KR101105610B1 - A conjugate of an antibody against ccr5 and an antifusogenic peptide - Google Patents

Abstract

The present invention relates to a conjugate comprising at least one anti-fusogenic peptide and an antibody against an HIV gp120 binding cell surface receptor, and to the pharmaceutical use of the conjugate, wherein the conjugate comprises one to eight antifusogenic peptides. Each conjugated to one terminus of the heavy and / or light chain of said antibody against HIV gp120 binding cell surface receptor.

Conjugate

Description

Conjugate of antibody and anti-fusogenic peptide to CCR5 {A CONJUGATE OF AN ANTIBODY AGAINST CCR5 AND AN ANTIFUSOGENIC PEPTIDE}

The present invention relates to conjugates of antibodies against CCR5 and antifusogenic peptides, wherein 1 to 8 antifusogenic peptides are each conjugated to one end of the heavy and / or light chain of the anti-CCR5 antibody. have. Antifusogenic peptides may be different, similar or identical in amino acid level.

Cell infection by human immunodeficiency virus (HIV) is performed by a process in which the membrane of the infected cell and the viral membrane are fused. A general scheme for this process is proposed: the viral envelope glycoprotein complex (gp120 / gp41) interacts with cell surface receptors located on the membrane of infected cells. When gp120 binds to the CD4 receptor, for example with a co-receptor such as CCR-5 or CXCR-4, a structural change of the gp120 / gp41 complex occurs. As a result of this structural change, the gp41 protein can be inserted into the membrane of the target cell. This insertion is the beginning of the membrane fusion process. Because of the naturally occurring polymorphism, the amino acid sequence of the gp41 protein is known to vary according to different HIV strains. However, the same domain form, more precisely the fusion signal, two heptad repeat domains (HR1, HR2) and transmembrane domains (from the N-terminus to the C-terminus) can be recognized. It is proposed that the fusion (or fusogenic) domain participates in insertion into and disruption of the cell membrane. The HR region consists of multiple stretches comprising seven amino acids (“heptad”) (see, eg, Shu, W., et al., Biochemistry 38 (1999) 5378-5385). In addition to the heptad, there are one or more leucine zipper-type motifs. The composition forms a coiled coil structure of gp41 protein and as well as peptides derived from the domain. A wound coil is generally an oligomer consisting of two or more interacting spirals. Peptides having amino acid sequences derived from the HR1 or HR2 domain of gp41 are effective in vitro and in vivo inhibitors of the acquisition of HIV into cells (eg, peptides are described, for example, in 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®, HR2 peptide), T651 (US 6,479,055), and T2635 (WO 2004/029074) are very potent inhibitors of HIV infection. For example, attempts have been made to enhance the efficacy of HR2 inducing peptides using 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 a peptide to a particular molecule can change its pharmacokinetic properties, for example, an increase in the serum half-life of the peptide conjugate. Conjugates include, for example, chimeric molecules including polyethylene glycol (PEG) and interleukin-6 (EP 0 442 724), PEG and erythropoietin (WO 01/02017), endostatin and immunoglobulins (US 2005/008649). For secreted antibody based fusion proteins (US 2002/147311), fusion polypeptides comprising albumin (US 2005/0100991; human serum albumin US 5,876,969), PEGylated polypeptides (US 2005/0114037), and interferon fusions Reported. Also known in the art are immunotoxins including gelonin and antibodies (WO 94/26910), modified transferrin-antibody fusion proteins (US 2003/0226155), antibody-cytokine fusion proteins (US 2003/0049227), and immunity Fusion proteins (US 2003/0103984) which consist of peptides and antibodies with stimulatory, membrane transport, or homoaffinity activity are described. In WO 2004/085505 a long acting biologically active conjugate consisting of a biologically active compound chemically linked to macromolecules is reported.

Co-receptor CCR5 is used by most HIV-1 primary isolates and is important for the establishment and maintenance of infection. In addition, CCR5 function is not necessary for human health. The mutant CCR5 allele “CCR5 Δ 32” encodes a truncated, non-functional protein (Samson, M., et al., Nature 382 (1996) 722-725; Dean, M., et al., Science 273 (1996) 1856-1862). Individuals homozygous for the mutation lack CCR5 expression and are strongly protected from HIV-1 infection. They do not show clear phenotypic results and are highly resistant to M-tropic HIV infection, while heterozygous individuals show delayed disease progression (Schwarz, MK and Wells, TN, Nat. Rev. Drug Discov. 1 (2002) 347-358. The lack of CCR5 has no obvious side effects, presumably CCR5 is part of a highly redundant chemokine network as receptors for α-chemokines MIP-1a, MIP-1β, and RANTES, sharing many overlapping functions, most of which replace Because they have receptors (Rossi, D. and Zlotnik, A., Annu. Rev. Immunol. 18 (2000) 217-242). The identification of CCR5 as an HIV-1 co-receptor was based on the ability of its ligands MIP-1α, MIP-1β, and RANTES to block infection by R5 except R5X4 or X4 isolates (Cocchi, F , Et al., Science 270 (1995) 1811-1815). CCR5 is also a receptor of "cluster" chemokines that is produced preferentially during the inflammatory response and regulates the recruitment of neutrophils (CXC chemokines), macrophages and subgroups of T cells (Th1 and Th2 cells, which are helper T cells). Th1 responses are typically reactions that include effective cell-mediated immunity against viruses and tumors, pro-inflammatory responses in charge of intracellular parasite killing, and persistent autoimmune responses, for example, while Th2 responses are important for allergies. I think. Thus, inhibitors of these chemokine receptors are believed to be useful as immunomodulators. In the Th1 response, for example, the hyperactive response is dampened in autoimmunity, including rheumatoid arthritis, or in the Th2 response, an asthma attack, or an allergic reaction, including atopic dermatitis, is reduced (eg, , 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. Disord. 2 (2002) 265-278; Lehner, T., Trends Immunol. 23 (2002) 347-351).

Antibodies to human CCR5 are described, for example, in 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 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, Are summarized in WO 01/58916, WO 01/58915, WO 01/43779, WO 01/42308, and EP 05007138.0.

Polyethylene glycol conjugates of antibodies to CCR5 are known from US 2003/0228306. US 2003/0215421 relates to chemokine-toxin conjugates.

WO 01/43779 relates to conjugates of anti-CD4 antibodies and anti-CCR5 antibodies, and conjugates of anti-CD4 antibodies and HIV-1 fusion inhibitory peptides. Conjugates of CCR5 antibodies and toxins are described in EP 1 346 731.

Summary of the Invention

The present invention relates to conjugates comprising 1 to 8 antifusogenic peptides and antibodies to HIV gp120 binding cell surface receptors, wherein 1 to 8, preferably 2 or 4 antifusogenic of each conjugate The peptide is characterized by conjugation to one terminus of the heavy and / or light chain of said antibody against HIV gp120 binding cell surface receptors (the number of antifusogenic peptides of eight per antibody only means that the antibody comprises eight termini). That is, for example, in the case of two heavy and two light chains; if the antibody comprises fewer C- and N-terminus, e.g. as an scFv , The corresponding number of maximally possible antifusogenic peptides in the conjugate is reduced, ie, reduced to less than eight).

The invention further comprises a conjugate comprising at least one anti-fusogenic peptide and an anti-CCR5 antibody (mAb CCR5), wherein up to 1 to 8 antifusogenic peptides each comprise a heavy chain and / or of said anti-CCR5 antibody Characterized by being conjugated to one end of the light chain (the number of 8 antifusogenic peptides per mAb CCR5 is only ie, for example, if mAb CCR5 comprises 8 ends, ie two heavy and two light chains). Possible if the mAb CCR5 comprises fewer C- and N-terminus, e.g., as scFv, the corresponding number of maximally possible antifusogenic peptides in the conjugate Reduced, ie, reduced to less than eight).

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

Preferably the conjugate is characterized by the following formula:

mAb CCR5-[linker] _m- [antifusogenic peptide] _n

Wherein m is independently 0 for each antifusogenic peptide (i.e., peptide bonds between mAb CCR5 and antifusogenic peptide) or 1 (i.e., 1 linker between mAb CCR5 and antifusogenic peptide) n is an integer from 1 to 8;

Preferred conjugates of the heavy and / or light and antifusogenic peptides of mAb CCR5 (“chain conjugates”) are selected from the group consisting of:

(1) [antifusogenic peptide]-[linker] _m- [heavy chain]

(2) [heavy chain]-[linker] _m- [antifusogenic peptide]

(3) [antifusogenic peptide]-[linker] _m- [heavy chain]-[antifusogenic peptide]

(4) [antifusogenic peptide]-[linker] _m- [light chain]

(5) [light chain]-[linker] _m- [antifusogenic peptide]

(6) [antifusogenic peptide]-[linker] _m- [light chain]-[antifusogenic peptide]

(7) [antifusogenic peptide]-[linker] _m- [heavy chain]-[linker] _m- [antifusogenic peptide]

(8) [antifusogenic peptide]-[linker] _m- [light chain]-[linker] _m- [antifusogenic peptide]

Wherein the linker may be the same or different in (in or between) the chain conjugate, m is an integer of 1 or 0, and m is independently in (in or between) the chain conjugate Same or different).

(The left side of the peptide or mAb CCR5 chain refers to the N-terminus and the right side refers to the C-terminus. Thus, in (1), the C-terminus of the antifusogenic peptide is derived from mAb CCR5 by peptide bonds or linkers. Connected to the N-terminus of the heavy chain).

Preferably the chain conjugate is assembled to a conjugate according to the invention comprising a mAb CCR5 (eg consisting of two light chains and two heavy chains including a constant Fc domain, scFv fragment, or Fab fragment).

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

Preferably the conjugate is characterized by comprising a variable heavy domain consisting of an immunoglobulin backbone selected from the group consisting of the heavy chain CDR3 sequence SEQ ID NOs: 16, 17 and a CDR3 region.

Preferably the conjugate is an immunoglobulin backbone, and a CDR3 region selected from the group consisting of SEQ ID NOs: 16, 17, a CDR2 region selected from the group consisting of SEQ ID NOs: 13, 14, 15, and a CDR1 sequence SEQ ID NO: 9, 10, 11, 12 characterized in that it comprises a variable heavy domain consisting of a CDR1 region selected from the group consisting of.

Preferably the conjugate is characterized in that it comprises a heavy chain variable domain selected from the group of heavy chain variable domains comprising SEQ ID NOs: 1, 3, 5, and 7.

Preferably the conjugate is from an immunoglobulin backbone, and a CDR1 region selected from SEQ ID NOs: 18, 19, 20, a CDR2 region selected from SEQ ID NOs: 21, 22, 23, and SEQ ID NOs: 24, 25 It comprises a variable light domain consisting of a selected CDR3 region.

Preferably the conjugate is a CDR of SEQ ID NO: 1 as a heavy chain CDR and a CDR of SEQ ID NO: 2 as a light chain CDR, a CDR of SEQ ID NO: 3 as a heavy chain CDR and a CDR of SEQ ID NO: 4 as a light chain CDR, A CDR of SEQ ID NO: 5 as a heavy chain CDR and a CDR of SEQ ID NO: 6 as a light chain CDR, or a CDR of SEQ ID NO: 7 as a heavy chain CDR and a CDR of SEQ ID NO: 8 as a light chain CDR do.

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

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

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

c) the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by amino acid sequence SEQ ID NO: 6;

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

Preferably the conjugate comprises a heavy chain (V _H ) variable domain defined by amino acid sequence SEQ ID NO: 1 and a light chain (V _L ) variable domain defined by amino acid sequence SEQ ID NO: 2; Or a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 3 and a light chain variable domain defined by amino acid sequence SEQ ID NO: 4; Or a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and a light chain variable domain defined by amino acid sequence SEQ ID NO: 6; Or a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 7 and a light chain variable domain defined by amino acid sequence SEQ ID NO: 8; Amino acids glycine (G) and asparagine (N), tripeptide GST, and a linker selected from the group consisting of SEQ ID NOs: 36-62 and SEQ ID NOs: 67-70; And an antifusogenic peptide selected from the group of peptides defined by SEQ ID NOs: 29-35 and SEQ ID NO: 73.

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

Preferably the conjugate is characterized by comprising an antifusogenic peptide (two antifusogenic peptides) at each C-terminus of the heavy chain or at each N-terminus of the light chain. Preferably the conjugate is characterized by comprising an antifusogenic peptide (four antifusogenic peptides) at each C-terminus of the heavy chain and at each N-terminus of the light chain.

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

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

The invention further comprises a conjugate comprising at least two antifusogenic peptides and an anti-CD4 antibody (mAb CD4), wherein two to eight, preferably two or four antifusogenic peptides are each Characterized in that it is conjugated to one end of the heavy and / or light chain of the anti-CD4 antibody (the number of eight antifusogenic peptides per mAb CD4 is only when mAb CD4 comprises eight ends, ie Only if, for example, it consists of two heavy and two light chains; if mAb CD4 contains fewer C- and N-terminus, for example, if it is as a scFv, the corresponding in the conjugate The maximum number of antifusogenic peptides is reduced, i.e. less than eight).

Preferably, with or without intermediate linkers, preferably by peptide bonds, the carboxy-terminal amino acid of the antibody chain is conjugated to the amino-terminal amino acid of the anti-fusogenic peptide, or the carboxy-terminal amino acid of the antifusogenic peptide Conjugated to the amino-terminal amino acid of the antibody chain.

Preferably, the conjugate is characterized by the following formula:

Antibodies-[linker] _m- [antifusogenic peptide] _n

Wherein m is independently 0 (ie, peptide bond between the antibody and anti-fusogenic peptide) or 1 (ie, one linker between the antibody and anti-fusogenic peptide) for each antifusogenic peptide, and n is an integer from 2 to 8, preferably even from 2 to 8, more preferably from 2 or 4. Preferably the antibody is mAb CD4.

Preferred conjugates of the heavy and / or light chains of the antibodies and antifusogenic peptides (“chain conjugates”) are selected from the group consisting of (from N-terminus to C-terminus):

(1) [antifusogenic peptide]-[linker] _m- [heavy chain]

(2) [heavy chain]-[linker] _m- [antifusogenic peptide]

(3) [antifusogenic peptide]-[linker] _m- [heavy chain]-[antifusogenic peptide]

(4) [antifusogenic peptide]-[linker] _m- [light chain]

(5) [light chain]-[linker] _m- [antifusogenic peptide]

(6) [antifusogenic peptide]-[linker] _m- [light chain]-[antifusogenic peptide]

(7) [antifusogenic peptide]-[linker] _m- [heavy chain]-[linker] _m- [antifusogenic peptide]

(8) [antifusogenic peptide]-[linker] _m- [light chain]-[linker] _m- [antifusogenic peptide]

Wherein the linker may be the same or different (in or between) in the chain conjugate, m is an integer of 1 or 0, and m is independently in (in or between) the chain conjugate Same or different].

(The left side of the peptide or antibody chain means the N-terminus and the right side means the C-terminus. Thus, in (1), the C-terminus of the anti-fusogenic peptide is determined by the peptide bond or linker of the heavy chain of the antibody. Connected to the N-terminus).

Preferably, the chain conjugate is assembled to a conjugate according to the invention comprising a mAb CD4 (eg consisting of two light chains and two heavy chains, including a constant Fc domain, scFv fragment, or Fab fragment). .

Particularly preferred chain conjugates are (2), (3), (4), and (7). Particularly preferred conjugates according to the invention are 2 x [mAb CD4 light chain] and 2 x (2), 2 x [mAb CD4 light chain] and 2 x (3), or 2 x [mAb CD4 heavy chain] and 2 x (4 ), Or 2 x [mAb CD4 light chain] and 2 x (7). The heavy and / or light chains preferably comprise constant regions.

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

Preferably the conjugate is an immunoglobulin backbone, 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 And a variable heavy domain consisting of a CDR1 region selected from the CDR1 sequences of SEQ ID NOs: 97, 98, or 99.

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

Preferably the conjugate is an immunoglobulin backbone, 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 SEQ ID NO: It comprises a variable light domain consisting of a CDR3 region selected from 94, 95, or 96.

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

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

a) a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 81, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 75;

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

c) a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 84, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 78;

d) a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 85, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 79;

e) a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 86, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 80.

Preferably the conjugate is a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 81, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 75; Or a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 82, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 76; Or a heavy chain variable domain defined by amino acid sequence SEQ ID NO: 84, and a light chain variable domain defined by amino acid sequence SEQ ID NO: 78; Heavy chain variable domain defined by amino acid sequence SEQ ID NO: 85, and light chain variable domain defined by amino acid sequence SEQ ID NO: 79, or heavy chain variable domain defined by amino acid sequence SEQ ID NO: 86, and amino acid sequence Light chain variable domains defined by SEQ ID NO: 80; Amino acids glycine (G) and asparagine (N), tripeptide GST, and a linker selected from SEQ ID NOs: 36-62 and SEQ ID NOs: 67-70; And an antifusogenic peptide selected from the antifusogenic peptides of SEQ ID NOs: 29-35 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 by comprising an antifusogenic peptide (two antifusogenic peptides) at each C-terminus of the heavy chain or at each N-terminus of the light chain. Preferably the conjugate is characterized by comprising an antifusogenic peptide (four antifusogenic peptides) at each C-terminus of the heavy chain and at each N-terminus of the light chain. Preferably the conjugate is characterized by comprising an antifusogenic peptide (four antifusogenic peptides) at the two C-terminus of the heavy chain and at the two N-terminus of the light chain.

Preferably the conjugate comprises two light chain variable domains of SEQ ID NO: 75, each heavy chain variable domain of SEQ ID NO: 81, a linker of SEQ ID NO: 69, and an antifusogenic peptide of SEQ ID NO: 73 Comprising two conjugates of type (2) comprising, or two light chain variable domains of SEQ ID NO: 76, each heavy chain variable domain of SEQ ID NO: 82, a linker of SEQ ID NO: 69, and SEQ ID 2 conjugates of type (2) comprising an anti-fusogenic peptide of NO: 73, or two light chain variable domains of SEQ ID NO: 78, each of which is a heavy chain variable domain of SEQ ID NO: 84, SEQ ID A linker of NO: 69 and two conjugates of type (2) comprising an antifusogenic peptide of SEQ ID NO: 73, or two light chain variable domains of SEQ ID NO: 79, each of SEQ ID NO A type comprising a heavy chain variable domain of: 85, a linker of SEQ ID NO: 69, and an antifusogenic peptide of SEQ ID NO: 73 (2) comprising two conjugates, or two light chain variable domains of SEQ ID NO: 80, each of which is a heavy chain variable domain of SEQ ID NO: 86, a linker of SEQ ID NO: 69, and SEQ ID NO: Two conjugates of type (2) comprising an anti-fusogenic peptide of 73.

Preferably the conjugate is characterized in that the antibody is of IgG1 subclass or IgG4 subclass. In addition, the antibody is preferably of IgG4 or IgG1 or IgG2 subclass with mutations in amino acid positions S228, L234, L235, and / or D265, and / or contains a PVA236 mutation. Preferably the conjugate is characterized in that the antibody of the IgG4 subclass has a S228P mutation and the antibody of the IgG1 subclass has a L234A and L235A mutation.

It is particularly preferred that the antibody is an anti-CD4 antibody or an anti-CCR5 antibody.

The invention comprises a process for the preparation of a conjugate according to the invention, which method comprises the following steps:

a) culturing a cell containing at least one plasmid containing at least one nucleic acid molecule encoding a conjugate according to the invention under conditions suitable for expression of the conjugate,

b) recovering the conjugate from the cells or supernatant.

In one embodiment, there are genes encoding the light and heavy chains of mAb CCR5 or mAb CD4 with or without linked antifusogenic peptides located on the same expression vector or different expression vectors.

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

The present invention includes the use of the conjugate according to the invention for the preparation of a medicament for the treatment of viral infections. Preferably said use is characterized in that the viral infection is an HIV infection.

The present invention includes the use of the conjugates according to the invention for antiviral treatment, preferably for the treatment of patients in need of anti-HIV treatment.

Detailed description of the invention

One aspect of the invention is a conjugate comprising one or more anti-fusogenic peptides and an anti-CCR5 antibody (mAb CCR5), wherein 1 to 8 antifusogenic peptides each comprise a heavy and / or light chain of said anti-CCR5 antibody. It is characterized by conjugation to one end. The number of antifusogenic peptides of eight per mAb CCR5 is only possible if mAb CCR5 comprises eight ends, ie, consisting of two heavy chains and two light chains. If mAb CCR5 comprises fewer C- and N-terminus, eg as scFv, the corresponding number of maximally possible antifusogenic peptides in the conjugate is reduced, i.e. 8 Reduced to less than. Another aspect of the invention is a conjugate comprising two or more anti-fusogenic peptides and an anti-CD4 antibody (mAb CD4), wherein 2 to 8 antifusogenic peptides each represent a heavy chain and / or of said anti-CD4 antibody. It is characterized in that it is conjugated to one end of the light chain.

Where reference is made below to generic antibodies or mAb CCR5 or mAb CD4, the same reference may be included for other mAbs and corresponding sequences, as appropriate.

An “antifusogenic peptide” is a peptide that inhibits a membrane fusion related event or the membrane fusion event itself, among others, a peptide that inhibits infection of uninfected cells by a virus due to membrane fusion. These antifusogenic peptides are preferably linear peptides. For example, they can be derived from gp41 ectodomains such as DP107, DP178. Examples of such peptides are 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 1996/19495, WO 1996/19495 / 59615, WO 2000/69902, and WO 2005/067960. For example, the amino acid sequence of the peptide is SEQ ID NOs: 1 to 10 of US 5,464,933; SEQ ID NOs: 1 to 15 of US 5,656,480; SEQ ID NOs: 1 to 10 and 16 to 83 of US 6,013,263; SEQ ID NOs from US 6,017,536: 1 to 10, 20 to 83 and 139 to 149; SEQ ID NOs: 1 to 10, 17 to 83, and 210 to 214 of US 6,093,794; SEQ ID NOs: 1 to 10, 16 to 83, and 210 to 211 of US 6,060,065; SEQ ID NOs: 1286 and 1310 of US 6,258,782; SEQ ID NOs: 1129, 1278-1309, 1311, and 1433 of US 6,348,568; SEQ ID NOs: 1 to 10 and 210 to 238 of US 6,479,055; SEQ ID NOs from US 6,656,906: 1 to 171, 173 to 216, 218 to 219, 222 to 228, 231, 233 to 366, 372 to 398, 400 to 456, 458 to 498, 500 to 570, 572 to 620, 622 To 651, 653 to 736, 739 to 785, 787 to 811, 813 to 823, 825, 827 to 863, 865 to 875, 877 to 883, 885, 887 to 890, 892 to 981, 986 to 999, 1001 to 1003 , 1006 to 1018, 1022 to 1024, 1026 to 1028, 1030 to 1032, 1037 to 1076, 1078 to 1079, 1082 to 1117, 1120 to 1176, 1179 to 1213, 1218 to 1223, 1227 to 1237, 1244 to 1245, 1256 To 1268, 1271 to 1275, 1277, 1345 to 1348, 1350 to 1362, 1364, 1366, 1368, 1370, 1372, 1374 to 1376, 1378 to 1379, 1381 to 1385, 1412 to 1417, 1421 to 1426, 1428 to 1430 , 1432, 1439 to 1542, 1670 to 1682, 1684 to 1709, 1712 to 1719, 1721 to 1753, 1755 to 1757; Or SEQ ID NOs: 5 to 95 of WO 2005/067960 or selected from the group consisting of these. The antifusogenic peptide has an amino acid sequence comprising 5 to 100 amino acids, preferably 10 to 75 amino acids and more preferably 15 to 50 amino acids. Particularly preferred antifusogenic peptides include 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 NOs: 29-35 and SEQ ID NO: 73) . In one embodiment, a conjugate according to the invention comprises at least one antifusogenic peptide and an antibody, wherein i) the antifusogenic peptide is a linear peptide having an amino acid sequence of 5 to 100 amino acids, ii) 1 8 to 8 antifusogenic peptides are conjugated to one end of the heavy and / or light chain of the antibody, respectively. In another embodiment, the conjugates according to the invention comprise one or more antifusogenic peptides and antibodies, for example anti-CCR5 antibodies (mAb CCR5), wherein i) the antifusogenic peptide is a gp41 ecto Derived from the domain and ii) each of 1 to 8 antifusogenic peptides is conjugated to one end of either the heavy and / or light chain of said antibody. The term “gp41 ectodomain” refers to an amino acid sequence starting with amino acid position 561 of HIV-1 gp160 and ending with amino acid position 620, or starting with amino acid position 50 of HIV-1 gp41 and ending with amino acid position 109 (SEQ ID NO: 66 (See also, eg, Bar, S. and Alizon, MJ Virol. 78 (2004) 811-820).

The term “antibody” includes various forms of antibody constructs, including whole antibodies and antibody fragments. The antibodies according to the invention are preferably human antibodies, humanized antibodies, chimeric antibodies, T cell antigen scavenging antibodies (WO 98/33523, WO 98/52976, and WO 00/34317). Antibody genetic engineering is described, for example, in Morrison, S.L., et al., Proc. Natl. Acad Sci. 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.

An "antibody fragment" comprises a portion of a full length antibody, preferably its variable domain or at least its antigen binding site. Examples of antibody fragments are, for example, single chain antibody molecules (scFv), Fab, F (ab) ₂ fragments, and the like as long as they retain the characteristics of the anti-CCR5 antibody. ScFv antibodies are described, eg, in Houston, JS, Methods in Enzymol. 203 (1991) 46-88. Huston also describes linkers and methods for linking polypeptides useful in the present invention.

"CCR5" is described, for example, in Oppermann, M., Cell Signal. 16 (2004) 1201-1210 and Swiss Prot P51681, means human CCR5. The terms “antibody that binds CCR5”, “anti-CCR5 antibody”, or “mAb CCR5” as used interchangeably within the present application specifically bind to CCR5 and preferably inhibit HIV fusion with target cells. Means an antibody. Binding can be tested in cell based in vitro ELISA assays (CCR5 expressing CHO cells). Binding is found if the antibody results in an S / N (signal / noise) ratio of at least 5, preferably at least 10, at an antibody concentration of 100 ng / ml. The term “inhibiting HIV fusion with a target cell” refers to contacting the target cell with the virus, eg, in the presence of an antibody at a concentration effective to inhibit membrane fusion between the target cell (eg PBMC) and the virus. Inhibition of HIV fusion with a target cell, as measured in an assay comprising measuring luciferase reporter gene activity or HIV p24 antigen concentration. The term "membrane fusion" refers to a fusion between a first cell co-expressing CCR5 and CD4 polypeptides, and a second cell or virus expressing an HIV env protein. Membrane fusion is measured by reporter gene assays (eg, luciferase reporter gene assays), by genetically engineered cells and / or viruses.

Preferred anti-CCR5 antibodies are disclosed 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. Particularly preferred anti-CCR5 antibodies are described in WO 2006/103100. Particularly preferred anti-CCR5 antibodies are characterized by comprising a variable heavy 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. Further preferred antibodies include an immunoglobulin backbone, and a CDR3 region selected from the group consisting of the CDR3 sequences SEQ ID NO: 16, 17, a CDR2 region selected from the group consisting of the CDR2 sequences SEQ ID NO: 13, 14, 15, and CDR1 SEQ ID NO: 9, 10, 11, 12 comprises a variable heavy chain region consisting of a CDR1 region selected from the group consisting of. Preferred heavy chain variable domains are shown in SEQ ID NOs: 1, 3, 5, 7. Preferred anti-CCR5 antibodies are also selected from the group consisting of an immunoglobulin backbone and a CDR1 region selected from the group consisting of CDR1 sequences SEQ ID NO: 18, 19, 20, CDR2 sequences SEQ ID NO: 21, 22, 23 And a variable light domain consisting of a CDR2 region and a CDR3 region selected from the group consisting of CDR3 sequence SEQ ID NOs: 24, 25. The anti-CCR5 antibody is preferably a CDR of SEQ ID NO: 1 as a heavy chain CDR and a CDR of SEQ ID NO: 2 as a light chain CDR, a CDR of SEQ ID NO: 3 as a heavy chain CDR and a CDR of SEQ ID NO: 4 as a light chain CDR. CDRs, CDRs of SEQ ID NO: 5 as heavy chain CDRs and CDRs of SEQ ID NO: 6 as light chain CDRs, or CDRs of SEQ ID NO: 7 as heavy chain CDRs and CDRs of SEQ ID NO: 8 as light chain CDRs.

CDR sequences can be determined according to the standard definition of Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). CDRs of SEQ ID NOs: 1-8 are shown in SEQ ID NOs: 9-25.

Anti-CCR5 antibodies preferably comprise variable heavy and light chain domains independently selected from the group consisting of:

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

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

c) the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by SEQ ID NO: 6;

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

"CD4" is described, for example, in Brady, R.L. and Barclay, A. N., Curr. Top. Microbiol. Immunol. 205 (1996) 1-18] and SwissProt PO 1730. As used interchangeably within this application, the terms “antibody that binds to CD4”, “anti-CD4 antibody”, or “mAb CD4” specifically binds to CD4 and preferably inhibits HIV fusion with target cells. Means an antibody. Binding can be tested in cell based in vitro ELISA assays (CD4 expressing CHO cells). Binding is found if the antibody results in an S / N (signal / noise) ratio of at least 5, preferably at least 10, at an antibody concentration of 100 ng / ml. The term “inhibiting HIV fusion with a target cell” refers to contacting the cell with the virus, eg, in the presence of an antibody at a concentration effective to inhibit membrane fusion between the target cell (eg, PBMC) and the virus. Inhibition of HIV fusion with a target cell, as measured in an assay comprising measuring luciferase reporter gene activity or HIV p24 antigen concentration. The term "membrane fusion" refers to a fusion between a first cell expressing a CD4 polypeptide and a second cell or virus expressing an HIV env protein. Membrane fusion is measured by reporter gene assays (eg, luciferase reporter gene assays), by genetically engineered cells and / or viruses. Illustrative anti-CD4 antibodies are described, 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, It is described in WO 91/009966.

"CXCR4" is described, for example, in Feng, Y., et al., Science 272 (1996) 809-810, or in Tamamura, H. and Fujii, N., Expert Opinion on Therapeutic Targets 9 (2005) 1267-1282 ] Human CXCR4 as described. As used interchangeably within this application, the terms “antibody that binds to CXCR4”, “anti-CXCR4 antibody”, or “mAb CXCR4” specifically bind CXCR4 and preferably inhibit HIV fusion with target cells. Means an antibody. Binding can be tested in cell based in vitro ELISA assays (CXCR4 expressing CHO cells). Binding is found if the antibody results in an S / N (signal / noise) ratio of at least 5, preferably at least 10, at an antibody concentration of 100 ng / ml. The term “inhibiting HIV fusion with a target cell” refers to contacting the cell with the virus in the presence of an antibody at a concentration effective to inhibit membrane fusion between the target cell (eg, PBMC) and the virus, eg, Inhibition of HIV fusion with a target cell, as measured in an assay comprising measuring luciferase reporter gene activity or HIV p24 antigen concentration. The term "membrane fusion" refers to a fusion between a first cell expressing a CXCR4 polypeptide and a second cell or virus expressing an HIV env protein. Membrane fusion is measured by reporter gene assays (eg, luciferase reporter gene assays), by genetically engineered cells and / or viruses. Exemplary anti-CXCR4 antibodies are described in Strizki, J.M., et al., J. Virol. 71 (1997) 5678-5683 (Ab 12G5), US 7,138,496.

"Antibody to HIV gp120 binding cell surface receptor" is used herein to bind to cell surface receptors used by HIV gp120 subunits that mediate the binding of viruses to the cell surface of a cell, thereby binding gp120 to said receptor. , And also antibodies that prevent HIV from binding to the cell surface. HIV gp120 is derived from proteolytic cleavage of the precursor HIV gp160 (see, eg, Brenneman, D.E., et al., Int. Rev. Neurobiol. 32 (1990) 305-353). The second segment of the cleavage is HIV gp41. These two segments non-covalently associate and mediate HIV and cell binding and cell fusion. Exemplary cell surface receptors are CD4 receptors or chemokine receptors such as CCR5 receptors, CXCR4 receptors, or CXCR5 receptors. Thus, in one embodiment, antibodies to HIV gp120 binding cell surface receptors are anti-CD4 antibodies, or anti-CCR5 antibodies, or anti-CXCR4 antibodies, or anti-CXCR5 antibodies.

The antibodies used in the conjugates according to the invention are preferably characterized in that the constant domains are of human origin. Such constant domains are well known in the art and are described, for example, by Kabat (see, eg, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218). ). For example, a useful human IgG1 heavy chain constant region (C _H 1 -hinge-C _H 2 -C _H 3) comprises an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 26, 27. For example, useful human kappa (κ) light chain constant domains comprise the amino acid sequence of kappa light chain constant domain (κ light chain constant domain, C _L ) of SEQ ID NO: 28. It is further preferred that the variable domain of the antibody is of mouse origin and comprises the antibody variable domain sequence framework of the mouse antibody according to Kabat (eg, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000). 214-218).

Preferred anti-CCR5 antibodies show CCR5 binding to the same epitope (s) of CCR5 as it is an antibody selected from the group consisting of antibodies A to E or CCR5 by antibodies A to E due to steric hindrance or competitive binding of the binding. The binding to is suppressed. Epitope binding is investigated using alanine scanning according to the method for epitope mapping described by Olson, WC, et al. (J. Virol. 73 (1999) 4145-4155). Signal reduction of at least 75% shows that the mutated amino acid (s) contribute to the epitope recognized by the antibody. If the amino acid contributing to the epitope is recognized by the antibody being investigated and antibodies A, B, C, D, or E, the antibody is found to bind to the same epitope. Antibody C, which exhibits a lower IC ₅₀ value than antibody 2D7 in HIV assay, binds to an epitope comprising an amino acid on the ECL2 domain of CCR5 that is different from the epitope recognized by antibody 2D7 (Lee, B., et al., J Biol.Chem. 274 (1999) 9617-9626) (2D7 binds to amino acids K171 and E172 of ECL2A, not to ECL2B amino acids 184-189). Epitope binding to antibody C appears to be 20% for CCR5 mutant K171A or E172A (glu 172 is mutated to ala). 100% epitope binding is defined for wild type CCR5. Further preferred anti-CCR5 antibodies bind the same epitope to which antibody C binds.

The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groups of molecules such as sugar side chains or amino acids, and usually have specific tertiary structural characteristics as well as specific charge characteristics. Structural and non-structural epitopes are distinguished in that binding to structural epitopes that are not non-structural epitopes is lost in the presence of denaturation solvents. Preferably the antibody according to the invention specifically binds to native CCR5 but not to denatured CCR5. The antibody preferably comprises a heavy chain CDR3 of SEQ ID NO: 17, and preferably also a heavy chain CDR selected from the group of CDRs of SEQ ID NO: 10, 11, 12, 14 and / or 15. Preferably the antibody is antibody B, C, D, or E, or comprises the variable domain of antibody B, C, D, or E. Preferably the antibody that binds denatured CCR5 is antibody A or comprises the variable domain of antibody A.

As used herein, the term “variable domain” (variable domain of light chain (V _L ), variable domain of heavy chain (V _H )) refers to each domain of a light and heavy chain domain pair that is directly involved in antibody binding to an antigen. The variable domains of the light and heavy chains have the same general structure, ie have an "immunoglobulin backbone", each domain comprising four "skeletal regions" (FR), the sequence of which is widely conserved, Connected by three “hypervariable regions” (or “complementarity determining regions”, CDRs). The framework region adopts a β-plate structure and the CDRs can form a loop connecting the β-plate structure. The CDRs in each chain are fixed in tertiary structure by the framework regions and together with the CDRs of the other chain form an antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity / affinity of the antibodies according to the invention, thus providing a further object of the invention.

As used herein, the term “antigen-binding portion of an antibody” or “antigen-binding site of an antibody” refers to an amino acid residue of an antibody that is responsible for antigen-binding. The antigen-binding site of an antibody comprises amino acid residues of a "complementarity determining region" or "CDR". "Framework" or "FR" regions are variable domain regions other than the hypervariable regions as defined herein. Thus, the light and heavy chain variable domains of the antibody comprise regions FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (immunoglobulin backbone) from N- to C-terminus. In particular, the CDR3 region of the heavy chain is the region that most contributes to antigen binding and defines the antibody. Preferably the anti-CCR5 antibody according to the invention is characterized by comprising a CDR3 sequence of SEQ ID NO: 16 or SEQ ID NO: 17 in the heavy chain variable domain. Complementarity determining regions (CDRs) and framework (FR) regions are standard definitions of Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). It depends on.

The “Fc portion” of an anti-CCR5 antibody is a portion that does not directly participate in binding to CCR5, but exhibits various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are classified into classes of IgA, IgD, IgE, IgG, and IgM, many of which are IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. Can be further classified into subclasses (isotypes). Depending on the heavy chain constant region, different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The antibody according to the invention is preferably of IgG type. "Fc portion of an antibody" is a term well known to those skilled in the art and is defined based on papain cleavage of the antibody. Antibodies according to the invention contain as human Fc moieties or Fc moieties derived from human origin. In a further embodiment of the invention, the Fc portion is an Fc portion of a human antibody of subclass IgG4 or an Fc portion of a human antibody of subclass IgG1, IgG2, or IgG3, wherein the Fcγ receptor (eg, It is modified such that neither FcγRIIIa) binding and / or C1q binding can be detected. Preferably the Fc portion is a human Fc portion, particularly preferably a human Fc portion of the human IgG4 subclass or a mutated Fc portion of the human IgG1 subclass. More preferred are the Fc portions of the human IgGl subclass with the mutations L234A and L235A. More 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 mutations S228P. IgG4 shows reduced Fcγ receptor (FcγRIIIa) binding, while antibodies of other IgG subclasses show strong binding. However, Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrates), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are similarly reduced Fcγ if modified Residues that provide receptor binding (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 the antibody according to the invention is mutated to L234, L235, and / or D265 and / or contains a PVA236 mutation with respect to Fcγ receptor binding of the IgG4 subclass or of the IgG1 or IgG2 subclass. Mutations S228P, L234A, L235A, L235E, and / or PVA236 at amino acid positions 233 to 236 of IgG1 (PVA236 is the amino acid sequence ELLG at amino acid positions 233 to 236 of IgG1 (one letter is given in amino acid code) or EFLG of IgG4 Preferably replaced with PVA). Particularly preferred are mutations S228P of IgG4 and mutations of L234A and L235A of IgG1. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by binding of the complement factor C1q to the Fc portion of most IgG antibody subclasses. C1q binding to the antibody is caused by protein-protein interactions defined at the so-called binding site. Such Fc partial binding sites are known in the art and are described, for example, in Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., MoI. Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., MoI. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; And EP 0 307 434. The Fc partial binding site is characterized by, for example, amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to Kabat's EU index). Subclass IgG1, IgG2, and IgG3 antibodies usually exhibit complement activation, including C1q and C3 binding, whereas IgG4 does not activate complement system and does not bind C1q and C3. Anti-CCR5 antibodies that do not bind Fcγ receptors and / or complement factor C1q do not induce antibody-dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC). Preferably, the antibody is characterized in that it binds to CCR5, contains an Fc moiety derived from human origin, and does not bind to the Fcγ receptor and / or complement factor C1q. More preferably, the antibody is a human, or humanized, or T-cell antigen scavenging antibody. C1q binding is described by Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184. “C1q binding” means that in the assay the optical density (OD) at 492-405 nm is 15 in the test antibody than the value at which the unmodified wild-type antibody Fc portion binds to human C1q at an antibody concentration of 8 μg / ml. If% is lower, it is found to be absent. ADCC can be measured as antibody binding to human FcγRIIIa on human NK cells. Binding is measured at an antibody concentration of 20 μg / ml. "No Fcγ receptor binding" or "no ADCC" means that binding to human FcγRIIIa on human NK cells at an antibody concentration of 20 μg / ml is 30% or less compared to binding of the same antibody as human IgG1 (SEQ ID NO: 26) do.

The antibodies used in the conjugates according to the invention also include antibodies (variant antibodies) with "conservative sequence modifications" which are amino acid sequence modifications which do not affect or modify the characteristics of the antibodies according to the invention mentioned above. do.

Modifications can be introduced by standard techniques known to those skilled in the art, such as site-specific mutations and PCR-mediated mutations. Conservative amino acid substitutions include substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Family of amino acid residues with similar side chains are defined in the art. These families include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), non-charged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine , Cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine), and aromatic side chains Amino acids with (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, the predicted non-essential amino acid residues in human anti-CCR5 antibodies may preferably be replaced with another amino acid residue of the same side chain family. Thus, herein “variant” anti-CCR5 antibodies are amino acids by at most 10, preferably about 2 to about 5 additions, deletions and / or substitutions in one or more of the variable domain regions outside the heavy chain CDR3 region of the parent antibody. It refers to a molecule whose sequence differs from the amino acid sequence of the “parent” anti-CCR5 antibody. Each other heavy chain CDR region comprises at most one amino acid addition, loss and / or substitution. The present invention includes a method of modifying the CDR amino acid sequence of a parent antibody that binds to CCR5, which comprises a heavy chain variable domain and / or from the group of heavy chain variable domains consisting of SEQ ID NO: 1, 3, 5, 7, 82, 83 Or selecting a light chain variable domain from the group of light chain variable domains consisting of SEQ ID NOs: 2, 4, 6, 8, 76, 77 and providing a nucleic acid encoding said first variable domain amino acid sequence, wherein one amino acid is Modified in heavy chain CDR1, one amino acid is modified in heavy chain CDR2, 1-3 amino acids are modified in light chain CDR1, 1-3 amino acids are modified in light chain CDR2, and / or 1-3 amino acids are light chain Modifying the nucleic acid to be modified at CDR3, expressing and incorporating the modified variable domain (s) amino acid sequence in an antibody structure, determining if the antibody binds to CCR5, and If the body is coupled to CCR5 it characterized in that the screening said modified variable domain (s) / CDR (s). Preferably, the modification is conservative sequence modification. Amino acid sequence modifications are described in Riechmann, L., et al., Nature 332 (1988) 323-327, and Queen, C, et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033, which may be performed by mutations based on molecular modeling as described.

As used herein, the term “linker” or “peptide linker” refers to a peptide linker of natural and / or synthetic origin. These are accumulated in linear amino acid chains where 20 naturally occurring amino acids are monomeric building blocks. The length of the chain is 1 to 50 amino acids, preferably 1 to 28 amino acids, particularly preferably 3 to 25 amino acids. The linker may contain a sequence of naturally occurring polypeptides, such as repeating amino acid sequences or hinge-functional polypeptides. The linker has the function of ensuring that the peptide conjugated to the anti-CCR5 antibody can perform its biological activity by ensuring that it is correctly folded and properly presented. Preferably the linker is a “synthetic peptide linker” designated as abundant in glycine, glutamine, and / or serine residues. These residues are arranged in small repeating units of up to 5 amino acids, for example GGGGS, QQQQG, or SSSSG. The small repeating unit may be repeated 2-5 times to form a multimeric unit. Up to six additional artificial, naturally occurring amino acids may be added at the amino- and / or carboxy-terminus of the multimeric unit. Another synthetic peptide linker consists of a single amino acid that is repeated 10-20 times, such as serine in the linker SSSSSSSSSSSSSSS. At each of the amino- and / or carboxy-terminus, up to six additional artificial, naturally occurring amino acids may be present. Preferred linkers are shown in Table 2. Linker [GQ ₄ ] ₃ GNN (SEQ ID NO: 40), LSLSPGK (SEQ ID NO: 36), LSPNRGEC (SEQ ID NO: 37), LSLSGG (SEQ ID NO: 61), LSLSPGG (SEQ ID NO: 62) , G ₃ [SG ₄ ] ₂ SG (SEQ ID NO: 69) is particularly preferred. All peptide linkers can be encoded by nucleic acid molecules and thus can be expressed recombinantly. Since the linker is the peptide itself, the antifusogenic peptide is linked to the linker through a peptide bond formed between two amino acids. The peptide linker is introduced between the anti-fusogenic peptide and the anti-CCR5 antibody chain to which the anti-fusogenic peptide is conjugated. Thus, there may be two or three possible sequences each (amino-terminal to carboxy-terminal):

a) antifusogenic peptide-peptide linker-anti-CCR5 antibody polypeptide chain, or

b) anti-CCR5 antibody polypeptide chains-peptide linkers-antifusogenic peptides, or

c) Anti-Fusogenic Peptides-Peptide Linkers-Anti-CCR5 Antibody Polypeptide Chains-Peptide Linkers-Anti-Fusogenic Peptides.

In one embodiment of the invention, the mAb CCR5-conjugate is characterized by comprising:

i) the heavy chain (V _H ) variable domain defined by amino acid sequence SEQ ID NO: 1 and the light chain (V _L ) variable domain defined by SEQ ID NO: 2; Or a heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 3 and a light chain variable domain defined by SEQ ID NO: 4; Or a heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 5 and a light chain variable domain defined by SEQ ID NO: 6; Or a heavy chain variable domain defined by the amino acid sequence SEQ ID NO: 7 and a light chain variable domain defined by SEQ ID NO: 8;

ii) a linker selected from the group consisting of amino acids glycine (G) and asparagine (N), tripeptide GST, and SEQ ID NOs: 36-62, and SEQ ID NOs: 67-70; And

iii) an antifusogenic peptide selected from the group of peptides defined by SEQ ID NOs: 29-35, or 73.

Preferred conjugates of the heavy and / or light and antifusogenic peptide (s) of mAb CCR5 ("chain conjugates") are: conjugate (1) [antifusogenic peptide]-[linker] _m- [heavy chain], (2) [heavy chain]-[linker] _m- [antifusogenic peptide], (3) [antifusogenic peptide]-[linker] _m- [heavy chain]-[antifusogenic peptide], (4) [anti Fusogenic peptide]-[linker] _m- [light chain], (5) [light chain]-[linker] _m- [antifusogenic peptide], (6) [antifusogenic peptide]-[linker] _m- [light chain] ]-[Antifusogenic peptide], (7) [antifusogenic peptide]-[linker] _m- [heavy chain]-[linker] _m- [antifusogenic peptide], (8) [antifusogenic peptide]- [Linker] _m- [light chain]-[linker] _m- [antifusogenic peptide] (wherein the linker can be the same or different both in and between said chain conjugates, m being an integer of 1 or 0 And m is within and between the conjugates Which may be the same or different at both). For example, in a conjugate comprising a chain conjugate (7) and a mAb CCR5 light chain, the two linkers in the chain conjugate (7) may be identical, ie, have the same amino acid sequence and length, or It may be different, that is, they may have different amino acid sequences and lengths, or they may be missing one or both. For example, in a conjugate comprising chain conjugates (2) and (4), the linker contained in chain conjugate (2) and the linker contained in chain conjugate (4) may be identical, ie They may have the same amino acid sequence and length, or may be different, ie they may have different amino acid sequences and / or lengths, or they may be absent. In a chain conjugate, the linker (s) may be present (m = 1) or absent (m = 0). Preferred chain conjugates are chain conjugates (2), (3), (4), and (7). One embodiment of the invention is a conjugate comprising 2 x [mAb CCR5 light chain] and 2 x chain conjugate (2). The conjugate comprises a non-conjugate anti-CCR5 antibody light chain and two anti-CCR5 antibody heavy chains optionally conjugated with an intermediate linker at the C-terminus of the anti-fusogenic peptide through the N-terminus.

Another embodiment of the invention is a conjugate comprising two mAb CCR5 light chains and two chain conjugates (3). Yet another embodiment is a conjugate comprising two mAb CCR5 heavy chains and two chain conjugates (4). A further embodiment of the invention is a conjugate comprising two mAb CCR5 light chains and two chain conjugates (7). The heavy and / or light chain preferably comprises a constant region (Fc).

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

The invention further provides for the use of the conjugates according to the invention in an effective amount, with a pharmaceutically acceptable carrier, for the manufacture of a pharmaceutical preparation, for the treatment of AIDS patients.

As used herein, the term "amino acid" means alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), Cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), Lysine (lys, K), Methionine (met, M), Phenylalanine (phe, F), Proline (pro, P), Serine (ser, S), Threonine (thr, T), Tryptophan (trp, W), Tyrosine group of naturally occurring carboxy α-amino acids including (tyr, Y), and valine (val, V).

Methods and techniques known to those skilled in the art useful in the practice of the present invention are described, for example, in Ausubel, F.M., ed., Current Protocols in Molecular Biology, Volume I to III (1997), Wiley and Sons; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

In the conjugate according to the invention, the carboxy-terminal amino acid of the antibody chain is bound to the amino-terminal amino acid of the anti-fusogenic peptide via peptide bonds, or the carboxy-terminal amino acid of the anti-fusogenic peptide is linked to the antibody chain via peptide bonds. Conjugated to the amino-terminal amino acid of. In one embodiment, the intermediate linker is between the antifusogenic peptide and the antibody chain. Thus, the conjugate according to the invention is characterized by the following general formula:

Antibodies-[linker] _m- [antifusogenic peptide] _n

Wherein m is independently 0 (ie, 1 direct peptide bond between the antibody and antifusogenic peptide) or 1 (ie, a linker is present between the antibody and antifusogenic peptide for each antifusogenic peptide) N is an integer of 1 to 8; In one embodiment n is an integer from 2 to 8. In another embodiment, n is an integer from 2 to 4. In another embodiment, n is an integer of 2 or 4. One embodiment of the invention includes a conjugate characterized by comprising an anti-fusogenic peptide at each C-terminus of the heavy chain of the antibody or at each N-terminus of the light chain. In this embodiment, two antifusogenic peptides are conjugated to one antibody. In another embodiment, conjugates comprising an antifusogenic peptide at each C-terminus of the heavy chain and at each N-terminus of the light chain are included. In this embodiment, four antifusogenic peptides are conjugated to one antibody. In one embodiment, the antibody is mAb CCR5 or mAb CD4.

Antifusogenic peptides introduced at the ends of mAb CCR5 heavy and / or light chain (s) are small in size compared to mAb CCR5. For example, the immunoglobulins of class G, which are minimal immunoglobulins, have a molecular weight of approximately 150 kDa; The antifusogenic peptides preferably have a size (molecular weight) of less than 12.5 kDa, which corresponds to about 100 amino acids, generally less than 7.5 kDa, which corresponds 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 acid residues, 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 that can be conjugated to the mAb CCR5 heavy chain and / or light chain (s) is a combined number of amino- and carboxy-terminus of 1 to anti-CCR5 antibody polypeptide chain. Because the present invention includes different anti-CCR5 antibodies, the number of antifusogenic peptides can vary. For anti-CCR5 antibodies comprising two heavy chains and two light chains, the combined number of amino-terminus (N-terminus) and carboxy-terminus (C-terminus) is 8, which is a conjugated antifusogenic peptide at the same time Total maximum number of; For example, for an anti-CCR5 antibody fragment, such as a single chain antibody (scFv), the combined number of ends and thus the maximum number of conjugated antifusogenic peptides is two. If a single antifusogenic peptide is conjugated to mAb CCR5, the peptide may occupy either end of the anti-CCR5 antibody chain. Likewise, if the maximum possible number of peptides are conjugated to mAb CCR5, all ends are occupied by a single peptide. If the number of peptides conjugated to mAb CCR5 is less than the maximum possible number, the distribution of peptides at the ends of the anti-CCR5 antibody chain may be different. For example, if four peptides are conjugated to G or E class immunoglobulins, five different combinations are possible (see Table 1). In two combinations, all of one terminus, ie all four amino-terminal or all four carboxy-terminus of the anti-CCR5 antibody chain, are each conjugated to one single antifusogenic peptide. The other end is not conjugated. This in one embodiment allows for the modification / conjugation to be placed within one region of the anti-CCR5 antibody. In other cases, the polypeptide is conjugated at both ends. Within this combination, the conjugated peptides are placed in different regions of the anti-CCR5 antibody. In any case, the sum of the conjugated ends is four.

Possible combination for the case where four peptides are conjugated at the ends of an anti-CCR5 antibody consisting of four polypeptide chains Number of amino-terminus Number of carboxy-terminus Total number of ends occupied 4 0 4 3 One 4 2 2 4 One 3 4 0 4 4

The present invention preferably includes a conjugate wherein at least two of the termini are conjugated to the antifusogenic peptide. The amino acid sequences of the antifusogenic peptides may be different, similar or identical. In one embodiment, amino acid sequence identity is in a range from 90% to less than 100%; These amino acid sequences and corresponding peptides are defined to be similar. In a preferred embodiment, the antifusogenic peptides are identical, ie have 100% amino acid identity.

The present invention includes a conjugate comprising at least one anti-fusogenic peptide and an anti-CCR5 antibody (mAb CCR5), wherein 1 to 8 antifusogenic peptides each comprise a heavy and / or light chain of said anti-CCR5 antibody. It is conjugated through a peptide bond at one end of either. In one embodiment, the conjugate according to the invention comprises two or more antifusogenic peptides and anti-CCR5 antibodies, wherein the 2 to 8 antifusogenic peptides each comprise a heavy chain and / or of said anti-CCR5 antibody Conjugated to one end of the light chain.

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

Conjugation between the antifusogenic peptide and the anti-CCR5 antibody is performed at the nucleic acid level. Thus, peptide bonds are formed between antifusogenic peptides and anti-CCR5 antibody chains, with or without intermediate linkers. Thus, the carboxy-terminal amino acid of the anti-fusogenic peptide is conjugated to the amino-terminal amino acid of the anti-CCR5 antibody chain with or without intermediate linker, or the carboxy-terminal amino acid of the anti-CCR5 antibody chain with or without intermediate linker. The amino-terminal amino acids of the anti-fusogenic peptides are conjugated, or both ends of the anti-CCR5 antibody chain are conjugated to the antifusogenic peptides, respectively, with or without intermediate linkers. For the recombinant preparation of antifusogenic peptide-anti-CCR5 antibody-conjugates according to the invention, one or more nucleic acid molecules encoding different polypeptides are required, preferably 2 to 8 nucleic acid molecules. These nucleic acid molecules encode different anti-CCR5 antibody polypeptide chains of the conjugate and are shown below as structural genes. They may be located on the same expression plasmid (vector) or alternatively may be located on different expression plasmids (vector). Assembly of the conjugate is preferably located within the expressing cell prior to secretion of the conjugate. Thus, nucleic acid molecules encoding the polypeptide chains of the conjugates are preferably expressed in the same host cell. If a conjugate mixture is obtained after recombinant expression, the conjugate can be isolated and purified by methods known to those skilled in the art. These methods are well established and widely used for immunoglobulin tablets and are used alone or in combination. The method may comprise, for example, affinity chromatography using microbial-derived proteins (eg, protein A or protein G affinity chromatography), ion exchange chromatography (eg, cation exchange (carboxymethyl resin) , Anion exchange (amino ethyl resin) and mixed-form exchange chromatography), thiothio adsorption (e.g., using beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. For example, phenyl-sepharose, aza-arenophilic resin, or m-aminophenylboronic acid, metal chelate affinity chromatography (eg, Ni (II)-and Cu (II)- Affinity materials), size exclusion chromatography, and preparation electrophoresis methods (eg gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102). Using recombinant manipulation methods known to those of skill in the art, the conjugates can be custom-made at the nucleic acid / gene level. Nucleic acid sequences encoding immunoglobulins are known and can be obtained, for example, from genomic databases. Likewise, nucleic acid sequences encoding antifusogenic peptides are known and can be easily deduced from their amino acid sequences. Members required for the construction of expression plasmids for expressing the conjugates of the present invention are, for example, native and / or modified and / or conjugated versions of the expression cassette for the anti-CCR5 antibody light chain, natural and / or modified thereof. And / or conjugated versions of the expression cassette for the anti-CCR5 antibody heavy chain (alternatively the anti-CCR5 antibody light chain and the anti-CCR5 antibody heavy chain are obtained in the same expression cassette as, for example, a bicistronic expression member. ), Selection markers, and E. coli replication as well as selection units. These expression cassettes include promoters, DNA segments encoding secretory signal sequences, structural genes, and terminator / polyadenylation signals. The members are assembled to be operatively linked to form on one plasmid that encodes all chains of the conjugate, or on two or more plasmids that encode one or more chains of the conjugate, respectively. For expression of the encoded polypeptide, the plasmid (s) are introduced into a suitable host cell. The protein is preferably produced in mammalian cells such as CHO cells, NSO cells, Sp2 / 0 cells, COS cells, HEK cells, K562 cells, BHK cells, PER.C6® cells and the like. Regulatory members of the plasmids should be selected such that they are functional in the selected host cell. For expression, host cells containing plasmids encoding one or more chains of the conjugate are cultured under conditions suitable for chain expression. The expressed conjugate chains are functionally assembled. Fully treated antifusogenic peptide-anti-CCR5 antibody-conjugate is secreted into the medium.

An "expression plasmid" is a nucleic acid encoding a polypeptide expressed in a host cell. Typically, expression plasmids, for example, for E. coli, have a transcriptional terminator comprising a prokaryotic plasmid proliferation unit comprising a replication origin, a resistance gene, a eukaryotic selection marker, and a promoter, a structural gene, and a polyadenylation signal. One or more expression cassettes for expression of the structural gene (s) comprising. Gene expression usually occurs under the control of a promoter and the structural gene is said to be "operably linked" to the promoter. Similarly, regulatory members and core promoters are operatively linked if they regulate the activity of the core promoter.

Thus, one aspect of the present invention is a method of making a conjugate according to the present invention, comprising the following steps:

a) culturing cells containing one or more expression plasmids, each under conditions suitable for expression of the conjugate, each containing one or more nucleic acid molecules encoding a conjugate according to the invention,

b) recovering the conjugate from the cells or supernatant.

The term "under conditions suitable for expression of a conjugate" refers to conditions that are used in the culturing of a cell expressing a polypeptide and are known or readily measurable to those skilled in the art. It is known to those skilled in the art that these conditions may vary depending on the type of polypeptide expressed and the type of cell being cultured. In general, cells are incubated at a temperature of 20 ° C. to 40 ° C., and for a time sufficient to allow the polypeptide conjugates to be produced effectively, eg, for 4 to 28 days.

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

Regardless of the route of administration chosen, the compounds of the invention and / or the pharmaceutical compositions of the invention which can be used in the form of suitable hydrates are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. Can be. The dosage level selected is the specific composition, route of administration, time of administration, rate of secretion of the specific compound used, other drugs, compounds and / or substances used in combination with the particular composition used, age of the patient being treated, It will depend on a variety of pharmacokinetic factors, including gender, weight, conditions, general health and well-known factors in previous medical history and medical fields.

The invention preferably comprises the use of the conjugate according to the invention for the treatment of a patient suffering from an immunodeficiency syndrome such as AIDS.

The following examples, sequence listing, drawings and depositions are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the process without departing from the spirit of the invention.

1: Plasmid map of mAb CCR5 κ- light chain expression vector 4900.

Figure 2: Plasmid map of mAb CCR5 γ1-heavy chain expression vector 4901.

Figure 3: Plasmid map of mAb CCR5 γ1-heavy chain conjugate expression vector 4995.

4: Plasmid map of mAb CD4 κ- light chain expression vector 6310.

5: Plasmid map of mAb CD4 γ1-heavy chain expression vector 6309.

Figure 6: Plasmid map of mAb CD4 γ1-heavy chain conjugate expression vector 6303.

term- CCR5  Antibody Deposit

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

Cell line Deposit number Deposit date m <CCR5> Pz01.F3 DSM ACC 2681 18.08.2004 m <CCR5> Pz02.1C11 DSM ACC 2682 18.08.2004 m <CCR5> Pz03.1C5 DSM ACC 2683 18.08.2004 m <CCR5> Pz04.1F6 DSM ACC 2684 18.08.2004

Antibody Name

<CCR5 >> Pz01.F3: Antibody A SEQ ID NOs: 1, 2

<CCR5> PzO2.1C11: Antibody B SEQ ID NOs: 3, 4

<CCR5> PzO3.1C5: Antibody C SEQ ID NOs: 5, 6

<CCR5> F3.1H12.2E5: Antibody D SEQ ID NO: 7, 8

<CCR5> PzO4.1F6: Antibody E

Variable domain 1 of mAb CD4 is recorded in SEQ ID NOs: 10, 15, 45, and 56 of US 5,871,732.

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

Variable domain 3 of mAb CD4 is recorded in FIGS. 3, 4, 12, and 13 of WO 91/009966.

Antibody sequence, Anti-fusogenic Peptide  Sequence and Peptide  Linker sequence

SEQ ID NO: 1 <CCR5> Pz01.F3 heavy chain, variable domain

SEQ ID NO: 2 <CCR5> Pz01.F3 light chain, variable domain

SEQ ID NO: 3 <CCR5> PzO2.1C11 heavy chain, variable domain

SEQ ID NO: 4 <CCR5> Pz02.1C11 light chain, variable domain

SEQ ID NO: 5 <CCR5> PzO3.1C5 heavy chain, variable domain

SEQ ID NO: 6 <CCR5> PzO3.1C5 light chain, variable domain

SEQ ID NO: 7 <CCR5> F3.1H12.2E5 heavy chain, variable domain

SEQ ID NO: 8 <CCR5> F3.1H12.2E5 light chain, variable domain

SEQ ID NO: 9 Heavy Chain CDR1 mAb CCR5

SEQ ID NO: 10 heavy chain CDR1 mAb CCR5

SEQ ID NO: 11 Heavy Chain CDR1 mAb CCR5

SEQ ID NO: 12 Heavy Chain CDR1 mAb CCR5

SEQ ID NO: 13 Heavy Chain CDR2 mAb CCR5

SEQ ID NO: 14 heavy chain CDR2 mAb CCR5

SEQ ID NO: 15 Heavy Chain CDR2 mAb CCR5

SEQ ID NO: 16 heavy chain CDR3 mAb CCR5

SEQ ID NO: 17 heavy chain CDR3 mAb CCR5

SEQ ID NO: 18 Light Chain CDR1 mAb CCR5

SEQ ID NO: 19 Light Chain CDR1 mAb CCR5

SEQ ID NO: 20 Light Chain CDR1 mAb CCR5

SEQ ID NO: 21 Light Chain CDR2 mAb CCR5

SEQ ID NO: 22 Light Chain CDR2 mAb CCR5

SEQ ID NO: 23 Light Chain CDR2 mAb CCR5

SEQ ID NO: 24 Light Chain CDR3 mAb CCR5

SEQ ID NO: 25 Light Chain CDR3 mAb CCR5

SEQ ID NO: 26 γ1 heavy chain constant region

SEQ ID NO: 27 γ4 heavy chain constant region

SEQ ID NO: 28 κ light 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 NOs: 36-62, 67-70 Linker Peptides

SEQ ID NO: 63 Amino acid sequence of mature mAb CCR5 κ- light chain

SEQ ID NO: 64 Amino acid sequence of mature mAb CCR5 γ1-heavy chain

SEQ ID NO: 65 Amino acid sequence of mature mAb CCR5 conjugate heavy chain

SEQ ID NO: 66 HIV-1 gp41

SEQ ID NO: 71 Amino acid sequence of mature mAb CD4 κ- light chain

SEQ ID NO: 72 Amino acid sequence of mature mAb CD4 γ-heavy chain

SEQ ID NO: 73 afp-1

SEQ ID NO: 74 Amino Acid Sequence of Mature mAb CD4 Conjugate Heavy Chain

SEQ ID NO: 75 VII and light chain variable domain 1 mAb CD4

SEQ ID NO: 76 Chimeric Light Chain Variable Domain 1 mAb CD4

SEQ ID NO: 77 Chimeric Light Chain 1 mAb CD4

SEQ ID NO: 78 Chimeric Light Chain Variable Domain 2 mAb CD4

SEQ ID NO: 79 Chimeric Light Chain Variable Domain 3 mAb CD4

SEQ ID NO: 80 Chimeric Light Chain Variable Domain 4 mAb CD4

SEQ ID NO: 81 VII and heavy chain variable domain 1 mAb CD4

SEQ ID NO: 82 Chimeric Heavy Chain Variable Domain 1 mAb CD4

SEQ ID NO: 83 Chimeric heavy chain 1 mAb CD4

SEQ ID NO: 84 Chimeric Heavy Chain Variable Domain 2 mAb CD4

SEQ ID NO: 85 Chimeric Heavy Chain Variable Domain 3 mAb CD4

SEQ ID NO: 86 Chimeric Heavy Chain Variable Domain 4 mAb CD4

SEQ ID NO: 87 Light Chain CDR1 mAb CD4

SEQ ID NO: 88 Light Chain CDR1 mAb CD4

SEQ ID NO: 89 Light Chain CDR1 mAb CD4

SEQ ID NO: 90 Light Chain CDR1 mAb CD4

SEQ ID NO: 91 Light Chain CDR2 mAb CD4

SEQ ID NO: 92 Light Chain CDR2 mAb CD4

SEQ ID NO: 93 Light Chain CDR2 mAb CD4

SEQ ID NO: 94 Light Chain CDR3 mAb CD4

SEQ ID NO: 95 Light Chain CDR3 mAb CD4

SEQ ID NO: 96 light chain CDR3 mAb CD4

SEQ ID NO: 97 Heavy Chain CDR1 mAb CD4

SEQ ID NO: 98 heavy chain CDR1 mAb CD4

SEQ ID NO: 99 Heavy Chain CDR1 mAb CD4

SEQ ID NO: 100 Heavy Chain CDR2 mAb CD4

SEQ ID NO: 101 heavy chain CDR2 mAb CD4

SEQ ID NO: 102 heavy chain CDR2 mAb CD4

SEQ ID NO: 103 Heavy Chain CDR3 mAb CD4

SEQ ID NO: 104 Heavy Chain CDR3 mAb CD4

SEQ ID NO: 105 Heavy Chain CDR3 mAb CD4

Materials & Methods

General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is found in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Is shown. The amino acids of the antibody chains are numbered according to EU numbering (Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

Recombination DNA  Technology :

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

Gene Synthesis:

Desired gene segments were prepared from oligonucleotides prepared by chemical synthesis. By annealing and ligation of oligonucleotides, including PCR amplification, long 100-600 bp long gene segments located next to one restriction endonuclease cleavage site are assembled, followed by A-overhang. It was cloned into pCR2.1-TOPO-TA cloning vector (Invitrogen Corp., USA). DNA sequences of subcloned gene segments were confirmed by DNA sequencing.

Protein Measurements:

Using the molar extinction coefficient calculated on the basis of the amino acid sequence, the protein concentration of the conjugate was determined by measuring the optical density (OD) at 280 nm.

Example  One

term- CCR5  Preparation of Antibody Expression Plasmids

Gene segments encoding anti-CCR5 antibody light chain variable domain (V _L ) and human kappa-light chain constant domain (C _L ), anti-CCR5 antibody heavy chain variable domain (V _H ) and human γ1-heavy chain constant domain (C _H) Joining was performed as in the gene segment for 1-hinge-C _H 2-C _H 3).

For mAb CCR5 of SEQ ID NO: 63/64, the heavy and light chain variable domains are derived from mouse antibodies and the heavy and light chain constant domains are derived from human antibodies (C-kappa and IgG1).

The gene segment encoding the complete anti-CCR5 antibody light chain is then inserted at the N- and / or C-terminus with a nucleic acid encoding an antifusogenic peptide, including a linker linker sequence, and / or a complete anti-CCR5 antibody Gene segments encoding heavy chains were inserted at the N- and / or C-terminus with nucleic acids encoding antifusogenic peptides, including linker linker sequences.

a) vector 4900

Vector 4900 is an expression plasmid for transient expression of mAb CCR5 light chain (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

In addition to the mAb CCR5 κ- light chain expression cassette, the vector contains:

Hygromycin resistance genes as selection markers,

Origin of replication of the Epstein-Barr virus (EBV), oriP,

The origin of replication of the vector pUC18 allowing the plasmid to be replicated in E. coli, and

Β-lactam antibiotic lactamase gene conferring ampicillin resistance in E. coli.

The transcription unit of mAb CCR5 κ- light chain gene consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

Synthetic 5'-untranslated region,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-Intron-L2]),

Murine anti-CCR5 antibody mature variable κ-light chain coding segment arranged with a unique BsmI restriction site (L2 signal sequence) at the 5'-end and a splice donor site and a unique NotI restriction site at the 3'-end,

Human / mouse κ-light chain hybrid intron 2,

Human κ- light chain gene constant domain,

Human immunoglobulin κ-polyadenylation (“poly A”) signal sequence, and

Unique restriction sites AscI and FseI at the 5'- and 3'-ends respectively.

A plasmid map of mAb CCR5 κ- light chain expression vector 4900 is shown in FIG. 1. The amino acid sequence of the mature (without signal sequence) mAb CCR5 κ- light chain is shown in SEQ ID NO: 63.

b) vector 4991

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

In addition to the mAb CCR5 γ1-heavy chain expression cassette, the vector contains:

Hygromycin resistance genes as selection markers,

Origin of replication of Epstein-Barr virus (EBV), oriP,

-This. Origin of replication of the vector pUC18, oriP, which allows the plasmid to be replicated in E. coli, and

-This. Beta-lactam antibiotic degrading enzyme gene conferring ampicillin resistance to E. coli.

The transcription unit of mAb CCR5 γ1-heavy chain consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

Synthetic 5'-untranslated region,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-Intron-L2]),

Murine anti-CCR5 antibody mature variable heavy chain coding segment arranged with a unique BsmI restriction site (L2 signal sequence) at the 5'-end and a splice donor site and a unique NotI restriction site at the 3'-end,

Human / mouse heavy chain hybrid intron 2, comprising mouse heavy chain enhancer members (partial JH ₃ , JH ₄ ) (Neuberger, MS, EMBO J. 2 (1983) 1373-1378)

Genomic human γ 1-heavy chain gene constant domain,

Human γ1-immunoglobulin polyadenylation (“poly A”) signal sequence, and

Unique restriction sites AscI and SgrAI at the 5'- and 3'-ends respectively.

A plasmid map of mAb CCR5 γ1-heavy chain expression vector 4901 is shown in FIG. 2. The amino acid sequence of the mature (without signal sequence) mAb CCR5 γ1-heavy chain is shown in SEQ ID NO: 64.

c) vector 4995

Vector 4995 is an expression plasmid for transient expression of chimeric peptide-anti-CCR5 antibody γ1-heavy chain conjugate (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

Vector 4995 is C-terminal with nucleic acid (SEQ ID NO: 33) and mAb CCR5 γ1-heavy chain encoding anti-fusogenic peptide T-2635 and peptide linker sequence [GQ ₄ ] ₃ GNN (SEQ ID NO: 40) Derived from plasmid 4991 to join in.

In addition to the chimeric peptide anti-CCR5 antibody γ1-heavy chain conjugate expression cassette, the vector contains:

Hygromycin resistance genes as selection markers,

Origin of replication of Epstein-Barr virus (EBV), oriP,

-This. Origin of replication of the vector pUC18, oriP, which allows the plasmid to be replicated in E. coli, and

-This. Beta (β) -lactam antibiotic degrading enzyme gene conferring ampicillin resistance to E. coli.

The transcriptional unit of the chimeric peptide-anti-CCR5 antibody γ1-heavy chain conjugate consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

Synthetic 5'-untranslated region,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-Intron-L2]),

Murine anti-CCR5 antibody mature variable heavy chain coding segment arranged with a unique BsmI restriction site (L2 signal sequence) at the 5'-end and a splice donor site and a unique NotI restriction site at the 3'-end,

Human / mouse heavy chain hybrid intron 2 (Neuberger, MS, EMBO J. 2 (1983) 1373-1378) comprising a mouse heavy chain enhancer member (partial JH ₃ , JH ₄ ),

Genomic human γ 1-heavy chain gene constant domain,

Antifusogenic peptide T-2635,

Peptide linker sequence [GQ ₄ ] ₃ GNN,

Human γ1-immunoglobulin polyadenylation (“poly A”) signal sequence, and

Unique restriction sites AscI and SgrAI at the 5'- and 3'-ends respectively.

A plasmid map of mAb CCR5 γ1-heavy chain conjugate expression vector 4995 is shown in FIG. 3. The amino acid sequence of the mature (without signal sequence) conjugate heavy chain is shown in SEQ ID NO: 65.

Example  2

Final term CCR5  Preparation of Antibody Expression Plasmids

Fusion genes (heavy and / or light chain antibody fusion genes), mAb CCR5 gene segments, any linker gene segments and antifusogenic peptide gene segments were assembled by linkage of the corresponding nucleic acid segments using known recombinant methods and techniques. The nucleic acid sequences encoding the peptide linker and the antifusogenic polypeptide are synthesized by chemical synthesis, respectively, and then subjected to amplification. Linked to E. coli plasmid. Subcloned nucleic acid sequences were confirmed by DNA sequencing.

Example  3

HEK293 EBNA  Immunoglobulins and Immunoglobulins in Cells Mutant  Transient manifestations

Recombinant anti-CCR5 antibodies and anti-CCR5 antibody-variants include 10% ultra-low IgG FCS (fetal bovine serum, Gibco), 2 mM glutamine (Gibco), 1% volume by volume (v / v) Attachment growth type HEK293-EBNA cells (Epstein-Bar-virus nuclear antigen) cultured in Dulbecco's modified Eagle's medium (Gibco) with non-essential amino acids (Gibco) and 250 μg / ml G418 (Roche Molecular Biochemicals) Was generated by transient transfection of human embryonic kidney cell line 293 (American type culture collection Accession No. ATCC # CRL-10852). For transfection, FuGENE ™ 6 transfection reagent (Roche Molecular Biochemicals) was used in a ratio of reagent (μl) to DNA (μg) in the range of 3: 1 to 6: 1. The light and heavy chains comprising the antifusogenic peptide-anti-CCR5 antibody conjugate light and heavy chains, respectively, were prepared from two different plasmids using a molar ratio of light chain to heavy chain encoding the plasmid in the range of 1: 2 to 2: 1. Expression. Antifusogenic peptide-anti-CCR5 antibody conjugates containing cell culture supernatants were harvested 4-11 days after transfection. For example, general information regarding recombinant expression of human immunoglobulins in HEK293 cells can be found in Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example  4

Immunoglobulin-specific antibodies The conjugate  Used SDS PAGE , Weston Blotting The Expression analysis using lancer and detection

Expressed and secreted antifusogenic peptide-anti-CCR5 antibody conjugates were treated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and isolated anti-CCR5-antibody and anti Fusogenic peptide-anti-CCR5-antibody-conjugate chains were transferred from the gel to the membrane and then detected by immunological methods.

SDS - PAGE  :

LDS sample buffer, 4-fold concentrate (4x): 4 g glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamine tetra 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) solution of phenol red, is added to water, total volume 10 ml Made with.

Cultures containing secreted antifusogenic peptide-anti-CCR5 antibody conjugates were centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was mixed with 1/4 volume (v / v) of 4 × LDS sample buffer and 1/10 volume (v / v) of 0.5 M 1,4-dithiothreitol (DTT). The samples were then incubated at 70 ° C. for 10 minutes and proteins separated by SDS-PAGE. NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast gel (pH 6.4) and NuPAGE® MOPS operating buffer (running buffer) were used.

Weston Blot  :

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

After SDS-PAGE, the isolated anti-fusogenic peptide-anti-CCR5 antibody conjugate chain was subjected to nitrocellulose according to Burnette's "Semidry-Blotting-Method" (Burnette, WN, Anal. Biochem. 112 (1981) 195-203). The filtration membrane (pore size: 0.45 μm) was transferred by electrophoresis.

Immunological Detection:

TBS-buffer: 50 mM TRIS-hydrochloride, 150 mM NaCl, adjusted to pH 7.5,

Blocking solution: 1% (w / v) Western blocking reagent (Roche Molecular Biochemicals) in TBS-buffer,

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

For immunological detection, western blotting membranes were incubated twice for 5 minutes with shaking at room temperature in TBS-buffer and once for 90 minutes in blocking solution.

Peptide  Immunoglobulins Conjugate  Detection of the chain:

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

Light Chain: The light chain of the antifusogenic peptide-anti-CCR5 antibody conjugate was detected using purified peroxidase conjugated rabbit anti-human kappa light chain antibody (DAKO, Code No. P 0129).

For visualization of antibody light and heavy chains, purified rabbit anti-human IgG conjugated to peroxidase in the case of heavy chains, first diluted 1: 10,000 in 10 ml blocking solution Incubation was carried out with the antibody or in the case of a light chain with purified peroxidase conjugated rabbit anti-human kappa light chain antibody at 4 ° C. overnight. After washing the membrane three times with TBTS-buffer and once with TBS buffer for 10 minutes at room temperature, the Western-blot membrane was developed with luminol / peroxide-solution producing chemiluminescence (Lumi-LightPLUS Western blotting Substrate, Roche Molecular Biochemicals). Thus, the membranes are incubated in 10 ml luminol / peroxide-solution for 10 seconds to 5 minutes, and then the light emitted is detected using LUMI-Imager F1 Analysator (Roche Molecular Biochemicals) and / or using x-ray film. Recorded. Spot intensity was quantified using LumiAnalyst Software (Version 3.1).

Immunoblot  Multi-dyeing:

The secondary peroxidase-labeled antibody conjugate used for detection was at 70 ° C. in 1 M TRIS-hydrochloride-buffer (pH 6.7) with 100 mM beta-mercaptoethanol and 20% (w / v) SDS. It can be removed from the stained blots by incubating the membrane for 1 hour. After this treatment, blots can be stained twice with different secondary antibodies. Before the second detection, blots are washed three times at room temperature with shaking for 10 minutes each in TBS-buffer.

Example  5

Peptide  Immunoglobulins Conjugate  Affinity Tablets, Dialysis and Concentrations

Expressed and secreted antifusogenic peptide-anti-CCR5 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 min at 10,000 g) and filtration through a 0.45 μm filter, the purified culture supernatant containing peptide immunoglobulin conjugates was added to PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO _4). , 137 mM NaCl and 2.7 mM KCl, pH 7.4), were placed on a Protein A-Sepharose ™ CL-4B column. Unbound protein was washed 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 the conjugate containing fractions were neutralized with 1 M TRIS-Base. The antifusogenic peptide-anti-CCR5 antibody conjugate was then dialyzed extensively against PBS buffer at 4 ° C, concentrated using an Ultrafree®-CL Centrifugal Filter Unit (Millipore Corp., USA) equipped with a Biomax-SK membrane, 0 ° C., stored in ice-water bath. Integrity of the conjugate was analyzed by SDS-PAGE in the presence and absence of a reducing agent and stained using Coomassie brilliant blue as described in Example 4. Aggregation of antifusogenic peptide-anti-CCR5 antibody conjugates was analyzed by analytical size exclusion chromatography.

Example  6

Peptide  Immunoglobulins Conjugate Deglycosylation

N-linked carbohydrates of the anti-CCR5 antibody and anti-fusogenic peptide-anti-CCR5 antibody conjugates were prepared using peptide-N-glycosidase F (PNGaseF, Roche Molecular Biochemicals, Mannheim, Germany or Prozyme, San Leandro, CA). It was cut by the used enzyme treatment. Thus, anti-CCR5 antibody and anti-fusogenic peptide-anti-CCR5 antibody conjugates were used at a protein concentration of about 2 mg / ml at 12 ° C. using 50 mU PNGaseF per mg of N-glycosylated protein in PBS buffer. Incubate for ˜ 24 hours. Peptide-N-glycosidase F was then isolated by preparative gel filtration in accordance with known methods. Briefly, PNGaseF treated anti-CCR5-antibody and antifusogenic peptide-anti-CCR5 antibody conjugates were prepared in PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl and 2.7 mM KCl, pH 7.4) on a Superose ™ 12 10/300 GL column (GE Healthcare former Amersham Bioscience, Sweden) equilibrated to 0.5 and using an Akta Explorer chromatography system from Amersham Bioscience (GE Healthcare former Amersham Bioscience, Sweden). Elution was carried out using equilibration buffer at a flow rate of ˜1.0 ml / min.

Example  7

Single-cycle antiviral activity Assay

For the production of pseudotyped NL-Bal viruses, the plasmids pNL4-3Δenv (HIV pNL4-3 genome construct with loss in the env gene) and pCDNA3.1 / NL-BAL env [containing the NL-BaI env gene pcDNA3.1 plasmid (obtained from NIBSC Centralized Facility for AIDS reagent)] was co-transfected into HEK 293FT cell line (Invitrogen), 10% fetal bovine serum (FCS), 100 U / mL penicillin, 100 μg / Cultures were incubated in Dulbecco's modified minimum medium (DMEM) with mL streptomycin, 2 mM L-glutamine and 0.5 mg / mL Genithycin (all media were from Invitrogen / Gibco). Supernatants containing Pseudotype virus were harvested 2 days after transfection and cell debris was removed by filtration through a 0.45 μm pore size PES (polyethersulfone) filter (Nalgene) and aliquots at −80 ° C. Stored in the form. For normalization in assay performance, virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells, resulting in approximately 1.5 × 10 ⁵ RLU (relative light units) per well. Obtained. Test antifusogenic peptide-anti-CCR5 antibody conjugates, control antibodies and control antifusogenic peptides (T-20, T-1249, T-651 and T-2635) were diluted in 96-well plates. Assays were performed in duplicates. Each plate contained cell control and virus control wells. Viral stock 1.5 × 10 ⁵ RLU equivalents were added to each well, then 2.5 × 10 ⁴ JC53-BL cells were added to each well and the final assay volume was 200 μl per well. After 3 days of incubation at 37 ° C., 90% relative humidity, and 5% CO ₂ , the medium was aspirated and 50 μl of Steady-Glo® Luciferase Assay System (Promega) was added to each well. After incubation for 10 minutes at room temperature, assay plates were read on a Luminometer (Luminoskan, Thermo Electron Corporation). % Luciferase activity inhibition was calculated for each dose point after subtracting the background, and IC ₅₀ and IC ₉₀ -values were measured using XLfit curve fitting software for Excel (version 3.0.5 Buildl 2; Microsoft). The results are shown in Table 3.

Antiviral Activity of Antifusogenic Polypeptides, Antibodies and Antifusogenic Peptide-Anti-CCR5 Antibody Conjugates compound Antiviral activity IC ₅₀ (ng / mL) IC ₉₀ (ng / mL) Control Antibody 1 (Inactive) Inert Inert Control Antibody 2 (Inactive) Inert Inert T-20 206 3955 T-1249 11 90 T-651 11 139 T-2635 14 161 mAb CCR5 (4901/4900) 114 2387 Chimeric Peptide mAb CCR5 Conjugate (4995/4900) 7 45

Example  8

Cell-cell fusion Assay

On day 1, gp160-expressing HeLa cells (2 × 10 ⁴ cells / 50 μl / well) were planted in white 96 well microtiter plates in DMEM medium with 10% FCS and 2 μg / ml doxycycline. On day 2, 100 μl of supernatant sample or antibody control per well was added to a clear 96 well microtiter plate. Next, 100 μl containing 8 × 10 ⁴ CEM-NKr-Luc suspension cells was added to the medium, and incubated at 37 ° C. for 30 minutes. HeLa cell culture medium was aspirated from a 96 well plate, 100 μl in 200 μl antibody / CEM-NKr-Luc mixture was added and incubated overnight at 37 ° C. On day 3, 100 μl / well Bright-Glo ™ luciferase assay substrate (1,4-dithiothreitol and sodium dithionite; Promega Corp., USA) is added and luminescence is at least 15 minutes at room temperature Measured after incubation.

material :

HeLa-R5-16 cells (cell lines for expressing HIV gp160 upon doxycycline induction) were cultured in DMEM medium with nutrients and 10% FCS with 400 μg / ml G418 and 200 μg / ml hygromycin B. CEM.NKR-CCR5-LUC (Cat. No. 5198, T-cell line available from NIH AIDS Research & Reference Reagent Program McKesson BioServices Corporation Germantown, MD 20874, USA). Cell type: CEM.NKR-CCR5 (Cat. # 4376) was transfected (electroporation) to express the luciferase gene under transcription conditions of HIV-2 LTR, 10% fetal bovine serum, 4 mM glutamine, Proliferation was in RPMI 1640 with penicillin / streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin), and 0.8 mg / ml Genithycin Sulfate (G418). Growth characteristics: round lymphocyte cells, not very diverse in shape. The cells can grow in suspension as single cells and form small clumps. Divide into 1: 10 twice a week. Special Features: Expression of luciferase activity after HIV-2 LTR interaction. 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). Cell lines are described in 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  9

Peripheral blood monocyte cells ( PBMC ) In  Antiviral activity Assay .

Human PBMCs were isolated from smokescreens (obtained at Stanford Blood Center) by Ficoll-Paque (Amersham, Piscataway, New Jersey, USA) density gradient centrifugation, according to the manufacturer's protocol. Briefly, blood was transferred from the smokescreen in 50 ml conical tubes and diluted in sterile Dulbecco's phosphate buffered saline (Invitrogen / Gibco) to a final volume of 50 ml. 25 ml of diluted blood were transferred to two 50 ml conical tubes and carefully lowered together with 12.5 ml of Ficoll-Plac Plus (Amersham Biosciences) and centrifuged for 20 minutes at room temperature without stopping at 450 xg. . The white cell layer was carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove remaining red blood cells, cells were incubated with ACK disruption buffer (Biosource) for 5 minutes at room temperature and washed one or more times with PBS. The number of PBMCs was counted and 10% FCS (Invitrogen / Gibco), 1% penicillin / streptomycin, 2 mM L-glutamine, 1 mM sodium-pyruvate, and 2 μg / ml phytohemagglutinin (Invitrogen) Incubate at 37 ° C. for 24 hours at a concentration of 2-4 × 10 ⁶ cells / ml in all RPMI1640. Prior to assay, cells were incubated with 5 units / ml human IL-2 (Roche Molecular Biochemicals) for a minimum of 48 hours. In 96 well round bottom plates, 1 × 10 ⁵ PBMCs were diluted in the presence of test peptide-immunoglobulin-conjugates, control immunoglobulins and control peptides (T-20, T-1249, T-651 and T-2635). Under infection with HIV-1 JR-CSF virus (Koyanagi, Y., et al., Science 236 (1987) 819-822). The amount of virus used was equivalent to 1.2 ng HIV-1 p24 antigen / well. Infection was duplicated in duplicates. Plates were incubated at 37 ° C. for 6 days. Virus generation was carried out in Microsoft Excel Fit (version 3.0.5 Build 12; equation 205; Microsoft) at the end of infection using p24 ELISA (HIV-1 p24 ELISA # NEK050B, Perkin Elmer / NEN). One binding site and a sigmoidal dose-response model were used to measure.

Example  10

term- CD4  Preparation of Antibody Expression Plasmids

The gene segments encoding the anti-CD4 antibody light chain variable domain (V _L ) and human kappa-light chain constant domain (C _L ) were divided into anti-CD4 antibody heavy chain variable domain (V _H ) and human γ1-heavy chain constant domain (C _H 1). The gene segments for the hinge-C _H 2-C _H 3) were joined as well.

For mAb CD4 of SEQ ID NO: 71/72, the heavy and light chain variable domains are described, eg, in Reimann, K.A., et al., Aids Res. Human Retrovir. 13 (1997) 933-943 or US Pat. No. 5,871,732, and the heavy and light chain constant domains were derived from human antibodies (C-kappa and IgG1).

The gene segment encoding the complete anti-CD4 antibody light chain was then joined at the N- and / or C-terminus with a nucleic acid encoding an antifusogenic peptide, including a linker sequence, and / or a complete anti-CD4 antibody Gene segments encoding heavy chains were joined at the N- and / or C-terminus with nucleic acids encoding antifusogenic peptides, including junction linker sequences.

a) vector 6310

Vector 6310 is an expression plasmid for transiently expressing mAb CD4 light chains (genomically organized expression cassettes; exon-intron organization) in expression plasmids, eg, HEK293 cells.

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

Neomycin resistance gene as a selection marker,

-This. Origin of replication of the vector pUC18 allowing the plasmid to be replicated in E. coli, and

-This. Β-lactam antibiotic degrading enzyme gene conferring ampicillin resistance in E. coli.

The transcription unit of mAb CD4 κ- light chain gene consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

The 5'-untranslated region of the human antibody germline gene,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [Ll-intron-L2]),

Humanized anti-CD4 antibody mature variable κ-light chain coding segment, arranged with a splice donor site and a unique BamHI restriction site at the 3'-end,

Cleaved human κ-light chain intron 2,

Human κ- light chain gene constant domain,

Bovine growth hormone (bGH) polyadenylation (“poly A”) signal sequence, and

Unique restriction sites AscI and SgrAI at the 3'-end.

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

b) vector 6309

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

In addition to the mAb CD4 γ1-heavy chain expression cassette, the vector contains:

-This. Origin of replication of the vector pUC18 allowing the plasmid to be replicated in E. coli, and

-This. Beta-lactam antibiotic degrading enzyme gene conferring ampicillin resistance in E. coli.

The transcription unit of mAb CD4 γ1-heavy chain consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

The 5'-untranslated region of the human antibody germline gene,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [Ll-intron-L2]),

Humanized anti-CD4 antibody mature variable heavy chain coding segment, arranged with the splice donor site and the unique XhoI restriction site at the 3'-end,

Mouse / human heavy chain hybrid intron 2,

Genomic human γ1-heavy chain gene constant domains containing L234A and L235A mutations,

Bovine growth hormone (bGH) polyadenylation (“poly A”) signal sequence, and

Unique restriction site SgrAI at the 3′-end.

A plasmid map of mAb CD4 γ 1-heavy chain expression vector 6309 is shown in FIG. 3. The amino acid sequence of the mature (without signal sequence) mAb CD4 γ 1-heavy chain is shown in SEQ ID NO: 72.

c) vector 6303

Vector 6303 is, for example, an expression plasmid for transient expression of chimeric peptide-anti-CD4 antibody γ1-heavy chain conjugate (genomically organized expression cassette; exon-intron organization) in HEK293 cells.

Vector 6303 was derived from vector 6309 by joining mAb CD4 γ1-heavy chain to the last amino acid except one C-terminal amino acid, i.e., removing the C-terminal lysine residue of the heavy chain, wherein the vector was antifusogenic There was a nucleic acid encoding peptide afp-1 (SEQ ID NO: 73) and peptide glycine-serine linker of SEQ ID NO: 69.

In addition to the chimeric peptide anti-CD4 antibody γ1-heavy chain conjugate expression cassette, the vector contains:

-This. Origin of replication of the vector pUC18, oriP, which enables replication of the plasmid in E. coli, and

-This. A beta (β) -lactam antibiotic degrading enzyme gene conferring ampicillin resistance to E. coli.

The transcription unit of the chimeric peptide-anti-CD4 antibody γ1-heavy chain conjugate consists of the following members:

Immediate early enhancers and promoters of human cytomegalovirus,

The 5'-untranslated region of the human antibody germline gene,

Murine immunoglobulin heavy chain signal sequence comprising a signal sequence intron (signal sequence 1, intron, signal sequence 2 [L1-Intron-L2]),

Humanized anti-CD4 antibody mature variable heavy chain coding segment, arranged with a splice donor site and a unique XhoI restriction site at the 3'-end,

Mouse / human heavy chain hybrid introns containing L234A and L235A mutations,

The peptide serine-glycine linker sequence of SEQ ID NO: 69,

Antifusogenic peptide afp-1 of SEQ ID NO: 73,

Bovine growth hormone (bGH) polyadenylation (“poly A”) signal sequence, and

Unique restriction site SgrAI at the 3′-end.

A plasmid map of mAb CD4 γ 1-heavy chain conjugate expression vector 6303 is shown in FIG. 6. The amino acid sequence of the mature (without signal sequence) conjugate heavy chain is shown in SEQ ID NO: 74.

Example  11

Final term CD4  Preparation of Antibody Expression Plasmids

Fusion genes (heavy and / or light chain antibody fusion genes) comprising a mAb CD4 gene segment, any linker gene segment, and an antifusogenic peptide gene segment were assembled by linking the nucleic acid segment in question using known recombinant methods and techniques. . Nucleic acid sequences encoding the peptide linker and the antifusogenic polypeptide were respectively synthesized by chemical synthesis and then linked to the amplification E. coli plasmid. Subcloned nucleic acid sequences were confirmed by DNA sequencing.

Example  12

HEK293 EBNA  Immunoglobulins and Immunoglobulins in Cells Mutant  Transient manifestations

Recombinant anti-CD4 antibodies and anti-CD4 antibody-variants include 10% ultra-low IgG FCS (fetal bovine serum, Gibco), 2 mM glutamine (Gibco), 1% volume by volume (v / v) Attachment growth type HEK293-EBNA cells (Epstein-Bar-virus nuclear antigen) cultured in Dulbecco's modified Eagle's medium (Gibco) with non-essential amino acids (Gibco) and 250 μg / ml G418 (Roche Molecular Biochemicals) Was generated by transient transfection of human embryonic kidney cell line 293 (American type culture collection Accession No. ATCC # CRL-10852). For transfection, FuGENE ™ 6 transfection reagent (Roche Molecular Biochemicals) was used in a ratio of reagent (μl) to DNA (μg) in the range of 3: 1 to 6: 1. The light and heavy chains comprising the antifusogenic peptide-anti-CD4 antibody conjugate light and heavy chains, respectively, were prepared from two different plasmids using a molar ratio of light chain to heavy chain encoding the plasmid in the range of 1: 2 to 2: 1. Expression. Antifusogenic peptide-anti-CD4 antibody conjugates containing cell culture supernatants were harvested 4-11 days after transfection. For example, general information regarding recombinant expression of human immunoglobulins in HEK293 cells can be found in Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example  13

Immunoglobulin-specific antibodies The conjugate  Used SDS PAGE , Weston Blotting The Expression analysis using lancer and detection

Expressed and secreted antifusogenic peptide-anti-CD4 antibody conjugates were treated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and isolated anti-CD4-antibody and antifuso Generic peptide-anti-CD4-antibody-conjugate chains were transferred from the gel to the membrane and then detected by immunological methods.

SDS - PAGE  :

LDS sample buffer, 4-fold concentrate (4x): 4 g glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamine tetra 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) solution of phenol red, is added to water to a total volume of 10 ml made.

Cultures containing secreted antifusogenic peptide-anti-CD4 antibody conjugates were centrifuged to remove cells and cell debris. Fractions of clarified supernatant were mixed with 1/4 volume (v / v) of 4 × LDS sample buffer and 1/10 volume (v / v) of 0.5 M 1,4-dithiothreitol (DTT). The samples were then incubated at 70 ° C. for 10 minutes and proteins separated by SDS-PAGE. NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast gel (pH 6.4) and NuPAGE® MOPS operating buffer (running buffer) were used.

Weston Blot  :

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

After SDS-PAGE, the isolated anti-fusogenic peptide-anti-CD4 antibody conjugate chain was removed according to Burnette's "Semidry-Blotting-Method" (Burnette, WN, Anal. Biochem. 112 (1981) 195-203). The filtration membrane (pore size: 0.45 μm) was transferred by electrophoresis.

Immunological Detection:

TBS-buffer: 50 mM TRIS-hydrochloride, 150 mM NaCl, adjusted to pH 7.5,

Blocking solution: 1% (w / v) Western blocking reagent (Roche Molecular Biochemicals) in TBS-buffer,

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

For immunological detection, western blotting membranes were incubated twice for 5 minutes with shaking at room temperature in TBS-buffer and once for 90 minutes in blocking solution.

Peptide  Immunoglobulins Conjugate  Detection of the chain:

Heavy Chain: For detection of the heavy chain of the antifusogenic peptide-anti-CD4 antibody conjugate, purified rabbit anti-human IgG antibody conjugated to peroxidase was used (DAKO, Code No. P 0214).

Light Chain: The light chain of the antifusogenic peptide-anti-CD4 antibody conjugate was detected using purified peroxidase conjugated rabbit anti-human kappa light chain antibody (DAKO, Code No. P 0129).

For visualization of antibody light and heavy chains, the purified rabbit anti-human IgG antibody conjugated to peroxidase in the case of heavy chains, first diluted 1: 10,000 in a 10 ml blocking solution Or incubated with purified peroxidase conjugated rabbit anti-human kappa light chain antibody in the case of light chains at 4 ° C. overnight. After washing the membrane three times with TBTS-buffer and once with TBS buffer for 10 minutes at room temperature, the Western-blot membrane was developed with luminol / peroxide-solution producing chemiluminescence (Lumi-LightPLUS Western blotting Substrate, Roche Molecular Biochemicals). Thus, the membranes are incubated in 10 ml luminol / peroxide-solution for 10 seconds to 5 minutes, and then the light emitted is detected using LUMI-Imager F1 Analysator (Roche Molecular Biochemicals) and / or using x-ray film. Recorded. Spot intensity was quantified using LumiAnalyst Software (Version 3.1).

Immunoblot  Multi-dyeing:

The secondary peroxidase-labeled antibody conjugate used for detection was at 70 ° C. in 1 M TRIS-hydrochloride-buffer (pH 6.7) with 100 mM beta-mercaptoethanol and 20% (w / v) SDS. It can be removed from the stained blots by incubating the membrane for 1 hour. After this treatment, blots can be stained twice with different secondary antibodies. Before the second detection, blots are washed three times at room temperature with shaking for 10 minutes each in TBS-buffer.

Example  14

Peptide  Immunoglobulins Conjugate  Affinity Tablets, Dialysis and Concentrations

The 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 min at 10,000 g) and filtration through a 0.45 μm filter, the purified culture supernatant containing peptide immunoglobulin conjugates was added to PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO _4). , 137 mM NaCl and 2.7 mM KCl, pH 7.4), were placed on a Protein A-Sepharose ™ CL-4B column. Unbound protein was washed with PBS equilibration buffer and 0.1 M citrate buffer, pH 5.5. Antifusogenic peptide-anti-CD4 antibody conjugates were eluted with 0.1 M citrate buffer, pH 3.0, and the conjugate containing fractions were neutralized with 1 M TRIS-Base. The antifusogenic peptide-anti-CD4 antibody conjugate was then dialyzed extensively against PBS buffer at 4 ° C., concentrated using an Ultrafree®-CL Centrifugal Filter Unit (Millipore Corp., USA) equipped with a Biomax-SK membrane, 0 ° C., stored in ice-water bath. The integrity of the conjugate was analyzed by SDS-PAGE in the presence and absence of a reducing agent and stained using Coomassie brilliant blue as described in Example 13. Aggregation of antifusogenic peptide-anti-CD4 antibody conjugates was analyzed by analytical size exclusion chromatography.

Example  15

Peptide  Immunoglobulins Conjugate Deglycosylation

N-linked carbohydrates of anti-CD4 antibody and anti-fusogenic peptide-anti-CD4 antibody conjugates were prepared using peptide-N-glycosidase F (PNGaseF, Roche Molecular Biochemicals, Mannheim, Germany or Prozyme, San Leandro, CA). Cleavage was by enzymatic treatment. Thus, anti-CD4 antibody and anti-fusogenic peptide-anti-CD4 antibody conjugate were used at a protein concentration of about 2 mg / ml at 12 ° C. using 50 mU PNGaseF per mg of N-glycosylated protein in PBS buffer. Incubate for ˜ 24 hours. Peptide-N-glycosidase F was then isolated by preparative gel filtration in accordance with known methods. Briefly, PNGaseF treated anti-CD4-antibody and antifusogenic peptide-anti-CD4 antibody conjugates were prepared in PBS buffer (10 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ , 137 mM NaCl and 2.7 mM KCl, pH 7.4) on a Superose ™ 12 10/300 GL column (GE Healthcare former Amersham Bioscience, Sweden) equilibrated to 0.5 and using an Akta Explorer chromatography system from Amersham Bioscience (GE Healthcare former Amersham Bioscience, Sweden). Elution was carried out using equilibration buffer at a flow rate of ˜1.0 ml / min.

Example  16

Single-cycle antiviral activity Assay

For the production of pseudotyped NL-Bal viruses, the plasmids pNL4-3Δenv (HIV pNL4-3 genome construct with loss in the env gene) and pCDNA3.1 / NL-BAL env [containing the NL-Bal env gene pcDNA3.1 plasmid (obtained from the NIBSC Centralized Facility for AIDS reagent)] was co-transfected into a HEK 293FT cell line (Invitrogen), 10% fetal bovine serum (FCS), 100 U / mL penicillin, 100 μg / Cultures were incubated in Dulbecco's modified minimum medium (DMEM) with mL streptomycin, 2 mM L-glutamine and 0.5 mg / mL Genithycin (all media were from Invitrogen / Gibco). Supernatants containing Pseudotype virus were harvested 2 days after transfection and cell debris was removed by filtration through a 0.45 μm pore size PES (polyethersulfone) filter (Nalgene) and aliquots at −80 ° C. Stored in the form. For normalization in assay performance, virus stock aliquots were used to infect JC53-BL (US NIH Aids Reagent Program) cells, resulting in approximately 1.5 × 10 ⁵ RLU (relative light units) per well. Obtained. Test antifusogenic peptide-anti-CD4 antibody conjugate, parent anti-CD4 antibody, control antibody and control antifusogenic peptide (T-651) were diluted in 96-well plates. Assays were performed in duplicates. Each plate contained cell control and virus control wells. Viral stock 1.5 × 10 ⁵ RLU equivalents were added to each well, then 2.5 × 10 ⁴ JC53-BL cells were added to each well and the final assay volume was 200 μl per well. After 3 days of incubation at 37 ° C., 90% relative humidity, and 5% CO ₂ , the medium was aspirated and 50 μl of Steady-Glo® Luciferase Assay System (Promega) was added to each well. After incubation for 10 minutes at room temperature, assay plates were read on a Luminometer (Luminoskan, Thermo Electron Corporation). % Luciferase activity inhibition was calculated for each dose point after subtracting the background, and IC ₅₀ and IC ₉₀ -values were measured using XLfit curve fitting software for Excel (version 3.0.5 Buildl 2; Microsoft). The results are shown in Tables 4 and 5, respectively.

Antiviral Activity (by Weight) of Antifusogenic Polypeptides, Antibodies and Antifusogenic Peptide-Anti-CD4 Antibody Conjugates compound Antiviral activity NL-BAL (R5) NL-4-3 (X4) IC ₅₀ (ng / mL) /
IC ₉₀ (ng / mL) IC ₅₀ (ng / mL) /
IC ₉₀ (ng / mL) Control Antibody 1 (Inactive) Inert / inactive Inert / inactive Control Antibody 2 (Inactive) Inert / inactive Inert / inactive T-651 21.5 / 181.9 169.2 / 1099.7 mAb CD4 (6310/6309) Maximum Suppression = 50% Maximum Suppression = 20% Anti-Fusogenic Peptide-Anti-CD4 Antibody Conjugates (6310/6303) 6.1 / 32.2 43.9 / 123.9

Antiviral Activity (Molarity) of Antifusogenic Polypeptides, Antibodies and Antifusogenic Peptide-Anti-CD4 Antibody Conjugates compound Antiviral activity NL-BAL (R5) NL-4-3 (X4) IC ₅₀ (nM) /
IC ₉₀ (nM) IC ₅₀ (nM) /
IC ₉₀ (nM) Control Antibody 1 (Inactive) Inert / inactive Inert / inactive Control Antibody 2 (Inactive) Inert / inactive Inert / inactive T-651 5.4 / 45 42/275 mAb CD4 (6310/6309) Maximum Suppression = 50% Maximum Suppression = 20% Anti-Fusogenic Peptide-Anti-CD4 Antibody Conjugates (6310/6303) 0.038 / 0.201 0.275 / 0.774

Example  17

Cell-cell fusion Assay

On day 1, gp160-expressing HeLa cells (2 × 10 ⁴ cells / 50 μl / well) were planted in white 96 well microtiter plates in DMEM medium with 10% FCS and 2 μg / ml doxycycline. On day 2, 100 μl of supernatant sample or antibody control per well was added to a clear 96 well microtiter plate. Next, 100 μl containing 8 × 10 ⁴ CEM-NKr-Luc suspension cells was added to the medium, and incubated at 37 ° C. for 30 minutes. HeLa cell culture medium was aspirated from a 96 well plate, 100 μl in 200 μl antibody / CEM-NKr-Luc mixture was added and incubated overnight at 37 ° C. On day 3, 100 μl / well Bright-Glo ™ luciferase assay substrate (1,4-dithiothreitol and sodium dithionite; Promega Corp., USA) is added and luminescence is at least 15 minutes at room temperature Measured after incubation.

material :

HeLa-R5-16 cells (cell lines for expressing HIV gp160 upon doxycycline induction) were cultured in DMEM medium with nutrients and 10% FCS with 400 μg / ml G418 and 200 μg / ml hygromycin B. CEM.NKR-CD4-LUC (Cat. No. 5198, 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) was transfected (electroporation) to express the luciferase gene under transcription conditions of HIV-2 LTR, 10% fetal bovine serum, 4 mM glutamine, Proliferation was in RPMI 1640 with penicillin / streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin), and 0.8 mg / ml Zenithin sulfate (G418). Growth characteristics: round lymphocyte cells, not very diverse in shape. The cells can grow in suspension as single cells and form small clumps. Divide into 1: 10 twice a week. Special Features: Expression of luciferase activity after HIV-2 LTR interaction. 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). Cell lines are described in 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

Peripheral blood monocyte cells ( PBMC ) In  Antiviral activity Assay .

Human PBMCs were isolated from smokescreens (obtained at Stanford Blood Center) by Ficoll-Paque (Amersham, Piscataway, New Jersey, USA) density gradient centrifugation, according to the manufacturer's protocol. Briefly, blood was transferred from the smokescreen in 50 ml conical tubes and diluted in sterile Dulbecco's phosphate buffered saline (Invitrogen / Gibco) to a final volume of 50 ml. 25 ml of diluted blood were transferred to two 50 ml conical tubes, carefully placed in the lower layer using 12.5 ml of Ficoll-Plac Plus (Amersham Biosciences), and centrifuged at room temperature for 20 minutes without stopping at 450 xg. . The white cell layer was carefully transferred to a new 50 ml conical tube and washed twice with PBS. To remove remaining red blood cells, cells were incubated with ACK disruption buffer (Biosource) for 5 minutes at room temperature and washed one or more times with PBS. The number of PBMCs was counted and 10% FCS (Invitrogen / Gibco), 1% penicillin / streptomycin, 2 mM L-glutamine, 1 mM sodium-pyruvate, and 2 μg / ml phytohemagglutinin (Invitrogen) Incubate at 37 ° C. for 24 hours at a concentration of 2-4 × 10 ⁶ cells / ml in all RPMI1640. Prior to assay, cells were incubated with 5 units / ml human IL-2 (Roche Molecular Biochemicals) for a minimum of 48 hours. In a 96 well round bottom plate, 1 × 10 ⁵ PBMC was diluted in the presence of test antifusogenic peptide-anti CD-4 antibody-conjugate, control immunoglobulin, parent anti-CD4 antibody and control peptide (T-651) Under infection with HIV-1 JR-CSF virus (Koyanagi, Y., et al., Science 236 (1987) 819-822). The amount of virus used was equivalent to 1.2 ng HIV-1 p24 antigen / well. Infection was duplicated in duplicates. Plates were incubated at 37 ° C. for 6 days. Virus generation was carried out in Microsoft Excel Fit (version 3.0.5 Build 12; equation 205; Microsoft) at the end of infection using p24 ELISA (HIV-1 p24 ELISA # NEK050B, Perkin Elmer / NEN). One binding site and a sigmoidal dose-response model were used to measure.

Antiviral Activity (by Weight) of Antifusogenic Polypeptides, Antibodies and Antifusogenic Peptide-Anti-CD4 Antibody Conjugates compound Antiviral activity NL-BAL NL-4-3 IC ₅₀ (ng / mL) /
IC ₉₀ (ng / mL) IC ₅₀ (ng / mL) /
IC ₉₀ (ng / mL) Control Antibody 1 (Inactive) Inert / inactive Inert / inactive Control Antibody 2 (Inactive) Inert / inactive Inert / inactive T-651 14.1 / 57.8 41.4 / 79.6 mAb CD4 (6310/6309) 917.6 / up to 54% suppression 35.6 / 1117.0 Anti-Fusogenic Peptide-Anti-CD4 Antibody Conjugates (6310/6303) 5.3 / 18.2 3.2 / 25.1

<110> F. Hoffmann-La Roche AG <120> A conjugate of an antibody against CCR5 and an antifusogenic          peptide <130> 23883 WO <150> EP 06017156.8 <151> 2006-08-17 <150> EP 07014335.9 <151> 2007-07-20 <150> EP 06020647.1 <151> 2006-09-29 <160> 105 <170> PatentIn version 3.2 <210> 1 <211> 119 <212> PRT <213> mouse <400> 1 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly   1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val          35 40 45 Ala Ser Ile Ser Thr Gly Asp Asn Thr Tyr Tyr Thr Asp Ser Val Arg      50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu  65 70 75 80 Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys Thr                  85 90 95 Arg Gly Arg Gly Asp Arg Gly Asp Leu Phe Gly Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ser         115 <210> 2 <211> 114 <212> PRT <213> mouse <400> 2 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly   1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Arg              20 25 30 Gly Asn Gln Met Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Thr Val Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Thr Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile             100 105 110 Lys arg         <210> 3 <211> 117 <212> PRT <213> mouse <400> 3 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Val Phe              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met      50 55 60 Ser Arg Leu Arg Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe  65 70 75 80 Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Ile Val Ser Ser         115 <210> 4 <211> 108 <212> PRT <213> mouse <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ser Ser Gly Asn Ile His Gly Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val          35 40 45 Tyr Asn Thr Lys Ala Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Asn Leu Gln Pro  65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His 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 <211> 117 <212> PRT <213> mouse <400> 5 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Ile Phe              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met      50 55 60 Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe  65 70 75 80 Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Ile Val Ser Ser         115 <210> 6 <211> 108 <212> PRT <213> mouse <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Gly Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val          35 40 45 Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Asp Leu Pro Arg                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 <210> 7 <211> 117 <212> PRT <213> mouse <400> 7 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Thr Phe              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Arg Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met      50 55 60 Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe  65 70 75 80 Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Ile Val Ser Ser         115 <210> 8 <211> 108 <212> PRT <213> mouse <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Gly Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val          35 40 45 Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His His Tyr Asp Leu Pro Arg                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 <210> 9 <211> 5 <212> PRT <213> mouse <400> 9 Thr Tyr Ala Met Ser   1 5 <210> 10 <211> 5 <212> PRT <213> mouse <400> 10 Val Phe Gly Val His   1 5 <210> 11 <211> 5 <212> PRT <213> mouse <400> 11 Ile Phe Gly Val His   1 5 <210> 12 <211> 5 <212> PRT <213> mouse <400> 12 Thr Phe Gly Val His   1 5 <210> 13 <211> 16 <212> PRT <213> mouse <400> 13 Ser Ile Ser Thr Gly Asp Asn Thr Tyr Tyr Thr Asp Ser Val Arg Gly   1 5 10 15 <210> 14 <211> 16 <212> PRT <213> mouse <400> 14 Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met Ser   1 5 10 15 <210> 15 <211> 16 <212> PRT <213> mouse <400> 15 Val Ile Trp Arg Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met Ser   1 5 10 15 <210> 16 <211> 11 <212> PRT <213> mouse <400> 16 Gly Arg Gly Asp Arg Gly Asp Leu Phe Gly Tyr   1 5 10 <210> 17 <211> 9 <212> PRT <213> mouse <400> 17 Val Asn Leu Ala Asp Ala Met Asp Tyr   1 5 <210> 18 <211> 17 <212> PRT <213> mouse <400> 18 Lys Ser Ser Gln Ser Leu Leu Tyr Arg Gly Asn Gln Met Asn Tyr Leu   1 5 10 15 Ala     <210> 19 <211> 11 <212> PRT <213> mouse <400> 19 Arg Ser Ser Gly Asn Ile His Gly Tyr Leu Ala   1 5 10 <210> 20 <211> 11 <212> PRT <213> mouse <400> 20 Arg Ala Ser Gly Asn Ile His Gly Tyr Leu Ala   1 5 10 <210> 21 <211> 7 <212> PRT <213> mouse <400> 21 Trp Ala Ser Thr Arg Glu Ser   1 5 <210> 22 <211> 7 <212> PRT <213> mouse <400> 22 Asn Thr Lys Ala Leu Ala Glu   1 5 <210> 23 <211> 7 <212> PRT <213> mouse <400> 23 Asn Thr Lys Thr Leu Ala Glu   1 5 <210> 24 <211> 9 <212> PRT <213> mouse <400> 24 Gln Gln Tyr Tyr Thr Tyr Pro Arg Thr   1 5 <210> 25 <211> 9 <212> PRT <213> mouse <400> 25 Gln His His Tyr Asp Leu Pro Arg Thr   1 5 <210> 26 <211> 330 <212> PRT <213> Homo sapiens <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys   1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr  65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 27 <211> 327 <212> PRT <213> Homo sapiens <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg   1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr  65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 28 <211> 107 <212> PRT <213> Homo sapiens <400> 28 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu   1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe              20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln          35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser      50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 29 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> C34 <400> 29 Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His   1 5 10 15 Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu              20 25 30 Leu Leu         <210> 30 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> T20 <400> 30 Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln   1 5 10 15 Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu              20 25 30 Trp Asn Trp Phe          35 <210> 31 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> T1249 <400> 31 Trp Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln   1 5 10 15 Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp              20 25 30 Ala Ser Leu Trp Glu Trp Phe          35 <210> 32 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> T651 <400> 32 Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu   1 5 10 15 Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu              20 25 30 Gln Glu Leu Leu          35 <210> 33 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> T2635 <400> 33 Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala Arg   1 5 10 15 Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn Glu              20 25 30 Ala Ala Leu Arg Glu Leu          35 <210> 34 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> N36 <400> 34 Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala   1 5 10 15 Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln              20 25 30 Ala Arg Ile Leu          35 <210> 35 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> DP107 <400> 35 Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu   1 5 10 15 Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu              20 25 30 Arg Tyr Leu Lys Asp Gln          35 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> linker 1 <400> 36 Leu Ser Leu Ser Pro Gly Lys   1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> linker 2 <400> 37 Leu Ser Pro Asn Arg Gly Glu Cys   1 5 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker 3 <400> 38 Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln   1 5 10 15 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker 4 <400> 39 Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly   1 5 10 15 <210> 40 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker 5 <400> 40 Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly   1 5 10 15 Asn Asn         <210> 41 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker 6 <400> 41 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly   1 5 10 15 <210> 42 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker 7 <400> 42 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn   1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker 8 <400> 43 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly   1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker 9 <400> 44 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly   1 5 10 15 <210> 45 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker 10 <400> 45 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly   1 5 10 15 Thr     <210> 46 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker 11 <400> 46 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly   1 5 10 15 Gly     <210> 47 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker 12 <400> 47 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly   1 5 10 15 Gly thro         <210> 48 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker 13 <400> 48 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly   1 5 10 15 Gly asn         <210> 49 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker 14 <400> 49 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly   1 5 10 15 Ala ser         <210> 50 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> linker 15 <400> 50 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly              20 25 <210> 51 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> linker 16 <400> 51 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly              20 25 <210> 52 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> linker 17 <400> 52 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly              20 25 <210> 53 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> linker 18 <400> 53 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ala Ser              20 25 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker 19 <400> 54 Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser   1 5 10 15 Gly     <210> 55 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> linker 20 <400> 55 Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser   1 5 10 15 Gly ala ser             <210> 56 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> linker 24 <400> 56 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Ala ser         <210> 57 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker 25 <400> 57 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 <210> 58 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> linker 26 <400> 58 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly              20 25 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> linker 27 <400> 59 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly     <210> 60 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> linker 28 <400> 60 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly              20 25 <210> 61 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> linker 29 <400> 61 Leu Ser Leu Ser Gly Gly   1 5 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> linker 30 <400> 62 Leu Ser Leu Ser Pro Gly Gly   1 5 <210> 63 <211> 214 <212> PRT <213> Homo sapiens <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Gly Tyr              20 25 30 Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val          35 40 45 Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Gly Asn Tyr Tyr Cys 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 Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu 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 Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 64 <211> 447 <212> PRT <213> Homo sapiens <400> 64 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Ile Phe              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met      50 55 60 Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe  65 70 75 80 Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His     210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn 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 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 65 <211> 503 <212> PRT <213> Homo sapiens <400> 65 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Pro Leu Gly Ile Phe              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Lys Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Met      50 55 60 Ser Arg Leu Arg Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe  65 70 75 80 Arg Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Lys Val Asn Leu Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Ile Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His     210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn 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 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly         435 440 445 Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gln Gln Gln Gln Gly Gn Asn     450 455 460 Asn Thr Thr Trp Glu Ala Trp Asp Arg Ala Ile Ala Glu Tyr Ala Ala 465 470 475 480 Arg Ile Glu Ala Leu Ile Arg Ala Ala Gln Glu Gln Gln Glu Lys Asn                 485 490 495 Glu Ala Ala Leu Arg Glu Leu             500 <210> 66 <211> 345 <212> PRT <213> Human immunodeficiency virus <400> 66 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly   1 5 10 15 Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln              20 25 30 Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile          35 40 45 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln      50 55 60 Leu Gln Ala Arg Ile Leu Ala 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 Ala                  85 90 95 Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu 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 Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys     130 135 140 Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser 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 Ala Val Leu Ser             180 185 190 Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr         195 200 205 His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu Gly Ile Glu Glu     210 215 220 Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Ile Arg Leu Val Asn Gly 225 230 235 240 Ser Leu Ala 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 Ala Leu Lys Tyr Trp Trp Asn Leu         275 280 285 Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu     290 295 300 Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu 305 310 315 320 Val Val Gln Gly Ala Cys Arg Ala 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 Sequence <220> <223> Linker 31 <400> 67 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Ser              20 <210> 68 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Linker 32 <400> 68 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser   1 5 10 15 Gly Gly Gly Ser Gly Gly Gly              20 <210> 69 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker 33 <400> 69 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Linker 34 <400> 70 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly   1 5 10 15 <210> 71 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> anti-CD4 antibody light chain <400> 71 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala 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 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu         115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe     130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu             180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser         195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 72 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> anti-CD4 antibody heavy chain <400> 72 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu 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 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys 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 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Pro Gly Lys     450 <210> 73 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> afp-1 <400> 73 Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Gln Tyr Thr Ser   1 5 10 15 Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn              20 25 30 Glu Gln Glu Leu Leu          35 <210> 74 <211> 503 <212> PRT <213> Artificial Sequence <220> <223> anti-CD4 antibody heavy chain-afp-1 antifusogenic peptide          conjugate <400> 74 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro         115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr     130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr             180 185 190 Val Pro Ser Ser Ser Leu 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 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys 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 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly     450 455 460 Ser Gly Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Gln Tyr 465 470 475 480 Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser 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 Ser Pro Ser Ser Leu Ala Val Ser Val Gly   1 5 10 15 Glu Lys Val Thr Met Ile Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala 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> 76 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chimeric light chain variable domain <400> 76 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gly Ser Leu Leu Tyr Ser              20 25 30 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala 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             100 105 110 <210> 77 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> chimeric light chain <400> 77 Met Asp Met Arg Val Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp   1 5 10 15 Leu Pro Gly Ala Arg Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser              20 25 30 Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser          35 40 45 Gly Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr      50 55 60 Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser  65 70 75 80 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly                  85 90 95 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala             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 Ala Ala Pro Ser Val Phe Ile     130 135 140 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150 155 160 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys                 165 170 175 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu             180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu         195 200 205 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr     210 215 220 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240 Cys     <210> 78 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chimeric light chain variable domain <400> 78 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala 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 Sequence <220> <223> chimeric light chain variable domain <400> 79 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr              20 25 30 Ile Ala Trp Tyr Gln His Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Cys Leu Gln Gln Tyr Asp Asn Leu Leu Phe                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg             100 105 <210> 80 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> chimeric light chain variable domain <400> 80 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Thr Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala 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> 81 <211> 122 <212> PRT <213> Mus musculus <400> 81 Glu Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Thr Val Val Val Ser Ser         115 120 <210> 82 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> humanized heavy chain variable domain <400> 82 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Val Thr Leu Asp Pro Ser Thr Asn Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 83 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> chimeric heavy chain <400> 83 Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly   1 5 10 15 Ala His Ser Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Val Lys Lys              20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe          35 40 45 Thr Ser Tyr Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu      50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp  65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Val Thr Leu Asp Pro Ser Thr Asn                  85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe         115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr     130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu                 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His             180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser         195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys     210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu                 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu             260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser         275 280 285 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu     290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser 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 Ser Lys Ala Lys Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Leu Gly Lys 465 <210> 84 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> chimeric heavy chain variable domain <400> 84 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 85 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> chimeric heavy chain variable domain <400> 85 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Asn Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Ala Ile Ser Asp His Ser Thr Asn Thr Tyr Tyr Pro Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe  65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Lys Tyr Gly Gly Asp Tyr Asp Pro Phe Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Pro Val Thr Val Ser Ser         115 120 <210> 86 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> chimeric heavy chain variable domain <400> 86 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu Asp Trp Ile          35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Asp Tyr Asp Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr Trp             100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 87 <211> 17 <212> PRT <213> Mus musculus <400> 87 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 88 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 88 Lys Ser Ser Gly Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 89 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 89 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Thr Asn Gln Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR <400> 90 Lys Ala 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   1 5 <210> 92 <211> 7 <212> PRT <213> Mus musculus <400> 92 Trp Ala Ser Thr Arg Glu Ser   1 5 <210> 93 <211> 11 <212> PRT <213> Mus musculus <400> 93 Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser   1 5 10 <210> 94 <211> 8 <212> PRT <213> Mus musculus <400> 94 Gln Gln Tyr Tyr Ser 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> Mus 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 5 <210> 98 <211> 5 <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   1 5 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 Lys Phe Lys   1 5 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   1 5 10 15 Gly     <210> 102 <211> 12 <212> PRT <213> Mus musculus <400> 102 Ala Ile Ser Asp His Ser Thr Asn Thr Tyr Tyr Pro   1 5 10 <210> 103 <211> 13 <212> PRT <213> Mus musculus <400> 103 Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr   1 5 10 <210> 104 <211> 13 <212> PRT <213> Mus musculus <400> 104 Glu Lys Asp Asn Tyr Ala Thr Gly Ala Trp Phe Ala Tyr   1 5 10 <210> 105 <211> 11 <212> PRT <213> Mus musculus <400> 105 Ala Arg Lys Tyr Gly Gly Asp Tyr Asp Pro Phe   1 5 10  

Claims (26)

A conjugate comprising at least one antifusogenic peptide and an antibody against an HIV gp120 binding cell surface receptor, wherein two or four antifusogenic peptides each comprise a heavy and / or light chain of said antibody against an HIV gp120 binding cell surface receptor. Conjugated at one terminus, and the antibody against the HIV gp120 binding cell surface receptor is an anti-CCR5 antibody, The anti-fusogenic peptide is a linear peptide and comprises an amino acid sequence of 5 to 100 amino acids, A conjugate comprising a heavy and / or light chain of mAb CCR5 and a conjugate of antifusogenic peptide (s) (“chain conjugate”) selected from the group consisting of: (1) [heavy chain]-[linker] _m- [antifusogenic peptide], and (2) [light chain]-[linker] _m- [antifusogenic peptide] Wherein the linkers may be the same or different, m is an integer of 1 or 0, and m may be independently the same or different). delete 2. The conjugate of claim 1, wherein at least two of the terminals of the anti-CCR5 antibody are conjugated to an antifusogenic peptide, respectively. delete delete delete 2. Conjugate according to claim 1, characterized in that it comprises a chain conjugate (1). The conjugate according to claim 1, which comprises two [mAb CCR5 light chains] and two chain conjugates (1). The conjugate of claim 1, wherein said anti-fusogenic peptide is selected from the group of peptides defined by SEQ ID NOs: 29-35 and SEQ ID NO: 73. 2. The conjugate of claim 1, wherein said anti-CCR5 antibody comprises a variable heavy domain consisting of an immunoglobulin backbone and a CDR3 region selected from the group consisting of heavy chain CDR3 sequences SEQ ID NOs: 16, 17. The anti-CCR5 antibody of claim 1, wherein the anti-CCR5 antibody comprises an immunoglobulin backbone and a CDR3 region, heavy chain CDR2 sequences SEQ ID NO: 13, 14, and 15 selected from the group consisting of heavy chain CDR3 sequences SEQ ID NO: 16 and 17 A conjugate comprising a variable heavy domain consisting of a CDR2 region selected from the group consisting of and a CDR1 region selected from the group consisting of heavy chain CDR1 sequences SEQ ID NO: 9, 10, 11, and 12. 2. The conjugate of claim 1, wherein said anti-CCR5 antibody comprises a heavy chain variable domain selected from the group of SEQ ID NOs: 1, 3, 5, and 7. The antibody of claim 1, wherein the anti-CCR5 antibody comprises an immunoglobulin backbone, and a CDR1 region selected from SEQ ID NOs: 18, 19, and 20, a CDR2 region selected from SEQ ID NOs: 21, 22, and 23, and SEQ ID NO: A conjugate comprising a variable light domain consisting of a CDR3 region selected from NO: 24 and 25. The antibody of claim 1, wherein the anti-CCR5 antibody comprises a CDR of SEQ ID NO: 1 as a heavy chain CDR and a CDR of SEQ ID NO: 2 as a light chain CDR, a CDR of SEQ ID NO: 3 as a heavy chain CDR and a SEQ ID as light chain CDR. CDR of SEQ ID NO: 8 as the heavy chain CDR and CDR of SEQ ID NO: 6 as the heavy chain CDR, CDR of SEQ ID NO: 6 as the heavy chain CDR, or CDR of SEQ ID NO: 7 as the heavy chain CDR Conjugate comprising a. The conjugate of claim 1, wherein the anti-CCR5 antibody comprises a variable heavy domain and a variable light domain independently selected from the group consisting of: a) the heavy chain (V _H ) variable domain defined by amino acid sequence SEQ ID NO: 1 and the light chain (V _L ) variable domain defined by SEQ ID NO: 2; b) the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 3 and the light chain variable domain defined by SEQ ID NO: 4; c) the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and the light chain variable domain defined by SEQ ID NO: 6; d) the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by SEQ ID NO: 8. The antibody of claim 1, wherein the anti-CCR5 antibody comprises a heavy chain (V _H ) variable domain defined by amino acid sequence SEQ ID NO: 1 and a light chain (V _L ) variable domain defined by amino acid sequence SEQ ID NO: 2, Heavy chain variable domain defined by amino acid sequence SEQ ID NO: 3 and light chain variable domain defined by amino acid sequence SEQ ID NO: 4, heavy chain variable domain defined by amino acid sequence SEQ ID NO: 5 and amino acid sequence SEQ ID NO The light chain variable domain defined by: 6, the heavy chain variable domain defined by amino acid sequence SEQ ID NO: 7 and the light chain variable domain defined by amino acid sequence SEQ ID NO: 8; Amino acids glycine (G) and asparagine (N), tripeptide GST, and a linker selected from the group consisting of SEQ ID NOs: 36-62 and SEQ ID NOs: 67-70; And A conjugate comprising an antifusogenic peptide selected from the group of peptides defined by SEQ ID NOs: 29-35 and SEQ ID NO: 73. The conjugate of claim 1, comprising an anti-fusogenic peptide selected from the group of SEQ ID NOs: 29-35 and SEQ ID NO: 73. 2. The light chain variable domain of claim 1, wherein each of the two light chain variable domains of SEQ ID NO: 2 comprises a heavy chain variable domain of SEQ ID NO: 1, a linker of SEQ ID NO: 40 and an antifusogenic peptide of SEQ ID NO: 33. 2 light chain variable domains of SEQ ID NO: 4, a heavy chain variable domain of SEQ ID NO: 3, a linker of SEQ ID NO: 40, and SEQ ID NO: 2 conjugates of type (2) comprising an anti-fusogenic peptide of 33, or two light chain variable domains of SEQ ID NO: 6, each of which is a heavy chain variable domain of SEQ ID NO: 5, SEQ ID NO: Two conjugates of type (2) comprising a linker of 40 and an antifusogenic peptide of SEQ ID NO: 33, or two light chain variable domains of SEQ ID NO: 8, each of SEQ ID NO: 7 Two of type (2) comprising a heavy chain variable domain of, a linker of SEQ ID NO: 40 and an antifusogenic peptide of SEQ ID NO: 33 Conjugate comprising the juice gate. The antibody of claim 1, wherein the anti-CCR5 antibody is an antibody of the IgG4 subclass, or an IgG1 or IgG2 subclass, having a mutation in amino acids S228, L234, L235, and / or D265, and / or containing a PVA236 mutation. Conjugate characterized in that. The conjugate of claim 19, wherein the anti-CCR5 antibody of the IgG4 subclass has a mutation S228P, and the anti-CCR5 antibody of the IgG1 subclass has mutations L234A and L235A. delete A pharmaceutical composition for the treatment of a viral infection comprising the conjugate of any one of claims 1, 3 and 7-20 together with a pharmaceutically acceptable excipient or carrier. 21. A medicament for the treatment of a viral infection, comprising the conjugate of any one of claims 1, 3 and 7-20. The agent according to claim 23, wherein the viral infection is an HIV infection. delete delete

Images