RU2774782C2 - DERIVATIVES OF PROTEIN FUSED WITH Fc WITH HIGH DOUBLE ACTIVITY: ANTIVIRAL ACTIVITY RELATIVELY TO HIV AND IMMUNOMODULATING ACTIVITY - Google Patents

Abstract

FIELD: immunology; medicine.

SUBSTANCE: group of inventions relates to immunology and medicine, in particular to derivatives of protein fused with Fc, directed against HIV. A derivative of protein fused with Fc contains, in the direction from N- to C-end: (a) extracellular domains D1 and D2 of human CD4, (b) Fc-site of human IgG1 containing at least one of M428L or N434S point mutations, (c) grouping selected from linker polypeptide (GGGGS)_n, where 1≤n≤10, SEQ ID NO:5-9 and combinations thereof, and (d) polypeptide originating from motifs of the heptad repeat 2 (HR2) gp41. Nucleic acid, a vector and a host cell expressing the specified derivatives of protein fused with Fc, as well as their therapeutic and diagnostic applications in healthcare are also proposed. Derivatives of protein fused with Fc are characterized by: (1) blocking the penetration of the human immunodeficiency virus (HIV) into host cells, (2) inducing effector functions due to the activation of natural killer (NK) and other cells of the immune system, (3) producing in mammalian cells with high yield, and (4) having prolonged antiviral and immunomodulating activity in vivo.

EFFECT: obtaining derivatives of protein fused with Fc with high double activity.

37 cl, 12 dwg, 14 ex

Description

FIELD OF THE INVENTION

The present invention relates to anti-HIV Fc fusion protein derivatives with improved yield when produced in mammalian cells, prolonged antiviral and immunomodulatory activities. The Fc fusion protein derivatives of the present invention are characterized by: (1) blocking entry of human immunodeficiency virus (HIV) into host cells, (2) inducing selective effector functions by activating natural killer (NK) and other cells of the immune system, ( 3) are produced in mammalian cells in high yield; and (4) have sustained activity in vivo. Various forms of these polypeptides are set forth and examples thereof are given. The scope of the present invention also includes isolated nucleic acids, vectors and host cells expressing these polypeptides, as well as their use in health care for therapeutic and diagnostic purposes.

PRIOR ART

HIV infection is one of the major public health threats of international concern. It is estimated that more than 78 million people worldwide have been infected with the human immunodeficiency virus since 1981. During that time, almost half of these infected individuals have died due to acquired human immunodeficiency syndrome (AIDS). See UNAIDS, http://www.unaids.org/, October 2015.

The creation of protective antibodies is the main mechanism for the development of vaccines against human pathogens. However, so far it has not been possible to create immunogens capable of inducing the production of such anti-HIV antibodies. The construction of such immunogens requires, firstly, the identification of conserved epitopes to provide a sustained and stable response and, secondly, the invention of new and more effective immunogens that properly present these epitopes. See Haynes B, Curr Opin Immunol. 2015; 35:39-47. In contrast to these modest advances in immunogen development, a large number of new effective broad spectrum antibodies (ie, broad spectrum neutralizing antibodies, bNAbs) against the HIV envelope glycoprotein isolated from HIV infected individuals have recently been identified. See Mascola J, et al., Immunol Rev. 2013; 254:225-244. In addition, synthetic molecules based on the structure of antibodies have also been proposed as new therapeutic agents. See Gardner M, et al., Nature 2015; 519:87-91. Indeed, highly effective antibodies can protect uninfected individuals from HIV infection or can be used in eradication strategies in HIV-infected patients. See Mascola, 2013, supra. The use of neutralizing antibodies against the envelope glycoprotein and its subunits has also been proposed to prevent HIV replication in vivo. See Yang X, et al., J. Virol. 2005; 79:3500-3508.

From a therapeutic point of view, antibodies are of particular importance due to their dual function: antiviral agents that can block HIV replication by binding to the HIV envelope glycoprotein, and activators of NK cells due to the interaction of constant regions of antibody chains (Fc) with the CD16 Fc receptor expressed on the surface of NK cells and other cells. This interaction allows CD16+ immune cells to kill infected cells through a mechanism known as antibody-dependent cell-mediated cytotoxicity (ADCC). See Milligan C, et al., Cell Host Microbe. 2015; 17:500-506. Since both activities are believed to be necessary to protect uninfected subjects, significant efforts have been made to develop neutralizing antibodies with enhanced antiviral activity and ADCC activity. Several IgG derivatives have been described that increase the affinity of human IgG1 for human CD16, thus increasing their ADCC activity. See Saxena A, et al., Frontiers Immunol 2016; 7(580):1-11. However, the clinical use of such modified Fc fusion proteins may be affected by the fact that they are produced in mammalian cells in low yield or that their effector functions are altered. Thus, there is a need in the art for neutralizing antibodies with improved ADCC activity, receptor selectivity, and high yield.

SUMMARY OF THE INVENTION

In a first aspect, this application relates to modified derivatives of the Fc fusion protein of the invention, containing in the N- to C-terminal direction:

(a) the D1 and D2 extracellular domains of the human CD4 receptor,

(b) Fc region of human IgG1 containing at least one of the point mutations M428L or N434S,

(c) a grouping selected from the group consisting of:

(1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10,

(2) SEQ ID NO: 5-9 and

(3) their combinations, and

(d) a gp41 derived polypeptide.

The Fc fusion protein derivatives of the invention have antiviral and ADCC activity superior to that of any comparable antibody known in the art. See Gardner, 2015, supra. The expression and duration of action of the Fc fusion protein derivatives of the invention is also higher than other comparable anti-HIV antibodies.

In a further aspect, the invention provides nucleic acids encoding derivatives of the Fc fusion protein of the invention, vectors containing said nucleic acids, and host cells containing the nucleic acids and vectors mentioned above.

In a further aspect, the invention relates to pharmaceutical compositions comprising Fc fusion protein derivatives, nucleic acids, vectors and host cells of the invention, or mixtures thereof.

In another aspect, the invention provides a combination comprising Fc fusion protein derivatives, nucleic acids, vectors, host cells, and pharmaceutical compositions of the invention, and at least one therapeutic agent.

In yet another aspect, the invention relates to the use of Fc fusion protein derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions and combinations of the invention, or mixtures thereof, as a medicament. In a further aspect of this aspect, the invention relates to the use of Fc fusion protein derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions and combinations of the invention, or mixtures thereof, in the treatment or prevention of HIV infection or AIDS. In an alternative form of said aspect, the invention relates to a method for treating or preventing HIV infection or AIDS in a subject, comprising administering to the subject a therapeutically effective amount of Fc fusion protein derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions and combinations of the invention, or mixtures thereof. . In another alternative form of this aspect, the invention relates to the use of Fc fusion protein derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions and combinations of the invention, or mixtures thereof, in the manufacture of a medicament for the treatment or prevention of HIV infection or AIDS.

In addition, the present invention relates to a method for derivatizing an Fc fusion protein of the invention, comprising the steps of (a) culturing a host cell containing a nucleic acid of the invention, (b) expressing a nucleotide sequence, and (c) isolating a derivative of an Fc fusion protein from host cell cultures.

In another aspect, the invention provides a method for inactivating HIV, comprising contacting a virus with an Fc fusion protein derivative of the invention.

In a further aspect, the invention provides a method for inducing gp120 expression in an HIV-infected cell, comprising the step of contacting the infected cell with Fc fusion protein derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions and combinations of the invention, or a mixture thereof.

In a further aspect, the invention relates to a method for detecting HIV in a sample comprising the steps of (a) contacting the sample with an Fc fusion protein derivative of the invention and (b) determining whether the Fc fusion protein derivative specifically binds to a molecule from the sample.

In yet another aspect, the invention provides a kit comprising Fc fusion protein derivatives, nucleic acids, vectors, host cells, and pharmaceutical compositions of the invention, or mixtures thereof.

BRIEF DESCRIPTION OF GRAPHICS

Fig. 1 Schematic representation of the general structure of the Fc fusion protein derivatives of the invention. In the N- to C-terminus direction, the molecules contain the D1 and D2 domains of human CD4, a human IgG1 constant region (e.g., wild-type, mutated), possibly a human CCR5 peptide, a flexible linker, and an HIV gp41 peptide (e.g., T-20, C34 or ENO).

Fig. 2 Schematic representation of screening procedures performed to improve the activity and yield of the Fc fusion protein. Various sets of mutations in the human IgG1 sequence were screened for improved production and effector functions. See Table 1. Various linkers and gp41 derived T20, C34 and EHO polypeptides were then screened.

Fig. 3 Production kinetics of various Fc fusion protein derivatives of the invention. All IgG1 mutations were introduced into the M7 IgG1 wt C34 molecule.

Fig. 4 Neutralizing activity of several Fc fusion protein derivatives of the invention. All mutations were introduced into the M7 IgG1 wt C34 molecule. A) IC ₅₀ values are shown for the following HIV isolates: NL4.3, BaL, AC10 and SVPB16. The geometric mean IC ₅₀ values for each Fc fusion protein derivative are shown. B) The effect of LL mutations on the neutralizing activity against the BaL isolate was shown. C) The influence of various combinations of mutations on the neutralizing activity against the BaL isolate is shown.

Fig. 5 ADCC activity of various mutated IgG fusion proteins of the invention. EC50 values for ADCC were normalized to the M1 reference molecule. Lower values indicate higher activity. A) ADCC activity of series A12-A141. B) The effect of LL mutations on ADCC activity is shown. C) The influence of various combinations of mutations on ADCC activity is shown.

Fig. 6 Binding to CD32a, CD32bc, Cd16a, CD16aVF, CD64 and C1q of various mutated IgG fusion proteins of the invention. The fold changes in the signals recorded upon binding in ELISA (enzyme immunoassay) were calculated for each compound using the wild-type IgG1 molecule M7AC34 (data not shown). The multiplicity of changes was subjected to the logarithmic transformation Log2 and divided into clusters.

Fig. 7 Binding affinity of several Fc fusion protein derivatives of the invention having sustained activity for the recombinant human neonatal Fc receptor (FcRn). ELISA was performed at pH 6.0 (upper panel) and pH 7.2 (lower panel).

Fig. 8 Effect of various linkers on the production, neutralization and ADCC activity of the fusion proteins of the invention. A) Production in 293T cells was assessed up to 7 days after transfection. B) Neutralizing activity against HIV-1 isolates BaL and AC10 was evaluated. B) ADCC activity is shown as in FIG. 5.

Fig. 9. Effect of various gp41 peptides on the production, neutralization and ADCC activity of the fusion proteins of the invention. A) Production in 293T cells was assessed up to 7 days after transfection. B) The neutralizing activity against the HIV-1 BaL isolate was evaluated. C) ADCC activity is represented by primary dose-response data (M7BC34, an IgG2 derivative, was used as a negative control).

Fig. 10. Effect of the fusion proteins of the invention in vivo. A) Schematic representation of in vivo experiments. B) Analysis of infected cells (HIV GAG+ cells) two weeks after infection in the spleen of untreated animals (CTROL) and animals treated (M20A16_4LLC34 and M21A16_4LLC34).

Fig. 11. Diagram of expression plasmid pM5A16T20. The main characteristics of the plasmid are shown, such as the selectable marker and open reading frames.

Fig. 12. Diagram of expression plasmid pM7A16LLC34. The main characteristics of the plasmid are shown, such as the selectable marker and open reading frames.

DEPOSIT OF MICROORGANISMS

7 B, D-38124 Braunschweig, ФРГ.Plasmids pM5A16T20 and pM7A16LLC34 were deposited on April 20, 2017 under registration numbers DSM 32496 and DSM 32497, respectively, in the DSMZ - German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen),

7B, D-38124 Braunschweig, Germany.

SEQUENCE LIST

The nucleotide and amino acid sequences shown in the accompanying sequence listing are shown using standard letter abbreviations and codes commonly used in the art. Only one strand of each nucleotide sequence is shown, however it is clear that a complementary strand is also included by reference to the strand shown. In the attached sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the D1 domain of the human CD4 receptor.

SEQ ID NO: 2 is the amino acid sequence of the D2 domain of the human CD4 receptor.

SEQ ID NO: 3 is the amino acid sequence of the Fc region of human IgG1.

SEQ ID NO: 4 is the amino acid sequence of the GGGGS linker polypeptide.

SEQ ID NO: 5 is the amino acid sequence of the N-terminal region of the human CCR5 receptor.

SEQ ID NO: 6 is the amino acid sequence of the M19 linker peptide.

SEQ ID NO: 7 is the amino acid sequence of the M20 linker peptide.

SEQ ID NO: 8 is the amino acid sequence of the M21 linker peptide.

SEQ ID NO: 9 is the amino acid sequence of the M22 linker peptide.

SEQ ID NO: 10 is the amino acid sequence of the T-20 polypeptide.

SEQ ID NO: 11 is the amino acid sequence of the C34 polypeptide.

SEQ ID NO: 12 is the amino acid sequence of the EHO polypeptide.

SEQ ID NO: 13 is the amino acid sequence of the Fc region of human IgG1 carrying a set of AM mutations (ie point mutations G236A, S239D, A330L and I332E).

SEQ ID NO: 14 is the amino acid sequence of the Fc region of human IgG1 carrying the A12 mutation set (ie F243L, R292P and Y300L point mutations).

SEQ ID NO: 15 is the amino acid sequence of the Fc region of human IgG1 carrying the A14 mutation set (ie point mutations F243L, R292P and P396L).

SEQ ID NO: 16 is the amino acid sequence of the Fc region of human IgG1 carrying the A16 mutation set (ie point mutations F243L, R292P, Y300L and P396L).

SEQ ID NO: 17 is the amino acid sequence of the Fc region of human IgG1 carrying the A18 mutation set (ie point mutations F243L, R292P, Y300L, P396L and V305I).

SEQ ID NO: 18 is the amino acid sequence of the Fc region of human IgG1 carrying the A41 mutation set (ie point mutations S298A, E333A and K334A).

SEQ ID NO: 19 is the amino acid sequence of the Fc region of human IgG1 carrying the set of LL mutations (ie point mutations M428L and N434S).

SEQ ID NO: 20 is the amino acid sequence of the Fc region of human IgG1 carrying the AM+LL mutation set (ie point mutations G236A, S239D, A330L, I332E, M428L and N434S).

SEQ ID NO: 21 is the amino acid sequence of the Fc region of human IgG1 carrying the A12+LL mutation set (ie point mutations F243L, R292P, Y300L, M428L and N434S).

SEQ ID NO: 22 is the amino acid sequence of the Fc region of human IgG1 carrying the A14+LL mutation set (ie point mutations F243L, R292P, P396L, M428L and N434S).

SEQ ID NO: 23 is the amino acid sequence of the Fc region of human IgG1 carrying the A16+LL mutation set (ie point mutations F243L, R292P, Y300L, P396L, M428L and N434S).

SEQ ID NO: 24 is the amino acid sequence of the Fc region of human IgG1 carrying the A18+LL mutation set (ie point mutations F243L, R292P, Y300L, P396L, V305I, M428L and N434S).

SEQ ID NO: 25 is the amino acid sequence of the Fc region of human IgG1 carrying the A41+LL mutation set (ie point mutations S298A, E333A, K334A, M428L and N434S).

SEQ ID NO: 26 is the amino acid sequence of the human IgG1 Fc region carrying the A16_1 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, Y300L, P396L, M428L and N434S).

SEQ ID NO: 27 is the amino acid sequence of the Fc region of human IgG1 carrying the A16_2 and LL mutation sets (ie point mutations F243L, R292P, Y300L, K322A, P396L, M428L and N434S).

SEQ ID NO: 28 is the amino acid sequence of the Fc region of human IgG1 carrying the A16_3 and LL mutation sets (ie point mutations F243L, R292P, Y300L, I332E, P396L, M428L and N434S).

SEQ ID NO: 29 is the amino acid sequence of the Fc region of human IgG1 carrying the A16_4 and LL mutation sets (i.e. point mutations F243L, R292P, Y300L, K322A, I332E, P396L, M428L and N434S).

SEQ ID NO: 30 is the amino acid sequence of the Fc region of human IgG1 carrying the A16_5 and LL mutation sets (i.e. point mutations F243L, R292P, Y300L, E333A, K334A, P396L, M428L and N434S).

SEQ ID NO: 31 is the amino acid sequence of the Fc region of human IgG1 carrying the A16_6 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, Y300L, K322A, I332E, K334A, P396L, M428L and N434S).

SEQ ID NO: 32 is the amino acid sequence of the Fc region of human IgG1 carrying the A41_1 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, S298A, E333A, K334A, M428L and N434S).

SEQ ID NO: 33 is the amino acid sequence of the Fc region of human IgG1 carrying the A41, A16 and LL mutation sets (i.e. point mutations F243L, R292P, S298A, E333A, K334A, M428L and N434S).

SEQ ID NO: 34 is the nucleotide sequence of the D1 domain of the human CD4 receptor.

SEQ ID NO: 35 is the nucleotide sequence of the D2 domain of the human CD4 receptor.

SEQ ID NO: 36 is the nucleotide sequence of the Fc region of human IgG1.

SEQ ID NO: 37 is the nucleotide sequence of the linker polypeptide.

SEQ ID NO: 38 is the nucleotide sequence of the 5' terminal region of the human CCR5 receptor.

SEQ ID NO: 39 is the nucleotide sequence of the M19 linker peptide.

SEQ ID NO: 40 is the nucleotide sequence of the M20 linker peptide.

SEQ ID NO: 41 is the nucleotide sequence of the M21 linker peptide.

SEQ ID NO: 42 is the nucleotide sequence of the M22 linker peptide.

SEQ ID NO: 43 is the nucleotide sequence of the T-20 polypeptide.

SEQ ID NO: 44 is the nucleotide sequence of the C34 polypeptide.

SEQ ID NO: 45 is the nucleotide sequence of the EHO polypeptide.

SEQ ID NO: 46 is the nucleotide sequence of the Fc region of human IgG1 carrying a set of AM mutations (ie point mutations G236A, S239D, A330L and I332E).

SEQ ID NO: 47 is the nucleotide sequence of the Fc region of human IgG1 carrying the A12 mutation set (ie F243L, R292P and Y300L point mutations).

SEQ ID NO: 48 is the nucleotide sequence of the Fc region of human IgG1 carrying the A14 mutation set (ie F243L, R292P and P396L point mutations).

SEQ ID NO: 49 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16 mutation set (ie point mutations F243L, R292P, Y300L and P396L).

SEQ ID NO: 50 is the nucleotide sequence of the Fc region of human IgG1 carrying the A18 mutation set (ie point mutations F243L, R292P, Y300L, P396L and V305I).

SEQ ID NO: 51 is the nucleotide sequence of the Fc region of human IgG1 carrying the A41 mutation set (ie point mutations S298A, E333A and K334A).

SEQ ID NO: 52 is the nucleotide sequence of the Fc region of human IgG1 carrying a set of LL mutations (ie point mutations M428L and N434S).

SEQ ID NO: 53 is the nucleotide sequence of the Fc region of human IgG1 carrying the AM+LL mutation set (ie point mutations G236A, S239D, A330L, I332E, M428L and N434S).

SEQ ID NO: 54 is the nucleotide sequence of the human IgG1 Fc region carrying the A12+LL mutation set (ie point mutations F243L, R292P, Y300L, M428L and N434S).

SEQ ID NO: 55 is the nucleotide sequence of the Fc region of human IgG1 carrying the A14+LL mutation set (ie point mutations F243L, R292P, P396L, M428L and N434S).

SEQ ID NO: 56 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16+LL mutation set (ie point mutations F243L, R292P, Y300L, P396L, M428L and N434S).

SEQ ID NO: 57 is the nucleotide sequence of the Fc region of human IgG1 carrying the A18+LL mutation set (ie point mutations F243L, R292P, Y300L, P396L, V305I, M428L and N434S).

SEQ ID NO: 58 is the nucleotide sequence of the Fc region of human IgG1 carrying the A41+LL mutation set (ie point mutations S298A, E333A, K334A, M428L and N434S).

SEQ ID NO: 59 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_1 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, Y300L, P396L, M428L and N434S).

SEQ ID NO: 60 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_2 and LL mutation sets (ie point mutations F243L, R292P, Y300L, K322A, P396L, M428L and N434S).

SEQ ID NO: 61 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_3 and LL mutation sets (ie point mutations F243L, R292P, Y300L, I332E, P396L, M428L and N434S).

SEQ ID NO: 62 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_4 and LL mutation sets (i.e. point mutations F243L, R292P, Y300L, K322A, I332E, P396L, M428L and N434S).

SEQ ID NO: 63 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_5 and LL mutation sets (i.e. point mutations F243L, R292P, Y300L, E333A, K334A, P396L, M428L and N434S).

SEQ ID NO: 64 is the nucleotide sequence of the Fc region of human IgG1 carrying the A16_6 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, Y300L, K322A, I332E, K334A, P396L, M428L and N434S).

SEQ ID NO: 65 is the nucleotide sequence of the Fc region of human IgG1 carrying the A41_1 and LL mutation sets (i.e. point mutations S239D, F243L, R292P, S298A, E333A, K334A, M428L and N434S).

SEQ ID NO: 66 is the nucleotide sequence of the Fc region of human IgG1 carrying the A41, A16 and LL mutation sets (i.e. point mutations F243L, R292P, S298A, E333A, K334A, M428L and N434S).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-HIV Fc fusion protein derivatives with improved dual antiviral and immunomodulatory activity. The Fc fusion protein derivatives of the present invention are characterized by enhanced ability to: (1) block entry of human immunodeficiency virus (HIV) into host cells and (2) induce effector functions through natural killer (NK) cell activation. The Fc fusion protein derivatives of the present invention are also characterized by (3) high production in mammalian cells and (4) prolonged activity in vivo.

1. Definitions of basic terms and expressions

The term "AC10" as used herein refers to a primary isolate of HIV characterized by its resistance to antibodies against the CD4 binding site. AC10 refers to isolates with level 2 (moderate) sensitivity to neutralizing antibodies. See Seaman M, et al., J. Virol. 2010; 84:1439-1452.

The term "adeno-associated virus" or "AAV" as used herein refers to a member of the Parvoviridae family of viruses that contains a linear, single-stranded DNA genome of approximately 5,000 nucleotides. At least 11 recognizable AAV serotypes (AAV1-11) are known in the art.

The term "AAV vector" as used herein refers to a nucleic acid having an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR flanking a sequence encoding a polypeptide operably linked to transcriptional regulatory elements ( eg promoters, enhancers) and a polyadenylation sequence. The AAV vector may include, optionally, one or more introns located between exons of the polypeptide coding sequence. See Samulski J, et al., Annu. Rev. Virol. 2014; 1:427-451.

The term "AIDS" as used herein refers to the symptomatic phase of HIV infection and includes both acquired immunodeficiency syndrome (known as AIDS) and "ARC", or AIDS-associated complex. See Adler M, et al., Brit. Med. J. 1987; 294:1145-1147. The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and malignancies resulting from immunodeficiency.

The term "amino acid" as used herein refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Amino acids in this specification may be referred to either by their well-known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides may be referred to by their common single letter codes.

The term "antibody drug conjugate" or "ADC" as used herein refers to an antibody, an antigen-binding fragment of an antibody, an Fc fusion protein derivative, an antibody complex, or an antibody fusion protein that is conjugated to a therapeutic agent. Conjugation may be covalent or non-covalent. Preferably, the conjugation is covalent.

The term "antiretroviral therapy" or "AT" as used herein refers to the administration of one or more antiretroviral drugs (ie, antiretroviral agents for the treatment of HIV) to inhibit HIV replication. Typically, AT involves the administration of at least one antiretroviral agent (or, usually, a cocktail of antiretroviral agents), such as a nucleoside reverse transcriptase inhibitor (eg, zidovudine (AZT), lamivudine (3TC), and abacavir), a non-nucleoside reverse transcriptase inhibitor (eg. , nevirapine, and efavirenz) and a protease inhibitor (eg, indinavir, ritonavir, and lopinavir). The term highly active antiretroviral therapy (HAART) refers to treatment regimens designed to aggressively suppress HIV replication and disease progression. HAART typically includes three or more different drugs such as, for example, two nucleoside reverse transcriptase inhibitors and a protease inhibitor.

The term "binding efficiency" as used herein refers to the affinity of the molecule for the CD4 receptor and preferably the D1 and D2 domains of said receptor. In the context of the invention, "affinity" refers to the strength with which an Fc fusion protein derivative binds, for example, to the CD4 binding site on gp120. As used herein, the term "binding" or "specifically binds" refers to an interaction between binding pairs (e.g., two proteins or compounds, preferably between (1) the CD4 binding site on gp120 and (2) the CD4 receptor or the D1 and D2 domains of the CD4 receptor In some embodiments, the interaction is characterized by an affinity constant of at most 10 ^-6 mol/l, at most 10 ^-7 mol/l, or at most 10 ^-8 mol/l In general, the expression "binding" or "specifically binds" refers to specific binding one compound to another, where the level of binding, as measured by any standard assay, is statistically significantly higher than the background control in the assay.

The term "C34" as used herein refers to a gp41-derived polypeptide of SEQ ID NO: 11 comprising part of the HR2 region of gp41. See Eggink D, et al., J. Virol. 2008; 82(13):6678-6688.

The term "CCR5" as used herein refers to the human C-C chemokine receptor type 5, also known as CD195, a receptor having 7 transmembrane domains associated with G proteins. CCR5 binds to various chemokines and acts as a co-receptor for HIV Env during HIV entry into target cells. Various regions of CCR5 are involved in the function of the HIV co-receptor; however, the first interaction occurs between the N-terminal extracellular region of CCR5 and the HIV Env co-receptor binding site located on the gp120 subunit. See Lagenaur L, et al., Retrovirology 2010; 7:11. The complete sequence of the human CCR5 protein has UniProt accession number P51681 (August 18, 2015).

The term "CD4" or "CD4 receptor" as used herein refers to cluster of differentiation 4, a glycoprotein expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. CD4 helps the T cell receptor (TCR) attach to antigen presenting cells. Using the part located inside the T cell, CD4 amplifies the signal generated by the TCR by recruiting an enzyme known as tyrosine kinase lck, which is essential for the activation of many molecules involved in the signaling cascade of the activated T cell. The complete human CD4 protein sequence is UniProt accession number P01730 (June 18, 2012).

The term "codon-optimized" as used herein refers to a change in the codons of nucleic acids, reflecting the typical use of codons by the host organism to improve the expression of the reference polypeptide without changing its amino acid sequence. There are several methods and programs known in the art for codon optimization. See Narum D, et al., Infect. Immun. 2001; 69(12):7250-7253), Outchkourov N, et al., Protein Expr. Purif. 2002; 24(1):18-24, Feng L, et al., Biochemistry 2000; 39(50):15399-15409 and Humphreys D, et al., Protein Expr. Purif. 2000; 20(2):252-264.

The term "complement" as used herein refers to the part of the innate immune system that enhances (complements) the ability of antibodies and phagocytic cells to remove microbes and damaged cells from the body, promotes inflammation, and attacks the plasma membrane of pathogens. See Janeway C, et al., "The complement system and innate immunity", Immunobiology: The Immune System in Health and Disease (Garland Science, New York, US, 2001).

The term "comprising" or "comprises" as used herein also encompasses "consisting of", in accordance with common patent practice.

The term "EHO" as used herein refers to the C34EHO peptide, the sequence of the HR2 fragment of the transmembrane subunit of the HIV-2 EHO isolate of SEQ ID NO: 12.

The term "Env" or "gp160" as used herein refers to a glycoprotein having either the antigenic specificity or biological function of the HIV outer envelope (Env) protein and comprising two subunits, the glycoproteins gp120 and gp41. Example sequences of wild-type (wt) gp160 polypeptides are available. See GenBank registration numbers AAB05604 and AAD12142.

The term "crystallizable immunoglobulin fragment" or "Fc region" as used herein refers to the tail region of an antibody that interacts with cell surface receptors, referred to as Fc receptors, and certain proteins of the complement system.

The term "functionally equivalent variant" as used herein refers to: (1) a polypeptide resulting from a modification, deletion, or insertion of one or more amino acids and substantially retaining the activity of its reference polypeptide, and (2) a polynucleotide resulting from the result of a modification, deletion, or insertion of one or more bases, and substantially retaining the activity of the polypeptide expressed by the reference nucleic acid. Functionally equivalent variants considered in the context of this invention encompass polypeptides showing at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% similarity or identity with sequences of SEQ ID NOs: 1-33, or polynucleotides showing at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% similarity or identity with the sequences SEQ ID NO: 34-66. The degree of identity or similarity between two polypeptides or two polynucleotides is determined using computer algorithms and methods well known in the art. Identity and similarity between two amino acid sequences is preferably determined using the BLASTP algorithm. See Altschul S, et al., "BLAST Manual" (NCBI NLM NIH, Bethesda, MD, USA, 2001).

The term "fusion protein" as used herein refers to genetically engineered proteins consisting of two or more functional domains derived from different proteins. The fusion protein can be produced by standard methods (eg, by gene expression of the nucleotide sequence encoding said fusion protein in a suitable cell).

T, et al., FEBS Lett. 2000; 477:145-149; Chan D, et al., Cell 1997; 89:263-273. В данном описании «gp41» также относится к трансмембранному гликопротеину оболочки других типов ВИЧ (например, ВИЧ-2, ВИОЧ, ВИО), помимо ВИЧ-1. Нуклеотидные и аминокислотные последовательности gp-41 многих ВИЧ находятся в публичном доступе. См. базу данных последовательностей ВИЧ http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html, апрель 2017.The term "gp41" as used herein refers to the envelope glycoprotein gp41 of the human immunodeficiency virus type 1. Gp41 is a subunit that, together with gp120, forms the HIV-1 Env glycoprotein. Env is a trimer consisting of three outer subunits (gp120) and three transmembrane subunits (gp41). The extracellular grouping of the gp41 protein contains three main functional regions: the fusion peptide (FP), the N-terminal heptad repeat (HR1), and the C-terminal heptad repeat (HR2). The HR1 and HR2 regions contain a number of leucine zipper-like motifs that tend to form helical structures. See Peisajovich S, Shai Y, Biochem. Biophys. Acta 2003; 1614:122-129;

T, et al., FEBS Lett. 2000; 477:145-149; Chan D, et al., Cell 1997; 89:263-273. As used herein, "gp41" also refers to a transmembrane envelope glycoprotein of other types of HIV (eg, HIV-2, HIV, SIV) in addition to HIV-1. Nucleotide and amino acid sequences of gp-41 of many HIV are in the public domain. See HIV sequence database http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html, April 2017.

The term "gp41 inhibitors" as used herein includes a range of polypeptides of various lengths that include the HR2 region of gp41. These inhibitors include, without limitation, T-20, C34, T-1249, T-2635, and EHO polypeptides derived from gp41.

The term “gp41-derived polypeptide” as used herein refers to a polypeptide derived from gp41 heptad repeat 1 (HR1) or heptad repeat 2 (HR2) motifs. The HR1 and HR2 sequences of gp41 are well known in the art. See Lupas A, Trends Biochem. sci. 1996; 21:375-382 and Chambers P, et al., J. Gen. Virol. 1990; 71:3075-3080. Preferably, the gp41-derived polypeptide is derived from HR2. Polypeptides derived from gp41 may contain additional exogenous amino acids located at their N- or C-terminus. Preferably, there are less than 10 exogenous amino acids, more preferably less than 5, and most preferably, less than 3.

The term "gp120" as used herein refers to a glycoprotein having either the antigenic specificity or the biological function of the HIV outer envelope (env) protein. "Protein gp120" is a molecule derived from the gp120 region of the Env polypeptide. The amino acid sequence of gp120 is approximately 511 amino acids. Gp120 is a highly N-glycosylated protein with an average molecular weight of 120 kDa. Gp120 contains five relatively conserved domains (C1-C5) interspersed with five variable domains (V1-V5). Variable domains contain significant amino acid substitutions, insertions and deletions. "gp120 polypeptide" includes both single subunits and multimers. The gp41 part is anchored in the membrane bilayer of the virion (and is transmembrane), while the gp120 segment protrudes into the surrounding space. The receptor-binding domain of gp120 is located in the N-terminal half of the protein. Behind it is a proline-rich region (PRR) that acts as a hinge or trigger, telling the fusion apparatus to bind to the receptor. The C-terminus of gp120 is highly conserved and interacts with gp41. See GenBank registration numbers AAB05604 and AAD12142.

The term "HIV" as used herein includes HIV-1 and HIV-2, simian and human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). "HIV-1" means human immunodeficiency virus type 1. HIV-1 encompasses, but is not limited to, extracellular viral particles and forms of HIV-1 associated with HIV-1 infected cells. The HIV-1 virus may be any of the known major subtypes (classes A, B, C, D, E, F, G, and H) or a distinct subtype (group O), including laboratory strains and primary isolates. "HIV-2" means human immunodeficiency virus type 2. HIV-2 encompasses, but is not limited to, extracellular viral particles and forms of HIV-2 associated with HIV-2 infected cells. The term "SIV" refers to simian immunodeficiency virus, which is an HIV-like virus that infects monkeys, chimpanzees, and other non-human primates. SIV encompasses, but is not limited to, extracellular viral particles and forms of SIV associated with SIV-infected cells.

The term "exposure to HIV" as used herein refers to contact of an uninfected subject with a subject having HIV infection or AIDS, or contact with body fluids of an HIV-infected subject, in which said fluids from an infected subject come into contact with a mucous membrane, cut or a scratch in a tissue (eg, needle stick, unprotected intercourse) or other surface of an uninfected subject in such a way that the virus can be transmitted from the infected subject or the body fluids of the infected subject to the uninfected subject.

The term "HIV infection" as used herein refers to evidence of the presence of the HIV virus in an individual, including asymptomatic seropositivity, AIDS-associated complex (ARC), and acquired immunodeficiency syndrome (AIDS).

The term "IC ₅₀ ", as used herein, refers to the amount of a particular active agent required to inhibit 50% of a given biological process or component of a biological process (ie, enzyme, cell, cell receptor, or microorganism).

The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned (with the introduction "gaps" if necessary) for maximum matching, not counting any conservative amino acid substitutions as part of the identical sequence. Percent identity can be determined using a sequence comparison program or algorithm, or by visual inspection. Various algorithms and programs are known in the art that can be used to align amino acid or nucleotide sequences. Examples of algorithms suitable for determining sequence similarity include, without limitation, the BLAST, Gapped BLAST, and BLAST 2.0, WU-BLAST-2, ALIGN, and ALIGN-2 algorithms. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402, Altschul S, et al., J. Mol. Biol. 1990; 215:403-410, Altschul S, et al., Meth. Enzymol. 1996; 266:460-480, Karlin S, et al., Proc. Natl. Acad. sci. USA 1990; 87:2264-2268, Karlin S, et al., Proc. Natl. Acad. sci. USA 1993; 90:5873-5877, Genentech Corp, South San Francisco, CA, US, https://blast.ncbi.nlm.nih.gov/Blast.cgi, April 2017. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, according to the Smith-Waterman local homology search algorithm, the Needleman-Wunsch homology region alignment algorithm, the Pearson-Lipman similarity search method, by computerized execution of these algorithms, or by manual alignment and visual inspection. See Smith T, et al., Adv. Appl. Math. 1981; 2:482-489, Needleman S, et al., J. Mol. Biol. 1970; 48:443-453, Pearson W, et al., Lipman D, Proc. Natl. Acad. sci. USA 1988; 85:2444-2448, GAP, BESTFIT, FASTA, and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA; Ausubel F, et al., Eds., "Short Protocols in Molecular Biology", 5th Ed. (John Wiley and Sons, Inc., New York, NY, USA, 2002).

The term "kit" as used herein refers to a product containing the various reagents needed to carry out the uses and methods of the invention, which are packaged in such a way that they can be transported and stored. Suitable materials for packaging the kit components include glass, plastic (eg polyethylene, polypropylene, polycarbonate), bottles, vials, paper, or envelopes.

The term "neutralizing antibody" as used herein is any antibody, antigen-binding fragment, or Fc fusion protein derivative that binds to an extracellular molecule (e.g., a protein or protein domain on the surface of a pathogenic virus) and interferes with the ability of the extracellular molecule to infect a cell or modulate its activity. In general, Fc fusion protein derivatives of the invention can bind to the surface of an extracellular molecule and are able to inhibit its binding to a cell by at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 60% , 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10% relative to the attachment of the extracellular molecule to the cell in the absence of said Fc fusion protein derivatives or in the presence of a negative control. Methods are described in the art for confirming whether a derivative of an Fc fusion protein is neutralizing. See Li M, et al., J. Virol. 2005; 79:10108-10125, Wei X, et al., Nature 2003; 422:307-312, and Montefiori D, Curr. Protocol. Immunol. 2005; Jan, Chapter 12:Unit 12.11. In the context of the invention, the pathogen is preferably HIV, and more specifically, the gp120 protein of the HIV viral envelope. In particular, the term "HIV neutralizing antibody" refers to an Fc fusion protein derivative with affinity for the CD4 binding site of gp120, such as IgGb12. The term "neutralizing antibodies" encompasses the bnAbs subclass. As used herein, a "broad spectrum neutralizing antibody" or "bnAb" is understood to mean an antibody obtained by any method that, when administered at an effective dose, can be used as a therapeutic agent for the prevention or treatment of HIV infection or AIDS against more than 7 strains of HIV. preferably more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more strains of HIV.

The term "NK cell" as used herein refers to a "natural killer cell", a type of cytotoxic lymphocyte critical to the innate immune system. NK cells provide rapid responses to virus-infected cells and respond to tumor formation for approximately 3 days after infection. Typically, immune cells recognize HLA (human leukocyte antigen) presented on the surface of infected cells, triggering the release of cytokines, causing lysis or apoptosis. NK cells are unique in that they have the ability to recognize stressed cells in the absence of antibodies and HLA, resulting in a much faster immune response. NK cells are defined as large granular lymphocytes and constitute the third cell type differentiated from the common lymphoid progenitor from which B and T lymphocytes are derived. It is known that NK cells differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus, from where they enter the circulation. In humans, NK cells typically express the surface markers CD16 (FcγRIII) and CD56.

The terms "NL4-3" and "BaL" as used herein refer to two different HIV isolates commonly used in the laboratory. The NL4-3 isolate was cloned from the NY5 and LAV proviruses. See Adachi A, et al., J. Virol. 1986; 59:284-291. The BaL isolate was obtained from a primary culture of adherent cells grown from a lung tissue explant. See Gartner S, et al., Science 1986; 233:215-219.

The terms "nucleic acid", "polynucleotide" and "nucleotide sequence" are used interchangeably herein to refer to any polymeric form of nucleotides of any length and consisting of ribonucleotides or deoxyribonucleotides. The terms cover both single and double stranded polynucleotides as well as modified polynucleotides (eg, methylated, protected). Typically, a nucleic acid is a "coding sequence", which herein refers to a DNA sequence that is transcribed and translated into a polypeptide in a host cell, when placed under the control of suitable regulatory sequences. The boundaries of the coding sequence are defined by an initiation codon at the 5' (amino) end and a translation termination codon at the 3' (carboxy) end. The coding sequence may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA cDNA, eukaryotic (eg, mammalian) genomic DNA sequences, and even synthetic DNA sequences. The translation termination sequence is usually located in the 3' direction relative to the coding sequence.

The term "operably linked" as used herein means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell). upon introduction of the vector into the host cell). See Auer H, Nature Biotechnol. 2006; 24:41-43.

The term "panel of HIV isolates" as used herein refers to a collection of reference HIV isolates intended to be used as Env pseudotyped viruses to facilitate standardized assessment of 2/3 levels of neutralizing antibody responses. See Mascola R, et al., J. Virol. 2005; 79(16):10103. Pseudoviruses exhibit a neutralizing phenotype that is characteristic of most primary isolates of HIV-1. The gp160 genes have been cloned from sexually acquired acute/early infections and include a wide range of genetic, antigenic and geographic diversity within the B subtype. These clones use CCR5 as a co-receptor. See Li, et al., J. Virol. 2005; 79(16):10108-10125.

The expression "parenteral administration" and "administered parenterally", as used herein, means methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal , intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

The term "pharmaceutically acceptable carrier" as used herein encompasses any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents physiologically compatible with Fc fusion protein derivatives, nucleic acids, vectors and host cells. according to the invention.

The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as polymers of naturally occurring amino acids and polymers of artificially occurring amino acids.

The terms "prevent", "preventive" and "prevention" as used herein refer to inhibiting the onset or reducing the incidence of disease in a subject. The warning may be complete (eg, complete absence of pathological cells in the subject). Prevention can also be partial, such as reducing the appearance of abnormal cells in a subject, for example. The warning also applies to reduced susceptibility to the clinical condition. In the context of this invention, the terms "prevent", "prevention" and "prevention" specifically refer to preventing or reducing the likelihood of HIV infection in a subject exposed to HIV.

The term "sample" as used herein refers to any biological fluid and, in particular, to blood, serum, plasma, lymph, saliva, serum of peripheral blood or tissue cells, semen, sputum, cerebrospinal fluid, tears, mucus, sweat, milk or brain extracts obtained from the subject. Body tissue may include thymus, lymph node, spleen, bone marrow, or tonsil tissue. The term "sample" also refers to non-biological samples (eg derived from water, beverages).

The term "subject" as used herein refers to an individual, plant, or animal, such as a human, non-human primate (eg, chimpanzees and other great and non-human apes); farm animals such as birds, fish, cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs. The term does not denote a specific age or gender. The term "subject" includes the embryo and fetus. In a preferred embodiment, the subject is a human.

The term "T-1249" as used herein refers to a gp41-derived polypeptide comprising part of the HR2 region of gp41. See Eggink, 2008, supra.

The term "T-20" as used herein refers to a gp41-derived polypeptide of SEQ ID NO: 10, comprising part of the HR2 region of gp41, also known as enfuvirtide. See CAS [159519-65-0] and US 5464933. This polypeptide has antiviral activity in the nanomolar range and has been used in therapy against HIV infection. See Zhang D, et al., Expert Opin Ther Pat. 2015; 25:159-173 and Eggink, 2008, supra.

The term "T-2635" as used herein refers to a gp41-derived polypeptide encompassing part of the HR2 region of gp41. See Eggink, 2008, supra.

The term "therapeutic agent" as used herein refers to an atom, molecule or compound useful in the treatment or prevention of a disease. Examples of therapeutic agents include, without limitation, antibodies, antibody fragments, antiretroviral agents for the treatment of HIV, drugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (e.g., DNases and RNases), hormones, immunomodulators, chelators, boron compounds, photoactive agents, or dyes. , radionuclides, oligonucleotides, interfering RNAs, siRNAs, RNAi, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, protein-binding enzymes, peptides, or combinations thereof.

The term "therapeutically effective amount" as used herein refers to the dose or amount of Fc fusion protein derivatives, nucleic acids, vectors, pharmaceutical compositions of the invention, or mixtures thereof, that elicit a therapeutic response or desired effect in a subject.

The terms "therapy" or "therapeutic" as used herein refer to the use of Fc fusion protein derivatives, nucleic acids, vectors, pharmaceutical compositions of the invention, or mixtures thereof, for the treatment or prevention of disease, including, but not limited to, HIV and AIDS. .

The term "treat" or "treatment" as used herein refers to administering a derivative of an Fc fusion protein, nucleic acid, vector, host cell, or pharmaceutical composition of the invention to control the progression of a disease after its clinical signs have been manifested. Control of disease progression refers to beneficial or desirable clinical outcomes, which include, but are not limited to, symptom reduction, disease duration reduction, disease stabilization (especially to avoid further deterioration), disease progression delay, disease improvement, and remission (both partial and complete) . Controlling the progression of the disease also includes an increase in survival compared to the survival expected in the absence of treatment. In the context of this invention, the terms "treat" and "treatment" specifically refer to arresting or slowing the infection and destruction of healthy CD4+ T cells in an HIV-infected subject. This also applies to reversing and slowing the onset of symptoms of acquired immunodeficiency disease, such as extremely low CD4+ T cell counts and recurrent infections caused by opportunistic pathogens. Beneficial or desirable clinical results include, but are not limited to, an increase in the absolute number of naïve CD4+ T cells (range 10-3520), an increase in the percentage of CD4+ T cells relative to total circulating immune cells (range 1-50%), or an increase in the number of CD4+ T cells. -cells as a percentage of the normal number of CD4+ T cells in an uninfected subject (range 1-161%). "Treatment" may also mean prolonging the survival of an infected subject, as compared to expected survival, if the subject is not receiving any targeted HIV treatment.

The term "vector" as used herein refers to a nucleic acid molecule, whether linear or circular, that contains a nucleic acid of the invention operably linked to additional segments that allow it to replicate autonomously in a host cell or according to the expression cassette of the nucleic acid molecule. acids.

2. Fc fusion protein derivatives

The present invention relates to derivatives of an Fc fusion protein containing in the N- to C-terminal direction:

(a) the D1 and D2 extracellular domains of the human CD4 receptor,

(b) a human IgG1 Fc region containing at least one of the G236A, S239D, A330L, I332E, F243L, R292P, S298A, Y300L, V305I, K322A, E333A, K334A, P396L, M428L, or N434S point mutations,

(c) a moiety selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9, and (3) combinations thereof, and

(d) a gp41 derived polypeptide.

The Fc fusion protein derivatives of the invention are characterized by enhanced antiviral activity and ADCC activity. The expression and duration of action (ie, t _1/2 ) of the Fc fusion protein derivatives of the invention is also higher than other comparable antibodies.

(a) D1 and D2 extracellular domains of the human CD4 receptor

In one embodiment, the D1 domain of the Fc fusion protein derivatives of the invention comprises amino acids 26-125 of the human CD4 receptor (ie UniProtKB accession number P01730) or a functionally equivalent variant thereof. In another embodiment, the D2 domain of the Fc fusion protein derivatives of the invention comprises amino acids 126-203 of the human CD4 receptor or a functionally equivalent variant thereof. Preferably, the D1 and D2 domains contain the sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or their functionally equivalent variant.

(b) Fc region of human IgG1

In a further embodiment, the human IgG1 Fc region comprises sets and subsets of mutation combinations that contain at least one of the F243L, R292P, S298A, Y300L, V305I, K322A, E333A, K334A, P396L, M428L, or N434S point mutations, or a functional equivalent thereof. option. In a further embodiment, the human IgG1 Fc region contains (1) at least one of the G236A, S239D, A330L, or I332E point mutations, or a functionally equivalent variant thereof.

(1) M428L and N434S point mutations (LL mutation set)

In a further embodiment of this embodiment, the human IgG1 Fc region contains at least one of the M428L or N434S point mutations, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains both the M428L or N434S point mutations, or a functionally equivalent variant thereof. A human IgG1 Fc region comprising SEQ ID NO: 19 or a functionally equivalent variant thereof is preferred.

In a further feature of this variant, the human IgG1 Fc region contains (1) at least one of the G236A, S239D, A330L, or I332E point mutations and (2) at least one of the M428L or N434S point mutations, or a functionally equivalent variant thereof. In one feature of this variant, the human IgG1 Fc region contains (1) at least one of the G236A, S239D, A330L, or I332E point mutations and the M428L point mutation, or a functionally equivalent variant thereof. In another feature of this variant, the human IgG1 Fc region contains (1) at least one of the G236A, S239D, A330L, or I332E point mutations and the N434S point mutation, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains point mutations G236A, S239D, A330L, or I332E, or a functionally equivalent variant thereof. A human IgG1 Fc region comprising SEQ ID NO: 19 or a functionally equivalent variant thereof is preferred.

In another feature of this variant, the human IgG1 Fc region contains at least one or more of the F243L or R292P point mutations, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains both F243L and R292P point mutations, or a functionally equivalent variant thereof. In a further feature, a human IgG1 Fc region contains at least one of the Y300L, P396L, S298A, E333A, or K334A point mutations, or a functionally equivalent variant thereof. A human IgG1 Fc region comprising SEQ ID NOs: 21-25 or a functionally equivalent variant thereof is preferred.

In a further characterization of this variant, the human IgG1 Fc region contains the K322A point mutation. A human IgG1 Fc region comprising SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or a functionally equivalent variant thereof, is preferred.

(2) F243L, R292P, Y300L, P396L, S298A, E333A and K334A (mutation sets A12, A14, A16 and A41)

In another embodiment of this embodiment, the human IgG1 Fc region contains at least one or more of the F243L or R292P point mutations, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains both F243L and R292P point mutations, or a functionally equivalent variant thereof. In a further feature of this variant, the human IgG1 Fc region contains at least one of the Y300L, P396L, S298A, E333A, or K334A point mutations, or a functionally equivalent variant thereof. A human IgG1 Fc region comprising SEQ ID NOs: 14-18 or a functionally equivalent variant thereof is preferred.

In a further characterization of this variant, the human IgG1 Fc region contains (1) at least one of G236A, S239D, A330L, or I332E point mutations, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains point mutations G236A, S239D, A330L, and I332E, or a functionally equivalent variant thereof. In a further characterization of this variant, the human IgG1 Fc region contains the K322A point mutation.

(3) K322A point mutation

In another embodiment of this embodiment, the human IgG1 Fc region contains the K322A point mutation, or a functionally equivalent variant thereof.

In another feature of this variant, the human IgG1 Fc region contains (1) at least one of G236A, S239D, A330L, or I332E point mutations and a K322 point mutation, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains point mutations G236A, S239D, A330L, or I332E, or a functionally equivalent variant thereof.

In a further embodiment of this embodiment, the human IgG1 Fc region contains at least one or more of the F243L or R292P point mutations, or a functionally equivalent variant thereof. Preferably, the human IgG1 Fc region contains both F243L and R292P point mutations, or a functionally equivalent variant thereof. In a further characterization of this variant, the human IgG1 Fc region contains at least one of the Y300L, P396L, S298A, E333A, or K334A point mutations, or a functionally equivalent variant thereof.

(c) Grouping

In another embodiment, the moiety of derivatives of the Fc fusion protein of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 5-9, (3) a combination of (1) and (2) and a functionally equivalent option (1), (2) and (3). In a further embodiment of this embodiment, the moiety of the Fc fusion protein derivatives of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 5, (3) a combination of (1) and (2) and a functionally equivalent option (1), (2) and (3). In a further embodiment of this embodiment, the moiety of the Fc fusion protein derivatives of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 6, (3) a combination of (1) and (2) and a functionally equivalent option (1), (2) and (3). In another embodiment of this embodiment, the moiety of derivatives of the Fc fusion protein of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 7, (3) a combination of (1) and (2) and a functionally equivalent variant of (1), (2) and (3). In a further embodiment of this embodiment, the moiety of the Fc fusion protein derivatives of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 8, (3) a combination of (1) and (2) and a functionally equivalent option (1), (2) and (3). In another embodiment of this embodiment, the moiety of derivatives of the Fc fusion protein of the invention is selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n (SEQ ID NO: 4) where 1≤n≤10, (2) SEQ ID NO: 9, (3) a combination of (1) and (2) and a functionally equivalent option (1), (2) and (3). In one embodiment of this embodiment, the moiety contains only a linker or human CCR5 receptor sequence, or a functionally equivalent variant thereof. In another embodiment of this embodiment, the moiety contains a combination of a linker and a human CCR5 receptor sequence or a functionally equivalent variant thereof. Preferably, the linker is attached to the C-terminus of the human CCR5 receptor sequence when the combination is used. Preferably, the moiety contains only a linker or a combination of a linker and a human CCR5 receptor sequence or a functionally equivalent variant thereof. Preferably, the human CCR5 receptor sequence comprises SEQ ID NO: 5 or a functionally equivalent variant thereof. In one embodiment of this embodiment, SEQ ID NOs: 5-9, or functionally equivalent variants thereof, are further modified to include alanine residues. Preferably, the linker modified with alanine residues is SEQ ID NO: 8 or its functional equivalent.

(d) gp41 polypeptide

In a further embodiment, the gp41 derived polypeptide comprises a T-20, T-4912, C34, T-2635, and EHO polypeptide, combinations thereof, or a functionally equivalent variant. Preferably, the gp41-derived polypeptide comprises T-20, C34, and EHO polypeptides, or a functionally equivalent variant thereof. More preferably, the T-20, C34 and EHO polypeptides contain the sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

In a further embodiment, the Fc fusion protein derivatives of the invention comprise the sequences of SEQ ID NOs: 13-33 or a functionally equivalent variant thereof. Preferably, the Fc fusion protein derivatives of the invention comprise the sequences of SEQ ID NOs: 19-33 or a functionally equivalent variant thereof. More preferably, the Fc fusion protein derivatives of the invention comprise the sequence SEQ ID NO: 20, or a functionally equivalent variant thereof.

Preferably, the Fc fusion protein derivatives of the invention comprise:

(1) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, and M428L, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) a T-20 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(2) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E and M428L, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) C34 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(3) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, and N434S, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) a T-20 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(4) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E and N434S, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) C34 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(5) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, M428L and N434S, (c) a grouping selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) a T-20 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(6) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, M428L and N434S, (c) a grouping selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NO: 5 and (3) combinations thereof, and (d) a C34 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(7) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations F243L, R292P, Y300L, K322A, I332E, P396L, M428L and N434S, (c) a grouping selected from a group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9 and (3) combinations thereof, and (d) a C34 polypeptide. The human IgG1 Fc region may further comprise at least one of the S298A, E333A, or K334A point mutations. In another variation of this embodiment, the moiety further includes alanine residues as in SEQ ID NO: 8.

(8) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations F243L, R292P, Y300L, K322A, I332E, P396L, M428L and N434S, (c) a grouping selected from a group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9 and (3) combinations thereof, and (d) an EHO polypeptide. The human IgG1 Fc region may further comprise at least one of the S298A, E333A, or K334A point mutations. In another variation of this embodiment, the moiety further includes alanine residues as in SEQ ID NO: 8.

(9) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E and N434S, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9 and (3) combinations thereof, and (d) a T-20 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(10) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, and N434S, (c) a grouping selected from the group consisting of (1 ) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9 and (3) combinations thereof, and (d) C34 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(11) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, M428L and N434S, (c) a grouping selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9, and (3) combinations thereof, and (d) a T-20 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

(12) (a) human CD4 receptor D1 and D2 extracellular domains, (b) human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, M428L and N434S, (c) a grouping selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n where 1≤n≤10, (2) SEQ ID NOs: 5-9, and (3) combinations thereof, and (d) a C34 polypeptide. The human IgG1 Fc region may further comprise at least one of F243L, R292P, Y300L, P396L, S298A, E333A, or K334A point mutations.

The Fc fusion protein derivatives of the invention are used to prevent (i.e., neutralize) the attachment of molecules (e.g., HIV) to the human CD4 receptor in cells expressing said cassette on their surface (e.g., T helper cells, monocytes, macrophages, dendritic cells). Preferably, the Fc fusion protein derivatives of the invention are used to prevent the attachment of gp160 proteins located in the HIV viral envelope to human CD4 receptors found on T helper cells. The neutralizing capacity of the Fc fusion protein derivatives of the invention may have an IC ₅₀ of 10 ng/ml or less, and preferably an IC ₅₀ of less than 5 ng/ml, less than 2.5 ng/ml, less than 1.25 ng/ml, less than 0.625 ng/ml ml, less than 0.312 µg/ml, less than 0.156 ng/ml, less than 0.07 ng/ml, or less than 0.035 ng/ml.

In a further embodiment, the Fc fusion protein derivatives of the invention may be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Methods for attaching polypeptides to polymers are known in the art. See US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Preferably, the polymers are polyoxyethylene polyols and polyethylene glycol (PEG). PEG is a water soluble polymer which has the general formula: R(O-CH ₂ -CH ₂ ) _n OR where R can be hydrogen or a protecting group such as an alkyl or alkanol group. Preferably the protecting group has 1 to 8 carbon atoms, more preferably it is methyl. Preferably, n is an integer from 1 to 1000, and more preferably from 2 to 500. PEG has a preferred average molecular weight of 1000 to 40000, more preferably 2000 to 20000 and most preferably 3000 to 12000.

In a further embodiment, Fc fusion protein derivatives of the invention are coupled to a therapeutic agent to form an antibody drug conjugate (ADC). For example, therapeutic agents used in the treatment of opportunistic diseases and conditions caused by or facilitated by AIDS, such as Kaposi's sarcoma, for example, can be treated with ADCs derived from an Fc fusion protein of the invention and interferon alfa, a liposomal anthracycline. (eg doxil) or paclitaxel. Additional ADCs effective for the treatment of other opportunistic diseases such as viral and bacterial infections (eg, herpes zoster, pneumonia, tuberculosis), skin diseases, and other AIDS-associated malignancies (eg, lymphoma) can also be constructed by combining an Fc fusion protein derivative of the invention and a corresponding therapeutic agent.

3. Nucleic acids, vectors and host cells

In another aspect, the present invention provides nucleic acids encoding an Fc fusion protein derivative of the invention and expression cassettes and vectors containing said nucleic acids.

Preferably, the nucleic acids are polynucleotides, including without limitation deoxyribonucleotides (DNA) and ribonucleotides (RNA), linked by internucleotide phosphodiester bonds. In a preferred embodiment, the nucleic acids of the invention comprise polynucleotides encoding the extracellular domains D1 and D2 of the human CD4 receptor (SEQ ID NO: 34, SEQ ID NO: 35), a human IgG1 Fc region containing point mutations G236A, S239D, A330L, I332E, M428L and N434S (SEQ ID NO: 53), linker polypeptide (SEQ ID NO: 37), human CCR5 receptor (SEQ ID NO: 38) and T-20 polypeptide (SEQ ID NO: 43), C34 polypeptide (SEQ ID NO : 44) or EHO polypeptide (SEQ ID NO: 45) or their functionally equivalent variants. Preferably, the nucleic acids of the invention encode Fc fusion protein derivatives comprising the sequences of SEQ ID NOs: 13-25 or a functionally equivalent variant thereof.

Functionally equivalent nucleic acid variants of the invention can be obtained by insertion, deletion or substitution of one or more nucleotides relative to their reference sequences. Preferably, polynucleotides encoding functionally equivalent nucleic acid variants of the invention are polynucleotides whose sequences allow them to hybridize under conditions of high stringency to their reference nucleic acids. Typical stringent hybridization conditions include incubation in 6 X SSC (1 X SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 40% formamide at 42° C. for 14 hours followed by one or more wash cycles using 0.5 X SSC, 0.1% SDS at 60°C. Alternatively, high stringency conditions include hybridization at approximately 50-55°C in 6X SSC and a final wash at 68°C in 1-3X SSC. Moderate stringency conditions include hybridization at about 50°C to about 65°C in 0.2 or 0.3 M NaCl, followed by washing at about 50°C to about 55°C in 0.2 X SSC, 0.1% SDS (sodium dodecyl sulfate). In a further embodiment, the nucleic acids of the invention are codon-optimized.

In another embodiment, a nucleic acid variant is used that has at least 80%, 85%, 90%, 95%, or 99% similarity to its reference nucleic acid, wherein said variant encodes an Fc fusion protein derivative of the invention or a functionally equivalent variant thereof.

Nucleic acids of the invention may require restriction enzyme treatment in order to be ligated into a suitable vector (eg, 1, 2, or 3 terminal nucleotides may be removed). In a further embodiment, the invention relates to said nucleic acids that have been cut at each end with a restriction enzyme.

In another embodiment, the present invention relates to an expression cassette containing the nucleic acid of the invention, a promoter sequence and a 3'-UTR (untranslated region), and optionally a selectable marker.

In another embodiment, the present invention relates to a vector containing the nucleic acid of the invention. In a further aspect of this embodiment, the nucleic acid of the invention is contained in an expression cassette contained in said vector. Suitable vectors of the present invention include, without limitation, prokaryotic vectors such as plasmids pUC18, pUC19 and Bluescript and their derivatives such as plasmids mp18, mp19, pBR322, pMB9, ColE1, pCR1 and RP4; phage and shuttle vectors such as the pSA3 and pAT28 vectors; yeast expression vectors such as 2 micron plasmid-type vectors; integrating plasmids; YEP vectors; centromeric plasmids and analogues; insect cell expression vectors such as the pAC series and the pVL series vectors; plant expression vectors such as the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and analogs; and expression vectors in higher eukaryotic cells, or based on viral vectors (e.g., adenoviruses, adeno-associated viruses, retroviruses, lentiviruses), as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion®, Life Technologies Corp., Carlsbad, CA, US), pcDNA3, pcDNA 3.1, pcDNA3.1/hyg, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl. Preferably, the vector is a pcDNA3.1 vector. More preferably, the vector is pABT-5, pABT-7 and pABT-8.

In a further embodiment, the virus-based vector is an AAV vector. AAV vectors encoding derivatives of the Fc fusion protein of the invention may be constructed in accordance with molecular biology techniques well known in the art. See Brown T, "Gene Cloning" (Chapman & Hall, London, GB, 1995); Watson R, et al., "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, N.Y., US, 1992); Alberts B, et al., "Molecular Biology of the Cell" (Garland Publishing Inc., New York, N.Y., US, 2008); Innis M, et al., Eds., "PCR Protocols. A Guide to Methods and Applications" (Academic Press Inc., San Diego, Calif., US, 1990); Erlich H, Ed., "PCR Technology. Principles and Applications for DNA Amplification" (Stockton Press, New York, N.Y., US, 1989); Sambrook J, et al., "Molecular Cloning. A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., US, 1989); Bishop T, et al., "Nucleic Acid and Protein Sequence. A Practical Approach" (IRL Press, Oxford, GB, 1987); Reznikoff W, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, Mass., US, 1987); Davis L, et al., "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, N.Y., US, 1986), Schleef M, Ed., "Plasmid for Therapy and Vaccination" (Wiley-VCH Verlag GmbH , Weinheim, Del., 2001).

For example, HEK-293 cells (expressing E1 genes), a helper plasmid carrying adenovirus functions, a helper plasmid providing rep genes from serotype 2 and cap genes from the desired AAV serotype (e.g., AAV8), and finally a backbone plasmid with ITR (Inverted Terminal Repeat) and construct of interest. To create an AAV vector expressing an Fc fusion protein derivative of the invention, the cDNA of the Fc fusion protein derivative can be cloned into an AAV backbone plasmid under the control of a universal (eg, CAG) or cell-specific promoter.

AAV vectors (viral vector particles) can be generated by transfecting HEK293 cells without helper virus using three modified plasmids. See Matsushita T, et al., Gene Ther. 1998; 5:938-945 and Wright J, et al., Mol. Ther. 2005; 12:171-178. Cells can be cultured to 70% confluency in spinner flasks (Corning Inc., Coining, NY, USA) in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% BFS (Fetal Bovine Serum) and then co-transfected with: 1) a plasmid carrying an expression cassette flanked by viral ITRs (described above); 2) a helper plasmid carrying AAV rep2 and the corresponding cap genes (cap1 and cap9), and 3) a plasmid providing adenovirus helper functions. The vectors can then be purified with two successive cesium chloride gradients using a standard protocol or an optimized protocol as previously described. See Ayuso E, et al., Gene Ther. 2010; 17:503-510. The vectors can be further dialyzed against PBS (phosphate buffered saline), filtered, quantified by qPCR (quantitative polymerase chain reaction) and stored at -80° C. until use.

In another embodiment, the present invention relates to a host cell containing the nucleic acid, expression cassette or vector of the invention. The host cell for use in the present invention may be any type of cell, including both eukaryotic cells and prokaryotic cells. Preferably the cells include prokaryotic cells, yeast cells or mammalian cells. More preferably, the host cells are HEK-293 and CHO cells.

4. Pharmaceutical compositions

In a further aspect, this invention relates to pharmaceutical compositions comprising at least one of: Fc fusion protein derivatives, nucleic acids, vectors or host cells of the invention (hereinafter individually or collectively referred to as "active(s) agent(s) according to the invention") or mixtures thereof, combined with a pharmaceutically acceptable carrier. These pharmaceutical compositions are used to treat HIV or AIDS in a subject or to prevent HIV infection in an uninfected subject. In one embodiment, the composition comprises a mixture of multiple (eg, two or more) Fc fusion protein derivatives, nucleic acids, vectors, or host cells of the invention. In one embodiment of the invention, the composition comprises an Fc fusion protein derivative comprising at least one of SEQ ID NOs: 13-25 or nucleic acids, vectors, or host cells expressing said Fc fusion protein derivatives, or a mixture thereof. Preferably, the composition comprises an Fc fusion protein derivative comprising at least one of SEQ ID NOs: 19-25 or nucleic acids, vectors, or host cells expressing said Fc fusion protein derivatives, or a mixture thereof. More preferably, the composition comprises an Fc fusion protein derivative comprising at least one of SEQ ID NO: 20 or nucleic acids, vectors, or host cells expressing said Fc fusion protein derivative, or a mixture thereof. The preparation of pharmaceutical compositions containing derivatives of the Fc fusion protein of the invention is known in the art. See McNally E, et al., Eds., "Protein Formulation and Delivery" (Marcel Dekker, Inc., New York, NY, USA, 2000), Hovgaard L, et al., Eds., "Pharmaceutical Formulation Development of Peptides and Proteins, 2nd ^Ed . (CRC Press, Boca Raton, FL, USA, 2012) and Akers M, et al., Pharm Biotechnol. 2002; 14:47-127.

Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the mode of administration, the active agent of the invention may be coated with a material to protect the agent from exposure to conditions that could lead to inactivation of the agent.

S, et al., J. Virol. 1998, 72:1497-1503; Mulligan M, Webber J, AIDS 1999; 13 (Suppl A):S105-S112; O'Hagan D, et al., J. Virol. 2001; 75:9037-9043 and Rainczuk A, et al., Infect. Immun. 2004; 72:5565-5573. Конкретный остов вектора, в который встроены нуклеиновые кислоты по изобретению, не важен, если указанная нуклеиновая кислота экспрессируется у субъекта надлежащим образом. Примеры подходящих векторов включают вирусы и плазмиды, но не ограничиваются ими. Предпочтительно, когда используется вирусный вектор, используют вектор AAV. Предпочтительно, когда используется плазмидный вектор, используют pcDNA3.1 и pVAX1 (Invitrogen, Carlsbad, СА, USA); последовательности ДНК доступны на сайте Invitrogen http://www.thermofisher.com/uk/en/home/brands/invitrogen.html, октябрь 2015); pNGVL (National Gene Vector Laboratory, University of Michigan, MI, USA); и p414cyc (регистрационный номер АТСС 87380) и p414GALS (регистрационный номер АТСС 87344). Более предпочтительно, в качестве плазмидного вектора используют плазмиду pcDNA3.1. Наиболее предпочтительно, плазмидный вектор представляет собой РМ5А16Т20 и pM7A16LLC34.In another embodiment, the present invention provides pharmaceutical compositions specifically suited for gene therapy ("passive immunization"). Said pharmaceutical compositions comprise at least one of the nucleic acids or vectors of the invention, or a mixture thereof, and are prepared according to methods known in the art. Cm.

S, et al., J. Virol. 1998, 72:1497-1503; Mulligan M, Webber J, AIDS 1999; 13(Suppl A):S105-S112; O'Hagan D, et al., J. Virol. 2001; 75:9037-9043 and Rainczuk A, et al., Infect. Immun. 2004; 72:5565-5573. The specific backbone of the vector into which the nucleic acids of the invention are inserted is not important as long as said nucleic acid is properly expressed in the subject. Examples of suitable vectors include, but are not limited to viruses and plasmids. Preferably, when a viral vector is used, an AAV vector is used. Preferably, when a plasmid vector is used, pcDNA3.1 and pVAX1 (Invitrogen, Carlsbad, CA, USA) are used; DNA sequences are available on the Invitrogen website http://www.thermofisher.com/uk/en/home/brands/invitrogen.html, October 2015); pNGVL (National Gene Vector Laboratory, University of Michigan, MI, USA); and p414cyc (ATCC Accession 87380) and p414GALS (ATCC Accession 87344). More preferably, the pcDNA3.1 plasmid is used as the plasmid vector. Most preferably, the plasmid vector is PM5A16T20 and pM7A16LLC34.

Schemes and use of passive immunization are known in the art. See Donnelly J, et al., Annu. Rev. Immunol. 1997; 15:617-648; Robinson H, Pertmer T, Adv. VirusRes. 2000; 55:1-74; Gurunathan S, et al., Annu. Rev. Immunol. 2000; 18:927-974 (2000) and Ulmer J, Curr. Opin. drug discov. devel. 2001; 4:192-197. Briefly, passive immunization in the context of the present invention is designed to direct the expression of an Fc fusion protein derivative in a subject in vivo. See Ulmer J, et al., Science 1993; 259:1745-1749. Typically, the nucleic acid is cloned into a bacterial plasmid that is optimized for expression in eukaryotes and consists of the following: (1) an origin of replication for propagation in bacteria, usually derived from E. coli, such as ColE1, (2) an antibiotic resistance gene , usually kanamycin, to select bacteria that integrate the plasmid, (3) a strong promoter for optimal expression in mammalian cells, such as the cytomegalovirus (CMV) or simian virus 40 (SV40) promoter, (4) a multiple cloning site 3' from the promoter to insert a gene of interest; and (5) an SV40 or bovine growth hormone (BGH) polyadenylation signal to stabilize the mRNA.

Another object of the present invention is the delivery of vectors using non-pathogenic or attenuated bacterial strains carrying plasmids capable of expressing Fc fusion protein derivatives of the invention, such as Escherichia spp., Salmonella spp., Shigella spp., Mycobacterium spp. and Listeria spp., not limited to those listed.

The use of a particular strain of Escherichia is not essential to the present invention. Examples of Escherichia strains that can be used in the present invention include Escherichia coli strains DH5a, HB 101, HS-4, 4608-58, 1184-68, 53638-C-17, 13-80 and 6-81, enteropathogenic E. coli , enteropathogenic E. coli and enterohemorrhagic E. coli. See Sambrook, 1989, supra; Sansonetti P, et al., Ann. microbiol. 1982; 132A:351-355); Evans D, et al., Infect. Immun. 1975; 12:656-667; Donnenberg S, et al., J. Infect. Dis. 1994; 169:831-838 and McKee M, O'Brien A, Infect. Immun. 1995; 63:2070-2074.

The use of a particular strain of Salmonella is not essential to the present invention. Examples of Salmonella strains that can be used in the present invention include S. typhi (ATCC Accession No. 7251), S. typhimurium (ATCC Accession No. 13311), S. galinarum (ATCC Accession No. 9184), S. enteriditis (ATCC Accession No. 4931 ), S. typhimurium (ATCC accession number 6994), S. typhi aroC double mutant, aroD (Hone D, et al., Vaccine 1991; 9:810-816) and S. typhimurium aroA mutant (Mastroeni D, et al. , Micro Pathol 1992; 13:477-491).

The use of a particular strain of Shigella is not essential to the present invention. Examples of Shigella strains that can be used in the present invention include S. flexneri (ATCC accession number 29903), S. flexneri CVD1203 (ATCC accession number 55556), S. flexneri 15D (Sizemore D, et al., Vaccine 1997; 15: 804-807; Sizemore D, et al., Science 1995, 270:299-302), S. sonnei (ATCC accession number 29930) and S. dysenteriae (ATCC accession number 13313).

The use of a particular strain of Mycobacterium is not essential to the present invention. Examples of Mycobacterium strains that can be used in the present invention include M. tuberculosis strain CDC1551 (Griffith T, et al., Am. J. Respir. Crit. Care Med. 1995; 152:808-811), M. tuberculosis strain Beijing (van Soolingen D, et al., J. Clin. Microbiol. 1995; 33:3234-3238), M. tuberculosis H37Rv strain (ATCC accession number 25618), M. tuberculosis pantothenate auxotrophic strain (Sambandamurthy V, Nat. Med. 2002; 8:1171-1174, M. tuberculosis rpoV mutant strain (Collins D, et al., Proc. Natl. Acad. Sci USA. 1995; 92: 8036, M. tuberculosis leucine auxotroph strain (Hondalus M , et al., Infect Immun 2000;68(5):2888-2898), BCG Danish strain (ATCC accession number 35733), BCG Japanese strain (ATCC accession number 35737), BCG Chicago strain (ATCC accession number 27289) , BCG Copenhagen Strain (ATCC No. 27290), BCG Pasteur Strain (ATCC Accession No. 35734), BCG Glaxo Strain (ATCC Accession No. 35741), BCG Connaught Strain (ATCC Accession No. er ATCC 35745) and BCG Montreal (ATCC registration number 35746).

The use of a particular strain of Listeria is not essential to the present invention. Examples of Listeria monocytogenes strains that can be used in the present invention include, without limitation, L. monocytogenes strain 10403S (Stevens R, et al., J. Virol. 2004; 78:8210-8218), L. ivanovii and L. seeligeri strains ( Haas A, et al., Biochim. Biophys. Acta. 1992; 1130:81-84) or L. monocytogenes mutant strains such as (1) actA plcB double mutant (Peters C, et al., FEMS Immunol. Med. Microbiol. 2003; 35:243-253 and Angelakopoulos H, et al., Infect. Immun. 2002; 70:3592-3601) or (2) a dal dat double mutant in the alanine racemase gene and the D-amino acid aminotranserase gene (Thompson R, et al., Infect Immun 1998;66:3552-3561).

Methods for delivering vectors using bacterial carriers are well known in the art. See US 6500419, US 5877159 and US 5824538; Shata M, et al., Mol. Med. Today 2000; 6:66-71; Hone D, Shata M, J. Virol. 2001; 75:9665-9670; Shata M, et al., Vaccine 2001; 20:623-629; Rapp U and Kaufmann S, Int. Immunol. 2004, 16:597-605; Dietrich G, et al., Curr. Opin. Mol. Ther. 2003; 5:10-19 and Gentschev I, et al., J. Biotechnol. 2000; 83:19-26. The type of plasmids delivered by said bacterial carriers for the expression of Fc fusion protein derivatives of the invention is not critical.

In a further embodiment, the use of an AAV vector to deliver the nucleic acids of the invention is also provided.

Pharmaceutical compositions of the present invention can be administered by various methods known in the art. As will be appreciated by one of skill in the art, the route or mode of administration will vary depending on the desired results. Active agents of the invention may be formulated with carriers that will protect the agent from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such compositions are well known in the art. See Robinson J, et al., Eds., "Sustained and Controlled Release Drug Delivery Systems" (Marcel Dekker, Inc., New York, NY, USA, 1978).

In order to administer the active agent of the invention via certain routes of administration, it may be necessary to coat the agent with a material or to co-administer the agent with a material to prevent its inactivation or to ensure its proper distribution in vivo. For example, the agent can be administered to the subject in a suitable carrier (eg, liposome) or diluent. Pharmaceutically acceptable diluents include, but are not limited to, saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes. See Strejan G, et al., J. Neuroimmunol. 1984; 7:27-41. Many methods for the production of liposomes are known in the art. See US 4,522,811, US 5,374,548, and US 5,399,331. Liposomes may contain one or more moieties that are selectively transported to specific cells or organs and thus improve targeted drug delivery. Typical targeting moieties include folate or biotin, mannosides, and surfactant protein A receptor. In one embodiment of the invention, the active agents of the invention are encapsulated in liposomes; in a more preferred embodiment, the liposomes are grouped for targeted delivery. In the most preferred embodiment, the active agents in liposomes are delivered by bolus injection.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media in the preparation of pharmaceutical compositions of the invention is contemplated in this application because their use is not incompatible with the active agents of the invention. Additional active compounds may also be included in pharmaceutical compositions.

Pharmaceutical compositions are generally sterile and stable under the conditions of manufacture and storage. The composition may be presented as a solution, microemulsion, liposome, or other ordered structure suitable for the concentration of the active agent. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol), or suitable mixtures thereof. Coatings such as lecithin can be used to maintain proper flow, for example, particle size deviations can be reduced, and surfactants can also be used. In many cases it is preferred to include isotonic agents such as, for example, sugars, polyhydric alcohols (eg mannitol, sorbitol) or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by the inclusion in the composition of compounds that slow absorption (for example, monostearate salts, gelatin).

Sterile injectable solutions can be prepared by incorporating the active agent of the invention in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as appropriate, followed by microfiltration sterilization. Typically, dispersions are prepared by incorporating the active agent into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (i.e., lyophilization), which allow the preparation of a powder of the active ingredient plus any additional desired ingredient from their previously sterile filtered solution.

Administration schedules are selected to provide the optimal desired response (eg, response to treatment). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased, depending on the therapeutic situation. For example, the Fc fusion protein derivatives of the invention can be administered once or twice a week by subcutaneous injection, or once or twice a month by subcutaneous injection.

For ease of administration and uniformity of administration, it is most expedient to formulate parenteral compositions in the form of a dosage form. Dosage form as used herein refers to physically discrete units presented as single doses for subjects to be treated, each unit containing a predetermined amount of the active agent calculated to provide the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of the dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active agent and the particular therapeutic effect to be achieved.

Examples of pharmaceutically acceptable antioxidants include, but are not limited to, water-soluble antioxidants (e.g., ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite), oil-soluble antioxidants (e.g., ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate , alpha-tocopherol) and metal chelating agents (eg citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid). Pharmaceutical compositions of the invention include those suitable for oral, nasal, topical (eg buccal and sublingual), rectal, vaginal or parenteral administration. For convenience, the formulations may be presented in unit dosage form and may be prepared by any means known in the art. The amount of active agent that can be combined with a carrier material to form a single dose dosage form will vary depending on the subject being treated and the particular route of administration. The amount of active agent that can be combined with a carrier material to form a single dose dosage form will generally be that amount of the composition that produces a therapeutic effect. Typically, this amount will range from about 0.001% to about 90% active agent, preferably from about 0.005% to about 70%, and most preferably from about 0.01% to about 30%.

Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or aerosol compositions containing such carriers as are known in the art to be suitable. Dosage forms for topical or transdermal administration of the compositions of the present invention include powders, aerosols, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active agent of the invention may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be required.

The pharmaceutical compositions of the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents (eg paraben, chlorobutanol, phenol sorbic acid). It may also be desirable to include isotonic agents (eg sugars, sodium chloride) in the compositions.

Actual dosage levels of the active agents in the pharmaceutical compositions of the present invention may vary to achieve the desired therapeutic response in the subject. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular agent of the invention used, its amount, route of administration, time of administration, rate of excretion or expression of the particular active agent used, duration of treatment, other drugs, compounds or materials used in combination with specific pharmaceutical compositions used, age, sex, weight, condition, general health and history of the subject being treated, and other such factors known in the medical field. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe a therapeutically effective amount of the active agent(s) required. For example, a physician or veterinarian may start with lower doses of the active agents of the invention used in a pharmaceutical composition than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, a suitable daily dose of a composition of the invention will be that amount of active agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Preferably, administration is parenteral, more preferably intravenous, intramuscular, intraperitoneal or subcutaneous. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally as dosage forms. While the active agent of the invention may be administered alone, it is preferred that the agent be administered as part of a pharmaceutical composition.

Pharmaceutical compositions of the invention may be administered using medical devices known in the art. For example, in a preferred embodiment, the pharmaceutical composition of the invention may be administered via a needleless hypodermic injection device. See US 5,399,163, US 5,383,851, US 5,312,335, US 5,064,413, US 4,941,880, US 4,790,824, or US 4,596,556. (e.g., US 4,447,233 (non-implantable, rate controlled), US 4,447,224 (implantable, variable rate), US 4,487,603 (implantable, rate controlled)), transcutaneous drug delivery devices (e.g., US 4,486,194), and osmotic drug delivery systems (eg, US 4439196 and US 4475196). Many other such implants, delivery systems and modules are known to those skilled in the art.

Pharmaceutical compositions of the invention must be sterile and fluid to the extent that delivery of the composition can be accomplished by syringe. In addition to water, the carrier may be isotonic buffered saline, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it is preferable to include isotonic agents, eg sugars, polyhydric alcohols (eg mannitol, sorbitol) and sodium chloride in the composition. Long-term absorption of injectable compositions can be achieved by including an absorption delaying agent (eg, aluminum monostearate and gelatin) in the composition.

5. Methods of treatment and prevention

In another aspect, the invention relates to a method for treating or preventing HIV infection or AIDS in a subject, comprising administering to said subject at least one of the Fc fusion protein derivatives, nucleic acids, vectors, host cells, or pharmaceutical compositions of the invention, or a mixture thereof. . Beneficial therapeutic or prophylactic effects of active agents and pharmaceutical compositions of the invention on symptoms of HIV infection or AIDS include, for example, preventing or delaying initial infection in a subject exposed to HIV, reducing the viral load in a subject infected with HIV, prolonging the asymptomatic phase of HIV -infections, maintaining low viral loads in HIV-infected subjects whose viral levels have been reduced by antiretroviral therapy (AT), increasing levels of CD4 T cells or reducing the decline in CD4 T cells, both HIV-1 specific and non-specific, in in drug-naive subjects and in subjects treated with AT, improvement in general health or quality of life in a subject with AIDS and increased life expectancy in a subject with AIDS. The physician or veterinarian can compare the effect of treatment with the condition of the subject prior to treatment, or with the expected condition of the untreated subject, to determine whether the treatment is effective in suppressing AIDS. In a preferred embodiment, the active agents and pharmaceutical compositions of the invention are used for the prevention of HIV infection or AIDS. In another preferred embodiment, the active agents and pharmaceutical compositions of the invention are used to treat HIV infection or AIDS.

The active agents and pharmaceutical compositions of the invention may find use in the treatment of HIV infection or AIDS. While all subjects that can be affected by HIV or its equivalents (e.g. chimpanzees, macaques, baboons or humans) can be treated in this way, the active agents and pharmaceutical compositions of the invention are intended in particular for their therapeutic use in humans. . Often, more than one administration may be required to achieve the desired therapeutic effect; the exact protocol (dosage and frequency of administration) can be established according to standard clinical procedures.

The present invention also relates to the reduction or elimination of symptoms associated with HIV infection or AIDS. These include symptoms associated with the minor symptomatic phase of HIV infection, including, for example, herpes zoster, skin rash and nail infections, mouth ulcers, recurrent infection of the nose and throat, and weight loss. In addition, other symptoms associated with the main symptomatic phase of HIV infection include, for example, oral candidiasis and vaginal candidiasis (Candida), persistent diarrhea, weight loss, persistent cough, and reactivated tuberculosis or recurrent herpes infections such as herpes simplex ( herpes simplex). Other symptoms of advanced AIDS that can be treated in accordance with the present invention include, for example, diarrhea, nausea and vomiting, oral stomatitis and ulcers, persistent, recurrent vaginal infections and cervical cancer, persistent generalized lymphadenopathy (PGL), severe skin infections , warts and ringworm, respiratory infections, pneumonia, especially pneumonia caused by Pneumocystis carinii (PCP), herpes zoster (or shingles), nervous system problems such as pain, numbness or "tingling" in the arms and legs, neurological disorders , Kaposi's sarcoma, lymphoma, tuberculosis, or other similar opportunistic infections.

In another preferred embodiment, the active agents or pharmaceutical compositions of the invention are administered to an HIV infected or exposed subject in combination with at least one therapeutic agent. Preferably, the therapeutic agent is indicated for the prevention or treatment of HIV or AIDS. Suitable therapeutic agents include, without limitation, drugs included in current antiretroviral therapy (AT) and highly active antiretroviral therapy (HAART) protocols, such as a non-nucleoside reverse transcriptase inhibitor (e.g., efavirenz, nevirapine, delavirdine, etravirine, rilpivirine), nucleoside analogue inhibitors reverse transcriptase (eg, zidovudine, tenofovir, lamivudine, emtricitabine) and protease inhibitors (eg, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir), hereinafter referred to independently or collectively as "antiretroviral(s) agent(s) for the treatment of HIV ". In one embodiment of this embodiment, at least one active agent or pharmaceutical composition of the invention and at least one anti-HIV antiretroviral agent are co-administered to a subject at the same time. In another embodiment, at least one active agent or pharmaceutical composition of the invention is administered to a subject prior to administration of any antiretroviral agent for the treatment of HIV. In yet another embodiment, at least one active agent or pharmaceutical composition of the invention is administered after the subject has been administered an anti-HIV antiretroviral agent, such as after an interruption of an AT or HAART protocol.

In addition, Fc fusion protein derivatives of the invention can also be administered with therapeutic agents that can induce HIV gp120 expression on the surface of latently infected cells, allowing NK cells to rapidly clear them. See Siliciano J, et al, J Allergy Clin Immunol. 2014; 134(1):12-19.

6. Neutralization and methods of determination

In a further aspect, the present invention relates to a method for inactivating HIV, which comprises the step of contacting the virus with at least one Fc fusion protein derivative of the invention. Preferably, the method is carried out on a sample containing HIV or suspected of having HIV. The method can be carried out under conditions that promote binding of the Fc fusion protein derivative to HIV, as described in the art. See Lu L, et al., Retrovirology 2012; 9(104), 1-14.

In a further aspect, the present invention relates to a method for detecting a molecule or fragment thereof (e.g., HIV, gp120) that attaches to the D1 and D2 domains of the human CD4 receptor in a sample, comprising the steps of (a) contacting the sample with a derivative of an Fc fusion protein at of the invention and (b) determining whether the Fc fusion protein derivative specifically binds to a molecule in the sample. The sample may be a biological sample, including, without limitation, blood, serum, urine, tissue, or other biological material from uninfected, infected, or potentially infected subjects (eg, subjects exposed to recurrent or episodic exposure to HIV). The sample can also be non-biological (eg derived from water, beverages). Preferably the molecule is HIV and more preferably the gp120 protein of the HIV viral envelope.

In a preferred embodiment of this aspect, the present invention relates to a method for detecting HIV in a sample comprising the steps of (a) contacting the sample with an Fc fusion protein derivative of the invention and (b) determining whether the Fc fusion protein derivative specifically binds to an HIV molecule. in the sample. Preferably, the sample is a plasma sample or a serum sample.

Initially, the sample can be manipulated to make it more suitable for the detection method. In a preferred embodiment, the contact between the sample and the Fc fusion protein derivative is prolonged (ie, incubation is carried out under conditions suitable for the stability of the sample and the Fc fusion protein derivative). Conditions at the stage of bringing into contact can be determined by a person skilled in the art in a routine manner. Suitable buffers that can be used in the contacting step include physiological buffers that do not interfere with the assay to be performed. For example, Tris or triethanolamine (TEA) buffer can be used. The pH of the buffer (and resulting lysis reagent, including the buffer solution) may range from about 2.0 to about 10.0, possibly from about 4.0 to about 9.0, preferably from about 7.0 to about 8.5, and even more preferably from about 7.5 to about 8.0, or about 7.0, about 7.5, about 8.0, or about 8.5. Typical conditions of "bringing into contact" may include incubation for 15 minutes to 4 hours (for example, 1 hour at 4°C, 37°C or at room temperature). However, they may vary as needed, for example according to the nature of the cooperating binding partners. Alternatively, the sample may be subjected to gentle rocking, agitation, or rotation. In addition, other suitable reagents may be added, such as blocking agents to reduce non-specific binding. For example, 1-4% BSA (bovine serum albumin) or other suitable blocking agent (eg milk) can be used. The conditions for bringing into contact can be varied and adapted depending on the purpose of the detection method. For example, if the incubation temperature is, for example, room temperature or 37° C., this may increase the ability to identify binders that are stable under these conditions (eg, stable under conditions found in the human body).

Preferably, the Fc fusion protein derivatives of the invention are brought into contact with the sample under conditions that allow the formation of a complex between the Fc fusion protein derivative and the molecule or fragment thereof present in the sample. The complex formation is then determined and measured by appropriate means, indicating, for example, the presence of HIV in the sample. These means of detection and measurement depend on the nature of the binding partners and include, but are not limited to, homogeneous and heterogeneous binding assays, such as, for example, radioimmunoassays (RIA), ELISA (enzyme immunoassay), immunofluorescence, immunohistochemistry, flow cytometry (e.g., FACS), BIACORE and Western blotting. Preferred methods of analysis, especially for large-scale analysis of subject sera and blood, as well as blood-derived products, are flow cytometry, ELISA, and Western blotting.

In a preferred embodiment, the measurement is performed by flow cytometry (eg, FACS). As used herein, the term "flow cytometry" refers to an assay in which the proportion of material in a sample is determined by labeling the material (for example, by binding a labeled antibody to the material) by causing a stream of liquid containing the material to pass through a beam of light, splitting the light emitted sample, into component wavelengths using a series of filters and mirrors, and detecting light. Flow cytometry provides sensitive detection and rapid quantification of certain individual cell characteristics, such as relative size variability and endogenous fluorescence, as well as quantification of any cellular compound that can be labeled with a fluorochrome. See Melamed M, et al., "Flow Cytometry and Cell Sorting", 2nd Ed. (Wiley-Liss, New York, NY, USA, 1990). One of the main advantages of flow cytometry is the rapid quantification of analytes on a large number of particles or cells. Typically, fluorochromes selected for use as detectable markers are selected based on their ability to fluoresce when excited by light at the wavelength used by the laser. When a fluorochrome is excited with a laser beam, it emits light which is then evaluated by the photomultiplier tubes of the flow cytometer. This method is capable of analyzing 10,000 cells/particles within 1-2 minutes. Flow cytometers have filters to detect emissivity from different fluorochromes that fluoresce at different wavelengths and allow four or more different fluorochromes to be used as detectable markers, meaning that at least 4 different molecules can currently be detected simultaneously. These sample analysis methods and devices are commercially available and well known in the art (eg, FACSCalibur flow cytometer; BD Biosciences Corp., Franklin Lakes, NJ, USA).

In a preferred embodiment, said measurement comprises analyzing a sample, preferably by flow cytometry, using a reporter capable of binding to an Fc fusion protein derivative, preferably the Fc region of said Fc fusion protein derivative. Preferably, the reporter contains a detectable moiety, and more preferably it is an Fc-specific secondary antibody linked to a detectable moiety.

Detectable moieties that can be used include fluorophores. By "fluorophore" (or "fluorochrome" or "chromophore") is meant a fluorescent compound that can re-emit light when excited by light. Fluorophores that can be used include biological (eg, proteins) and chemical fluorophores. Examples of biological fluorophores include T-sapphire, Cerulean, mCFPm, CyPet, EGFP, PA-EGFP, Emerald, EYFP, Venus, mCitrine, mKO, mOrange, DSRed, JRed, mStrawberry, mCherry, PA-mCherry, mRubl, Tomato, mPuby, mPub mKate, mKatushka, Kaede, Halotag and super ecliptic fluorine. Examples of chemical fluorophores include Alexafluor, rhodamine, BODIPY, tetramethylrhodamine, cyanine dyes, fluorescein, quantum dots, IR dyes, FM dyes, ATTO dye. The secondary antibody can also be labeled with enzymes that can be used for detection, such as, for example, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase or glucose oxidase. When an antibody is labeled with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to catalyze a reaction whose product can be distinguished. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. The antibody can also be labeled with biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be labeled with an enzyme or a fluorescent label.

Preferably, the Fc secondary antibody used is specific to the species from which the primary antibody is derived (eg, human). In one embodiment, said Fc-specific secondary antibody is selected from the group consisting of IgA (eg, IgA1, IgA2), IgD, IgE, IgG (eg, IgG1, IgG2, IgG3, IgG4) and IgM. Preferably, the secondary antibody is IgG and more preferably IgG1. Said Fc-specific secondary antibody may be any vertebrate antibody, preferably any mammalian antibody, and more preferably any non-human antibody (e.g. rabbit, mouse, rat, goat, horse, sheep or donkey).

For use as reagents in the aforementioned assays, it may be convenient to have Fc fusion protein derivatives of the invention bound to the inner surface of the microtiter plate wells. The Fc fusion protein derivatives of the invention can be directly coupled to the well of a microtiter plate. However, maximum binding of the Fc fusion protein derivatives to the wells can be achieved by pre-treating the wells with polylysine before adding the Fc fusion protein derivatives. In addition, the antibody derivatives of the invention may be covalently attached to the wells by methods known in the art. Typically, Fc fusion protein derivatives of the invention are used for immobilization on wells in the concentration range of 0.01 to 100 μg/mL, although they can also be used in higher or lower amounts. Samples are then added to the wells coated with the Fc fusion protein derivative of the invention.

7. Sets

In a further aspect, this invention relates to kits containing at least one of: antibody derivatives, nucleic acids, vectors, host cells, pharmaceutical compositions or combinations of the invention, or mixtures thereof. Optionally, the components of the kits of the invention may be packaged in suitable containers and labeled for the detection, inactivation, diagnosis, prevention, or treatment of HIV or AIDS or related conditions. The components of the kits can be stored in single-dose or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, reconstitution formulation. The containers may be made from various materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a hypodermic stopper). The kits may further comprise more containers containing a pharmaceutically acceptable carrier. They may further include other materials that are commercially and user-friendly, including, but not limited to, buffers, diluents, filters, needles, syringes, culture media for one or more suitable host cells, or other active agents. The kits may contain instructions commonly included in commercial packaging of diagnostic and therapeutic products that contain information such as indications, use, dosage, manufacture, administration, contraindications, or warnings regarding the use of such diagnostic and therapeutic products.

All publications cited in this document are incorporated by reference in their entirety. A better understanding of the invention described above in general terms will allow the following examples, which are given by way of illustration and are not intended to limit the present invention, unless otherwise indicated.

General Methods

1. Construction of Fc fusion protein derivatives

The two extracellular domains of the human CD4 molecule (D1 and D2) were fused to the Fc region of human IgG1 containing point mutations G236A, S239D, A330L and I332E, including the hinge, CH2 and CH3 domains, to obtain a modified CD4-IgG1 molecule. Based on the modified CD4-IgG1 framework, Fc-fusion protein derivatives were created with the following characteristics:

(a) M428L and N434S point mutations were made in the Fc chain to prolong the activity of the Fc fusion protein derivatives.

(b) A K322A point mutation was made to modulate complement activation.

(c) F243L and R292P point mutations in the Fc chain were made to improve the production of Fc fusion protein derivatives.

(d) at least one or more Y300L, P396L, S298A, E333A, or K334A point mutations in the Fc chain have been made to further improve the production of Fc fusion protein derivatives.

(e) adding the N-terminal human CCR5 extracellular sequence (SEQ ID NO: 5) at the C-terminus of the Fc chain.

(e) adding the sequence T-20 (SEQ ID NO: 10), C34 (SEQ ID NO: 11) or EHO (SEQ ID NO: 12) at the C-terminus of the Fc chain.

(g) sequential addition of (1) CCR5 sequence (SEQ ID NO: 5), (2) T-20 sequence (SEQ ID NO: 10), C34 (SEQ ID NO: 11) or EHO sequence (SEQ ID NO: 12) and (3) a variable number of flexible units (GGGGS) (SEQ ID NO: 4) and/or SEQ ID NO: 6-9 at the C-terminus of the Fc chain.

All polynucleotides expressing derivatives of the Fc fusion protein of the invention were synthesized using the GeneArt process and expression plasmids pcDNA3.1 and pcDNA3.4.

Plasmids were dissolved in 10 mM Tris buffer, pH 8, to a concentration of 0.5 µg/µl. Chemically competent E. coli One Shot TOP10 (Life Technologies Corp., Carlsbad, CA, USA) were transformed using 1 μl of the plasmid following the manufacturer's instructions. Briefly, 1 μl of the plasmid was added to the bacteria vial and incubated on ice for 15 minutes. After that, the tube was incubated at 42°C for 30 seconds and immediately placed on ice for two minutes. Bacteria were resuspended in 250 μl of SOC medium (Life Technologies Corp., Carlsbad, CA, USA) and incubated for 1 hour at 37°C and 225 rpm in an Innova 4000 shaker incubator (New Brunswick Scientific Co., Inc. ., Enfield, CT, USA). Thereafter, 100 μl of the cell culture at a 1/100 dilution was spread on LB agar plates containing selection ampicillin (100 μg/ml). The plates were incubated at 37°C for 16-24 hours in a Heraeus incubator (Thermo Fisher Scientific, Waltham, MA, USA). A single colony was isolated and inoculated into 5 ml of LB medium containing ampicillin for selection (100 μg/ml) and incubated for 8 hours at 37°C and 225 rpm in an Innova 4000 shaker incubator (New Brunswick Scientific Co. , Inc., Enfield, CT, USA). Thereafter, 500 ml of LB medium containing ampicillin for selection (100 μg/ml) were inoculated with 500 μl of the culture described above and incubated at 37° C. with stirring at 225 rpm for 16 hours as described previously. Bacteria were harvested by centrifugation at 3000 g for 45 minutes at room temperature in an Eppendorf 5810R centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and plasmids were isolated using a Qiagen Plasmid Maxi kit (Qiagen NV, Venlo, NL) according to the manufacturer's instructions. Quantitative spectrophotometric analysis of purified plasmids was performed using a nanodrop 1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA).

2. Protein preparation, quantitation and purification

HEK-293 cells were transfected with plasmids encoding various derivatives of the Fc fusion protein of the invention using a Calphos transfection kit (Clontech®, Takara Bio Inc., Otsu, JP) following the manufacturer's instructions. After 48 hours, the supernatant was collected, purified by filtration through a 0.45 μm filter (EMD Millipore, Merck KGaA, Darmstadt, DE) and stored at -20°C until use.

Quantitative analysis of derivatives of the Fc fusion protein was carried out by ELISA. Briefly, Maxisorp 96-F plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were incubated overnight at 4°C with 100 µl/well F(ab)2 goat anti-human IgG (Fc-specific) ( Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) at 1 µg/ml in PBS. After blocking with PBS / 10% FBS / 0.05% tween20 and washing, serial dilutions of the culture supernatant (containing the recombinant protein) in blocking buffer (100 µl/well) were added to the plate and incubated overnight at 4°C. The plates were washed again and a secondary HRP-F(ab)2 goat anti-human IgG antibody (Fc-specific) (Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) at 1/10000 dilution in blocking buffer (100 µl/well) was added and incubated at room temperature for one hour. The plates were washed and bound antibodies were detected using the substrate OPD (o-phenylenediamine dihydrochloride), the reaction was stopped by adding 4N. _{H2SO4 .} The product was measured at 492 nm using an ELISA plate reader.

Proteins were purified using a Protein A Sepharose column (GE Healthcare, Inc., Stamford, CT, USA). Proteins were obtained by transient transfection using serum free medium as described above. The supernatants were collected, centrifuged at 3000 g for 10 minutes and filtered through a 0.45 µm filter to remove cell debris. The purified supernatant was added to pre-washed Sepharose beads with protein A and incubated overnight at 4°C in the bottom-to-lid rotation mode. Protein A was washed with Tris buffered saline (TBS) and the bound protein was eluted with 4M MgCl ₂ . Alternatively, proteins were purified using CaptureSelect FcXL Affinity Matrix columns (Thermo Fisher Scientific, Waltham, MA, USA) and eluted with pH 3.5 glycine buffer. Purified proteins were dialyzed against PBS, concentrated by ultrafiltration, quantitated by ELISA or spectrophotometry, and stored at -80° C. until use.

3. Neutralization studies

S, et al., Vaccine 2011; 29:5250-5259. Бесклеточную нейтрализацию вируса производными слитого с Fc белка исследовали в стандартном анализе на основе TZM-bl. См. Li, 2005, выше. Вкратце, в 96-луночном культуральном планшете 100 мкл предварительно разведенных производных слитого с Fc белка предварительно инкубировали с 50 мкл псевдовируссодержащего материала, используя 200 TCID₅₀ (инфекционные дозы для тканевой культуры) при 37°С в течение одного часа. Затем в каждую лунку добавляли по 10000 клеток-мишеней TZM-bl с люциферазным репортером в объеме 100 мкл. Планшеты культивировали при 37°С и 5% СО₂ в течение 48 часов. Исследовали серийные разведения производных слитого с Fc белка от 1000 до 0,1 нг/мл. Репортерные клетки TZM-bl обрабатывали декстраном (Sigma-Aldrich, Сент-Луис, Миссури, США) для усиления инфекционности. Для считывания использовали субстрат люциферазы Britelite Plus (PerkinElmer, Inc., Waltham, MA, USA). Для аппроксимации данных нейтрализации нелинейной функцией в расчетах использовали нормализованные значения, которые аппроксимировали кривой ингибирования одного сайта с переменным угловым коэффициентом Хилла. Весь статистический анализ и аппроксимацию нелинейной функцией выполняли с использованием программного обеспечения GraphPad Prism v5.0.HIV isolates NL4-3, BaL, AC10, SVBP6, SVBP8, SVBP11, SVBP12, SVBP14, SVBP15, SVBP17, SVBP18 and SVBP19 were generated as pseudoviruses using Env expression plasmids and the pSG3 vector. Cm.

S, et al., Vaccine 2011; 29:5250-5259. Cell-free neutralization of virus by Fc fusion protein derivatives was examined in a standard TZM-bl based assay. See Li, 2005, supra. Briefly, in a 96-well culture plate, 100 μl of pre-diluted Fc fusion protein derivatives were pre-incubated with 50 μl of pseudovirus material using 200 TCID ₅₀ (tissue culture infectious doses) at 37° C. for one hour. Then, 10,000 TZM-bl target cells with a luciferase reporter were added to each well in a volume of 100 μl. The plates were cultured at 37°C and 5% CO ₂ for 48 hours. Serial dilutions of the Fc fusion protein derivatives were tested from 1000 to 0.1 ng/mL. TZM-bl reporter cells were treated with dextran (Sigma-Aldrich, St. Louis, MO, USA) to enhance infectivity. Britelite Plus luciferase substrate (PerkinElmer, Inc., Waltham, MA, USA) was used for reading. To approximate the neutralization data with a non-linear function, normalized values were used in the calculations, which approximated the single-site inhibition curve with a variable Hill slope. All statistical analysis and non-linear function fitting was performed using GraphPad Prism v5.0 software.

4. ADCC studies

To evaluate the ability of various Fc fusion protein derivatives to activate NK cell-mediated destruction of HIV-infected cells by NK cells, the ADCC assay was performed according to Alpert M, et al., J. Virol. 2012, 86:12039-12052. Briefly, the KHYG-1 CD16+ NK cell line was used as the source of effector cells and the CEM.NKR.CCR5+ Luc cell line as the source of target cells. Five days prior to the study, target cells were infected with material containing a highly infectious BaL isolate and cultured in R10 medium (RPMI supplemented with 10% fetal bovine serum) at 37°C. For analysis, 10 ⁴ target cells (more than 40% of them productively infected) were cultured with 10 ⁵ effector cells in R10 medium supplemented with 10 u/ml recombinant IL-2 (interleukin-2) in the presence of various concentrations of Fc fusion protein derivatives or antibody based molecules in a total volume of 200 µl in round bottom 96 well plates. After 8 hours of incubation at 37° C., cells were resuspended and 150 μl of cell suspension was mixed with 50 μl of luciferase substrate, Britelite Plus (PerkinElmer, Inc., Waltham, MA, USA). Luciferase units were used for reading. Since target cells express luciferase during HIV infection, a decrease in luciferase activity is a direct indicator of antibody- and NK-mediated cell killing.

5. Binding to Fc receptors (assay using ELISA)

To evaluate the binding affinity of Fc fusion protein derivatives to human Fc receptors, various ELISA assays were performed using recombinant soluble human CD64, CD32, CD16 and FcRn proteins (R&D Systems, Bio-Techne Ltd, Abingdon, GB). Briefly, Maxisorp 96-F plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were incubated overnight at 4°C with 100 μl/well of HIS.H8 clone mouse anti-6xHis antibody (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) at 1 µg/ml in PBS. After blocking with PBS/1% BSA/0.05% tween20 and washing, serial dilutions of Fc fusion protein immune complexes dissolved in blocking buffer (100 μl/well) were added to the plate and incubated overnight at 4°C. Immune complexes were generated by incubation of Fc fusion proteins (1 µg), biotin-conjugated mouse anti-human CD4 antibody (1.4 µg) (clone SK3, Biolegend) and streptavidin (0.125 µg) (Jackson ImmunoResearch Labs, Inc., West Grove , PA, USA) for two hours at room temperature. The plates were washed again and goat anti-human IgG secondary HRP-F(ab)2 (Fc-specific) (Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) was added at a 1/10,000 dilution in blocking buffer (100 µl /well) and incubated at room temperature for one hour. The plates were washed and bound antibodies were detected using the OPD substrate, the reaction was stopped by adding 4N. _{H2SO4 .} The product was measured at 492 nm using an ELISA plate reader.

6. Binding to C1q (study by ELISA)

To evaluate the binding affinity of Fc fusion protein derivatives to human C1q, ELISA studies were performed using soluble human C1q (Bio-Rad Labs, Inc., Hercules, CA, USA). Briefly, Maxisorp 96-F plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were incubated overnight at 4° C. with 100 μl/well human C1q (1 μg/ml in PBS). After blocking with PBS/1% BSA/0.05% tween20 and washing, serial dilutions of the Fc fusion protein were added and incubated overnight at 4°C. The plates were washed again and goat anti-human IgG secondary HRP-F(ab)2 (Fc-specific) (Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) was added at a 1/10,000 dilution in blocking buffer (100 µl /well) and incubated at room temperature for one hour. The plates were washed and bound antibodies were detected using the OPD substrate, the reaction was stopped by adding 4N. _{H2SO4 .} The product was measured at 492 nm using an ELISA plate reader.

Example 1

Construction of Fc-fusion Protein Derivatives

Mouse human CD4-IgG1 fusion protein was prepared as previously described. This fusion protein has been used in the past to identify antibodies to the CD4 binding site. See Carrillo J, et al., PLOS One 2015; 10(3):0120648; Fig. 1. However, since CD4-IgG1 molecules are known to have limited therapeutic potential, several changes have been made to the CD4-IgG1 protein sequence to enhance its antiviral activity and ADCC activity. See Jacobson J, et al., Antimicrob Agents Chemother. 2004; 48(2):423-429.

First, the Fc region of human IgG1 was mutated at positions G236A, S239D, A330L and I332E (ie the AM mutation set) as described in the art to enhance ADCC-mediated responses. See Bournazos, 2014, supra. Further modifications to enhance interaction with the HIV Env included the addition of a 29 amino acid sequence corresponding to the N-terminal extracellular region of CCR5 (SEQ ID NO: 5) and the addition of a T-20 sequence (SEQ ID NO: 10) at the C-terminus Fc chains with a flexible optimal linker. Thus, a derivative of the Fc fusion protein (M5) was constructed containing the sequences CCR5 and T-20. See FIG. 1 and 2. Also constructed molecules lacking the CCR5 sequence and having an extended linker (M7).

Secondly, several sets of mutations were introduced into the IgG1 sequence to improve the activity of the M5 and M7 molecules. The sets of mutations tested were: (1) A12 (i.e. F243L, R292P and Y300L; SEQ ID NO: 14), (2) A14 (i.e. F243L, R292P and P396L; SEQ ID NO: 15), (3) A16 (i.e. F243L, R292P, Y300L and P396L; SEQ ID NO: 16), (4) A18 (i.e. F243L, R292P, Y300L, P396L and V305I; SEQ ID NO: 17), (5) A41 (i.e. S298A, E333A and K334A; SEQ ID NO: 18), (6) LL (M428L and N434S; SEQ ID NO: 19) and combinations thereof (e.g. SEQ ID NO: 20-33 ). See FIG. 1 and 2., Tab. one.

For comparison purposes, an eCD4-IgG1 (M1) fusion protein was also prepared as previously described. The eCD4-IgG1 fusion protein is currently one of the most effective engineered anti-HIV antibodies known in the art. See Gardner, 2015, supra. To provide a better basis for comparison, the CD4-IgG1 Fc fusion protein strand was further mutated at positions G236A, S239D, A330L and I332E to form the M6 molecule. An additional control molecule was obtained by replacing the IgG1 sequence of the M7IgG1C34 molecule with a human IgG2 sequence. See FIG. 2. IgG2 lacks the ability to mediate ADCC and was therefore used as a negative control in the ADCC study.

Example 2

Derivatives of the Fc-fused protein

A standard transfection assay was performed to evaluate the yield of various molecules in mammalian cells (eg, the HEK293 cell line). The level of Fc fusion proteins released into the culture supernatant was analyzed by ELISA as described above and the dynamics of production was determined for 6 days after transfection. To evaluate the effect of mutations in the IgG1 sequence, a number of molecules based on the M7IgG1C34 derivative were analyzed. The molecules included wild-type IgG1 and several different sets of mutations. See Table 1. While the wild-type IgG1 derivative was obtained in high yield, the AM variant showed a distinct decrease in yield (ie, a decrease of approximately 90%). In contrast, all mutants containing the F243L and R292P point mutations (ie A12, A14, A16 and A18) and the A41 series mutants were produced in a yield comparable to the wild-type IgG1 molecule. See FIG. 3A. The introduction of LL mutations did not significantly change the yield of molecules. See FIG. 3B. On the contrary, the combination of mutations strongly influenced the yield. Indeed, the addition of the S239D and I332E mutations (contained in the AM series) to the background A16 genotype had a negative effect on production. See FIG. 3C.

Example 3

Neutralizing capacity of Fc fusion protein derivatives

A standard neutralization assay was performed to evaluate the effect of the A12, A14, A16, A18 and A41 mutation sets on the neutralizing ability of the Fc fusion protein derivatives of the invention. Four different viruses were used for analysis: (1) HIV-1 NL4-3 strain, (2) X4 monotropic laboratory isolate, (3) HIV-1 BaL strain, R5 monotropic laboratory isolate, and (4) two primary HIV-1 isolates. 1 (i.e. AC10 and SVPB16), both are type 2 viruses of the susceptibility level, which are particularly difficult to neutralize.

Briefly, in a 96-well culture plate, 100 μl of pre-diluted plasma samples were pre-incubated with 50 μl of pseudovirus material using 200 TCID ₅₀ at 37° C. for one hour. Then, 10,000 TZM-bl target cells with a luciferase reporter were added to each well in a volume of 100 μl. The plates were cultured at 37°C and 5% CO ₂ for 48 hours. Serial dilutions of the Fc fusion protein derivatives were tested from 1000 to 0.1 ng/mL. TZM-bl reporter cells were treated with dextran (Sigma-Aldrich, Saint Louis, MO, USA) to enhance infectivity. Britelite Plus luciferase substrate (PerkinElmer, Inc., Waltham, MA, USA) was used for reading. To fit the neutralization data with a non-linear function, normalized values were used in the calculations, which approximated a single-site inhibition curve with a variable Hill slope, and obtained for each molecule IC ₅₀ values against each virus. All statistical analysis and non-linear function fitting was performed using GraphPad Prism v5.0 software.

All molecules tested, including wild type IgG1 and Fc fusion protein derivatives containing the AM, A12, A14, A16, A18 and A41 mutation sets, showed similar efficacy in blocking HIV infectivity. Significant differences in the geometric mean IC ₅₀ values were not observed. See FIG. 4A. The introduction of LL mutations and combinations of different mutations did not significantly modify the neutralizing activity of the molecules against the HIV-1 BaL isolate. See FIG. 4B and 4C.

Example 4

Antibody-dependent cellular cytotoxic (ADCC) activity of Fc fusion protein derivatives

One of the most important features of the Fc fusion protein derivatives of the invention is their ability to activate NK cells and mediate ADCC. This mechanism is important for the efficient and rapid removal of HIV-infected cells and contributes to the protection of uninfected people from exposure to HIV and infection. See Euler Z, et al., AIDS Res Hum Retroviruses 2015; 31(1):13-24.

The ADCC activity of the Fc fusion protein derivatives of the invention was evaluated using a test previously described in the art. See Alpert M, et al., J. Virol. 2012, 86:12039-12052. In this assay, the killing capacity of NK cells is measured by the reduction in luciferase expression in an HIV-infected reporter cell line. Dose-response curve analysis against cells infected with BaL HIV isolate was performed by normalizing IC ₅₀ values against M1. Molecules carrying the AM mutation set (eg, M6 and M7AMC34) destroyed HIV-infected cells with the lowest IC ₅₀ values, consistent with binding data from ELISA studies. See FIG. 5A. On the other hand, molecules carrying the A16 and A18 mutation sets showed similar IC ₅₀ values, while molecules carrying the A41 mutation set and wild-type IgG1 molecules (eg, M7A41C34 and M1) showed lower activity. See FIG. 5A. The introduction of LL mutations did not significantly change the ADCC activity of the molecules. See FIG. 5V. On the contrary, the combination of mutations improved ADCC activity in some cases. Indeed, combinations of A16_4LL and A6_6LL showed the highest activity, which was comparable to the reference molecule M6. See FIG. 5C.

Example 5

Binding affinity of Fc fusion protein derivatives to human Fc receptors and C1q

The binding affinities of the Fc fusion protein derivatives of the invention to various Fc receptors were studied. These molecules were designed with the specific aim of increasing their binding affinity for human CD16. However, the introduced sets of mutations could also affect the binding affinities of the Fc fusion protein derivatives of the invention to other Fc receptors and cause undesirable side effects. Among human Fc receptors, CD64, CD32 and CD16 are the most important for mediating the effector functions of antibodies and Fc fusion proteins. See Vogelpoel L, et al., Front. Immunol. 2015; 6(79):1-11. Thus, the binding affinities of several derivatives of the Fc fusion protein of the invention to recombinant human CD64, CD32a, CD32b/c and CD16 (both V and F isoforms) were examined by ELISA.

The binding affinities of the Fc fusion protein derivatives of the invention to C1q were examined to evaluate possible complement activation. Thus, the binding affinities of several derivatives of the Fc fusion protein of the invention to recombinant human complement component C1q were examined by ELISA.

All ELISA data were normalized to the wild-type M7AC34 molecule containing unmutated human IgG1. The signal ratios were subjected to a Log2 transformation. After normalizing the data, the molecules and receptors were divided into clusters using the Euclidean distance as a similarity criterion. See FIG. 6. This global analysis revealed minimal and homogeneous changes in the affinity of various molecules for CD64. For CD32a and CD32b/c, there were minimal changes, except for the A16_1 molecules, which showed the highest affinity. In contrast, some molecules showed low or no affinity for C1q (molecules A16_4 and AM, respectively). See FIG. 6. All molecules increased affinity for CD16 to varying degrees, the most powerful derivatives were grouped into a cluster, including molecules A16_1, A16_3, Am, A16_6 and A16_4.

Example 6

A combination of mutations that enhance ADCC and prolong activity

The Fc region of the molecules, containing the wild-type IgG1 sequence and the AM and A16 mutation sets, was further modified to include the LL mutation set (ie, M428L and N434S). A set of LL mutations has been associated with prolonged activity of recombinant antibodies in human plasma. See Roopenian D, et al., Nature Reviews Immunology. 2007; 7:715-725.

First, different combinations of mutations were tested in transfection assays to evaluate their potential impact on production yield. The addition of the M428L and N434S mutations to the M7AC34 wild-type IgG1 molecule or to the M7AMC34 and M7A16C34 molecules did not affect their production yield, neutralizing activity, or ADCC activity. See FIG. 3-5.

The above results suggest that the introduced set of LL mutations did not adversely affect the antiviral activity against HIV and the ADCC activity of the Fc fusion protein derivatives of the invention.

Example 7

Effect of the LL mutation set on the binding affinity of Fc fusion protein derivatives to human neonatal Fc receptors (FcRn)

To analyze changes in affinity for FcRn receptors induced by a set of LL mutations in Fc fusion protein derivatives of the invention, ELISA studies were performed. Studies were carried out using two different pH conditions: pH 6.0 and pH 7.2. All molecules carrying the LL mutation set (M7ALLC34, M7A16LLC34, and M7A41LLC34) showed significantly higher affinity for FcRn receptors than their wild-type counterparts (M7AC34, M7A16C34, and M7A41C34) at pH 6.0 (10-fold mean increase), while both at pH 7.2 the effect was significantly lower. See FIG. 7. Thus, these data suggest that the LL mutation set maintains both the neutralizing and ADCC activity of the Fc fusion protein derivatives of the invention, while prolonging their activity.

Example 8

Analysis of the effect of linker sequences on protein production and activity

Although the M7A16_4LLC34 molecule showed an acceptable production, neutralizing and ADCC activity profile, the Fc fusion protein derivatives were further modified to include the CCR5 sequence (SEQ ID NO: 5) and various linkers with varying flexibility. To do this, on the basis of the M7A16_4LLT20 molecule, an M7A16T20 molecule was constructed, including the T20 sequence (SEQ ID NO: 10) or the C34 sequence (SEQ ID NO: 11). Similarly, the linker peptides M19, M20, M21 and M22 (SEQ ID NOS: 6-9) were also tested to form the molecules M19A16_4LLT20, M20A16_4LLT20, M21A16_4LLT20 and M22A16_4LLT20 and their respective C34 derivatives.

In terms of production, all T20-derivatives showed a worse yield than C34-analogues. The different linkers also affected the production, as the molecules carrying the M21 and M22 linkers showed the highest yield. See FIG. 8A. In addition, although the neutralizing ability was approximately the same for all molecules, the M22 derivatives were consistently less effective in neutralizing BaL or AC10 HIV-1 isolates. See FIG. 8B.

With respect to ADCC activity, a similar ranking was observed, where the M22 derivative was less potent than its M19-M21 counterparts. Derivatives M19, M20 and M21 unexpectedly showed higher activity than the reference molecule M6. See FIG. 8C.

Example 9

Analysis of the effect of gp41 sequences on protein production and activity

To improve yield and activity profile, the Fc fusion protein derivatives were further modified to include the HIV-2 EHO peptide (SEQ ID NO: 12) at the C-terminus. Derivatives M19A16_4LLEHO, M20A16_4LLEHO and M21A16_4LLEHO were constructed and compared with their C34 counterparts.

In terms of production, molecules carrying the EHO peptide showed the highest yield, with production rates comparable to those of the wild-type M7AC34 molecule. See FIG. 9A. Notably, this yield improvement was accompanied by high neutralizing activity and ADCC activity. See FIG. 9B and 9C.

Example 10

Virus inactivating activity of Fc fusion protein derivatives

To assess the irreversible inactivation of HIV induced by Fc fusion protein derivatives of the invention, highly infectious BaL HIV virus material was incubated in DMEM culture medium with serial dilutions (concentration range: 0.7 μg/mL to 0.7 pg/mL) of fusion fusion protein derivatives. Fc of the protein according to the invention (concentration range: from 1 μg/ml to 1 pg/ml). After 1 hour incubation at 37° C., samples were diluted with DMEM and viruses and soluble proteins were separated by adding the LentiX Concentrator reagent (Takara/Clontech Laboratories, Inc., Mountain View, CA, USA). The mixture was incubated for 30 minutes at 4°C and then centrifuged at 1500 g for 45 minutes at 4°C. The supernatants were removed and the virus pellets were resuspended in DMEM. The infectivity of the treated viruses was analyzed by titration in TZM-bl cells. See Li M, et al., J. Virol. 2005; 79:10108-10125. For each preparation, a TCID ₅₀ value was calculated to assess the residual infectivity of the treated viruses.

Example 11

AAV mediated expression of Fc fusion protein derivatives in vivo

Mice NSG (Jackson Laboratory, Bar Harbor, ME, USA) were kept under conditions of crossing between sibs and the absence of specific pathogens (SPF).

To induce stable expression of Fc fusion protein derivatives in these animals, their sequences were cloned into AAV8 expression plasmids (CBATEG, Universitat Autonoma de Barcelona, Barcelona, ES). 1×10 ¹¹ virus particles were diluted in 40 μl of buffer containing 100 mm sodium citrate, 10 mm Tris, pH 8, and injected into the calf muscle of mice at the age of eight weeks. At the same time, mice were humanized by intraperitoneal injection of 10 million peripheral blood mononuclear cells (PBMCs) isolated from healthy humans. Two weeks later, mice were infected by intraperitoneal injection of 10,000 TCDI ₅₀ NL4-3 HIV isolate. Blood samples were collected weekly and analyzed for CD4+ and CD8+ T cell counts by flow cytometry using Perfect Count particles (Cytognos SL, Salamanca ES) in combination with the following antibodies to human antigens: CD45-V450, CD3-APC/Cy7, CD4- APC, CD8-V500, CD14-PerCP/Cy5.5, CD56-PE, CD16-Fitc, and anti-mouse CD45-PE/Cy7. Samples were analyzed using an LSR II flow cytometer (BD Biosciences Corp., Franklin Lakes, NJ, USA). The samples were also tested for levels of Fc fusion protein derivatives using the ELISA approach described above. Mice were sacrificed 3 weeks after infection and blood and tissue samples were taken. Samples were analyzed by flow cytometry and viral load and total HIV DNA were determined by qPCR.

Example 12

Protocol for passive immunization with plasmid vectors

Mice NSG (Jackson Laboratory, Bar Harbor, ME, USA) were kept under conditions of crossing between sibs and the absence of specific pathogens (SPF).

Plasmids pM5A16T20 and pM7A16LLC34 were generated under endotoxin-free conditions using EndoFree Plasmid kits (Qiagen NV, Venlo, NL). For transient expression of Fc fusion protein derivatives in vivo, plasmids were administered to NGS mice via intramuscular or intravenous injections. Following plasmid administration, blood samples were collected weekly and levels of Fc fusion protein derivatives were examined using the ELISA approach described above. Mice were sacrificed 4 weeks after plasmid administration, and blood and tissue samples were taken and levels of Fc fusion protein derivatives and anti-idiotypic antibodies were analyzed.

Example 13

Activity in vivo

Fc fusion protein derivatives were generated in high volume HEK-293T cell cultures by transient transfection. The recombinant protein was purified by loading the supernatants onto CaptureSelect FcXL Affinity Matrix columns (Thermo Fisher Scientific, Waltham, MA, USA) and eluting the bound protein with pH 3.5 glycine buffer. After pH neutralization, dialysis and concentration, a high purity material was obtained at a concentration of 5 mg/mL.

Immunocompromised NSG mice were humanized by intraperitoneal injection of 10 million PBMC isolated from healthy humans. Two weeks later, mice were infected by intraperitoneal injection of 100 TCID ₅₀ NL4-3 HIV isolate. 24 hours prior to infection, mice were injected with 1 mg of purified Fc fusion protein derivative in 200 μl or the same volume of PBS via intraperitoneal injection. Blood samples were collected in the first and second weeks after infection by maxillary vein puncture and processed to obtain plasma samples. See FIG. 10A. In plasma samples, viremia levels were examined using Abbott Real Time HIV-1 (Abbott Laboratories, Abbott Park, IL, USA), while productive infection of blood cells and splenocytes was analyzed by assessing the percentage of HIV-1 GAG+ cells. Molecules M20A16_4LLC34 and M21A16_4LLC34 maintained the percentage of infected cells below the detection limit two weeks after infection. In contrast, in the control group, the median value of GAG-positive cells was 3.3%.

Example 14

HIV Env Glycoprotein Detection

To evaluate the ability of the Fc fusion protein derivatives of the invention to identify HIV proteins in a sample, MOLT cells chronically infected with NL4-3 or BaL HIV isolates were incubated with increasing amounts of the Fc fusion protein derivatives. Molecules bound to these cells were detected with a mouse anti-human IgG antibody coupled to fluorochrome phycoerythrin (PE/Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and analyzed by flow cytometry on an LSRII BD Biosciences flow cytometer. Corp., Franklin Lakes, NJ, USA).

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SEQUENCE LISTING

<110> AlbaJuna Therapeutics, S.L.

Carrillo Molina, Jorge

Clotet Sala, Bonaventura

Blanco Arbues, Julian M.

<120> Fc-fusion protein derivatives with high dual HIV antiviral and

immunomodulatory activity

<130> P1607WO

<150> US62/504411

<151> 2017-05-10

<160> 66

<170>PatentIn version 3.5

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Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 14

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A12 mutation set

(i.e. point mutations F243L, R292P and Y300L)

<400> 14

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 15

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the A14 mutation set

(i.e. point mutations F243L, R292P and P396L)

<400> 15

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 16

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the A16 mutation set

(i.e. point mutations F243L, R292P, Y300L and P396L)

<400> 16

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 17

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the A18 mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L and V305I)

<400> 17

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Ile Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 18

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the A41 mutation set

(i.e. point mutations S298A, E333A and K334A)

<400> 18

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 19

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the LL mutation set

(i.e. point mutations M428L and N434S)

<400> 19

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 20

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying the AM+LL mutation set

(i.e. point mutations G236A, S239D, A330L, I332E, M428L and N434S)

<400> 20

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Ala Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Leu Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 21

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A12+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, M428L and N434S)

<400> 21

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 22

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A14+LL mutation set

(i.e. point mutations F243L, R292P, P396L, M428L and N434S)

<400> 22

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 23

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A16+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L, M428L and N434S)

<400> 23

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 24

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A18+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L, V305I, M428L

and N434S)

<400> 24

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Ile Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 25

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Human IgG1 Fc region carrying A41+LL mutation set

(i.e. point mutations S298A, E333A, K334A, M428L and N434S)

<400> 25

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 26

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_1 and LL mutations

(i.e. point mutations S239D, F243L, R292P, Y300L, P396L, M428L

and N434S)

<400> 26

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 27

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_2 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, K322A, P396L, M428L

and N434S)

<400> 27

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 28

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_3 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, I332E, P396L, M428L

and N434S)

<400> 28

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 29

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_4 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, K322A, I332E, P396L,

M428L and N434S)

<400> 29

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 30

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_5 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, E333A, K334A, P396L,

M428L and N434S)

<400> 30

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 31

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A16_6 and LL mutations

(i.e. point mutations S239D, F243L, R292P, Y300L, K322A, I332E,

P396L, M428L and N434S)

<400> 31

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Leu Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Glu Glu Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Leu Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 32

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of A41_1 and LL mutations

(i.e. point mutations S239D, F243L, R292P, S298A, E333A, K334A,

M428L and N434S)

<400> 32

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 33

<211> 232

<212> PRT

<213> Human IgG1

<220>

<221> PEPTIDE

<222> (1)..(232)

<223> Fc region of human IgG1 carrying a set of mutations A41, A16 and LL

(i.e. point mutations F243L, R292P, S298A, E333A, K334A, M428L and

N434S)

<400> 33

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Leu Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Pro Glu Glu Gln

65 70 75 80

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 34

<211> 300

<212> DNA

<213> Human CD4 receptor

<220>

<221> gene

<222> (1)..(300)

<223> Human CD4 receptor D1 domain

<400> 34

aagaaagtgg tgctgggcaa gaaaggcgac accgtggaac tgacctgcac cgccagccag 60

aagaagtcca tccagttcca ctggaagaac agcaaccaga tcaagatcct gggcaaccag 120

ggcagcttcc tgaccaaggg ccccagcaag ctgaacgaca gagccgactc tcggcggagc 180

ctgtgggacc agggcaattt cccactgatc atcaagaacc tgaagatcga ggacagcgac 240

acctacatct gcgaggtgga agatcagaaa gaagaggtgc agctgctggt gttcggcctg 300

<210> 35

<211> 234

<212> DNA

<213> Human CD4 receptor

<220>

<221> gene

<222> (1)..(234)

<223> D2 domain of the human CD4 receptor

<400> 35

accgccaact ccgacaccca tctgctgcag ggccagagcc tgaccctgac actggaaagc 60

cctccaggca gcagccccag cgtgcagtgt agaagcccca gaggcaagaa catccagggc 120

ggcaagaccc tgagcgtgtc ccagctggaa ctgcaggata gcggcacctg gacctgtacc 180

gtgctgcaga accagaaaaa ggtggaattc aagatcgaca tcgtggtgct agcc 234

<210> 36

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region

<400> 36

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gttcccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 37

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> flexible linker GGGGS

<220>

<221> gene

<222> (1)..(15)

<223> flexible linker GGGGS

<400> 37

ggggggggag gctcc 15

<210> 38

<211> 87

<212> DNA

<213> Human CCR5 receptor

<220>

<221> gene

<222> (1)..(87)

<223> N-terminal region of the human CCR5 receptor

<400> 38

accgattatc aggtgtccag ccccatctac gacatcaact actacaccag cgagccctgc 60

cagaaaatca acgtgaagca gatcgcc 87

<210> 39

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> linker peptide M19

<220>

<221> gene

<222> (1)..(126)

<223> linker peptide M19

<400> 39

ggtggcggag gtaccgctga ggcctggtac aatctgggca acgcctacta caagcaggggc 60

gactaccaga aggccatcga gtattatcag aaggcccttg agctggaccc caacaatgct 120

gaagct 126

<210> 40

<211> 111

<212> DNA

<213> Artificial Sequence

<220>

<223> linker peptide M20

<220>

<221> gene

<222> (1)..(111)

<223> linker peptide M20

<400> 40

ggtggcggag gtaccgctga agccgccgct aaagaagccg ctgcaaaaga ggctgccgcc 60

aaagaggcag ctgctaaaga agcagcagcc aaagaagccg ccgcaaaggc t 111

<210> 41

<211> 150

<212> DNA

<213> Artificial Sequence

<220>

<223> linker peptide M21

<220>

<221> gene

<222> (1)..(150)

<223> linker peptide M21

<400> 41

ggcggcggag gtaccgctgg atctgctgct ggaagcggag caggctctgc aggttctgca 60

gcaggatctg gtgcaggttc cgctggtagt gcagctggtt ctggcgctgg ctctgctggt 120

agcgctgcag gcagcggagc tgctggatcc 150

<210> 42

<211> 183

<212> DNA

<213> Artificial Sequence

<220>

<223> linker peptide M22

<220>

<221> gene

<222> (1)..(183)

<223> linker peptide M22

<400> 42

ggcggcggag gtaccggagc tggtgctggt ggcggaggaa tgtttggatc tggcggcgga 60

ggtggcggca caggatctac aggacctggc tacagcttcc ctcactacgg ctttccaacc 120

tacggcggca tcacatttca ccccggcacc accaagtcta acgccggcat gaagcacgga 180

tcc 183

<210> 43

<211> 108

<212> DNA

<213> Human immunodeficiency virus type 1

<220>

<221> gene

<222> (1)..(108)

<223> T-20 polypeptide

<400> 43

tacacaagcc tgatccacag cctgatcgag gaaagccaga accagcagga aaagaacgag 60

caggaactgc tggaactgga caagtgggcc atcctgtgga attggttt 108

<210> 44

<211> 102

<212> DNA

<213> Human immunodeficiency virus type 1

<220>

<221> gene

<222> (1)..(102)

<223> C34 polypeptide

<400> 44

tggatggaaa tggacagaga gatcaacaat tacaccagcc tgatccactc cctgatcgag 60

gaaagccaga accagcagga aaagaacgag caggaactgc tg 102

<210> 45

<211> 102

<212> DNA

<213> Human immunodeficiency virus type 2

<220>

<221> gene

<222> (1)..(102)

<223> EHO polypeptide

<400> 45

tggcagcagt gggaaagaca agtgcggttc ctggacgcca acatcaccaa gctgctggaa 60

gaggcccaga tccagcaaga gaagaatatg tacgagctgc ag 102

<210> 46

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying the AM mutation set

(i.e. G236A, S239D, A330L and I332E point mutations)

<400> 46

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

gccggacccg acgtgttcct gttcccccca aagcccaagg acaccctgat gatctcccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctctgcccga ggaaaagacc 360

atcagcaagg ccaagggcca gcccagggaa ccccaggtgt acacactgcc ccccagcaga 420

gatgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ttacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgaccgt ggacaagagc 600

agatggcagc agggcaacgt gttcagctgc tccgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 47

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A12 mutation set

(i.e. point mutations F243L, R292P and Y300L)

<400> 47

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 48

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the A14 mutation set

(i.e. point mutations F243L, R292P and P396L)

<400> 48

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

gccggacccg acgtgttcct gttcccccca aagcccaagg acaccctgat gatctcccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctctgcccga ggaaaagacc 360

atctccaagg ccaagggcca gcccagggaa ccccaggtgt acacactgcc ccccagcaga 420

gatgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ttacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgaccgt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 49

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the A16 mutation set

(i.e. point mutations F243L, R292P, Y300L and P396L)

<400> 49

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 50

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the A18 mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L and V305I)

<400> 50

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccatc ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 51

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the A41 mutation set

(i.e. point mutations S298A, E333A and K334A)

<400> 51

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gttcccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacgcca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgccgccacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 52

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the LL mutation set

(i.e. point mutations M428L and N434S)

<400> 52

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gttcccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 53

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying the AM+LL mutation set

(i.e. point mutations G236A, S239D, A330L, I332E, M428L and N434S)

<400> 53

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

gccggacccg acgtgttcct gttcccccca aagcccaagg acaccctgat gatctcccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctctgcccga ggaaaagacc 360

atctccaagg ccaagggcca gcccagggaa ccccaggtgt acacactgcc ccccagcaga 420

gatgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ttacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgaccgt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 54

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A12+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, M428L and N434S)

<400> 54

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 55

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A14+LL mutation set

(i.e. point mutations F243L, R292P, P396L, M428L and N434S)

<400> 55

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

gccggacccg acgtgttcct gttcccccca aagcccaagg acaccctgat gatctcccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacagca cctaccgggt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctctgcccga ggaaaagacc 360

atctccaagg ccaagggcca gcccagggaa ccccaggtgt acacactgcc ccccagcaga 420

gatgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ttacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg actccgacgg ctcattcttc ctgtactcca agctgaccgt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 56

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A16+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L, M428L and N434S)

<400> 56

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 57

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A18+LL mutation set

(i.e. point mutations F243L, R292P, Y300L, P396L, V305I,

M428L and N434S)

<400> 57

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccatc ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 58

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Human IgG1 Fc region carrying A41+LL mutation set

(i.e. point mutations S298A, E333A, K334A, M428L and N434S)

<400> 58

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gttcccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcccag agaggaacag 240

tacaacgcca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgccgccacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 59

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_1 and LL mutations

(i.e. point mutations S239D, F243L, R292P, Y300L, P396L, M428L

and N434S)

<400> 59

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggacctg acgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 60

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_2 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, K322A, P396L, M428L

and N434S)

<400> 60

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcgc agtgtccaac aaggccctgc ctgcccccat cgagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 61

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_3 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, I332E, P396L, M428L and

N434S)

<400> 61

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccga ggagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 62

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_4 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, K322A, I332E, P396L,

M428L and N434S)

<400> 62

gagcctaaga gctgcgacaa gacccacacc tgtcctccat gtcctgctcc agaactgctc 60

ggcggacctt ccgtttttct gctgcctcct aagcctaagg acaccctgat gatctccaga 120

acacccgaag tgacctgcgt ggtggtggat gtgtcccacg aagatcctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga caaagcctcc tgaggaacag 240

tacaacagca ccctgagagt ggtgtccgtg ctgacagtgc tgcaccagga ttggctgaac 300

ggcaaagagt acaagtgcgc cgtgtccaac aaggccctgc cagctccaga ggaaaagacc 360

atctccaagg ccaagggcca gcctagagaa ccccaggtgt acacactgcc tccaagcaga 420

gatgagctga ccaagaacca ggtgtccctg acctgtctgg tcaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaatgga cagcccgaga acaactacaa gacaacccct 540

ctggtgctgg actccgacgg ctcattcttc ctgtactcca agctgaccgt ggacaagagc 600

agatggcagc agggcaacgt gttcagctgc tctgtgctgc acgaagccct gcacagccac 660

tacacccaga agtccctgtc tctgagccct ggaaaa 696

<210> 63

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_5 and LL mutations

(i.e. point mutations F243L, R292P, Y300L, E333A, K334A, P396L,

M428L and N434S)

<400> 63

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgccgccacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccca ggcaaa 696

<210> 64

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A16_6 and LL mutations

(i.e. point mutations S239D, F243L, R292P, Y300L, K322A, I332E,

P396L, M428L and N434S)

<400> 64

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggacctg acgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacagca ccctgcgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcgc agtgtccaac aaggccctgc ctgcccccga ggagaaaacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

ctggtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 65

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of A41_1 and LL mutations

(i.e. point mutations S239D, F243L, R292P, S298A, E333A, K334A,

M428L and N434S)

<400> 65

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacgcca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgccgccacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<210> 66

<211> 696

<212> DNA

<213> Human IgG1

<220>

<221> gene

<222> (1)..(696)

<223> Fc region of human IgG1 carrying a set of mutations A41, A16 and LL

(i.e. point mutations F243L, R292P, S298A, E333A, K334A, M428L

and N434S)

<400> 66

gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tgaactgctg 60

ggcggaccta gcgtgttcct gctgccccca aagcccaagg acaccctgat gatcagccgg 120

acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagccccc tgaggaacag 240

tacaacgcca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 300

ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgccgccacc 360

atcagcaagg ccaagggcca gccccgcgaa ccccaggtgt acacactgcc ccctagcagg 420

gacgagctga ccaagaacca ggtgtccctg acctgtctcg tgaagggctt ctacccctcc 480

gatatcgccg tggaatggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 540

cctgtgctgg acagcgacgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600

cggtggcagc agggcaacgt gttcagctgc agcgtgctgc acgaggccct gcactcccac 660

tacacccaga agtccctgag cctgagccct ggaaaa 696

<---

Claims (41)

1. A derivative of an Fc fusion protein directed against HIV, which contains in the direction from the N- to the C-terminus: (a) extracellular domains D1 and D2 of human CD4, (b) a human IgG1 Fc region containing at least one of the point mutations M428L or N434S, (c) a moiety selected from the group consisting of (1) a linker polypeptide with the sequence (GGGGS) _n , where 1≤n≤10, (2) SEQ ID NO:5-9, and (3) combinations thereof, and (d) a polypeptide derived from gp41 heptad repeat 2 (HR2) motifs. 2. The Fc fusion protein derivative of claim 1, wherein the human IgG1 Fc region further comprises at least one of G236A, S239D, A330L, or I332E point mutations. 3. An Fc fusion protein derivative according to claim 1 or 2, wherein the human IgG1 Fc region further comprises the F243L point mutation. 4. Derivative fusion with Fc protein according to any one of paragraphs. 1-3, where the Fc region of human IgG1 further contains the R292P point mutation. 5. Derivative fusion with Fc protein according to any one of paragraphs. 1-4, wherein the human IgG1 Fc region further contains the Y300L point mutation. 6. Derivative fusion with Fc protein according to any one of paragraphs. 1-5, where the Fc region of human IgG1 further contains the P396L point mutation. 7. Derivative fusion with Fc protein according to any one of paragraphs. 1-6, where the Fc region of human IgG1 further contains at least one of the point mutations S298A, E333A or K334A. 8. Derivative fusion with Fc protein according to any one of paragraphs. 1-7, where the Fc region of human IgG1 additionally contains a point mutation K322A or V305I. 9. Derivative fusion with Fc protein according to any one of paragraphs. 1-8, where grouping (c) has the sequence SEQ ID NO:5. 10. Derivative fusion with Fc protein according to any one of paragraphs. 1-9, wherein the gp41 derived polypeptide contains a T-20, C34 or EHO polypeptide. 11. An isolated nucleic acid encoding an Fc fusion protein derivative of claim 1. 12. Nucleic acid according to claim 11, which is codon-optimized. 13. Expression vector containing the nucleic acid according to paragraphs. 11, 12. 14. A host cell expressing a Fc fusion protein derivative according to any one of paragraphs. 1-10, where the specified host cell contains a derivative of the protein fused with Fc according to any one of paragraphs. 1-10, the nucleic acid according to claims 11, 12 or an expression vector according to claim 13. 15. Pharmaceutical composition for the treatment or prevention of HIV infection or AIDS, containing a therapeutically effective amount of an Fc fusion protein derivative according to any one of paragraphs. 1-10, the nucleic acid according to claims 11, 12, an expression vector according to claim 13, a host cell according to claim 14, or mixtures thereof. 16. Derivative fusion with Fc protein according to any one of paragraphs. 1-10 for use as a drug for the treatment or prevention of HIV infection or AIDS. 17. Derivative fusion with Fc protein according to any one of paragraphs. 1-10 for use in the treatment of HIV infection or AIDS. 18. Derivative fusion with Fc protein according to any one of paragraphs. 1-10 for use in the prevention of HIV infection or AIDS. 19. Nucleic acid according to paragraphs. 11, 12 for use as a medicament for the treatment or prevention of HIV infection or AIDS. 20. Nucleic acid according to paragraphs. 11, 12 for use in the treatment of HIV infection or AIDS. 21. Nucleic acid according to paragraphs. 11, 12 for use in the prevention of HIV infection or AIDS. 22. An expression vector according to claim 13 for use as a drug for the treatment or prevention of HIV infection or AIDS. 23. An expression vector according to claim 13 for use in the treatment of HIV infection or AIDS. 24. An expression vector according to claim 13 for use in the prevention of HIV infection or AIDS. 25. A host cell according to claim 14 for use as a drug for the treatment or prevention of HIV infection or AIDS. 26. A host cell according to claim 14 for use in the treatment of HIV infection or AIDS. 27. A host cell according to claim 14 for use in the prevention of HIV infection or AIDS. 28. Pharmaceutical composition according to claim 15 for use as a drug for the treatment or prevention of HIV infection or AIDS. 29. A pharmaceutical composition according to claim 15 for use in the treatment of HIV infection or AIDS. 30. Pharmaceutical composition according to claim 15 for use in the prevention of HIV infection or AIDS. 31. Combination for the treatment or prevention of HIV infection or AIDS, containing a derivative of the protein fused with Fc according to any one of paragraphs. 1-10, the nucleic acid according to claims 11, 12, an expression vector according to claim 13, a host cell according to claim 14, or a pharmaceutical composition according to claim 15, and at least one therapeutic agent. 32. The combination of claim 31 wherein the therapeutic agent is an antiretroviral agent against HIV. 33. The method of obtaining a derivative fused with Fc protein according to any one of paragraphs. 1-10, including the stage (a) cultivation of the host cell containing the nucleic acid according to paragraphs. 11, 12, (b) expression of the nucleic acid and (c) isolation of the antibody derivative from the host cell culture. 34. A method for inactivating HIV, comprising the step of bringing the virus into contact with a Fc fusion protein derivative according to any one of paragraphs. 1-10. 35. A method for inducing gp120 expression in an HIV-infected cell, comprising the step of bringing the infected cell into contact with a Fc fusion protein derivative according to any one of paragraphs. 1-10, nucleic acid according to claims. 11, 12, the expression vector of claim 13, the host cell of claim 14, the pharmaceutical composition of claim 15, the combination of claims 31, 32 or their mixture. 36. A method for determining HIV in a sample, comprising the steps of (a) bringing the sample into contact with a derivative of a protein fusion with Fc according to any one of paragraphs. 1-10 and (b) determining if the antibody derivative specifically binds to a molecule in the sample. 37. A set for the treatment or prevention of HIV infection or AIDS, including a derivative of a protein fused with Fc according to any one of paragraphs. 1-10, the nucleic acid according to claims 11, 12, the expression vector of claim 13, the host cell of claim 14, the pharmaceutical composition of claim 15, the combination of claims 31, 32 or a mixture thereof and instructions for use.

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