression cassette

γ1-heavy chain anti-CD4 antibody this vector contains:

- an origin of replication of the pUC18 vector that allows replication

coagulation of this plasmid in E. coli cells, and a beta (P) -lactamase gene that confers ampicillin resistance in E. coli.

The transcriptional unit of the anti-CD4 antibody heavy chain γ1-peptide chimeric conjugate is composed of the following elements: - is the immediately early cytoplasmic enhancer and promoter.

human megalovirus,

-a 5 'untranslated region of a human antibody germline,

-a murine immunoglobulin heavy chain signal sequence including a signal sequence intron (signal sequence 1 ， intron, signal sequence 2 [L1-intron-L2]),

-the mature variable heavy chain of the human anti-CD4 antibody encoding the segment arranged with a splice donor site and a 3 'end Xhol restriction site Linomic, - a mouse heavy chain hybrid intron / human

containing the L234A and L235A mutagens,

-the serine glycine peptide linker sequence of SEQ ID NO:

69,

afp-1 antifusogenic peptide of SEQ ID NO: 73, the hormone polyadenylation ("poly A") signal sequence

bo vino growth (bGH) and,

-the SgrAI kinetic restriction site at the 3 'end. The plasmid map of the y1-heavy chain CD4 mAb conjugate expression vector 6303 is shown in Figure 6. The amino acid sequence of the mature heavy chain conjugate (no signal sequence) is shown in SEQ ID NO: 74. Example 11

Production of final anti-CD4 antibody expression plasmids

Fusion genes (antibody heavy and / or light chain fusion genes) comprising a CD4 mAb genomic segment, an optional ligand gene segment and an antifungal gene peptide segment have been assembled by recombinant methods and techniques known to the art. connection of concordant nucleic acid segments. Nucleic acid sequences encoding peptide ligands and antifusogenic polypeptides were synthesized by chimeric synthesis and then ligated into an E. coli plasmid for amplification. Subcloned nucleic acid sequences were verified by DNA sequencing. Example 12

Transient expression of immunoglobulin and immunoglobulin variants in EBNA HEK293 cells

Recombinant anti-CD4 antibodies and anti-CD4 antibody variants were generated by transient transfection of adherent cultured HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr virus nuclear antigen American type culture collection number deposit (ATCC # CRL-10852) grown in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) supplemented with 10% FCS ultra-low IgG (fetal bovine serum, Gibco), 2 mM Glutamine (Gibco), nonessential amino acids at 1% volume by volume (v / v) (Gibco) and G418 250 pg / ml (Roche Molecular Biochemicals). For transfection, FuGENE® 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a ratio of reagent (μΙ) to DNA (pg) ranging from 3: 1 to 6: 1. Ieve and heavy chains including the Ieve and heavy chains of the anti-CD4 peptide-antibody conjugate were expressed on two different plasmids using a mole ratio of plasmid encoding the Ieve chain to heavy chain ranging from 1: 2 to 2: 1, respectively. Cell-length supernatants containing antifusogenic peptide-anti-CD4 antibody conjugates were collected on day 4 to 11 post transfection. General information regarding recombinant expression of human immunoglobulins, for example in HEK293 cells, is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203. Example 13

Expression analysis using SDS-PAGE. Western blotting transfer and detection with immunoglobulin-specific antibody conditons.

The expressed and secreted antifusogenic peptide-anti-CD4 antibody conjugates were processed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and the separate anti-CD4 and anti-CD4 antibody chains. antifusogenic peptide of the antifusogenic peptide-anti-CD4 antibody conjugate were transferred from the gel to a membrane and subsequently detected by an immunological method.

SDS-PAGE:

LDS Sample Buffer, 4x Concentrate (4x): 4g glycerol, 0'682g TRIS-base, TRIS-hydrochloride 0'666g, 0.8g LDS (lithium dodecyl sulfate), 0'006g EDTA (ethylenediaminetetraacetic acid), 0.75 ml of a 1 wt% solution of Serva Blue G250 in water, 0.75 ml of a 1 wt% solution of phenol red and water to make a total volume of 10 ml.

The cuvette broth containing antifusogenic peptide-anti-CD4 antibody secreted conjugate was centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was mixed with 1/4 volume (v / v) 4xLDS sample buffer and 1/10 volume (v / v) 0.5 M 1，4-dithiothreitol (DTT). The samples were then incubated for 10 min at 70 ° C and the protein separated by SDS-PAGE. The NuPA-GE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast Gels (pH 6.4) and a NuPAGE® MOPS Running Buffer were used. Western blot:

Transfer Buffer: 39 mM glycine, 48 mM TRIS hydrochloride, 0.04 wt% (w / w) SDS and 20% volume (v / v) ethanol.

After SDS-PAGE, separate strands of antifusogenic peptide-anti-CD4 antibody conjugate were electrophoretically transferred to a nitrocellulose filter membrane (pore size: 0.45 μηη) according to ο "Semi-Blotting Method". Burnette section (Burnette, WN, Anal. Biochem. 112 (1981) 195-203). Immunological Detection:

TBS Buffer: 50 mM TRIS hydrochloride, 150 mM NaCl, pH adjusted to 7.5.

Blocking Solution: Western Blocking Reagent 1% (w / v) (Molecular Molecular Biochemicals) in TBS buffer.

TBST Buffer: 1x TBS buffer with 0.05% Tween-20 by volume.

me (v / v).

For immunological detection western blotting membranes were incubated with shaking at room temperature twice for 5 minutes in TBS buffer and once for 90 minutes in blocking solution. Detection of peptide and immunoglobulin conjugate chains: Heavy chain: For conjugate heavy chain detection

of antifusogenic peptide-anti-CD4 antibody a purified rabbit anti-human IgG peroxidase conjugated antibody (DAKO1 Code No. P.0214) was used.

Ieve Chain: The Ieve chain of the anti-spindle-peptide-anti-CD4 antibody conjugate was detected with a purified peroxidase conjugated human anti-Ieve kappa rabbit antibody (DAKO1 Code No. P.0129).

For visualization of antibody Ieve and heavy chains, Iavated and blocked Western blot membranes were first incubated in the case of a heavy chain with a purified peroxidase-conjugated rabbit anti-human IgG antibody or an Ieve1 chain with a purified perisidase conjugated anti-human Ieve kappa chain antibody at a 1: 10,000 dilution in 10 ml blocking solution at 4 ° C with overnight stirring. After washing the membranes three times with TBST buffer and once with TBS buffer for 10 min at room temperature, Western blot membranes were treated with a chemiluminescent Luminol / peroxide solution (LurrH-Lightpujs Western Blotting Substrate, Roche). Molecular Biochemicals). Then the membranes were incubated in 10 ml Luminol / peroxide solution for 10 seconds to 5 minutes and the emitted Light was subsequently detected with a LUMI-Imager Fl Analyzer (Roche Molecular Biochemicals) and / or recorded with a ray film. X. Point intensity was quantified with ο LumiAnaIyst Software (Version 3.1). Myoblast immunoblot marching:

Peroxidase labeled secondary antibody conjugate used for detection can be removed from the stained blot by incubating the membrane for one hour at 70 ° C in 1M TRIS-hydrochloride buffer (pH 6.7) containing 100 mM beta-mercaptoethanol and SDS 20% (w / v). After this treatment the blot may be stained with a different secondary antibody a second time. Prior to the second detection, the blot is washed three times at room temperature with shaking in TBS buffer for 10 minutes each. Example 14

Affinity Purification, Dialysis, and Concentration of Peptide-Immunoglobulin Conjugates

Expressed and secreted antifusogenic peptide-anti-CD4 antibody conjugates were purified by affinity chromatography using Protein A-Sepharose® CL-4B (GE Healthcare former Amersham Bioscience, Sweden) according to known methods. Briefly, after centrifugation (10,000 g for 10 minutes) and filtration through a 0.45 μηι filter, clarified culture supernatants containing the peptide-immunoglobulin conjugate were applied to a Protein A-Sepharose column. ® CL-4B equilibrated with PBS buffer (10 mM Na 2 HPO 4, 1 mM KH 2 PO 4, 137 mM NaCl and 2.7 mM KCI, pH 7.4). Unlinked proteins were removed with PBS equilibration buffer and 0，1M citrate buffer, pH 5.5. Antifusogenic peptide-anti-CD4 antibody conjugates were eluted with 0.1 M citrate buffer, pH 3.0 and fragments containing the conjugate ο were neutralized with 1M TRIS-Base. Then antifusogenic peptide-anti-CD4 antibody conjugates were intensively dialyzed against PBS buffer at 4 ° C, concentrated with an Ultrafree®-CL Centrifugal Filter Unit equipped with a Biomax-SK membrane (Millipore Corp., USA). and stored in an ice-water bath at 0 ° C. The integrity of the conjugates was analyzed by SDS-PAGE in the presence and absence of a reducing agent and stained with bright Coomassie blue as described in Example 13. Aggregation of aryphusogenic peptide-anti-CD4 antibody conjugates was analyzed by analytical chromatography by exclusion of

size.

Example 15

Deglycosylation of peptide and immunoglobulin conjugates N-Alloyed anti-CD4 antibody carbohydrates and conjugates of

antifusogenic peptide-anti-CD4 antibody were cleaved by enzymatic treatment with Peptide-N-Glycosidase F (PNGaseF, Roche Molecular Bio-chemicals, Mannheim, Germany or Prozyme, San Leandro, CA). Next, anti-CD4 antibodies and antifusogenic peptide-anti-CD4 antibody conjugates were incubated at 37 ° C for 12 to 24 h using PNGaseF 50 mU per mg N-glycosylated protein in PBS buffer with a pratein concentration of about of 2 mg / ml. Then Peptide-N-Glycosidase F was separated by preparative gel filtration according to known methods. Briefly, anti-CD4 antibodies and antifusogenic peptide-anti-CD4 antibody conjugates were applied to a Superose® 12 10/300 GL column (GE Healthcare former Amersham Bioscience, Sweden) equilibrated with PBS buffer (10 mM Na2HPO4, KH2PO4). at 1 mM, 137 mM NaCl and 2.7 mM KCI, pH 7.4) and then eluted with equilibration buffer at a flow rate of 0.5 to 1.0 ml / min using the exploratory chromatography system Akta from Amersham Bioscience (GE Healthcare former Amersham Bioscience, Sweden). Example 16

Single-cycle antiviral activity assay

For the production of pseudotyped NL-Bal virus, plasmid pNL4-3Aenv (HIV pNL4-3 genomic construct with an env gene delegation) and pCDNA3.1 / NL-BAL env [plasmid pcDNA3.1 containing ο NL-BaI env gene (obtained from NIBSC Centralized Facility for AIDS Reagents)] were co-transfected into HEK 293FT cell line (Invitrogen) 1 cuffed in Dulbecco's Minimal Modified Medium (DMEM) containing 10% fetal bovine serum (FCS) ), 100 U / ml Penicillin, 100 pg / ml Streptomycin, 2 mM L-glutamine and 0.5 mg / ml geniticin (all Invitro-

gen / Gibco). Supernatants containing the pseudotyped viruses were collected two days after transfection and cell debris removed by filtration through a 0.45 µm PES (polyethersulfone) filter (NaIgene) and stored at -80 ° C in aliquots. . For normalization of assay performance, virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells generating approximately 1.5 χ 105 RLU (relative light units) per well. Antifusogenic peptide-anti-CD4 antibody test conjugates, parental anti-CD4 antibody, reference antibodies, and reference antifusogenic peptide (T-651) were serially diluted in 96-well plates. The assay was performed in quadruplicates. Each plate contained control cell and control virus wells. The equivalent of 1.5 x 105 RLU of virus stocks was added to each well, then 2.5 x 104 JC53-BL cells were added to each well, with a final assay volume of 200 μΙ per well. After 3 days incubation at 37 ° C, 90% Relative Humidity and 5% CO 2, the media was aspirated and 50 μΙ Steady-Glo® Luciferase Assay System (Promega) was added to each well. Assay plates were read on a Luminometer (Luminoskan, Thermo Electron Corporation) after 10 min incubation at room temperature. The percentage inhibition of Iuciferase activity was calculated for each dose after background subtraction and the IC50 and IC50 values were determined by using the Excel XLfit curve fitting program (version 3.0.5 Build 12; Microsoft). Results are shown in Tables 4 and 5 respectively. Table 4: Antiviral activity of antifusogenic polypeptides, antibodies and antifusogenic peptide-anti-CD4 antibody conjugates (by weight)

Compound Antiviral activity NL-BAL (R5) NL-4-3 (x4) IC50 [ng / ml] / IC90 [ng / ml] IC50 [ng / ml] / IC90 [ng / ml] Reference Antibody 1 (inert) inactive / inactive inactive / inactive Reference antibody 2 (inert) inactive / inactive inactive / inactive T-651 21.5 / 181.9 169.2 / 1099.7 Compound Antiviral activity NL-BAL (R5) NL-4-3 (x4) IC50 [ng / ml] / IC90 [ng / ml] IC50 [ng / ml] / IC90 [ng / ml] mAb CD4 (6310/6309) maximal inhibition = 50% maximal inhibition = 20% Antifusogenic peptide conjugate anti-CD4 antibody (6310/6303) 6.1 / 32.2 43.9 / 123.9

Table 5: Antiviral activity of antifusogenic polypeptides, antibodies and antifusogenic peptide-anti-CD4 antibody conjugates (by molarity)

Compost。 Antiviral Activity NL-BAL (R5) NL-4-3 (x4) IC50 _ / IC90 _ IC50 _ / IC90 _] Reference Antibody 1 (Inert) Inactive / Inactive Inactive / Inactive Reference Antibody 2 (Inert) Inactive / inactive inactive / inactive T-651 5.4 / 45 42/275 mAb CD4 (6310/6309) maximal inhibition = 50% maximal inhibition = 20% antifusogenic peptide-anti-CD4 antibody conjugate (6310/6303) 0.038 / 0.201 0.275 / 0.774

Example 17

Cell-Cell Fusion Test

On day 1, gp160-expressing HeLa cells (2 x 104 cells / 50 μΙ / well) are seeded in a white 96 microtiter plate in DMEM medium supplemented with 10% FCS and 2 pg / ml doxicillin. On day 2, 100 μΙ per well of supernatant sample or control antibody is added to a clean 96 microtiter plate. Then, 100 μΙ containing 8x10 4 CEM-NKr-Luc cells suspended in medium are added and incubated for 30 min at 37 ° C. HeLa cell culture medium is aspirated from the 96-well plate, 100 μΙ of the 200 μΙ antibody / CEM-NKr-Luc mixture is added and incubated overnight at 37 ° C. On day 3，100 μΙ / well Bright-Glo® Luciferase Test Substrate (1,4-dithiothreitol and sodium dithionite; Promega Corp., USA) are added and Luminescence is measured after a minimum of 15 min incubation in OK. Materials:

HeLa-R5-16 cells (cell line to express HIV gp160 after doxicillin induction) are grown in DMEM medium containing nutrients and 10% FCS with 400 pg / ml G418 and 200 pg / ml Hygromycin B. CEM NKR-CD4-LUC (Catalog Number: 5198, a T cell line available from NIH AIDS Research & Reference Reagent Program McKesson BioServices Corporation Germantown, MD 20874, USA). Cell Type: CEM.NKR-CD4 (Cat. # 4376) and transfected (electroporation) to express the uciferase gene under transcriptional control of HIV-2 LTR and propagated in RPMI 1640 containing 10% fetal bovine serum, glutamine at 4 mM penicillin / streptomycin (100 U / ml Penicillin 1 Streptomycin 100 pg / ml), and 0.8 mg / ml geniticin sulfate (G418). Growth Characteristics: Round lymphoid cells, morphology not very variable. The cells grow in suspension as single cells, which can form small clumps. Raise 1:10 twice a week. Special Features: Express Iuciferase activity after HIV LTR transactivation. Suitable for infection with primary HIV isolates, for neutralization and drug sensitivity assays Spenlehauer, C, et al., Virology 280 (2001) 292-300; Trkola, A., et al., J. Virol. 73 (1999) 8966-8974). The cell line was obtained from the NIH AIDS Research and Reference Reagent Program, NIAID, NIH from Drs. John Moore and Catherine Spenlehauer. Bright-Glo® Luciferase Assay Buffer (Promega Corp. USA, Part No. E2264B), Bright-Glo®, Luciferase Assay Substrate (Promega Corp. USA, part No. EE26B). Example 18

Antiviral activity assay in peripheral blood mononuclear cells (PBMC).

Human PBMC are isolated from the white cell layer

(obtained from Stanford Blood Center) by Ficoll-Paque density gradient centrifugation (Amersham, Piscataway1 New Jersey, USA) according to the manufacturer's protocol. Briefly, the blood is transferred from the white cell layer to 50 ml conical tubes and diluted with Dulbecco's phosphate buffered saline (Invitrogen / Gibco) to a final volume of 50 ml. Twenty-five ml of the diluted blood is transferred to two 50 ml conical tubes, carefully covered with 12.5 ml Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at room temperature for 20 min at 450 g with Ienta stop. The white cell layer is carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove the remaining red blood cells, the cells are incubated for 5 min at room temperature with ACK Lysis Buffer (Biosource) and washed again with PBS. PBMC are counted and incubated in a concentration of 2 to 4 χ 106 cells / ml in RPMI1649 containing 10% FCS (Invitrogen / Gibco), 1% penicillin / streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 2 Mg / ml Phytohemagglutinin (Invitrogen) for 24 h at 37 ° C. Cells are incubated with 5 Units / ml human IL-2 (Roche Molecular BiocherrH-cals) for a minimum of 48 h prior to assay. In a 96-well round bottom plate 1 x 105 PBMC they are infected with the HIV-I JR-CSF virus (Koyanagi, Y., et al., Science 236 (1987) 819-822) in the presence of conjugates. antifusogenic peptide-anti-CD4 antibody, reference immunoglobulin, parental anti-CD4 antibody and reference peptide (T-651) serially diluted. The amount of virus used is equivalent to 1.2 ng of p24 HIV / well antigen. Infections are done in quadruplicates. The plates are incubated for 6 days at 37 ° C. Virus production is measured at the end of infection using p24 ELISA (p24 HIV-I ELISA # NEK050B, Perkin Elmer / NEN) using the dose-response sigmoid format model with a Microsoft Excel Fit site ligation (versa). 3.0.5

Build 12; equation 205; Microsoft). Table 6: Antiviral activity of antifusogenic polypeptides, antibodies and antifusogenic peptide-anti-CD4 antibody conjugates (by weight)

Compound Antiviral activity NL-BAL (R5) NL-4-3 (x4) IC50 [ng / ml] / IC90 [ng / ml] IC50 [ng / ml] / IC90 [ng / ml] Reference Antibody 1 (inert) inactive / inactive inactive / inactive Reference antibody 2 (inert) inactive / inactive inactive / inactive T-651 14.1 / 57.8 41.4 / 79.6 anti-CD4 antibody (6310/6309) 917.6 / inhibition max 54% 35.6 / 1117.0 Antifusogenic Peptide Chimeric Anti-CD4 Antibody Conjugate (6310/6303) 5.3 / 18.2 3.2 / 25.1 Sequence Listing

<110> F. Hoffmann-La Roche AG

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Glu Val Lys Leu Val Glu Ser Gly Gly

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Ser Leu Lys Le Ser Ser Cys Wing Ser Ser 2S

Wing Met Ser Trp Val Arg Gln Thr Pro 40

Wing Ser lie Ser Thr Gly Asp Asn Thr 50 55

GXy Arg Phe Thr lie As Arg Asp Asn

65 70

Gln Met Be Ser Read Arg Be Glu Asp 85

Gly Leu Val Lys Pro Gly Gly 15

Gly Phe Tbr Phe Be Thr Tyr 30

Glu Lys Arg Leu Glu Trp Val 45

Tyr Tyr Thr Asp Ser Val Arg 60

Arg Wing Asn lie Leu Tyr Leu 75 80

Thr Wing Met Tyr Phe Cys Thr 90 95

Arg Gly Arg Gly Asp Arg Gly Asp Read Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Read Val Thr Val Ser Ser 115

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mouse

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Asp Val Val Ser GXn Ser Pro Ser Ser Leu Ala. Val Ser Val Giy IS 10 15

Glu Lys Val Thr Met Be Cys Lys Be Be Gln Be Read Leu Tyr Arg 25 30

Gly Asn Gln Met Asn Tyr Read Wing Trp Tyr Gln Gln Lys Pro Gly Gin 40 45

Ser Pro Lys Leu Leu lie Tyr Trp Wing Ser Thr Arg Glu Ser Gly Val SO 55 60

Pro Asp Arg Phe Thr Gly Be Gly Be Gly Thr Glu Phe Thr Leu Thr 65 70 75 80

lie Ser Ser Val Lys Wing Glu Asp Leu Thr Val Tyr Tyr Cys Gln Gin 90 95

Tyr Tyr Thr Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie 100 105 110

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Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Arg Pro Be Gln 1 5 10 15

Ser Leu Ser Lie Thr Cys Thr Go Gly Phe Pro Leu Gly Val Phe 25 3 0 Gly Val His Trp Val Arg Gln Be Pro Gly Lys Gly Leu Glu Trp Leu 40 45

Gly Val Ile Trp Lys Gly Gly Gly Asn Thr Asp Tyr Asn Wing Phe Met 50 55 60

Be Arg Read Arg Ile Be Lys Asp Asn Be Lys Be Gln Val Phe Phe 65 70 75 80

Arg Met Asn Be Read Gln Thr Asp Asp Thr Wing Ile Tyr Tyr Cys Wing 85 30 95

Lys Val Asn Leu Wing Asp Wing Met Asp Wing Tyr Trp Gly Gln Gly Thr Ser 100 105 110

Val Ile Val Ser Ser 115

<210> 4

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<213> mouse

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Asp Ile Gln Met Thr Gln Be Pro AIa Be Read Ala Be Val Gly 10 15

Glu Thr Val Thr Ile Thr Cys Arg Be Gly Asn Ile His Gly Tyr 25 30

Leu Wing Trp Phe Gln Gln Lys Gln Gly Lyo Ser Pro Gln Leu Leu Val 40 45

Tyr Asn Thr Lys Wing Read Leu Glu Wing Gly Val Pro To Be Arg Phe To Be Gly

50 55 60

Be Gly Be Gly Thr Gln Phe Be Read Lys Ile Asn Asn Read Gln Pro 65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His Tyr Asp Leu Pro Arg 85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 5

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Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Arg Pro Be Gln 10 15

Be Leu Be Ile Thr Cys Thr Val Be Gly Phe Pro Be Read Gly Ile Phe

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Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 40 45

Gly Val Ile Trp Lys Gly Gly Gn Asn Thr Asp Tyr Asn Wing Phe Met 50 55 60

Be Arg Read Arg Ile Thr Lys Asp Asn Be Lys Be Gln Val Phe Phe 65 70 75 80

Arg Met Asn Be Read Gln Thr Asp Asp Thr Wing Ile Tyr Tyr Cys Wing 85 90 95

Lys Val Asn Leu Wing Asp Wing Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Ile Val Ser Ser 115

<210> 6

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Asp Ile Gln Met Thr Gln Be Pro Wing Be Read Be Wing Be Val Gly 1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Wing Be Gly Asn Ile His Gly Tyr 25 30

Leu Wing Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 40 45 Tyr Asn Thr Lys Thr Leu Wing Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Be Gly Be Gly Thr Gln Phe Be Read Lys Ile Asn Be Read Gln Pro 65 70 7S 80

Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Tyr Asp Leu Pro Arg 85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105

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Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Arg Pro Be Gln 10 15

Be Read Be Ile Thr Cys Thr Val Be Gly Phe Pro Be Read Gly Thr Phe 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 40 45

Gly Val Ile Trp Arg Gly Gly Gn Asn Thr Asp Tyr Asn Wing Phe Met 50 55 60

Be Arg Read Arg Ile Thr Lys Asp Asn Be Lys Be Gln Val Phe Phe 65 70 75 80

Arg Met Asn Be Read Gln Thr Asp Asp Thr Wing Ile Tyr Tyr Cys Wing 85 90 95

Lys Val Asn Leu Wing Asp Wing Met Asp Wing Tyr Trp Gly Gln Gly Thr Ser 100 105 110

Val Ile Val Ser Ser 115

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Asp Ile Gln Met Thr Gln Be Pro Be Be Read Be Be Be Val Gly 10 15

Glu Thr Val Thr Ile Thr Cys Arg Wing Be Gly Asn Ile His Gly Tyr 25 30

Leu Wing Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 40 45

Tyr Asn Thr Lys Thr Read Wing Glu Gly Val Pro Be Arg Phe Be Gly 50 55 60

Be Gly Be Gly Thr Gln Phe Be Read Lys Ile Asn Be Read Gln Pro 65 70 75 80

Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Tyr Asp Leu Pro Arg 85 90 95

Thr Phe Gly Gly Gly Thr Lvs Leu Glu Ile Lys Arg 100 "105

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Thr Tyr Wing Net Ser 1 5

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Val Phe Gly Val His 1 5

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Thr Phe Gly Val His 1 5

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Ser Ile Ser Thr Gly Asp Asn Thr Tyr Tyr Thr Asp Ser Val Arg Gly 10 15

<210> 14

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Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asri Wing Phe Met Ser 1 1 10 10 15

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Val Ile Trp Arg Gly Gly Asn Thr Asp Tyr Asn

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Gly Arg Gly Asp Arg Gly Asp Read Phe GIy Tyr 10 <21C> 17

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Val Asn Read Asp Wing Met Asp Wing Tyr ι 5

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Lys Ser Ser Gln Ser Leu Tyr Arg Gly Asn Gln Met Asn Tyr Leu 10 15

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Arg Be Ser Gly Asn Ile His Gly Tyr Leu Wing i 5 10

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Arg Wing Be Gly Asn lie His Gly Tyr Leu Wing 1 5 10

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Trp Wing Ser Thr Arg Glu Ser 1 5 <210 »22

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Asn Thr Lys Wing Leu Wing Glu 1 5

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<400> 23

Asn Thr Lys Thr Leu Wing Glu 1 5

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Gln Gln Tyr Tyr Thr Tyr Pro Arg Thr 1 5

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<400> 25

Gln His His Tyr Asp Read Pro Arg Thr 1 S

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Wing Be Thr Lys Gly Pro Be Val Phe Pro Read Wing Pro Be Ser Lys 1 5 10 15

Ser Thr Be Gly Gly Thr Wing Ward Read Gly Cys Leu Val Lys Asp Tyr 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Wing Leu Thr Ser 40 45

Gly Val His Thr Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Read Be Ser Val Val Thr Val Pro Be Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro To Be Asn Thr Lye Val Aep Lys

85 "90 95

Lys Val Glu Pro Lys Be Cys Asp Lys Thr His Cys Pro Pro Cys 100 105 110

Pro Wing Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125

Lys Pro Lys Asp Thr Read Met Ile Be Arg Thr Pro Glu Val Thr Cys

13C 135 140

Val Val Val Asp Val Be His Glu Asp Pro Glu Val Ly3 Phe Asn Trp 145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Be Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Read Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Wing Leu Pro Wing Pro Ile Glu Lys Thr Ile Ser Lys Wing Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Being Arg Asp Glu 225 230 235 240

Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Be Asp Ile Wing Val Glu Trp Glu Be Asn Gly Gln Pro Be Glu Asn

260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Read Asp Be Asp Gly Be Phe Phe 275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lya Ser Arg Trp Gln Gln Gly Acn 290 295 300

Val Phe Ser Cys Ser Val et Glu Wing Read His Asn His Tyr Thr 305 310 315 320

Gln Lys Be Read Be Read Be Pro Gly Lys 325 330

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Wing Be Thr Lys Gly Pro Be Val Phe Pro Read Wing Pro Cys Be Arg 10 15

Be Thr Be Glu Be Thr Wing Ward Leu Gly Cys Leu Val Lys Asp Tyr 25 30

Phe Pro Glu Pro Val Thr Val Ser Tr Asn Ser Gly Wing Leu Thr Ser 40 45

Gly Val His Thr Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Read Ser Be Val Val Thr Val Pro Be Ser Leu Gly Thr Lys Thr 65 70 75 80

Tyr Thr Cys Asn Val Αερ His Lys Pro Being Asn Thr Lys Val Asp Lys 85 90 95

Arg Val Glu Be Lys Tyr Gly Pro Pro Cys Pro Be Cys Pro Wing Pro 100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Lvs Pro Lys 115 120 125

Asp Thr Leu Ket Ile Be Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Be Gln Glu Asp Pro Olu Val Gln Phe Asn Trp Tyr Val Asp 145 ISD 155 160

Gly Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175

Asn Be Thr Tyr Arg Val Val Be Val Leu Thr Val Leu His Gln Asp 180 185 190

Trp Read Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lye Gly Leu 195 200 205

Pro Be Ser Ile Glu Lys Thr Ile Ser Lys Wing Lys Gly Gln Pro Arg 210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Being Gln Glu Glu Met Thr Lys 225 230 235 240

Asn Gln Val Ser Read Thr Cys Read Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255

lie Val Glu Wing Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Le Tyr Ser 275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe I Know 290 295 300

Cys Ser Val Met His Glu Wing Read His Asn His Tyr Thr Gln Lys Ser 305 310 315 320

Read Be Read Read Be Gly Lys 325

c21Q> 28

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<40Q> 28

Arg Thr Val Wing Ward Pro Be Val Phe Ile Phe Pro Pro Asp Glu 10 IS Gln Read Lys Be Gly Thr Wing Be Val Val Cys Read Leu Asn Pn 25 30

Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn Wing Leu Gln 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60

Thr Tyr Be Read Be Ser Thr Thr Read Read Thr Read Be Lys Wing Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95

Pro Val Thr Lys Be Phe Asn Arg Gly Glu Cys 100 105

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Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Be Leu Ile Hxs

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Being Leu Ile Glu Glu Being Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 25 30

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Glu Lys Asn Glu Gln Glu Read Leu Glu Read Le Asp Lys Trp Wing Ser Leu 25 30

Trp Asn Trp Phe 35

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Trp Gln Glu Trp Olu Gln Lys Ile Thr Wing Read Leu Glu Gln Wing Gln 10 15

Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp 25 30

Ala Ser Leu Trp Glu Trp Phe 35

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Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Be Leu 10 15

Ile His Ser Leu Ile Glu Glu Being Gln Asn Gln Gln Gln Lys Asn Glu 25 30

Gln Glu Leu Leu 35

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Thr Thr Trp Glu Wing Trp Asp Arg Wing Xle Wing Glu Tyr Wing Wing Arg 10 15

Ile Glu Wing Leu Ile Arg Wing Wing Gln Glu Gln Gln Gln Lys Asn Glu 25 30

Wing Wing Wing Leu Arg Glu Leu 35

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Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Read Leu Arg Wing Ile Glu Wing 10 15

Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln

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Arg Wing Ile Leu 35

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Asn Asn Leu Leu Arg Wing Ile Glu Wing Gln Gln His Leu Leu Gln Leu 10 15

Thr Val Trp Gly Ile Lys Gln Leu Gln Wing Arg Ile Leu Wing Val Glu 25 30 Arg Tyr Leu Lys Asp Gln

35

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Read Be Read Read Be Pro Gly Lys 1 5

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Read Ser Asn Arg Gly Glu Cys 1 5

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Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln IS 10 15

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Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly 10 15

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Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly 10 15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asn 10 15

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Be Gly Gly Gly Be Gly Gly Gly Be Gly Gly Gly Be Gly Gly Gly Gly 1 5 * 10 15 <210> 44

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<223> Ligant 9 <400> 44

Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Glv GIy 10 15

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<223> Ligant 10 <400> 45

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

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Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10

Gly

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Be Gly Gly Gly Gly Be Gly Gly Gly Gly Gly Be Gly Gly Gly Gly Gly 10 15

Gly Ttir

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Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Gly

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Gly asn

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Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Gly

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Ala Wing

<210> 50

<211> 25

<212> PRT

<213> Artificial

<220>

<223>! Binder 15

<400> 50

Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be 10 15 Gly Gly Gly Gly Be Gly Gly Gly Gly

25

<210> 51

<211> 26

<212> PRT

<213> Artificial

<220>

<2 2 3> Through 16 <400> 51

Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Glv Gly Ser 10 15

Glv Gly Gly Gly Being Gly Gly Gly Gly Gly 25

<210> 52 <211> 27 <212> PRT <213> Artificial <220> <223> ligand 17 <400> 52

Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Be 1 5 10 * 15

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 25

<210> 53 <211> 28 <212> PRT <213> Artificial <220> <223> Ligant 18 <400> 53

Be Oly Gly Gly Gly Be Gly Gly Gly Gly Be Gly Gly Gly Gly Gly Ser 10 15

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Wing 25 <210> 54

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Ligante 19

<400> 54

Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 10 10 15

Gly

<210> 55

<211> 19

<212> PRT

<213> Artificial

<220>

<223> Ligant 20

<400> 55

Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 10 IS

Gly Wing Ser

<210> 56 <211> 18 <212> PRT <213> Artificial <220> <223> Ligand 24 <400> 56

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

Ala Wing

<210> 57

<211> 16

«212> PRT

<213> Artificial <220>

<223> Ligante 25

<400> 57

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

<210> 58 <211> 26 <212> PRT <213> Artificial <220> <223> Ligand 26 <400> 58

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

Gly Gly Gly Gly Gly Gly Gly Gly Gly 25

<2Ι0> 59 <211> 17 <212> PRT <213> Artificial <220> <223> Ligand 27 <400> 59

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

Gly

<210> 60

<211> 27

<212> PRT

<213> Artificial

<220>

<223> Ligant 28

<40Ο> 60

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 Gly Gly Gly Gly Gly Gly Gly Gly 25

<210> 61

<211> 6

<212 »PRT

<213> Artificial

<22Q>

<223> binder 29

<400> 61

Read It Be Read It Be Gly Gly 1 5

<210> 62

<211> 7

<212> PRT

<213> Artificial

<220>

<223> binder 30

<400> 62

Read It Be Read It Be Pro Gly Gly

1 5

<210> 63

<211> 214

<212> PRT

<213> Homo sapiens

<400> 63

Asp Ile Gln Met Thr Gln Be Pro Be Be Read Be Be Be Val Gly 10 15

Glu Thr Val Thr Ile Thr Cye Arg Wing Gly Asn Ile His Gly Tyr 25 30

Leu Wing Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu goes 40 45

Tyr Asn Thr Lys Thr Read Wing Glu Gly Val Pro Be Arg Phe Be Gly

50 55 60

Being Gly Being Gly Thr Gln Phe Being Read Lys Ile Asn Being Read Gln Pro 65 70 75 80 Glu Asp Phe Gly Asn Tyr Cyr Gln His His Tyr Asp Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Wing Wing

100 105 110

Pro Be Go Phe Ile Phe Pro Be Asp Glu Gln Read Lys Ser Gly 115 120 125

Thr Wing Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Wing

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Wing Read Gln Ser Gly Asn Ser Gln 145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His Lys Val Tyr 18C 185 190

Cys Wing Glu Val Thr His Gln Gly Read Ser Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 64

<211> 44 7

<212> PRT

<213> Homo sapiens

<400> 64

Gln Val Gln Leu Lys Gln Be Gly Pro Gly Leu Val Arg Pro Be Gln 1 5 10 15

Be Read Be Ile Thr Cys Thr Val Be Gly Phe Pro Read Gly Ile Phe 25 30

Glv Val His Trp Val Arg Gln Ser Pro Gly Lys Glv Leu Glu Trp Leu 40 45

Gly Val Tle Trp Lys Gly Gly Gn Asn Thr Asp Tyr Asn Ala Wing Phe Met 50 55 60 Be Arg Leu Arg Ile Thr Lys Asp Asn Be Lys Be Gln Val Phe Phe

65 70 75 80

Arg Met Asn Be Read Gln Thr Asp Asp Thr Wing Ile Tyr Tyr Cys Wing 85 90 95

Lys Val Asn Leu Wing Asp Wing Met Asp Wing Tyr Trp Gly Gln Gly Thr Ser 100 105 110

Val Ile Val Ser Be Wing Be Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125

Pro Wing Be Ser Lys Be Thr Be Glv Gly Thr Wing Wing Read Gly Cys 130 135 140

Read Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160

Gly Wing Leu Thr Be Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 180 190

Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

Thr Lys Val Asp Lys Lys VaI Glu Pro Lvs Ser Cys Asp Lys Thr His 210 215 220

Thr Cys Pro Pro Cys Pro Wing Pro Glu Read Leu Gly Gly Pro Ser Val 225 230 235 240

Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Be Arg Thr 245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Be His Glu Asp Pro Glu 260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Wing Lys 275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Aen Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320

Cya Lys Val Ser Aan Lys Wing Pro Leu Pro Wing Ile Glu Lys Thr Ile 325 330 335

Be Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln Go Tyr Thr Leu Pro 340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gla Val Ser Leu Thr Cys Leu 355 360 365

Val Lys Gly Phe Tyr Pro Be Asp Ile Wing Val Glu Trp Glu Ser Asn 37C 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Will Read Asp Ser 385 390 395 400

Asp Gly Be Phe Phe Read Tyr Be Lys Read Thr Val Agp Lys Be Arg 405 410 415

? rp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Wing Leu 420 425 430

His Asn Kis Tvr Thr Gln Lys Be Read Be Read Be Pro Gly Lys 435 * 440 445

<210> 65 <211> 503 <212> PRT <213> Homo sapiens <400> 65 Gln Val Gln Leu Lys

15

Be Read Be Ile Thr Cys Thr Val Be Gly Phe Pro Read Gly Ile Phe 25 30

Gly Val Kis Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 40 4S

Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Wing Phe Met 50 55 60 Ser Arg Read Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Arg Met Asn Be Read Gln Thr Asp Asp Thr Wing Ile Tyr Tyr Cys Wing 85 90 95

Lys Val Asn Leu Wing Asp Wing Met Asp Tyr Trp Gly Gln Gly Thr Ser

IOC 105 lio

Val Ile Val Ser Be Wing Be Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125

Wing Pro Be Lys Be Thr Be Gly Gly Thr Wing Wing Read Gly Cys 130 135 140

Read Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160

Gly Wing Leu Thr Be Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 180 190

Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn Hls Lys Pro Ser Asn 195 200 205

Thr Lys Val Asp Lys VaI Glu Pro Lys Be Cys Asp Lys Thr His 210 215 220

Thr Cys Pro Pro Cys Pro Wing Pro Glu Read Leu Gly Gly Pro Ser Val 225 230 235 240

Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu Met Ile Be Arg Thr 245 250 255

Pro Glu Go Thr Cys Go Go Go Asp Val Be His Glu Asp Pro Glu 260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Wing Lys 275 280 285 Thr Lys Pro Arg Glu Glu

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320

Cys Lys Val Ser Asn Lys Wing Leu Pro Wing Pro Ile Glu Lys Thr Ile 325 330 335

Ser Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350

Pro Ser Axg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365

Val Lys Gly Phe Tyr Pro Asp Ile Wing Val Glu Trp Glu Ser Asn 370 375 360

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400

Asp Gly Be Phe Phe Read Tyr Be Lys Read Thr Val Asp Lys Be Arg 405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Wing Leu 420 425 430

His Asri His Tyr Thr Gln Lys Be Read Be Read Be Pro Gly Lys Gly 435 440 445

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gly Asn 450 455 460

Asn Thr Thr Trp Glu Wing Trp Asp Arg Wing Ile Wing Glu Tyr Wing Wing 465 470 475 480

Arg Ile Glu Wing Leu Ile Arg Wing Wing Gln Glu Gln Gln Gln Glu Lys Asn 485 490 495

Glu Wing Wing Leu Arg Glu Leu 500

<210> 66 <21i> 345 <212> PRT <213> Human Immunodeficiency Virus <400> 66

Val Gly Wing Ile Gly Wing Leu Phe Leu Gly Phe Leu Gly Wing Gly Wing 10 15

Be Thr Met Gly Wing Ward Be Met Thr Read Val Thr Wing Gln Arg Wing Gln

25 30

Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 40 45

Glu Wing Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln 50 55 60

Leu Gln Arg Wing Ile Leu Wing Val Glu Arg Tyr Leu Lys Asp Gln Gln 65 70 75 80

Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Wing 85 90 95

Val Pro Trp Asn Wing Be Trp Be Asn Lys Be Read Glu Gln Ile Trp 100 105 110

Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 115 120 125

Ser Leu Ile His Ser Leu lie Glu Glu Ser Gln Asn Gln Gln Glu Lys 130 135 140

Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Wing To Be Leu Trp Asn

145 150 155 160

Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile Met 165 170 175

Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Wing Val Leu Ser 180 185 190

Ile Val Asn Arg Val Arg Gln Gly Tyr To Be Pro Read To Be Phe Gln Thr 195 200 205

His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu 210 210 220 Glu Gly Gly Gly Glu Arg Asp Arg Asp Arg Ser Ile Arg Leu Val Asn Oly 225 230 235 240

Ser Leu Wing Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser 245 250 255

Tyr His Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu 260 265 270

Leu Leu Gly Arg Arg Gly Trp Glu Wing Leu Lys Tyr Trp Trp Asn Leu 275 280 285

Read Gln Tyr Trp Be Gln Glu Read Lys Asn Be Wing Will Be Read Leu

290 295 300

Asn Wing Thr Ile Wing Wing Val Val Wing Glu Gly Thr Wing Asp Arg Val Ile Glu 305 310 315 320

Val Val Gln Gly Cys Wing Arg Wing Ile Arg His Ile Pro Arg Arg Ile 325 330 335

Arg Gln Gly Leu Glu Arg Ile Leu Leu 340 345

<210> 67 <211> 20 «212> PRT <213> Artificial <220> <223> ligange 31 <400> 67

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly G X 5 10 15

Gly Gly Gly Ser 20

<21ü> 68

<2ll> 23

<212> PRT

<213> Artificial

<220>

<223> ligange 32 <4QÜ> 68

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

Gly Gly Gly Ser Gly Gly Gly 20

<210> 69 <211> 15 PRT <213> Artificial c220> <223> ligange 33 <40G> 69

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 10 10 15

<210> 70

<211> 16

<212> PRT

<213> Artificial

<22G>

<223> ligange 34

<400> 70

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 * 10 15

<21Q> 71

<211> 219

<212> PRT

<213> Artificial

<220>

<223> Anti-CD4 antibody light chain <400> 71

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Read Ala Val Val Read Gly 10 IS

Glu Arg Val Thr Met Asn Cys Lys Be Ser Gln Be Read Leu Tyr Ser 25 30

Thr Asn Gln Lys Asn Tyr Leu Wing Trp Tyr Gln Gln Lys Pro Gly Gln 40 45 Ser Pro Lys Leu Read Ile Tyr Trp Wing Being Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read 65 65 75 80

Ile Ser Ser Val Gln Wing Glu Asp Val Wing Val Tyr Tyr Cys Gln Gln 85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys IOC 105 110

Arg Thr Val Wing Ward Pro Be Val Phe Ile Phe Pro Pro Asp Glu 115 120 125

Gln Leu Lys Be Gly Thr Wing Be Val Val Cys Leu Leu Asn Asn Phe 130 135 140

Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn Wing Leu Gln 145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Olu Gln Asp Ser Lys Asp Ser 165 170 175

Thr Tyr Be Read Be Ser Thr Thr Read Read Thr Read Be Lys Wing Asp Tyr Glu

160 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205

Pro Val Thr Lys Be Phe Aen Arg Gly Glu Cys 210 215

<21Q> 72

<211> 452

<212> PRT

«213> Artificial

<220>

<223> Anti-CD4 antibody heavy chain

<4 QQ> 72

Gln Val Gln Leu Gln Gln Ser Gly Fro Glu Val Val Lys Pro Gly Wing 1 5 10 15 Ser Val Lvs Met Ser Cys Lys Wing Gly Tyr Thr Phe Thr Ser Tyr 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Read Asp Trp Ile 40 45

Gly Tyr lie Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lya Gly Lya Ward Thr Read Thr Be Asp Thr Be Thr Be Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Le Be Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp 100 105 110

Gly Gln Gly Thr Read Val Val Val Be Ser Wing Be Thr Lys Gly Pro 115 120 125

Be Val Phe Pro Read Wing Pro Be Ser Lys Ser Thr Be Gly Gly Thr 130 135 140

Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160

Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His Thr Phe Pro 165 170 175

Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Thr 180 185 190

Val Pro Be Ser Be Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220

Cys Asp Lys Thr His Hir Cys Pro Pro Cys Pro Wing Pro Glu Wing Wing 225 230 235 240

Gly Gly? Ro Be Val Phe Valu Phe Pro Lys Pro Lys Pro Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Cys Val Val Val Asp Val Ser 260 265 270

His Glu Aep Pro Glu Val Lyo Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300

Tyr Arg Val Val Ser Val Val Read Thr Val Val Read His Gln Asp Trp Le Val Asn 305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Wing Pro Leu Pro 325 330 335

Ile Glu Lys Thr Ile Be Lys Wing Lys Gly Gln Pro

34Q 345 350

Val Tyr Thr Leu Pro Pro Being Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Aap Ile Wing Val 370 375 380

Glu Trp Glu Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400

Pro Val Leu Asp Be Asp Gly Be Phe Phe Leu Tvr Be Lys Leu Thr 405 410 415

Val Asp Lys Be Arg Trp Gln Gln Gly Asn Val Phe Be Cys Be Val 420 425 430

Met His Glu Wing Read His Asn His Tyr Thr Gln Lys Be Read Be Read 435 440 445

Ser Pro Gly Lys 450

<210> 73

«211> 37

<212> PRT

<213> Artificial

<220> <223> afp-l <400> 73

Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Gln Tyr Thr Ser 10 15

Leu Ile His Ser Leu Ile Glu Glu Being Gln Asn Gln Gln Glu Lys Asn 25 30

Glu Gln Glu Leu Leu 35 <21Q> 74 <211> 503 «212> PRT <213> Artificial <22Q> <223> Heavy Chain

afp-l antifusogenic agent <400> 74

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Wing 10 15

Be Val Lys Het Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Read Aap Trp Ile 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe 50 55 60

Lys Gly Lys Wing Thr Read Thr Be Asp Thr Be Thr Be Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Le Be Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp 100 105 no

Gly Gln Gly Thr Read Val Val Val Be Ser Ala Be Thr Lys Gly Pro 115 120 125 Be Val Phe Pro Read Ala Pro Val Be Ser Lys Be Thr Be Gly Gly Thr 130 13S 140

Wing Wing Leu Oly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160

Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His Thr Phe Pro 165 170 175

Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Thr 180 165 190

Val Pro Be Ser Be Read Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Wing Pro Glu Wing Wing 225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu 245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300

Tyr Arg Val Val Ser Val Val Read Thr Val Val Read His Gln Asp Trp Le Val Asn 305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Wing Pro Leu Pro 325 330 335

Ile Glu Lys Thr Ile Be Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350

Val Tyr Thr Leu Pro Pro Be Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Be Asp Ile Val Wing 370 375 380

Glu Trp Glu Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400

Pro Val Leu Asp Be Asp Gly Be Phe Phe Leu Tyr Be Lys Leu Thr 405 410 415

Val Asp Lys Be Arg Trp Gln Gln Gly Asn Val Phe Be Cys Be Val 420 425 430

Met His Glu Wing Read His Asn His Tyr Thr Gln Lys Be Read Be Read 435 440 445

Be Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

Ser Gly Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Gln Tyr 465 470 475 480

Thr Be Read Ile His Be Read Ile Glu Glu Be Gln Asn Gln Gln Glu 485 490 495

Lys Asn Glu Gln Glu Leu Leu 500

<210> 75

<211> 112

<212> PRT

<213> Mus musculus

<400> 75

Asp Ile Val Met Thr Gln Be Pro Be Be Read Ala Will Be Val Gly

10 15

Glu Lys Val Thr Met Ile Cys Lys Be Ser Gln Be Read Leu Tyr Ser 25 30

Thr Asn Gln Lys Asn Tyr Leu Wing Trp Tyr Gln Lys Pro Gly Gln

40 45

Be Pro Lys Leu Read Ile Tyr Trp Wing Be Thr Arg Glu Be Gly Val SO 55 60 Pro Asp Arg Phe Thr Gly Be Gly Be Gly Thr Asp Phe Thr Leu Thr 65 70 75 80

Ile Ser Ser Val Lys Glu Wing Asp Leu Val Wing Tyr Tyr Cys Gln Gln 85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lvs Leu Glu Ile Lys 100 105 lio

<21Q> 76

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Chimeric light chain variable domain <4Q0> 76

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Wing Val Ser Leu Gly 10 15

Glu Arg Wing Wing Ile Asn Cys Lys Be Ser Gly Be Read Leu Tyr Ser 25 30

Thr Asn Gln Lys Asn Tyr Leu Wing Trp Tyr Gln Lys Pro Gly Gln 40 45

Pro Pro Lys Read Leu Ile Tyr Trp Wing Be Thr Arg Glu Be Gly Val 50 55 60

Pro Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read 65 65 75 80

Ile Ser Ser Leu Gln Wing Glu Asp Val Wing Val Tyr Tyr Cys Gln Gln 85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys 100 105 lio

<210> 77

<211> 241

<212> PRT

<213> Artificial

<220> <223>

Chimeric Light Chain <400> 77

Met Asp Met Arg Val Pro Wing Gln Leu Leu Gly Leu Leu Leu Leu Trp 10 15

Read Pro Gly Wing Arg Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser 25 30

Leu Wing Val Ser Leu Gly Glu Arg Wing Thr Ile Asn Cys Lys Ser Ser 40 45

Gly Ser Leu Read Tyr Be Thr Asn Gln Lys Asn Tyr Leu Wing Trp Tyr 50 55 60

Gln Gln Lys Pro Gly Gln Pro Lys Read Leu Ile Tyr Trp Wing Ser 65 70 75 80

Thr Arg Glu Be Gly Val Pro Asp Arg Phe Be Gly Be Gly Be Gly

85 90 95

Thr Asp Phe Thr Read Le Thr Ile Be Ser Leu Gln Wing Glu Asp Val Wing 100 105 110

Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Arg Thr Phe Gly Arg Gly 115 120 125

Thr Lys Leu Glu Ile Lys Arg Thr Val Ward Wing Pro Ser Val Phe Ile 130 135 140

Phe Pro Pro Be Asp Glu Gln Read Lys Be Gly Thr Wing Be Val Val 145 150 155 160

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys 165 170 175

Val Asp Asn Wing Read Le Gln Be Gly Asn Be Gln Glu Be Val Thr Glu 180 185 190

Gln Asp Be Lys Asp Be Thr Tyr Be Read Be Be Thr Read Le Thr 195 200 205

Ser Lys Asp Wing Tyr Glu Lys His Lys Val Tyr Wing Cys Glu Val Thr 210 215 220 His Gln Gly Leu Being Ser Pro Val Thr Lys Being Phe Asn Arg Gly Glu 225 230 235 240

Cys

<210> 78

<211> 112

<212> PRT

<213> Artificial

<220>

<223> Chimeric light chain variable domain <400> 78

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Wing Val Ser Leu Gly 10 15

Glu Arg Val Thr Met Asn Cys Lys Be Ser Gln Be Read Leu Tyr Ser 25 30

Thr Asn Gln Lye Asn Tyr Leu Wing Trp Tyr Gln Lys Pro Gly Gln

40 45

Be Pro Lys Read Leu Ile Tyr Trp Wing Be Thr Arg Glu Be Gly VaI 50 55 60

Pro Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read 65 65 75 80

Ile Ser Ser Val Gln Wing Glu Asp Val Wing Val Tyr Tyr Cys Gln Gln 85 90 95

Tyr Tyr Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110

<210> 79

<211> 108

<212> PRT

<213> Artificial

<220>

<223> Chimeric light chain variable domain <40O> 79

Asp Ile Gln Met Thr Gln Be Pro Be Be Read Ala Be Val Ser Gly 10 15 Asp Arg Val Thr Ile Thr Cys Alys Be Glri Asp Ile Asn Asn Tyr 25 30

Ile Wing Trp Tyr Gln His Thr Pro Gly Lys Wing Pro Lys Leu Leu Ile 40 45

His Tyr Thr Be Thr Read Gln Pro Gly Val Pro Be Arg Phe Be Gly 50 55 60

Being Gly Being Gly Thr Asp Tyr Thr Phe Thr Ile Being Being Read Gln Pro 65 70 75 80

Glu Asp Ile Wing Thr Tyr Cys Leu Gln Gln Tyr Asp Asn Leu Phe 85 90 95

Thr Phe Gly Gln Gly Thr Lys Read Gln Ile Thr Arg 100 105

<210> 80 <211> 112 <212> PRT <213> Artificial <220> <223> Variable Domain <400> 80

Asp Ile Val Met Thr Gln Be Pro Asp Be Read Ala Wing Val Be Read Gly 1 5 10 15

Glu Arg Val Thr Met Asn Cys Lys Be Ser Gln Be Read Leu Tyr Ser 25 30

Thr Asn Gln Lys Asn Tyr Leu Wing Trp Tyr Gln Lys Pro Gly Gln 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Wing Ser Thr Arg Glu Ser Gly Val 50 55 60

Pro Asp Arg Phe Be Gly Be Gly Be Gly Thr Asp Phe Be Read 65 65 75 80

Ile Ser Ser Val Gln Wing Glu Asp Val Val Wing Val Tyr Tyr Cys Gln Gln 85 30 95 Tyr Tyr Be Tyr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 HO

<210> 81

<211> 122

<212> PRT

<213> Mus musculus

<400> 81

Glu Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Ly3 Pro Gly Wing 10 15

Be Val Lys Met Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Aep Trp Ile 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe 50 55 60

Lys Gly Lys Wing Thr Thr Read Asp Lys Be Wing Being Thr Tyr 65 70 75 80

Met Glu Read Be Ser Read Thr Be Glu Asp Be Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp 100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120

<210> 82

<211> 122

<212> PRT

<213> Artificial

<220>

<223> Humanized heavy chain variable domain <400> 82

Gln Val Gln Leu Gln Glu Ser Gly Wing Glu Val Lys Lys Pro Gly Wing IS 10 15

Ser Val Lys Val Ser Cys Lys Wing Ser Gly Tyr Thr Phe Thr Ser Tyr 25 30 Val Ile His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu Glu Trp Ile 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe 50 55 60

Lys Gly Lys Wing Thr Val Thr Read Asp Pro Be Thr Asn Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Le Be Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp

100 105 110

Gly Gln Gly Thr Read Val Val Val Ser Ser 115 120

<210> 83

<211> 467

<212> PRT

<213> Artificial

<220>

<223> Chimeric heavy chain

«400> 83

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Val Wing Val Pro Gly 10 15

HiS Wing Gln Val Gln Leu Gln Glu Be Gly Glu Wing Val Lys Lys 25 30

Pro Gly Wing Be Val Lys Val Be Cys Lys Wing Be Gly Tyr Thr Phe 40 45

Thr Ser Tyr Val Ile His Trp Val Arg Gln Pro Wing Gly Gln Gly Leu

50 55 SO

Glu Trp Ile Gly Tyr Asle Pro Tyr Asn Asp Gly Thr Asp Tvr Asp 65 70 75 80

Glu Lys Phe Lys Gly Lys Wing Thr Val Thr Read Asp Pro Be Thr Asn 85 90 95 Thr Wing Tyr Met Glu Read Be Ser Leu Arg Be Glu Asp Thr Wing Val 100 105 110

Tyr Tyr Cys Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe 115 120 125

Wing Tyr Trp Gly Gln Gly Thr Read Val Val Val Ser Ser Wing Al Ser Thi 130 135 140

Lys Gly Pro Be Val Phe Pro Read Wing Pro Cys Be Arg Be Thr Be 145 150 155 160

Glu Ser Thr Wing Wing Read Gly Cys Read Val Val Lys Asp Tyr Phe Pro Glu 165 170 175

Pro Val Thr Val Ser Trp Asn Ser Gly Wing Read Thr Ser Gly Val His 180 185 190

Thr Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Le Ser Ser 195 200 205

Val Val Thr Val Prq Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 210 215 220

Asn Val Asp His Lys Pro Being Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240

Be Lys Tyr Gly Pro Pro Cys Pro Be Cys Pro Wing Pro Glu Phe Leu 245 250 255

Gly Gly Pro Ser Val Phe Leu Phe Pro Lys Pro Lys Asp Thr Leu 260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285

Gln Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Goes Glu 290 295 300

Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320

Tyr Arg Val Val Ser Val Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 340 345 350

Ile Glu Lys Thr Ile Be Lys Wing Lys Gln Pro Arg Glu Pro Gln Val 355 360 365

Tyr Thr Leu Pro Pro Gln Glu Glu Mefc Thr Lys Asn Gln Val Ser 370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Wing Val Glu 385 390 395 400

Trp Glu Be Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415

Val Leu Asp Be Asp Gly Be Phe Phe Leu Tyr Be Arg Leu Thr Val 420 425 430

Asp Lys Be Arg Trp Gln Glu Gly Asn Val Phe Be Cys Be Val Met 435 440 445

His Glu Ala Read His Asn His Tyr Thr Gln Lys Be Read Be Read Be 450 455 460

Read Gly Lys 465

<210> 84

<211> 122

<212> PRT

<213> Artificial

<220>

<223> Chimeric heavy chain variable domain <400> 84

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Wing 10 15

Be Val Lys Met Be Cys Lys Wing Be Gly Tyr Thr Phe Thr Be Tyr 25 30

Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Read Asp Trp Ile 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe 50 5S 60

Lys Gly Lys Wing Thr Read Thr Be Asp Thr Be Thr Be Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Arg Be Glu Asp Thr Wing Val Tyr Tyr Cys

85 9D 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp 100 105 110

Gly Gln Gly Thr Read Val Val Val Ser Ser 115 120

<210> 85

«211> 120

<212> PRT

<213> Artificial

<2 2 0>

<223> Chimeric heavy chain variable domain <4Q0> 85

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 10 15

Be Read Arg Read Be Cys Be Wing Be Gly Phe Thr Phe Be Asn Tyr 25 30

Wing Met Ser Trp Val Arg Gln Pro Wing Gly Lys Gly Leu Glu Trp Val 40 45

Wing Wing Ile Be Asp His Be Thr Asn Thr Tyr Tyr Pro Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Be Arg Asp Asn Be Lys Asn Thr Read Phe 65 70 75 80

Read Gln Met Asp Be Read Arg Pro Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Lys Tyr Gly Gly Asp Tyr Asp Pro Phe Asp Tyr Trp Gly Gln 100 105 110 GXy Thr Pro Val Thr Val Ser Ser 115 120

<210> 86

<211> 122

<212> PRT

<213> Artificial

<220>

<223> Chimeric heavy chain variable domain

«40Q> 86

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Wing 10 15

Ser Val Lyo Met Ser Cyo Lys Wing Ser Gly Tyr Thr Phe Thr Ser Tyr 25 OJ

Val Ue His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe

50 55 60

Lya Gly Lys Ward Thr Read Thr Be Asp Thr Be Thr Be Thr Wing Tyr 65 70 75 80

Met Glu Read Be Ser Read Le Be Glu Asp Thr Wing Val Tyr Tyr Cys 85 90 95

Wing Arg Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr Trp 100 105 110

Gly Oln Gly Thr Read Val Val Val Ser Ser 115 120

<210> 87

<211> 1?

<212 »PRT

<213> Mus musculus

<400 · »87

Lys Be Ser Gln Be Read Leu Tvr Be Thr Asn Gln Lys Asn Tyr Leu 10 15

Wing <21Q> 88

<211> 17

<212> PRT

<213> Artificial

<220>

<223> CDR

<400> 88

Lys Be Ser Gly Be Read Leu Tyr Be Thr Asn Gln Lys Asn Tyr Leu 10 15

Allah

<210> 89

<211> 17

<212> PRT

<213> Artificial

<2 2 0>

<223> CDR

<400> 89

Lys Be Ser Gln Be Read Leu Tyr Be Thr Asn Gln Lys Asn Tyr Leu 10 15

Allah

<210> 90

<211> 9

<212> PRT

<213> Artificial

<220>

<223> CDR

<400> 30

Lys Wing Ser Gln Asp Ile Asn Asn Tyr 1 5

<210> 91

<211> 7

<212> PRT

<213> Mus musculus

<400> 91 Trp Ala Ser Thr Arg Glu Ser

<210> 92

<211> 7

<212> PRT

<213> Mus musculus

<4Q0> 92

Trp Wing Be Thr Arg Glu Ser 1 5

<210> 93

<211> 11

<212> PRT

<213> Hus musculus

<40Q> 93

Tyr Thr Be Thr Read Gln Pro Gly Val Pro Ser 10

<21Q> 94

<211> 8

<212> PRT

<213> Mus musculus

<4 The Q> 94

Gln Gln Tyr 'Iyr Be Tyr Arg Thr 1 5

<210> 95

<211> 8

<212> PRT

<213> Mus musculus

<400> 95

Gln Gln Tyr Tyr Ser Tyr Arg Thr 1 5

<210> 96

<211> 6

<212> PRT

<213> Mu3 musculus

<400> 96

Tyr Asp Asn Leu Leu Phe 1 5 <210> 97

<211> 5

<212> PRT

<213> Mus musculus

<400> 97

Ser Tyr Val Ile His

1 s

<210> 98

<211 »S

<212> PRT

<213> Mus musculus

<400> 98

Ser Tyr Val Ile His

1 5

<210> 99

<211> 10

<212> PRT

<213> Mus musculus

<400> 99

Gly Phe Thr Phe Ser Asn Tyr Ala Met Ser 10

<210> 100

<211> 17

<212> PRT

<213> Mus musculus

<400> 100

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lye Phe Lys 10 15

Gly

<210> 101

«211> 17

«212> PRT

<213> Mus musculus

<400 »101

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe Lys 10 15 Gly

<210> 102

<211> 12

<212> PRT

<213> Mus musculus

<400> 102

Wing Xle Be Asp His Be Thr Asn Thr Tyr Tyr Pro 10

<210> 103

<211> 13

<212> PRT

<213> Mus musculus

<400 »103

Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr 10

<210> 104 <211 »13 <212> PRT <213» Mus musculus <400> 104

Glu Lys Asp Asn Tyr Wing Thr Gly Wing Trp Phe Wing Tyr 10

<210> 105

<211> 11

<212> PRT

<213> Mus musculus

<4Ü0> 105

Wing Arg Lys Tyr Gly Gly Asp Tyr Asp Pro Phe 10

Claims (25)

1. Conjugate comprising one or more antifungal peptides and an antibody against an HIV gp120-binding cell surface receptor, characterized in that two to four antifungal peptides are each conjugated to a termination of the heavy and / or light chains of said antibody against a cell surface receptor that binds to HIV gp120. Conjugate according to Claim 1, characterized in that the antibody against an HIV gp120-binding cell surface receptor is an anti-CCR5 (mAB CCR5) antibody. Conjugate according to Claim 2, characterized in that at least two of the terminations of said anti-CCR5 antibody are each conjugated to an antifusogenic peptide. Conjugate according to Claim 2, characterized in that said antifusogenic peptides are linear peptides and comprise an amino acid sequence of 5 to 100 amino acids. Conjugate according to any one of claims 2 to 4, characterized in that mAb CCR5 - [linker] m- [antifungal peptide], wherein m is independently for each antifusogenic peptide, or 0 or 1, and η is an integer at most between 1 to 8. Conjugate according to claim 5, characterized in that it comprises a heavy and / or light chain conjugate of mAB CCR5 and the selected antifusogenic peptide (s) ("chain conjugate") from the group consisting of: (1) [antifusogenic peptide] - [ligand] m- [heavy chain] (2) [heavy chain] - [ligand] m- [antifusogenic peptide] (3) [antifusogenic peptide] - [ligand ] m- [heavy chain] - [antifusogenic peptide] (4) [antifusogenic peptide] - [ligand] m- [light chain] (5) [light chain] - [ligand] m- [antifusogenic peptide] (6 ) [antifusogenic peptide] - [ligand] m- [light chain] - [antifusogenic peptide] (7) [antifusogenic peptide] - [ligand] m- [heavy chain] - [ligand] m- [antifusogenic peptide] ( 8) [antifusogenic peptide] - [ligand] m- [light chain] - [ligand] m - [antifusogenic peptide] where the Ligand may be the same or different, where m is an integer from 1 to 0 in can independently be the same or different. Conjugate according to Claim 6, characterized in that it comprises a chain conjugate (2), (3), (4), or (7). Conjugate according to claim 6, characterized in that it comprises 2x [mR CCR5 light chain] and 2 χ (2), 2x [mAB CCR5 light chain] and 2x (3), 2x [mAB CCR5 heavy chain] and 2x (4) or 2x [mAB CCR5 light chain] and 2x (7). Conjugate according to any one of claims 2 to 8, characterized in that said antifusogenic peptide is known from the group of peptides defined by SEQ ID NO: 29 to 35 and SEQ ID NO: 73. Conjugate according to any one of claims -2 to 9, characterized in that said anti-CCR5 antibody comprises a heavy chain variable domain consisting of an immunoglobulin structure and a CDR3 region selected from the group consisting of the heavy chain CDR3 sequences SEQ ID NO: 16, 17. Conjugate according to any one of claims 2 to 10, characterized in that said anti-CCR5 antibody comprises a heavy chain variable domain consisting of an immunoglobulin structure and a CDR3 region selected from the group consisting of in the heavy chain CDR3 sequences SEQ ID NO: 16 and 17, a CDR2 region selected from the group consisting of the heavy chain CDR2 sequences of SEQ ID NO: 13, 14 and 15 and a selected CDR1 region of the group consisting of heavy chain CDR1 sequences SEQ ID NO: 9,10,11 and 12. Conjugate according to any one of claims -2 to 11, characterized in that said anti-CCR5 antibody comprises a heavy chain variable domain selected from the group of SEQ ID NO: 1, 3, 5e7. Conjugate according to any one of claims -2 to 12, characterized in that said anti-CCR5 antibody comprises a light chain variable domain consisting of an immunoglobulin backbone and a selected CDR1 region of SEQ ID. NO: 18, 19 and 20, a selected CDR2 region of SEQ ID NO: 21, 22 and 23 and a selected CDR3 region of SEQ ID NO: 24 and 25. Conjugate according to any one of claims -2 to 13, characterized in that said anti-CCR5 antibody comprises 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 are 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. A conjugate according to any one of claims -2 to 14, characterized in that said anti-CCR5 antibody comprises a heavy chain variable domain and a light chain variable domain independently selected from the group consisting of a ) the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the light chain variable domain (V1) defined by SEQ ID NO: 2; b) the heavy chain variable domain defined by the 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 the 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 the amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by SEQ ID NO: 8. Conjugate according to any one of claims -2 to 15, characterized in that said anti-CCR5 antibody comprises the heavy chain variable domain (Vh) defined by the amino acid sequence SEQ ID NO: 1 and the light chain variable domain (V1) defined by SEQ ID NO: 2, the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and the light chain variable domain defined by SEQ ID NO: 4 , the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 6 or the heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by the amino acid sequence SEQ ID NO: 8; a ligand selected from the group consisting of the amino acids glycine (G) and asparagine (Ν), the tripeptide GST and SEQ ID NO: 36 to 62 and SEQ ID NO: 67 to 70; and an antifusogenic peptide selected from the group of peptides defined by SEQ ID NO: 29 to 35 and SEQ ID NO: 73. Conjugate according to any one of claims -2 to 15, characterized in that it comprises an antifusogenic peptide selected from the group of SEQ ID NO: 29 to 35 and SEQ ID NO: 73. A conjugate according to any one of claims -2 to 17, characterized in that it comprises 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 ligand of SEQ ID NO: 40 and an antifusogenic peptide of SEQ ID NO: 33, comprising two light chain variable domains of SEQ ID NO: 4, two conjugates of type (2) each one comprising a heavy chain variable domain of SEQ ID NO: 3, a ligand of SEQ ID NO: 40 and an antifungal peptide of SEQ ID NO: 33, 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 ligand of SEQ ID NO: 40, and an antifusogenic peptide of SEQ ID NO: 33 or by comprise 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 ligand of SEQ ID NO: 40, and an antifusogenic peptide of SEQ ID NO: 33. A conjugate according to any one of claims 2 to 18, characterized in that said anti-CCR5 antibody is of the IgG4 subclass, or of the IgGI or IgG2 subclass, with a mutation at amino acid S228, L234, L235, and / or D265 and / or contain the PVA236 mutation. Conjugate according to claim 19, characterized in that said anti-CCR5 antibody of subclass lgG4 has the S228P mutation and said anti-CCR5 antibody of subclass IgGI has mutations L234A and L235A. . A method for producing a conjugate as defined in any one of claims 1 to 20, characterized in that the method comprises a) culturing a cell containing one or more plasmids containing one or more nucleic acid molecules encoding a conjugate according to claims 1 to 20 under conditions suitable for expression of the conjugate, b) recovering the conjugate from the cell or cell culture supernatant. Pharmaceutical composition, containing a conjugate as defined in any one of claims 1 to 20, together with a pharmaceutically acceptable excipient or carrier. Use of a conjugate as defined in any one of claims 1 to 20 for the manufacture of a medicament for treating viral infections. Use according to claim 23, characterized in that the viral infection is an HIV infection. Use of a conjugate as defined in any one of claims 1 to 20 for treating a patient in need of antiviral treatment.