EA042588B1 - MODIFIED IMMUNOGLOBULINS WITH ALTERED FcRn BINDING - Google Patents

Abstract

The invention relates to compositions and methods for antibody-mediated therapy. In particular, the invention relates to modified immunoglobulins with altered half-life.

Description

Technical field

The present invention relates to compositions and methods for antibody-mediated therapy. In particular, the present invention relates to modified immunoglobulins with altered half-life.

State of the art

Antibodies are immunological proteins that bind to a specific antigen. In most mammals, including humans and mice, antibodies are composed of paired heavy and light polypeptide chains. Each chain is made up of individual immunoglobulin domains (Ig), and accordingly the generic term immunoglobulin is used for such proteins. Each chain consists of two distinct regions, called variable and constant regions. The sequences of the variable regions of the light and heavy chains differ significantly between antibodies and are responsible for binding to the target antigen. The constant regions are characterized by less sequence diversity and are responsible for binding to a number of natural proteins to trigger important biochemical events. There are five different classes of antibodies in humans, including IgA (which includes the IgA1 and IgA2 subclasses), IgD, IgE, IgG (which includes the IgG1, IgG2, IgG3, and IgG4 subclasses), and IgM. A distinguishing feature of these antibody classes is their constant regions, although there may be more subtle differences in the V region.

In FIG. 1 shows an IgG1 antibody used herein as an example to describe the general structural features of immunoglobulins. IgG antibodies are tetrameric proteins consisting of two heavy chains and two light chains. The heavy chain of IgG consists of four immunoglobulin domains connected in the direction from the N-terminus to the C-terminus in the order VH-CH1-CH2-CH3, which means heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2 and constant heavy chain domain 3 (also referred to as VH-Cy1-Cy2-Cy3, meaning heavy chain variable domain, constant γ domain 1, constant γ domain 2, and constant γ domain 3, respectively). The IgG light chain consists of two immunoglobulin domains connected in the direction from the N-terminus to the C-terminus in the order V _{L -CL} which means the light chain variable domain and the light chain constant domain, respectively.

The variable region of an antibody contains the antigen-binding determinants of the molecule and therefore determines the specificity of the antibody for its target antigen. The variable region is so named because it differs most significantly in sequence from other antibodies of the same class. The strongest sequence variability is observed in complementarity determining regions (CDRs). There are six CDRs in total, three each for the heavy chain and the light chain, which are designated CDR1 V _H , CDR2 V _H , CDR3 V _H , CDR1 V _L , CDR2 V _L and CDR3 V _L . The variable region outside the CDR is called the framework region (FR). Despite the fact that FRs are not as diverse as CDRs, the sequence of the framework regions can vary between different antibodies. In general, this characteristic antibody structure provides a stable scaffold (FR region) from which the immune system can explore a significant variety of antigen binding sites (CDRs), providing specificity for a wide range of antigens. A number of high resolution structures are available for various fragments of the variable regions of different organisms, some in unbound form and some in complex with an antigen. The sequence and structural features of antibody variable regions are well characterized (Morea et al., 1997, Biophys Chem. 68:9-16; Morea et al., 2000, Methods, 20:267-279, which are incorporated herein by reference in their entirety) , and conserved antibody features have allowed the development of a variety of antibody modification techniques (Maynard et al., 2000, Annu. Rev. Biomed. Eng. 2:339-376, which is incorporated herein by reference in its entirety). For example, it is possible to graft a CDR from one antibody, such as a mouse antibody, onto the framework region of another antibody, such as a human antibody. This technique, referred to in the art as humanization, produces less immunogenic antibody therapeutics from antibodies from non-human species. Variable region fragments may exist in the absence of other regions of the antibody, including, for example, an antigen binding fragment (Fab) containing V _h -Cy1 and V _H -C _L , a variable fragment (Fv) containing VH and V _L , single chain variable fragment (scFv) containing V _H and V _L connected together in the same chain, as well as many other fragments of the variable regions (Little et al., 2000, Immunol Today, 21:364-370, which is fully included in this document by reference).

The Fc region of an antibody interacts with a number of Fc receptors and ligands to provide a number of important functionalities referred to as effector functions.

In IgG, a site on the Fc region, containing amino acids from both the C _H 2 domain and the C _H 3 domain, mediates interaction with the neonatal Fc receptor (FcRn), a receptor that mediates the return of antibody after endocytosis from the endosome back into the circulation (Raghavan et al ., 1996, Annu Rev. Cell Dev. Biol 12:181-220; Ghetie et al., 2000, Annu Rev. Immunol 18:739-766, which are incorporated herein by reference in their entirety). This process, combined with filtering prevention

- 1 042588 by the kidneys due to the large size of the full-sized molecule, provides favorable half-life of antibodies in the blood serum for one to three weeks. Fc binding to FcRn also plays a key role in antibody transport within and between cells. The binding site for FcRn on Fc is also the site to which bacterial proteins A and G bind. Strong binding to these proteins is generally used as a means to purify antibodies using protein A or protein G carrier affinity chromatography during protein purification. Therefore, the quality of said region on Fc is important both for the clinical properties of the antibodies and for their purification.

FcRn plays a key role in several immunological and non-immunological processes as it mediates the transfer of maternal IgG to the fetus, regulates serum IgG and albumin stability, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation and cross-presentation. Unlike TRIM21, which is found in the cytosol, FcRn is a transmembrane receptor that resides predominantly in acidified endosomes and transports its ligands via recycling or transcytoplasmic pathways across layers of polarized cells. However, FcRn can also enhance the processing of immune complexes with subsequent presentation of antigenic peptides to T cells. A distinctive feature of the FcRn-IgG interaction is that FcRn binding to Fc IgG is strongly pH dependent, in particular binding occurs at acidic pH and no binding or release at neutral pH, which is a prerequisite for FcRn-mediated intracellular and extracellular transport. Modification of the FcRn-Fc IgG interaction resulted in the production of antibodies with a shorter or longer half-life from serum or with an altered ability to transport through cell layers. FcRn is widely expressed and as such is found at various cellular positions in both hematopoietic and non-hematopoietic cells.

There is a need for improved therapeutic IgGs that exhibit increased pH-dependent binding to FcRn.

Brief description of the invention

The present invention relates to compositions and methods for antibody-mediated therapy. In particular, the present invention relates to modified immunoglobulins with altered half-life.

For example, in some embodiments, the present invention relates to a composition comprising a therapeutic immunoglobulin with an altered binding affinity for FcRn, said immunoglobulin containing at least one mutation in the Fc region of the immunoglobulin. In some embodiments, the immunoglobulin is of the IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments, the immunoglobulin is a fusion of the Fc region of one of the immunoglobulin variants described herein.

In some embodiments of the present invention, the immunoglobulin contains mutations at one or more positions selected from, for example, 311, 434, 428, 438, or 435. In some embodiments of the present invention, the mutation is, for example:

IgG1-Q311R/N434W/M428E, IgG1-Q311R/N434W, IgG1-Q311R,

IgG1-N434W, IgG3(b)-Q311R/N434W/M428E, IgG3(b)-Q311R/N434W/M438E/R435H,

IgG1-M252S/Q311R/N434W/M428E, IgG1-Q311R/N434P/M428E,

IgG1-Q311R/N434W/M428D, IgG1-Q311R/N434W/M428E/H433K,

IgG1-L309K/Q311R/N434W/M428E, IgG1-L309R/Q311R/N434W/M428E,

IgG1-L309S/Q311R/N434W/M428E or IgG3(b)-Q311R/N434W/M428E/R435H (for example, the immunoglobulin constant region contains an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or sequences that are at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16).

According to additional embodiments of the present invention, a composition is provided, comprising: an immunoglobulin containing at least one mutation in the Fc region, wherein said immunoglobulin is characterized by altered binding to FcRn, and said mutation is selected, for example, from:

IgG1-Q311R/N434W/M428E, IgG1-Q311R/N434W, IgG1-Q311R,

IgG1-N434W, IgG3(b)-Q311R/N434W/M428E, IgG3(b)-Q311R/N434W/M438E/R435H,

IgG1-M252S/Q311R/N434W/M428E, IgG1-Q311R/N434P/M428E,

IgG1-Q311R/N434W/M428D, IgG1-Q311R/N434W/M428E/H433K, IgG1-L309K/Q311R/N434W/M428E, IgG1-L309R/Q311R/N434W/M428E, b)-Q311R/N434W/M428E/R435H.

In other embodiments, the present invention provides an IgG1 immunoglobulin with altered FcRn binding, said immunoglobulin containing mutations Q311R, N434W, and M428E.

- 2 042588

In some embodiments, the antibody binds to a target selected from, for example, a cancer marker, a cytokine, an infectious disease marker, or a growth factor.

According to other embodiments of the present invention, the use of the immunoglobulins described herein is provided for the treatment or prevention of a disease in a subject in need thereof. In some embodiments of the present invention, the disease is, for example, cancer, an autoimmune disease, an inflammatory disease, transplant rejection, or an infectious disease, but other diseases are specifically included within the scope of the present invention. In some embodiments, an antibody binds to a target selected from, for example, a cancer marker, a cytokine, an infectious disease marker, or a growth factor, but other targets are specifically included within the scope of the present invention.

In other embodiments, the present invention provides a method for treating or treating or preventing a disease in a subject, comprising providing the immunoglobulins described herein to a subject in need thereof.

In further embodiments, the present invention provides vaccine compositions comprising the immunoglobulin variants or Fc region hybrids described herein (eg, hybridized to an immunogen) and their use to induce an immune response in a subject.

Additional embodiments of the present invention are described in this document.

Brief description of the drawings

In FIG. 1 shows enzyme-linked immunosorbent assay (ELISA) results showing binding of a panel of Fc-modified h9C12 IgG1 variants to human FcRn at pH 6.0 (upper panel) and pH 7.4 (lower panel).

In FIG. Figure 2 shows sensograms obtained by the surface plasmon resonance (SPR) method, indicating the binding of a panel of Fc-modified variants of h9C12 IgG1 to human FcRn at pH 6.0 (7.8-4000.0 nM) and pH 7.4 (4000.0 nM). IgG1-M252Y/S254T/T256E was included as a control.

In FIG. 3 shows the amino acid sequence mapping of IgG1 variants. Amino acid sequence of the constant domain of the Fc-modified variants of IgG1.

In FIG. 4 shows sequences from FIG. 3.

In FIG. 5 shows the results of ELISA, indicating the binding of the panel

Fc-modified variants of h9C12 IgG1 and IgG3 with human FcRn at pH 6.0 and 7.4.

In FIG. 6 shows the SPR sensorgrams, indicating panel bonding

Fc-modified variants of h9C12 IgG1 with human FcRn at pH 6.0 (1000-15.6 nM) (A-D) and pH 7.4 (1000 nM) (E-F).

In FIG. Figure 7 shows in vivo half-life data (residual, %) of (A) h9C12 and (B) NIP IgG1 variants in Tg32-Alb'/' mice transgenic for the human FcRn gene. NIP = 4-hydroxy3-iodo-5-nitrophenylacetic acid.

In FIG. 8 shows the sequences of IgG variants.

Definitions

As used herein, the term antibody is used in its broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. . The term also includes antibody fragments containing an Fc region and fusion proteins that contain a region equivalent to the Fc region of an immunoglobulin.

The term antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , single chain antibody molecules (eg, scFv), diabodies, and polyspecific antibodies formed from antibody fragments.

As used herein, the term Fc region is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variants of Fc regions. In one embodiment of the present invention, the human IgG heavy chain Fc region is located from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region follows the EC numbering system, also referred to as the EC index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

In this document, the term effector functions is used in relation to those types of bio

- 3 042588 logical activities related to the Fc region of the antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody dependent cellular cytotoxicity (ADCC), phagocytosis; secretion of cytokines, immune complex-mediated uptake of antigen by antigen presenting cells, downregulation of cell surface receptors (eg, B cell receptor), and activation of B cells.

The term wild type, when used in relation to a protein, refers to proteins encoded by the genome of a cell, tissue, or organism, excluding a protein that is processed to produce synthetic proteins.

The term antigen-binding domain refers to the portion of an antigen-binding molecule that contains a region that specifically binds and is complementary to the portion or full-length antigen. In the case of a large antigen, the antigen-binding molecule can only bind to a certain part of the antigen, this part is called an epitope. An antigen binding domain may be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (V _L ) and an antibody heavy chain variable region (VH).

The term chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. For example, for chimeric antibodies, non-antigen binding components can be obtained from a wide variety of species, including primates such as chimpanzees and humans. Humanized antibodies are a particularly preferred form of chimeric antibodies.

The term antibody class refers to the type of constant domain or constant region that an antibody heavy chain contains. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA ₂ . The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, β, δ, ε, γ, and μ.

The term immunoglobulin Fc region equivalent region is intended to include naturally occurring allelic variants of an immunoglobulin Fc region, as well as variants containing alterations that introduce substitutions, additions, or deletions, but that do not significantly reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity) . For example, one or more amino acids can be removed from the N-terminus or C-terminus of the Fc region of an immunoglobulin without significant loss of biological function. Such options can be selected according to general rules known in the art so as to have minimal effect on activity (see, for example, Bowie, J. U. et al., Science, 247:1306-10 (1990)).

The term framework or FR refers to variable domain residues other than hypervariable region (HVR) residues (or CDRs). The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR are usually located in the following sequence in VH (or V _L ): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

As used herein, the terms full-length antibody, intact antibody, and whole antibody are used interchangeably to refer to an antibody having a structure that is substantially similar to that of a native antibody, or containing heavy chains that contain the Fc region defined herein.

The human consensus framework is a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the V _L or VH sequences of a human immunoglobulin are selected from a subset of variable domain sequences. Typically, a subgroup of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication, 91-3242, Bethesda Md. (1991), vols. 1-3. According to one embodiment of the present invention, in the case of VL, the subgroup is the kappa I subgroup described in Kabat et al., supra. In one embodiment of the present invention, in the case of VH, the subgroup is subgroup III as described in Kabat et al., supra.

A humanized antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments of the present invention, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or essentially all FRs correspond to the FRs of the human antibody. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A humanized form of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

As used herein, the term hypervariable region or HVR refers to each of

- 4 042588 regions of the variable domain of the antibody, the sequences of which are hypervariable and/or form structurally defined loops (hypervariable loops). Typically, native four-chain antibodies contain six HVRs; three in V _H (H1, H2, H3) and three in V _L (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from complementarity determining regions (CDRs), with CDRs having the highest sequence variability and/or being involved in antigen recognition. With the exception of CDR1 in V _H , CDRs typically contain amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used interchangeably herein to refer to portions of the variable region that form antigen-binding regions. This particular site has been described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlaps or subgroups of amino acid residues when compared to each other. However, the use of any definition to refer to the CDR of an antibody or variants thereof is included within the term defined and used herein. The corresponding amino acid residues, which include the CDR, as defined in each of the documents cited above, for comparison are presented in table. 1. The exact residue numbers that include a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which residues a particular CDR contains based on the amino acid sequence of an antibody's variable region.

The term variant and mutant, as applied to a polypeptide, refers to an amino acid sequence that differs in one or more amino acids from another, usually related, polypeptide. The variant may have conservative changes in which the substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains includes serine and threonine; a group of amino acids having amide-containing side chains includes asparagine and glutamine; a group of amino acids having aromatic side chains includes phenylalanine, tyrosine and tryptophan; unnatural amino acids such as p-aminophenylalanine; the group of amino acids having basic side chains includes lysine, arginine and histidine; and a group of amino acids containing side chains includes cysteine and methionine. Preferred groups of conservative amino acid substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Less commonly, a variant may contain non-conservative changes (eg, replacement of glycine with tryptophan). Similar minor changes may also include deletions or insertions of amino acids (ie additions) or both. Guidance for determining which and how many amino acid residues can be replaced, inserted, or removed without eliminating biological activity can be obtained using computer programs well known in the art, such as the DNAStar software. Variants can be tested in functional quantitative studies. Preferred variants have less than 10% and preferably less than 5% and even more preferably less than 2% changes (whether they are substitutions, deletions, etc.). To replace amino acids, the following nomenclature is used: the original amino acid, the position that replaces the amino acid. Accordingly, the replacement of lysine with alanine at position 573 is designated as K573A, and the replacement of lysine with proline at position 573 is designated as K573P. Multiple mutations are separated by adding marks (+) or /, for example Gly205Arg + Ser411Phe or G205R/S411F, which represent mutations at positions 205 and 411 with the replacement of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

The similarity between two amino acid sequences or between two nucleotide sequences is described by the identity parameter. For the purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is used in the Needle program of the EMBOSS package (EMBOSS: European Open Molecular Biology Software, Rice et al., 2000, Trends in Genetics, 16:276-277), preferably version 3.0.0 or later. Optional 11644.000-EP7 parameters used include a gap opening penalty of 10, a gap expansion penalty of 0.5, and an EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62). The result of the Needle program labeled as the longest identification (obtained using the -nobrief function) is used as the percent identity and is calculated as follows:

(Identical remainders of 100)/(Match length - total number of spaces in the match).

The expression amino acid position corresponding to a position in a reference sequence and a similar expression are intended to identify an amino acid residue that, in the primary or spatial structure, corresponds to a particular position in the reference sequence. One skilled in the art will appreciate that this can be done by comparing

- 5 042588 identification of a specific sequence with a reference sequence and identification of an amino acid residue that maps to a specific position in the reference sequence.

The expression Xnnn means the amino acid residue X located at the position corresponding to position nnn in the HSA, and the expression XnnnY means the replacement of any amino acid X located at the position corresponding to position nnn in the HSA with the amino acid residue Y.

As used herein, the term affinity refers to a measure of the strength of the binding between two members of a binding pair, such as an immunoglobulin and an FcRn. Kd is the dissociation constant and has the unit of molarity. The affinity constant is the reciprocal of the dissociation constant. Affinity constant is sometimes used as a general term to describe a chemical compound. It is a direct measure of binding energy. The natural logarithm of K is linearly related to the binding free energy, the Gibbs energy, through the equation AG ₀ = -RTLN(K), where R is the gas constant and the temperature is in degrees Kelvin. Affinity can be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR blocks (GE Healthcare).

As used herein, the term under conditions under which a specified subject develops an immune response refers to any qualitative or quantitative induction, generation and/or stimulation of an immune response (eg, innate or acquired).

As used herein, the term immune response refers to the response of a subject's immune system. For example, immune responses include, but are not limited to, a detectable change (e.g., increase) in Toll receptor activation, expression and/or secretion of a lymphokine (e.g., cytokine (e.g., Th1 or Th2 type cytokines) or chemokine), macrophage activation, activation dendritic cells, T cell activation (eg, CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (eg, antibody production and/or secretion). Additional examples of immune responses include the binding of an immunogen (eg, an antigen (eg, an immunogenic polypeptide)) to an MHC molecule and induction of a cytotoxic T lymphocyte (CTL) response, induction of a B cell response (eg, antibody production), and/or a helper T response. -lymphocytes, and/or a delayed-type hypersensitivity (DTH) response to the antigen from which the immunogenic polypeptide is derived, proliferation (e.g., increase in cell population) of cells of the immune system (e.g., T cells, B cells (e.g., at any stage of development (e.g., plasma cells) and enhancing antigen processing and presentation by antigen-presenting cells.The immune response can be targeted to immunogens that the subject's immune system recognizes as foreign (e.g., foreign antigens of microorganisms (e.g., pathogens) or self-antigens recognized as foreign). , it should be understood that, as used herein, the term immune response refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., T β-cell responses (eg, antigen-specific T cells) and non-specific cells of the immune system) and humoral immune responses (eg, B-cell mediated responses (eg, through the production and secretion of antibodies into blood plasma, lymph, and/or tissue fluids). The term immune response includes all aspects of the ability of a subject's immune system to respond to antigens and/or immunogens (eg, initial response to an immunogen (eg, pathogen) as well as acquired responses (eg, memory) that result from an adaptive immune response).

As used herein, the term immunity refers to protection from a disease (eg, prevention or mitigation (eg, suppression) of a sign, symptom, or condition of a disease) when exposed to a microorganism (eg, pathogen) capable of causing a disease. Immunity can be innate (eg, maladaptive (eg, non-acquired) immune responses that exist in the absence of prior exposure to an antigen) and/or acquired (eg, immune responses that are mediated by B and T cells following previous exposure to an antigen (eg, B- and T-cells, which show increased specificity and reactivity for the antigen)).

As used herein, the term immunogen refers to an agent (e.g., a microorganism (e.g., bacteria, virus, or fungus) and/or a part or component thereof (e.g., a protein antigen)) that is capable of inducing an immune response in a subject. According to some embodiments of the present invention, immunogens elicit immunity against an immunogen (eg, a microorganism (eg, pathogen or pathogenic product)).

The term test (test) compound refers to any chemical compound, pharmaceutical preparation, drug, and the like, which can be used to treat or prevent a disease, disease, ailment, or dysfunction of the body, or can otherwise change the physiological or cellular status of the sample. Test compounds include both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. Known therapeutic compound refers to therapeutics

- 6 042588 tic compound, which has been shown (for example, using animal studies or previous experience in humans) is effective in such treatment or prevention.

In this document, the term pattern is used in its broadest sense. In one embodiment, the term may refer to a tissue sample. In another embodiment, the term is intended to include a sample or culture obtained from any source, including biological. Biological samples can be obtained from animals (including humans) and include liquids, solids, tissues and gases. Biological samples include, but are not limited to, blood products such as plasma, serum, and the like. These examples should not be considered as limiting the types of samples applicable according to the present invention. A sample suspected of containing a human chromosome or sequences associated with a human chromosome may contain a cell, chromosomes isolated from a cell (e.g., at metaphase chromosome segregation), genomic DNA (in solution or bound to a solid support such as a Southern blot support). ), RNA (in solution or attached to a solid support such as a Northern blot support), cDNA (in solution or attached to a solid support), and the like. A sample suspected of containing a protein may contain a cell, a piece of tissue, an extract containing one or more proteins, and the like.

Detailed description of the invention

The present invention relates to compositions and methods for antibody-mediated therapy. In particular, the present invention relates to modified immunoglobulins with altered half-life.

An immunoglobulin molecule consists of two identical heavy and two identical light polypeptide chains held together by interchain disulfide bonds. Each individual light and heavy chains fold into regions containing approximately 110 amino acids, suggesting a conserved three-dimensional conformation. The light chain contains one variable region (called V _L ) and one constant region (C _L ), while the heavy chain contains one variable region (VH) and three constant regions (C _H 1, CH2 and C _H 3). Pairs of regions are associated with the formation of discrete structures. In particular, the variable regions of the light and heavy chains, V _L and V _H , are associated with the formation of the FV region, which contains the antigen-binding site.

The variable regions of both the heavy and light chains show significant variability in structure and amino acid composition across antibody molecules, while the constant regions show little variability. Each antibody recognizes and binds an antigen through a binding site defined by the binding of the heavy and light chain variable regions to the FV region. The VL light chain variable region and the VH heavy chain variable region of a particular antibody molecule have specific amino acid sequences that allow the antigen binding site to adopt a conformation that binds to an antigenic epitope recognized by that particular antibody.

Within the variable regions are areas in which the amino acid sequence is very different in different antibodies. Within each heavy and light chain are three of these so-called hypervariable regions or complementarity determining regions (CDRs). Three CDRs from the light chain and three CDRs from the corresponding heavy chain form an antigen-binding site.

The amino acid sequences of many immunoglobulin heavy and light chains have been determined and two important features of antibody molecules have been identified. First, each chain consists of a number of similar, but not identical, sequences, each approximately 110 amino acids long. Each of these repeats corresponds to a discrete, compactly folded region of the protein structure known as the protein domain. The light chain consists of two such immunoglobulin domains, while the heavy chain of an IgG antibody contains four domains.

The second important feature revealed by comparison of amino acid sequences is that the amino-terminal sequences of both heavy and light chains vary greatly between different antibodies. Sequence variability is limited to approximately the first 110 amino acids corresponding to the first domain, while the remaining domains are constant across immunoglobulin chains of the same isotype. The amino-terminal variable domain or the V domain of the heavy and light chains (V _H and V _L , respectively) together make up the V region of an antibody and confer its ability to bind to a specific antigen, while the constant domains (C-domains) of the heavy and light chains (CH and CL, respectively) constitute the C-region. Many of the heavy chain C domains are numbered from amino to carboxy, eg C _H 1 , CH 2 , etc.

The protein domains described above associate to form larger globular domains. Accordingly, a fully folded and assembled antibody molecule contains three globular portions of equal size connected by a flexible portion of the polypeptide chain known as the hinge region. Each arm of said Y-shaped structure is formed by complexing the light chain with the amino-terminal half of the heavy chain, while the Y backbone is formed by carboxy pairing

- 7 042588 end halves of two heavy chains. The relationship between the heavy and light chains is such that the VH and V _L domains are paired, as are the CH1 and _CL domains. The CH3 domains pair with each other, but the CH2 domains do not interact; the carbohydrate side chains attached to the CH2 domains lie between the two heavy chains. The two antigen-binding sites are formed by paired VH and _VL domains at the ends of two Y arms.

Proteolytic enzymes (proteases), which cleave polypeptide sequences, are used to analyze the structure of antibody molecules and to determine which parts of the molecule are responsible for its various functions. Limited protease digestion with papain cleaves antibody molecules into three fragments. The two fragments are identical and have antigen-binding activity. They are called Fab fragments, i.e. antigen-binding fragments (Fragment antigen binding). Fab fragments correspond to two identical arms of an antibody molecule that contain complete light chains paired with the VH and CH1 domains of the heavy chains. The other fragment does not have antigen-binding activity, but in the initial experiments it was found to crystallize easily and was therefore named the Fc fragment, ie. Fragment crystallizable. This fragment corresponds to the paired CH2 and CH3 domains and is part of the antibody molecule that interacts with effector molecules and cells. Functional differences between heavy chain isotypes are due mainly to the Fc fragment. The hinge region that connects the Fc and Fab portions of the antibody molecule is in fact a flexible cable that allows the two arms of the Fab to move independently, rather than a rigid hinge.

The present invention relates to compositions and methods for antibody-mediated immunity against viral targets. In particular, the present invention relates to modified immunoglobulins with antiviral activity.

The present invention provides mutated immunoglobulins containing one or more mutations, preferably in the Fc or hinge region of the immunoglobulin. According to preferred embodiments of the present invention, the mutation alters the function of the immunoglobulin in the desired manner. For example, some embodiments of the present invention provide the use of an immunoglobulin variant comprising at least one mutation in the Fc region to modulate FcRn binding to increase the in vivo half-life of an antibody. An extended half-life is preferred for therapeutic antibodies.

In some embodiments, the immunoglobulin exhibits altered binding (eg, increased or decreased binding affinity) to FcRn compared to an immunoglobulin that does not contain the mutation. In some embodiments, the immunoglobulin is of the IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments of the present invention, the immunoglobulin contains mutations at one or more positions selected from, for example, 311, 428, 434, 435, or 438, numbered according to the Kabat system. Reference is made throughout the text to the numbering system described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)). The referenced Kabat collections list many amino acid sequences for antibodies for each subclass and list the most frequently occurring amino acids for each residue position in that subclass. Kabat uses a method of assigning a residue number to each amino acid in a listed sequence, and this method of assigning residue numbers has become common in the art. In the present description, the Kabat numbering scheme is used. In the context of the present invention, to assign a residue number to an amino acid sequence of a candidate antibody that is not included in the Kabat collection, the following steps should be followed. In general, a candidate sequence is aligned with any immunoglobulin sequence or any consensus sequence listed in Kabat et al. The matching can be done manually or by computer using conventional computer programs; an example of such a program is the Align 2 program. Alignment can be facilitated by using some amino acid residues that are common to most Fab sequences. For example, each of the light and heavy chains typically contains two cysteines that have the same residue numbers; in the V _L domain, two cysteines typically have residue numbers 23 and 88, and in the V _H domain, two cysteine residues typically have residue numbers 22 and 92. Framework residues usually, but not always, have approximately the same residue numbers, however, CDRs will vary in size. For example, in the case of a CDR from a candidate sequence that is longer than the CDR in the sequence described in Kabat et al. to which it is matched, subscripts are typically added to the residue number to indicate the insertion of additional residues. For candidate sequences that, for example, match the sequence described in Kabat et al. for residues 34 and 36, but do not contain a residue between them to match with residue 35, the number 35 is simply not assigned to the residue.

In some embodiments of the invention described herein, constant chain variants (eg, Fc) are provided. The immunoglobulins described in

- 8 042588 herein are not limited to specific CDR or variable region sequences and can be generated or designed to recognize the desired antigen or epitope. Therefore, according to some embodiments of the present invention, the mutation is selected, for example, from:

IgG1-Q311R/N434W/M428E, IgG1-Q311R/N434W, IgG1-Q311R,

IgG1-N434W, IgG3(b)-Q311R/N434W/M428E, IgG3(b)-Q311R/N434W/M438E/R435H, IgG1-M252S/Q311R/N434W/M428E, IgG1-Q311R/N434P/M428E, IgG1-Q311R/N434P/M428E, IgG1-Q311R/N434P/M428E N434W/M428D, IgG1-Q311R/N434W/M428E/H433K,

IgG1-L309K/Q311R/N434W/M428E, IgG1-L309RQ311RN434W/M428E,

IgG1-L309S/Q311R/N434W/M428E or IgG3(b)-Q311R/N434W/M428E/R435H (for example, the immunoglobulin constant region contains an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or sequences that are at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16).

According to some specific embodiments of the present invention, the immunoglobulin contains mutations Q311R, N434W and M428E.

The mutations described above may be introduced into any suitable immunoglobulin molecule. According to some embodiments of the present invention, the immunoglobulin is a monoclonal antibody and preferably obtained using recombinant techniques.

Monoclonal antibodies to target antigens (e.g., a cell surface protein, e.g., receptors) are generated by a variety of techniques, including conventional monoclonal antibody production techniques such as the somatic cell hybridization techniques described by Kohler and Milstein, Nature, 256:495 (1975) . However, according to some embodiments of the present invention, somatic cell hybridization procedures are preferred, and other techniques for producing monoclonal antibodies (eg, viral or oncogenic transformation of B lymphocytes) are also included within the scope of the present invention.

The preferred animal system for producing hybridomas is the mouse system.

Obtaining hybridomas in mice is a common procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion procedures are also known.

Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice carrying the complete human immune system, but not the mouse system. Splenocytes from transgenic mice are immunized with an antigen of interest and used to generate hybridomas that secrete human mAbs with specific affinity for human protein epitopes. (See, for example, Wood et al., WO 91/00906; Kucherlapati et al., WO 91/10741; Lonberg et al., WO 92/03918; Kay et al., WO 92/03917 incorporated herein by reference), N. Lonberg et al., Nature, 368:856-859 (1994); L. L. Green et al., Nature Genet., 7:13-21 (1994); S. L. Morrison et al. , Proc. Nat. Acad. Sci. USA, 81:6851-6855 (1994); Bruggeman et al., Immunol., 7:33-40 (1993); Tuaillon et al., Proc. Nat. Acad. Sci. USA, 90:3720-3724 (1993) and Bruggernan et al., Eur J. Immunol., 21:1323-1326 (1991)).

Monoclonal antibodies can also be produced by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, called the combinatorial antibody library display method, has been developed to identify and isolate antibody fragments having a particular antigenic specificity, which can be used to generate monoclonal antibodies. (See, for example, Sastry et al., Proc. Nat. Acad. Sci. USA, 86:5728 (1989); Huse et al., Science, 246:1275 (1989) and Orlandi et al., Proc. Nat Acad. Sci. USA, 86:3833 (1989)). After immunizing the animal with the immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Known methods for obtaining a DNA sequence of variable regions of a diverse population of immunoglobulin molecules using a mixture of oligomeric primers and PCR. For example, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or backbone sequence 1 (FR1) and a primer for the conserved 3' constant region can be used to PCR amplify the heavy and light chain variable regions from a number of mouse antibodies. (See, for example, Larrick et al., Biotechniques, 11:152-156 (1991)). A similar strategy can also be used to amplify human heavy and light chain variable regions from human antibodies (see, for example, Larrick et al., Methods: Companion to Methods in Enzymology, 2:106-110 (1991)).

Chimeric mouse-human monoclonal antibodies can be produced using recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule is cleaved with restriction enzymes to remove the region encoding the mouse Fc and replaced with an equivalent portion of the gene encoding the human Fc constant region (see, e.g., Robinson et al., PCT/US86/02269, EP Application 184187, EP Application 171496,

- 9 042588

EP 173494, WO 86/01533, US 4816567, EP application 125023 (each document incorporated herein by reference in its entirety), Better et al., Science, 240:1041-1043 (1988); Liu et al., Proc. Nat. Acad. sci. USA 84:3439-3443 (1987); Liu et al., J. Immunol., 139:3521-3526 (1987); Sun et al., Proc. Nat. Acad. sci. USA 84:214-218 (1987); Nishimura et al., Canc. Res. 47:999-1005 (1987); Wood et al., Nature, 314:446-449 (1985) and Shaw et al., J. Natl. Cancer Inst., 80:1553-1559 (1988)).

The chimeric antibody can be further humanized by replacing Fv variable region sequences that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are presented by S.L. Morrison, Science, 229:1202-1207 (1985) and Oi et al., Bio. Techniques 4:214 (1986). Said methods include isolating, processing and expressing nucleic acid sequences that encode all or part of the Fv variable regions of an immunoglobulin from at least one heavy chain or light chain. Sources of a suitable nucleic acid are well known to those skilled in the art and, for example, can be obtained from 7E3, an anti-GPIIbIIIa antibody-producing hybridoma. The recombinant DNA encoding the chimeric antibody or fragment thereof can then be cloned into an appropriate expression vector.

Suitable humanized antibodies can alternatively be generated by CDR substitution (e.g., US 5225539 (which is incorporated herein by reference in its entirety), Jones et al., Nature, 321:552-525 (1986); Verhoeyan et al., Science, 239 :1534 (1988) and Beidler et al., J. Immunol., 141:4053 (1988)). All of the CDRs of a particular human antibody may be replaced with at least a portion of the CDRs from a non-human species, or only some of the CDRs may be replaced with CDRs from a non-human species. Only the amount of CDR needed to bind the humanized antibody to the Fc receptor needs to be changed.

An antibody can be humanized by any method that allows at least a portion of the CDR from a human antibody to be replaced by a CDR derived from an antibody from a non-human species. Human CDRs may be replaced by CDRs from a species other than human; using site-directed mutagenesis of the oligonucleotide.

Also included within the scope of the present invention are chimeric and humanized antibodies in which certain amino acids have been substituted, removed or added. In particular, preferred humanized antibodies contain amino acid substitutions in the framework region, such as those that improve antigen binding. For example, in a humanized antibody containing mouse CDRs, amino acids located in the human framework region can be replaced with amino acids located at the corresponding positions in the mouse antibody. It is known that in some cases, such substitutions improve the binding of humanized antibodies to the antigen.

In some embodiments, the monoclonal antibody is a mouse antibody or fragment thereof. According to other preferred embodiments of the present invention, the monoclonal antibody is a bovine antibody or a fragment thereof. For example, a mouse antibody can be produced using a hybridoma that includes a B cell derived from a transgenic mouse having a genome containing a heavy chain transgene and a light chain transgene fused to an immortalized cell. Antibodies can be of various isotypes, including, but not limited to, IgG (eg, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4); IgM; IgA1; IgA2; IgAsec; IgD and IgE. In some preferred embodiments of the present invention, the antibody is an IgG isotype. According to other preferred embodiments of the present invention, the antibody is an IgM isotype. Antibodies may be full-length (eg, IgG1, IgG2, IgG3, or IgG4 antibody) or may include only the antigen-binding portion (eg, Fab, F(ab') ₂ , Fv, or single chain Fv fragment).

According to preferred embodiments of the present invention, the immunoglobulin is a recombinant antibody (eg, a chimeric or humanized antibody), a subunit or antigen-binding fragment thereof (eg, contains a variable region or at least a complementarity determining region (CDR)).

In some embodiments of the present invention, the immunoglobulin is monovalent (eg, contains one pair of heavy and light chains or antigen-binding portions thereof). In other embodiments, the immunoglobulin is bivalent (eg, contains two pairs of heavy and light chains, or antigen-binding portions thereof).

In some embodiments, the present invention provides vaccine compositions comprising a variant of an immunoglobulin or a hybrid of its Fc and an immunogen. The present invention is not limited to the specific composition of the vaccine composition. In fact, the vaccine composition of the present invention may contain one or more different agents in addition to the fusion protein. Suitable agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelators, oils, salts, therapeutic agents, drugs, biologically active substances, antibacterial

- 10 042588 agents and antimicrobial agents (eg antibiotics, antivirals, etc.). According to some embodiments of the present invention, the vaccine composition containing the fusion protein contains an agent and/or cofactor that enhances the ability of the immunogen to induce an immune response (eg, an adjuvant). According to some preferred embodiments of the present invention, the presence of one or more cofactors or agents reduces the amount of immunogen required to induce an immune response (eg, protective immune response (eg, protective immunization)). According to some embodiments of the present invention, the presence of one or more cofactors or agents can be used to shift the immune response to a predominantly cellular (eg, T cell mediated) or humoral (eg, antibody mediated) immune response. The present invention is not limited by the type of cofactor or agent used in the therapeutic agent of the present invention.

Adjuvants are described in general terms in Vaccine Design the Subunit and Adjuvant Approach, ed. by Powell and Newman, Plenum Press, New York, 1995. The present invention is not limited by the type of adjuvant used (eg, for use in a composition (eg, pharmaceutical composition)). For example, in some embodiments of the present invention, suitable adjuvants include an aluminum salt such as aluminum hydroxide gel (alum) or aluminum phosphate. In some embodiments of the present invention, the adjuvant may be a calcium, iron, or zinc salt, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.

In general, the immune response to an antigen is produced as a result of the interaction of the antigen with the cells of the immune system. Immune responses can be broadly divided into two categories: humoral and cell-mediated immune responses (eg, historically characterized by antibody and cellular effector defense mechanisms, respectively). These response categories have been termed Th1 type (cell mediated response) and Th2 type (humoral response) immune responses.

Stimulation of an immune response may be the result of a direct or indirect response of a cell or component of the immune system to an intervention (eg exposure to an immunogen). Immune responses can be measured in many ways, including the activation, proliferation, or differentiation of cells of the immune system (eg, B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.); activation and suppression of the expression of markers and cytokines; stimulation of IgA, IgM or IgG titers; splenomegaly (including increased cellularity of the spleen); hyperplasia and mixed cellular infiltrates in various organs. Other responses, cells and components of the immune system that can be assessed for immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments of the present invention, the compositions and methods of the present invention induce the expression and secretion of cytokines (e.g., macrophages, dendritic cells and CD4+ T cells). Modulation of the expression of a particular cytokine may occur locally or systemically. It is known that cytokine profiles can determine the regulatory and effector functions of T cells in immune responses. According to some embodiments of the present invention, Th1-type cytokines can be induced, and therefore, the immunostimulatory compositions of the present invention can stimulate an antigen-specific Th1-type immune response, including cytotoxic T cells (for example, thereby avoiding unwanted Th2-type immune responses (for example, production of Th2-type cytokines (eg, IL-13) involved in increasing the severity of the disease (eg, induction of mucus production by IL-13))).

Cytokines play a role in directing the response of T cells. Helper (CD4+) T cells direct the mammalian immune response by producing soluble factors that act on other cells of the immune system, including B cells and other T cells. Most mature CD4+ T helper cells express one of two cytokine profiles: Th1 or Th2. Th1 type CD4+ T cells secrete IL-2, IL-3, IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. Th1-type cytokines stimulate cell-mediated immunity and humoral immunity, which is characterized by an immunoglobulin class switch to IgG2a in mice and IgG1 in humans. Th1 responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Cytokines of the Th2 type primarily induce humoral immunity and induce class switching to IgG1 and IgE. Antibody isotypes associated with Th1 responses are generally capable of neutralization and opsonization, while those associated with Th2 responses are more associated with allergic reactions.

Several factors have been shown to influence the shift in the immune response, predominantly in

- 11 042588 answer type Th1 or Th2. The most well-characterized regulators are cytokines. IL-12 and IFN-γ are positive regulators of Th1 and negative regulators of Th2. IL-12 stimulates the production of IFN-γ, and IFN-γ provides a positive feedback for IL-12. IL-4 and IL-10 appear to be important in establishing the Th2 cytokine profile and reducing the production of Th1 cytokines.

Accordingly, according to preferred embodiments of the present invention, there is provided a method for stimulating a Th1 immune response in a subject, comprising administering to the subject a composition comprising an immunogen. However, in other embodiments, the present invention provides a method of stimulating a Th2-type immune response in a subject (eg, if a balanced T-cell mediated response is desired) comprising administering to the subject a composition comprising an immunogen. According to further preferred embodiments of the present invention, adjuvants may be used (eg, may be administered in conjunction with a composition of the present invention) to shift the immune response predominantly towards a Th1 or Th2 type immune response. For example, adjuvants that induce Th2 responses or attenuate Th1 responses include, but are not limited to, alum, saponins, and SB-As4. Adjuvants that induce Th1 type responses include, but are not limited to, MPL, MDP, ISCOMS, IL-12, IFN-γ, and SB-AS2.

Several other types of Th1 type immunogens (eg, as an adjuvant) may be used in the compositions and methods of the present invention. These include, but are not limited to, the following immunogens. According to some embodiments of the present invention, monophosphoryl lipid A is used (for example, in particular, 3-de-O-acylated monophosphoryl lipid A (3D-MPL)). 3D-MPL is a known adjuvant manufactured by Ribi Immunochem, Montana, USA. It is often supplied as a mixture of chemical compounds of 3-de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. In some embodiments of the present invention, diphosphoryl lipid A and its 3-O-deacylated variants are used. Each of these immunogens can be purified and obtained by the methods described in GB 2122204B, which is incorporated herein by reference in its entirety. Other purified and synthetic lipopolysaccharides have been described (see, for example, US Patent No. 6005099 and EP 0729473, Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al. , 1987, Immunology, 60(1):141-6 and EP 0549074, each of which is incorporated herein by reference in its entirety). According to some embodiments of the present invention, 3D-MPL is used in the form of a particulate composition (eg, a composition containing particles with a diameter of less than 0.2 μm, described in EP 0689454, which is incorporated herein by reference in its entirety).

In some embodiments of the present invention, saponins are used as the immunogen (eg, Th1 type adjuvant) in the composition of the present invention. Saponins are well known adjuvants (see, for example, Lacaille-Dubois and Wagner (1996), Phytomedicine, vol. 2 p. 363-386). Examples of saponins include Quil A (obtained from the bark of the South American tree Quillaja Saponaria Molina) and fractions thereof (see, for example, US Pat. No. 5,057,540, Kensil, Crit. Rev. Ther. Drug. Carrier Syst., 1996, 12(1-2 ):1-55 and EP 0362279, each of which is incorporated herein by reference in its entirety). The present invention also contemplates that the hemolytic saponins QS7, QS17 and QS21 are suitable (Quil A fractions purified by HPLC; see e.g. Kensil et al. (1991), J. Immunology, 146, 431-437, patent US No. 5057540; WO 96/33739; WO 96/11711 and EP 0362279, each of which is fully incorporated herein by reference). The present invention also contemplates that combinations of QS21 and polysorbate or cyclodextrin are suitable (see, for example, WO 99/10008, which is incorporated herein by reference in its entirety).

In some embodiments of the present invention, an immunogenic oligonucleotide containing unmethylated CpG dinucleotides (CpG) is used as an adjuvant. CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG is known in the art as an adjuvant for both systemic and mucosal administration (see, for example, WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2):870876 ; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6 and US Pat. No. 20050238660, each of which is incorporated herein by reference in its entirety). For example, in some embodiments of the present invention, the immunostimulatory sequence is purine-purine-C-G-pyrimidine-pyrimidine; where the CG motif is unmethylated.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, according to some embodiments of the present invention, the presence of one or more CpG oligonucleotides activates various subgroups of immune cells, including natural killer cells (which produce IFN-γ) and macrophages. In some embodiments of the present invention, CpG oligonucleotides are added to a composition according to the present invention.

- 12 042588 shaving to induce an immune response. In some embodiments of the present invention, a free-form CpG solution is administered along with an antigen (e.g., present in the solution (see, for example, WO 96/02555, which is incorporated herein by reference). In some embodiments of the present invention, the CpG oligonucleotide is covalently conjugated with an antigen (see, for example, WO 98/16247, which is incorporated herein by reference) or prepared with the addition of a carrier such as aluminum hydroxide (see, for example, Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

In some embodiments of the present invention, adjuvants such as complete Freund's adjuvant and incomplete Freund's adjuvant, cytokines (e.g., interleukins (e.g., IL-2, IFN-γ, IL-4, etc.), macrophage colony stimulating factor, necrosis factor tumors, etc.), detoxified mutated variants of a bacterial ADP-ribosylating toxin such as cholera toxin (CT), pertussis toxin (PT), or heat-labile E. coli toxin (LT), in particular LT-K63 (where the wild-type amino acid at position 63 is replaced by lysine), LT-R72 (where the wild-type amino acid at position 72 is replaced by arginine), CT-S109 (where the wild-type amino acid at position 109 is replaced by serine), and PT-K9/G129 (where the wild-type amino acid at position 9 replaced by lysine and amino acid at position 129 replaced by glycine) (see, for example, WO 93/13202 and WO 92/19265, each of which is incorporated herein by reference), as well as other immunogenic substances (for example, those that increase the effectiveness compositions according to the present invention) are used with a composition containing an immunogen according to the present invention.

Additional examples of adjuvants that can be used in the present invention include poly-di(carboxylatephenoxy)phosphazene (PCPP polymer, Virus Research Institute, USA), lipopolysaccharide derivatives such as monophosphoryl lipid A (MPL, Ribi ImmunoChem Research, Inc., Hamilton, Montana , USA), muramyl dipeptide (MDP, Ribi) and threonyl-muramyl dipeptide (t-MDP, Ribi), OM-174 (glucosamine disaccharide related to lipid A, OM Pharma SA, Meyrin, Switzerland) and Leishmania extension factor (purified Leishmania protein, Corixa Corporation, Seattle, Washington, USA).

Adjuvants may be added to the composition containing the immunogen, or the adjuvant may be formulated with carriers, eg liposomes or metal salts (eg aluminum salts (eg aluminum hydroxide)), prior to combination or co-administration with the composition.

According to some variants of implementation of the present invention, the composition containing the immunogen contains one adjuvant. According to other embodiments of the present invention, the composition contains two or more adjuvants (see, for example, WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241 and WO 94 /00153, each of which is incorporated herein by reference in its entirety).

According to some embodiments of the present invention, the composition containing the immunogen contains one or more mucoadhesives (see, for example, US patent No. 20050281843, which is fully incorporated herein by reference). The present invention is not limited by the type of mucoadhesive used. In fact, according to the present invention, it is envisaged that a variety of different mucoadhesives can be used in the present invention, including, but not limited to, cross-linked polyacrylic acid derivatives (for example, carbopol and polycarbophil), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides (for example, alginate and chitosan), hydroxypropylmethylcellulose, lectins, fimbriae proteins and carboxymethylcellulose. Although an understanding of the mechanism is not necessary for the implementation of the present invention and the present invention is not limited to any particular mechanism of action, according to some embodiments of the present invention, the use of a mucoadhesive (for example, in a composition containing an immunogen) enhances the induction of an immune response in a subject ( for example, when administering a composition of the present invention) due to the increased duration and/or intensity of immunogen exposure experienced by the subject when the mucoadhesive is used compared to the duration and/or intensity of immunogen exposure without the use of the mucoadhesive.

In some embodiments of the present invention, the immunoglobulin and/or vaccine compositions are used in combination with appropriate salts and buffers to deliver the compositions to a subject. Buffers are also used when the composition is administered to a patient. Aqueous compositions contain an effective amount of the composition dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions are also referred to as inoculums.

The expression pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not cause unwanted, allergic or other adverse reactions when administered to an animal or human body. As used herein, the term "pharmaceutically acceptable carrier" includes any and all diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, and the like. Except when any normal environments or agent are incompatible with Vecto

- 13 042588 ramie or cells according to the present invention, their use in therapeutic compositions is included within the scope of the present invention. Additional active ingredients may also be included in the compositions.

According to some embodiments of the present invention, the active compositions include classical pharmaceutical preparations. The introduction of the mentioned compositions in accordance with the present invention is carried out using any conventional route of administration, provided that the target tissue is available for this route. Suitable routes of administration include oral, nasal, buccal, rectal, vaginal or topical routes. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

The compositions may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be obtained in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, such preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for administration by injection include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it may be preferable to include isotonic agents, for example sugars or sodium chloride. Sustained absorption of injectable compositions can be achieved by the use of absorption delaying agents in the compositions, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in a suitable solvent with various other ingredients listed above, if necessary, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are the vacuum drying and freeze-drying methods, which make it possible to obtain a powder of the active ingredient with any additional desired ingredient from its previously sterilized solution by filtration.

Once prepared, the compositions are administered in a manner compatible with the dosage form and in such amount as is therapeutically effective. The formulations can be readily administered in a variety of dosage forms such as injectable solutions, drug release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. Said specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermolysis fluid or injected at a suggested infusion site (see, for example, Remington's Pharmaceutical Sciences, ^15th Edition, pages 1035-1038 and 15701580). According to some embodiments of the present invention, the active particles or agents are formulated as a therapeutic mixture containing from about 0.0001 to 1.0 mg, or from about 0.001 to 0.1 mg, or from about 0.1 to 1.0 mg, or even about 10 mg per dose or so. Multiple doses may be administered.

Additional formulations that are suitable for other routes of administration include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, the suppositories soften, melt, or dissolve in the fluids of the cavity. In general, in the case of suppositories, conventional binding agents and carriers may include, for example, polyalkylene glycols or triglycerides; suitable suppositories may be prepared from mixtures containing the active ingredient in an amount ranging from 0.5% to 10%, preferably 1-2%. Vaginal suppositories or pessaries are usually spherical or ovoid in shape and weigh approximately 5 g each. Vaginal medications are available in various physical forms, such as

- 14 042588 creams, gels or liquids that deviate from the classic suppository concept.

Treatment, as used herein, means alleviating, in whole or in part, the symptoms associated with a disorder or disease, or slowing, inhibiting, or stopping the further progression or worsening of these symptoms, or preventing or preventing a disease or disorder in a subject at risk for developing a disease or disorder. Therefore, for example, treating a viral infection may include inhibiting or preventing viral replication in a subject, or reducing symptoms of a viral infection in a subject.

As used herein, a therapeutically effective amount of a compound of the present invention refers to an amount of a compound that alleviates, in whole or in part, the symptoms associated with a disorder or disease, or slows, inhibits, or halts the further progression or worsening of these symptoms, or prevents or prevents a disease or disease, or disorder in a subject at risk for developing a disease or disorder.

A subject is any animal that can benefit from the administration of a compound described herein. According to some embodiments of the present invention, the subject is a mammal, such as a human, primate, dog, cat, horse, cow, pig, rodent, such as, for example, a rat or mouse. Typically, the subject is a human.

A therapeutically effective amount of a compound described herein for use in the present invention may vary depending on the route of administration and dosage form. Effective amounts of the compounds of the present invention are typically in the range of about 0.001 to 100 mg/kg/day, and more typically in the range of about 0.05 to 10 mg/kg/day. Typically, the compound or compounds used in the present invention are selected to provide a formulation that has a high therapeutic index. The therapeutic index is the ratio of doses that cause toxic and therapeutic effects, which can be expressed as the ratio of LD ₅₀ and ED ₅₀ . LD ₅₀ is the lethal dose for 50% of the population, and ED ₅₀ is the therapeutically effective dose for 50% of the population. LD ₅₀ and ED ₅₀ are determined using conventional pharmaceutical procedures in animal cell cultures or experimental animals.

The present invention also provides pharmaceutical compositions and medicaments which can be prepared by combining one or more of the compounds described herein, their pharmaceutically acceptable salts, their stereoisomers, their tautomers or their solvates, with pharmaceutically acceptable carriers, adjuvants, binders agents, diluents or the like. for inhibiting or treating varieties of primary and/or metastatic prostate cancer. Said compositions may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions or solutions. Soluble compositions can be formulated for various routes of administration, eg oral, parenteral, topical, rectal, nasal, or for administration via an implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular injections. The following dosage forms are given by way of example and should not be construed as limiting the present invention.

For oral, buccal and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gel capsules and capsules are acceptable as solid dosage forms. They can be obtained, for example, by mixing one or more compounds according to the present invention or their pharmaceutically acceptable salts or tautomers with at least one additive, such as starch or other additive. Suitable additives include sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms may contain other ingredients to facilitate administration such as an inactive diluent or lubricants such as magnesium stearate or preservatives such as paraben or sorbic acid or antioxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent , binding agents, thickeners, buffers, sweeteners, flavoring agents or flavors. Tablets and pills may be further treated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions and solutions which may contain an inactive diluent such as water. Pharmaceutical formulations and drugs can be prepared as liquid suspensions or solutions using a sterile liquid such as, but not limited to, oil, water, alcohol, and combinations thereof. Pharmaceutically acceptable surfactants, suspending agents, emulsifying agents may be added if necessary.

- 15 042588 oral or parenteral administration.

As noted above, suspensions may include oils. Suitable oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suspension formulations may also contain fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may contain alcohols such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerin, and propylene glycol. Ethers such as, but not limited to, polyethylene glycol, petroleum hydrocarbons such as mineral oil and petroleum jelly can also be used in suspension formulations; and water.

Dosage forms for injection typically include aqueous suspensions or oily suspensions, which can be prepared using a suitable dispersing agent or wetting agent and a suspending agent. Forms for injection may be present in the liquid phase or in the form of a suspension, which is prepared using a solvent or diluent. Acceptable diluents or carriers include sterilized water, Ringer's solution, or isotonic aqueous saline. Alternatively, sterile oils may be used as solvents or suspending agents. Typically, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di-, or triglycerides.

In the case of administration by injection, the pharmaceutical composition and/or drug may be a powder suitable for reconstitution using an appropriate solution as described above. Examples of suitable powders include, but are not limited to, lyophilized powders, rotary evaporator-dried or spray-dried powders, amorphous powders, granules, precipitates, or particles. When administered by injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations thereof.

For rectal administration, the pharmaceutical compositions and medicaments may be in the form of a suppository, ointment, enema, tablet or cream to release the compound in the intestine, sigmoid flexure of the colon and/or rectum. Rectal suppositories are prepared by mixing one or more compounds of the present invention or pharmaceutically acceptable salts or tautomers of the compound with acceptable carriers, such as cocoa butter or polyethylene glycol, which is present in the solid phase at normal storage temperatures and is present in the liquid phase at temperatures that are suitable for release. drug inside the body, such as in the rectum. Oils can also be used to prepare soft gelatin type formulations and suppositories. Water, saline, aqueous dextrose, and solutions of related sugars and glycerols can be used to prepare suspension formulations, which may also contain suspending agents such as pectins, carbomers, methylcellulose, hydroxypropylcellulose or carboxymethylcellulose, as well as buffers and preservatives.

The compounds of the present invention can be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation administration include solutions, sprays, dry powders or aerosols containing any suitable solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers. and their combinations. Formulations for inhalation contain as excipients, for example, lactose, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Aqueous and non-aqueous aerosols are generally used to deliver the compounds of the present invention by inhalation.

Typically, an aqueous aerosol is prepared by preparing an aqueous solution or suspension of the compound with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers may vary depending on the requirements of the particular compound, but typically include non-ionic surfactants (tweens, pluronic acids, or polyethylene glycol), non-toxic proteins such as serum albumin, sorbitol esters, oleic acid, lecithin, amino acids, such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are usually obtained from isotonic solutions. A non-aqueous suspension (eg, in a fluorocarbon propellant) can also be used to deliver the compounds of the present invention.

Aerosols containing compounds for use in accordance with the present invention can be conveniently delivered using an inhaler, nebulizer, sealed package or aerosol dispenser and a suitable propellant, for example, including, but not limited to, pressurized dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, nitrogen , air or carbon dioxide. In the case of a pressurized aerosol, the dosage form may be controlled by a valve to deliver a metered amount. Capsules and cartridges, for example, made from gelatin for use in an inhaler or insufflator, can be prepared using a powder mixture of the compound and a suitable powder base such as lactose or starch.

- 16 042588

Delivery of aerosols according to the present invention using ultrasonic nebulizers is preferred because aerosol nebulizers minimize the effect of shear on the agent, which can lead to degradation of the compound.

In the case of nasal administration, the pharmaceutical formulations and medicaments may be a spray, nasal drops or aerosol containing a suitable solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surface -active substances, bioavailability modifiers and their combinations. For administration as nasal drops, the compounds may be formulated in oily solutions or as a gel. Any suitable propellant may be used to administer the nasal aerosol, including compressed air, nitrogen, carbon dioxide, or a low-boiling hydrocarbon-based solvent.

Dosage forms for topical (including buccal and sublingual) or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and with any preservatives or buffers that may be required. Powders and sprays can be prepared, for example, with auxiliary substances such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Transdermal patches have the added benefit of providing a controlled delivery of a compound of the present invention into the body. Suitable dosage forms can be obtained by dissolving or dispersing the agent in an appropriate medium. Absorption enhancers can also be used to increase the flux of a compound of the present invention through the skin. The rate of such flow can be controlled by a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

In addition to the examples of dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are therefore included within the scope of the present invention. Suitable excipients and carriers are described, for example, in the manual Remingtons Pharmaceutical Sciences Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

The compositions of the present invention can be designed to provide short acting, fast release, long acting and sustained release as described below. Therefore, pharmaceutical formulations can also be formulated for controlled release or slow release.

The compositions of the present invention may also contain, for example, micelles or liposomes or some other encapsulated form, or may be administered in sustained release form to allow for long term storage and/or delivery. Therefore, pharmaceutical formulations and drugs can be compressed into granules or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Suitable implants may use known inert materials such as silicones and biodegradable polymers.

Specific dosages may be adjusted depending on disease conditions, age, body weight, general health, sex and diet of the subject, dosing intervals, routes of administration, release rates, and drug combinations. Any of the above dosage forms containing effective amounts are within the normal range of experimentation and therefore are within the scope of the present invention.

In some embodiments, the present invention provides methods and compositions for the treatment and prevention of any number of disorders.

Therapeutic antibodies can be used in the treatment of, for example, cancer, an autoimmune disease, an infection (eg, a viral infection), and the like.

In some embodiments, the immunoglobulins described herein are administered in combination with another therapeutic agent (eg, an anticancer agent, an immunosuppressant, or an antiviral agent). In some embodiments of the present invention, treatment with an immunoglobulin described herein precedes or follows treatment with another agent at intervals ranging from minutes to weeks. In embodiments of the present invention in which the other agent and the immunoglobulin are administered separately to the cell, it is generally necessary to ensure that the time interval between each delivery is not significant so that several treatments can have a beneficial combined effect on the cell. In such cases, it is assumed

- 17 042588 cells are expected to come into contact with both modes of exposure for about 12-24 hours between them, and more preferably for about 6-12 hours between them, with the most preferred delay time being only about 12 hours. In some situations it may be desirable to significantly increase the period of time for treatment, when between the respective introductions passes from several days (from 2 to 7) to several weeks (from 1 to 8).

In some embodiments of the present invention, more than one administration of an immunotherapeutic composition of the present invention or other agent is used. Various combinations may be used where the immunoglobulin is A and the other agent is B, as shown below: A/B/A, B/A/B, B/B/A, A/A/B, B/A/A , A/B/B, B/B/B/A, B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B /A/A, B/A/B/A, B/A/A/B, B/B/B/A, A/A/A/B, B/A/A/A, A/B/A /A, A/A/B/A, A/B/B/B, B/A/B/B, B/B/A/B.

Other combinations are included within the scope of the present invention.

According to some embodiments of the present invention, one or more compounds of the present invention and an additional active agent are administered to a subject, more often a human, in sequences and for a time interval that allows the compound to act in conjunction with another agent to provide a greater benefit than the benefit obtained when administered in a different way. For example, additional active agents may be co-administered by co-preparation, administered simultaneously, or administered sequentially in any order at different time points; however, if these agents are not administered simultaneously, they should be administered one after the other rapidly enough so as to provide the desired therapeutic or prophylactic effect. According to some embodiments of the present invention, the compound and additional active agents act at time intervals that overlap. Each additional active agent may be administered individually, in any suitable form, and by any suitable route. According to other embodiments of the present invention, the compound is administered before, simultaneously with, or after the administration of additional active agents.

In various examples, the compound and additional active agents are administered at intervals of less than about 1 hour, at intervals of about 1 hour, at intervals of from about 1 to about 2 hours, at intervals of from about 2 to about 3 hours, at intervals of from about 3 to about 4 hours, at about 4 to about 5 hours, at about 5 to about 6 hours, at about 6 to about 7 hours, at about 7 to about 8 hours, at about 8 to about 9 hours, at about 9 to about 10 hours, at about 10 to about 11 hours, at about 11 to about 12 hours, at no more than 24 hours, or at most 48 hours. active agents are administered simultaneously. In other examples, the compound and additional active agents are administered simultaneously by co-preparation.

In other examples, the compound and additional active agents are administered about 2 to 4 days apart, about 4 to 6 days apart, about 1 week apart, about 1 to 2 weeks apart, or more than 2 weeks apart.

In some examples, a compound of the present invention and optionally additional active agents are cyclically administered to a subject. Cyclic therapy involves administering a first agent over a specified period of time, followed by administering a second agent and/or a third agent over a specified period of time, and repeating said sequential administration. Cyclic therapy can provide many benefits, such as reducing the development of resistance to one or more treatments, avoiding or reducing the side effects of one or more treatments, and/or increasing the efficacy of a treatment.

In other examples, one or more compounds according to some embodiments of the present invention and optionally an additional active agent is administered using a cycle lasting less than about 3 weeks, about once every two weeks, about once every 10 days, or about once a week. One cycle may include administering a compound of the present invention and optionally a second active agent by infusion over about 90 minutes of each cycle, about 1 hour of each cycle, about 45 minutes of each cycle, about 30 minutes of each cycle, or about 15 minutes of each cycle. Each cycle may include at least 1 week of dosing off, at least 2 weeks of dosing off, at least 3 weeks of dosing off. The number of cycles administered is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.

Treatments may be administered to a subject at the same time, ie. individual doses of the additional active agents are administered separately, but over such a time interval that the compound of the present invention can act in conjunction with the additional active agents. For example, one component can be administered once a week in combination with others.

- 18 042588 components that can be administered once every two weeks or once every three weeks. In other words, dosing regimens are carried out simultaneously, even if the therapeutic agents are not administered at the same time or on the same day.

Additional active agents may act additively or, more typically, synergistically with the compound(s) of the present invention. In one example, one or more compounds of the present invention are administered simultaneously with one or more additional active agents in the same pharmaceutical composition. In another example, one or more compounds of the present invention are administered simultaneously with one or more additional active agents in separate pharmaceutical compositions. In another example, one or more compounds of the present invention are administered before or after administration of the second active agent. Included within the scope of the present invention is the administration of the compound of the present invention and the second active agent using the same or different routes of administration, eg oral and parenteral routes. According to some embodiments of the present invention, if a compound of the present invention is administered concurrently with a second active agent that has the potential to cause side effects, including but not limited to toxicity, the second active agent may preferably be administered at a dose that is below the threshold dose that causes unwanted side effect.

Virtually any antigen can be targeted by IgG variants, including, but not limited to, proteins, subunits, domains, motifs, and/or epitopes belonging to the following list of target antigens, which includes soluble factors such as cytokines and membrane-bound factors , including transmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine receptor A1, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-1 antitrypsin, a-V/e-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC , ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), VACE, VACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2) , BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A , cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10 , CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13 , CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45 , CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF ,CTLA-4,CX3CL1,CX3CR1,CXCL,CXCL1,CXCL2,CXCL3,CXCL4,CXCL5,CXCL6,CXCL7,CXCL8,CXCL9,CXCL10,CXCL11,CXCL12,CXCL13,CXCL14,CXCL15,CXCL16,CXCR,CXCR1,CXCR3CR2 , CXCR4, CXCR5, CXCR6, tumor associated cytokeratin antigen, DAN, DCC, DcR3, DC-SIGN, DAF factor, des(1-3)-IGF-I (IGF-1 brain), Dhh, digoxin, DNAM-1, DNase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1 , ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkin, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP- 14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC- 1), GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-a2, GFR-a3, GITR, glucagon, GLUT-4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-sar or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), Herpes simplex virus (HSV) glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma-associated antigen (hMW-MAA) , HIV gp120, V3-loop gp 120 HIV IIIB, HLA, HLA-DR, HM1.24, HMFG REM, HRG, Hrk,

- 19 042588 human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-γ, Ig, IgA receptor , IgE, IGF, IGF-binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-IR, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL- 23, interferon (IFN)-a, IFN-β, IFN-γ, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin a2, integrin a3, integrin a4, integrin α4/β7, integrin a5 (aV), integrin a5/β1, integrin a5/b3, integrin a6, integrin β1, integrin β2, interferon-γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11 , kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF- 1, latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L- selectin, LT-a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, lymphotoxin-β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF , MDC, Mer, metalloproteases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-a, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP -12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Muc1 ), MUC18, Mueller inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, neprilysin, neurotrophin-3, -4 or -6, neuroturin, neuronal growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF , PCAD, P-cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (eg, T-receptor a/β cells), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-a, TGF-β, polyspecific antibody to TGF-β, TGF-β R1 (ALK-5), TGF-β RII, TGF-β RIIb, TGF-β RIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone , Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-a, TNF-a-β, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R3Apo-2, DR4 ), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID), TNFRSF10D (TRAIL R4DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1 ), TNFRSF12 (TWEAK RFN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 ( OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2 ), TNFSF11 (TRANCE/RANK ODF ligand, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (ligand LIGHT HVEM, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC , p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, ART1 ligand), TNFSF7 (CD27 ligand CD70 ), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF , Trk, TROP-2, TSG, TSLP, tumor associated antigen CA 125, tumor associated antigen expressing carbohydrate, related Lewis Y, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4 , VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XEDAR, XIAP, XPD and receptors for hormones and growth factors.

One skilled in the art will appreciate that the above list of targets refers not only to specific proteins and biomolecules, but to the biochemical pathway or pathways of which they are elements. For example, reference to CTLA-4 as a target antigen implies that ligands and receptors that are involved in the T cell costimulation pathway, including CTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands or receptors , which are associated with the specified

- 20 042588 proteins are also targets. Therefore, as used herein, a target refers not only to a particular biomolecule, but also to the set of proteins that interact with said target and to the elements of the biochemical pathway to which said target belongs. One skilled in the art will also appreciate that any of the aforementioned target antigens, the ligands or receptors that bind to them, or other elements of their respective biochemical pathway, can be operably linked to the Fc variants of the present invention to produce an Fc fusion protein. Thus, for example, an Fc fusion that targets EGFR can be constructed by operably linking the Fc variant to EGF, TGF-β, or any other ligand, whether or not discovered, that binds to EGFR. Accordingly, an Fc variant of the present invention may be operably linked to EGFR to produce an Fc fusion that binds to EGF, TGF-β, or any other ligand, whether or not discovered, that binds to EGFR. Accordingly, virtually any polypeptide, whether it is a ligand, a receptor, or some other protein or protein domain, including but not limited to the targets listed above, and proteins that are constituents of their respective biochemical pathways, can be operably linked. with Fc variants of the present invention to develop an Fc hybrid.

The choice of an appropriate antigen depends on the desired application. For the treatment of cancer, it is desirable to have a target whose expression is restricted to cancer cells. Some targets that are particularly suitable for antibody treatment include targets that have signaling functions. Other therapeutic antibodies exert their effect by blocking signaling through the receptor, by inhibiting the binding of the receptor and its cognate ligand. Another mechanism of action of therapeutic antibodies is the suppression of receptor activity. Other antibodies act in ways other than signaling through the target antigen. In some cases, antibodies directed against infectious agents are used.

In one embodiment of the present invention, the Fc variants of the present invention are incorporated into an anti-cytokine antibody. In another embodiment, the Fc variants are hybridized or conjugated to a cytokine. As used herein, the term cytokine is a general term for proteins released by one population of cells that act on another cell as intercellular mediators. For example, as described in Penichet et al., 2001, J. Immunol. Methods, 248:91-101, which is expressly incorporated herein by reference, cytokines can be hybridized to an antibody to provide a set of desired properties. Examples of suitable cytokines include lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; a Mueller inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TRO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β; insulin-like growth factors I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocytic macrophage CSF (GM-CSF) and granulocytic CSF (G-CSF); interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12; IL-15, tumor necrosis factor such as TNF-α or TNF-β; C5a; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active native sequence cytokine equivalents.

Cytokines and soluble targets, such as members of the TNF superfamily, are preferred targets for use in embodiments of the present invention. For example, antibodies targeting VEGF, CTLA-4, and TNF, or fragments thereof, are particularly suitable antibodies for use with Fc variants that increase binding to FcRn. Therapies directed against these targets are often used in the treatment of autoimmune diseases and require multiple injections over a long period of time. Accordingly, the longer serum half-lives and less frequent treatments provided by embodiments of the present invention are particularly preferred.

A number of antibodies and Fc hybrids that are approved for use, are being studied in clinical trials, or are being developed may benefit from the use of the Fc variants of the present invention. In this document, such antibodies and Fc hybrids are referred to as clinical products and candidates. Accordingly, in a preferred embodiment, the Fc polypeptides of the present invention can be used in a variety of clinical products and candidates.

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For example, a number of antibodies that target CD20 may benefit from the use of the Fc polypeptides of the present invention. For example, the Fc polypeptides of the present invention can be used in an antibody that is substantially similar to rituximab (Rituxan®, IDEC/Genentech/Roche) (see, for example, US Pat. No. 5,736,137), an anti-CD20 chimeric antibody approved for the treatment non-Hodgkin's lymphoma; in HuMax-CD20, an anti-CD20 antibody developed by Genmab, in an anti-CD20 antibody described in US Pat. PCT/US2003/040426 entitled Immunoglobulin Variants and Uses).

A number of antibodies that target members of the epidermal growth factor receptor family, including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), may benefit from the use of polypeptides. Fc according to the present invention. For example, the Fc polypeptides of the present invention can be used in an antibody that is substantially similar to trastuzumab (Herceptin®, Genentech) (see, for example, US Pat. No. 5,677,171), a humanized Her2/neu antibody approved for the treatment of cancer. mammary gland; pertuzumab (rhuMab-2C4, Omnitarg™), which is developed by Genentech; an anti-Her2 antibody described in US Pat. No. 4,753,894; cetuximabe (Erbitux®, Imclone) (US Pat. No. 4,943,533, PCT WO 96/40210), a chimeric anti-EGFR antibody that has been studied in clinical trials for the treatment of various cancers; ABX-EGF (US patent No. 6235883), which was developed by Abgenix-Immunex-Amgen; HuMax-EGFr (US 10/172317), which is developed by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5558864, Murthy et al., 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J. Cell Biochem. 35 (4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); ICR62 (Cancer Research Institute) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(13):129-46; Modjtahedi et al., 1993, Br. J. Cancer. 1993 , 67(2):247-53 Modjtahedi et al., 1996, Br. J. Cancer, 73(2):228-35 Modjtahedi et al., 2003, Int. J. Cancer, 105(2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (US Patent No. 5891996); No. 6506883; Mateo et al., 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Cancer Research Institute, Memorial Sloan Kettering) (Jungbluth et al., 2003, Proc. Natl. Acad. Sci. USA, 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Research Institute) (PCT WO 01/62931 A2); and SC100 (Scancell) (PCT WO 01/88138).

In another preferred embodiment, the Fc polypeptides of the present invention can be used in alemtuzumab (Campath®, Millenium), a humanized monoclonal antibody currently approved for the treatment of chronic B-cell lymphocytic leukemia. The Fc polypeptides of the present invention can be used in various Fc antibodies or fusions that are substantially the same as other clinical products and candidates, including, but not limited to, muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), anti-CD33 (p67 protein) developed by Celltech/Wyeth, alefacept (Amevive ®)), an Fc region fusion targeting LFA-3 developed by Biogen, abciximab (Reopro (ReoPro®)) developed by Centocor/Lilly, basiliximab (Simulect®) developed by Novartis, palivizumab (Synagris®) ), Med immun, Infiximab (Remicade®), Anti-TNF-α Antibody by Centocor, Adalimumab (Humira®), Anti-TNF-α Antibody by Abbott, Humicade™, Anti-TNF-α Antibody , developed by Celltech, etanercept (Enbrel®), an Fc fusion targeting TNF-α developed by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody developed by Abgenix, ABX-IL8, an anti-IL-8 antibody which developed by Abgenix, ABX-MA1, anti-MUC18 developed by Abgenix, pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 developed by Antisoma, Therex (R1550), anti-MUC1 developed by Antisoma, AngioMab (AS 1405) , which is developed by Antisoma, HuBC-1, which is developed by Antisoma, thiplatin (AS 1407), which is developed by Antisoma, antegren (Antegren®) (natalizumab), an antibody to α-4-β-1 (VLA-4) and α-4 -β-7 by Biogen, anti-VLA-1 mAb, anti-VLA-1 integrin by Biogen, mAb LTBR, anti-lymphotoxin-β receptor (LTBR) by Biogen, CAT-152, anti-TGF antibody -e2 by Cambridge Antibody Technology, J695, anti-IL-12 antibody by Cambridge Antibody Technology and Abbott, CAT-192, anti-TGF-β! which is developed by Cambridge Antibody Technology and Genzyme, CAT-213, an antibody to eotaxin 1 which is developed by Cambridge Antibody Technology, LymphoStat-B™, an antibody to Blys which is developed by Cambridge Antibody Technology and Human Genome Sciences Inc., mAb to TRAIL-R1, TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences, Inc., Avastin™ (bevacizumab, rhuMAb-VEGF), VEGF antibody developed by Genentech, HER receptor family antibody developed by Genentech, anti-Tissue factor (ATF) developed by Genentech, Xolar™ (omalizumab), an anti-IgE antibody developed by Genentech, Raptiva™ (efalizumab), an anti-CD11a antibody developed by

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Genentech and Hota, MLN-02 (formerly LDP-02) antibody developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, anti-CD4 antibody developed by Genmab, HuMax-IL 15, anti-IL-15 antibody developed by Genmab and Amgen, HuMax-inflam, which is developed by Genmab and Medarex, HuMax-Cancer, an anti-heparanase I antibody, which is developed by Genmab and Medarex, and Oxford GcoSciences, HuMax-lymphoma, which is developed by Genmab and Amgen, HuMax-TAC, which is developed by Genmab, IDEC- 131, anti-CD40L antibody developed by IDEC Pharmaceuticals, IDEC-151 (clenoliximab), anti-CD4 antibody developed by IDEC Pharmaceuticals, IDEC-114, anti-CD80 antibody developed by IDEC Pharmaceuticals, IDEC-152, anti-CD23 antibody developed by IDEC Pharmaceuticals, anti-microbial migration factor (MIF) antibody developed by IDEC Pharmaceuticals, BEC2, anti-idiotypic antibody developed by Imclone, IMC-1C11, anti-KDR antibody developed by Imclone, DC101, anti-flk-1 antibody developed by Imclone, anti-VE-cadherin antibody developed by Imclone, CEA-Cide™ (labetuzumab), anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics, LymphoCide™ (epratuzumab), anti-CD22 antibody developed by Immunomedics, AFP-Cide developed by Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide developed by Immunomedics, MDX-010, anti-CTLA4 antibody developed by Medarex, MDX-060, anti-CD30 antibody developed by Medarex, MDX-070 developed by Medarex, MDX-018 developed by Medarex, Osidem™ (IDM-1) and anti-Her2 antibody developed by Medarex and ImmunoDesigned Molecules, HuMax™-CD4, anti-CD4 antibody developed by Medarex and Genmab, HuMax-IL 15, antibody to IL-15 developed by Medarex and Genmab, CNTO 148, anti-TNF-α antibody developed by Medarex and Centocor/J&J, CNTO 1275 anti-cytokine antibody developed by Centocor/J&J, MOR101 and MOR102, antibodies to intracellular molecule Adhesion 1 (ICAM-1) (CD54) developed by MorphoSys, MOR201, Anti-Fibroblast Growth Factor 3 (FGFR-3) developed by MorphoSys, Nuvion® (visilizumab), Anti-CD3 developed by Protein Design Labs, HuZAF ™, anti-interferon-γ antibody by Protein Design Labs, anti-α5β1 integrin by Protein Design Labs, anti-IL-12 by Protein Design Labs, ING-1, anti-Ep-CAM by Xoma , and MLN01, an anti-β2 integrin antibody developed by Xoma, all of the above references in this paragraph are expressly incorporated herein by reference.

The Fc polypeptides of the present invention may be incorporated into the aforementioned clinical candidates and products, or into antibodies and Fc fusions that are substantially similar thereto. The Fc polypeptides of the present invention may be incorporated into variants of the aforementioned clinical candidates and products that are humanized, affinity matured, engineered, or otherwise modified.

According to one embodiment of the present invention, the Fc polypeptides of the present invention are used to treat autoimmune, inflammatory, or transplant indications. Target antigens and clinical products and candidates that are relevant to such diseases include, but are not limited to, antibodies to α4β7 integrin such as LDP-02, antibodies to β2 integrin such as LDP-01, antibodies to the complement component ( C5) such as 5G1.1, CD2 antibodies such as BTI-322, MEDI-507, CD3 antibodies such as OKT3, SMART CD3 antibodies, CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A, CD11a antibodies, CD14 antibodies such as IC14, CD18 antibodies, CD23 antibodies such as IDEC 152, CD25 antibodies such as Zenapax, CD40L antibodies such as 5c8, Antova, IDEC-131, antibodies anti-CD64 such as MDX-33, anti-CD80 such as IDEC-114, anti-CD147 such as ABX-CBL, anti-E-selectin such as CDP850, anti-gpIIb/IIIa such as reopro/abcixima , ICAM-3 antibodies such as ICM3, ICE antibodies such as VX-740, FcR1 antibodies such as MDX-33, IgE antibodies such as rhuMab-E25, IL-4 antibodies such as SB -240683, IL-5 antibodies such as SB-240563, SCH55700, IL-8 antibodies such as ABX-IL8, interferon-γ antibodies, anti-TNF antibodies (TNF, TNF-α, TNF-α), such as CDP571, CDP870, D2E7, infliximab, MAK-195F and anti-VLA-4 antibodies such as antegren.

Fc variants of the present invention, such as those with increased FcRn binding, can be used in TNF inhibitor molecules to provide improved properties. Suitable TNF inhibitor molecules include any molecule that inhibits the action of TNF-α in a mammal. Suitable examples include Fc hybrid, Enbrel® (etanercept) and Humira® (adalimumab) and Remicade® (infliximab) antibodies. Monoclonal antibodies (such as Remicade and Humira) engineered using the Fc variants of the present invention to increase FcFn binding may provide improved efficacy by increasing half-life.

In some embodiments of the present invention, antibodies against infectious diseases are used. Antibodies against eukaryotic cells include antibodies directed against yeast cells, including but not limited to Saccharomyces cerevisiae, Hansenula

- 23 042588 polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, Plasmodium falciparium, and Yarrowia lipolytica.

Also suitable are antibodies against additional fungal cells, including target antigens associated with Candida strains, including Candida glabrata, Candida albicans, C. krusei, C. lusitaniae, and C. maltosa, as well as Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces species. and Penicillium, among others.

Antibodies directed against protozoan-associated target antigens include, but are not limited to, antibodies associated with Trypanosoma, Leishmania species, including Leishmania donovanii; Plasmodium spp., Pneumocystis carinri, Cryptosporidium parvum, Giardia lamblia, Entamoeba histolytica and Cyclospora cayetanensis.

Antibodies against prokaryotic antigens are also suitable, including antibodies against suitable bacteria such as pathogenic and non-pathogenic prokaryotes, including but not limited to Bacillus, including Bacillus anthracis; Vibrio, eg V. cholerae; Escherichia, eg Enterotoxigenic E. coli, Shigella, eg S. dysenteriae; Salmonella, eg S. typhi; Mycobacterium, eg M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium, eg C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, eg S. aureus; Haemophilus, eg H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, eg Y. lamblia, Y. pestis, Pseudomonas, eg P. aeruginosa, P. putida; Chlamydia, eg C. trachomatis; Bordetella, eg B. pertussis; Treponema, eg T. palladium; B. anthracis, Y. pestis, Brucella spp., F. tularensis, B. mallei, B. pseudomallei, B. mallei, B. pseudomallei, C. botulinum, Salmonella spp., V. cholerae toxin B (SEB), E coli O157:H7, Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma sp. and so on.

In some aspects, the antibodies are directed against viral infections; such viruses include, but are not limited to, orthomyxoviruses (e.g. influenza virus), paramyxoviruses (e.g. respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus), parvoviruses, poxviruses (eg, variola virus, vaccinia virus), enteroviruses (eg, poliovirus, coxsackievirus), hepatitis viruses (including A, B, and C), herpesviruses (eg, herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein virus -Barr), rotaviruses, Norfolk viruses, hantavirus, arenavirus, rhabdovirus (for example, rabies virus), retroviruses (including HIV, HTLV-I and II), papovaviruses (for example, papillomavirus), polyomaviruses and picornaviruses, and the like.

experimental part

Example 1

Fc modification to improve binding to FcRn.

The neonatal Fc receptor (FcRn) regulates the three-week half-life of IgG in humans. Modification of IgG antibodies to improve pH-dependent binding to FcRn has resulted in exemplary antibodies with extended half-life/altered pharmacokinetics and cell transport properties.

Materials and methods.

Cell culture. The HEK293E cell line was maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin at 37° C. in a humidified 5% CO ₂ /95% O ₂ atmosphere in an incubator.

Preparation of soluble recombinant human FcRn. The construction of a eukaryotic vector pcDNA3 encoding a recombinant truncated wild-type human FcRn that contains a cDNA encoding three extracellular domains (α1-α3) fused to the C-terminus of a cDNA encoding glutathione-S-transferase (GST) from Schistosoma japonicum has been described. (Berntzen, G., et al., J. Immunol. Methods, 2005. 298(1-2), p. 93-104; Andersen, J. T., et al., FEBS J., 2008. 275(16), pp. 4097-110). The vector also contains cDNA encoding human β2-microglobulin and an origin of replication sequence from the Epstein-Barr virus. GST-labeled hFcRn was generated by transient transfection of human mesonephros 293E (HEK293E) cells with polyethyleneimine Max (Polysciences) and the receptor was purified from harvested supernatant using a GSTrap FF column as described (Berntzen et al., supra; Andersen et al. ., see above).

Monomeric His-tagged human FcRn was generated using a baculovirus vector expression system (Kim, JK, et al., FcRn. Eur. J. Immunol., 1999, 29(9), p. 2819-25). The stock virus suspension encoding a His-tagged human FcRn was donated by Dr. Sally Ward (University of Texas, Southwestern Medical Center, Dallas, Texas, USA). In general terms, human FcRn was purified using a HisTrap HP column supplied in Ni ²⁺ charged form (GE Healthcare). The column was pre-equilibrated using 1x phosphate-buffered saline (PBS) with the addition of 0.05% sodium azide, the pH of the supernatant was adjusted using 1x PBS, 0.05% sodium azide (pH 10.9) to pH 7.2 and then applied per HisTrap HP column with flow rate

- 24 042588 ml/min. The column was washed with 200 ml 1x PBS and then 50 ml 25 mM imidazole, 1x PBS (pH 7.3) and human FcRn was eluted with 250 mM imidazole, 1x PBS (pH 7.4). The collected protein was buffer exchanged for 1x PBS using Amicron Ultra-10 filters (Millipore) followed by isolation of the monomeric fraction. A HiLoad 26/600 Superdex 200 prep grade column (GE Healthcare) was used to isolate the monomer fraction before protein concentration using Amicon Ultra columns (Millipore) and storage at 4°C.

Construction and production of Fc-modified variants of IgG1. Vectors encoding humanized 9C12 IgG1 variants were based on the pLNOH2/pLNOk expression system (Norderhaug, L., et al., J. Immunol. Methods, 1997, 204(1), p. 77-87). In particular, genes encoding heavy (H) and light (L) chain variable (V) regions derived from the TC31-9C12.C9 hybridoma cell line (Bank of Hybridomas for Developmental Research, University of Iowa) (Varghese, R., et al ., J. Virol., 2004, 78(22), p. 12320-32) was synthesized as a cloning cassette flanked by restriction sites recognized by BsmI/BsiWI endonucleases. The gene fragments were then subcloned into pLNOH2- ^NIP hIgG1-WT-oriP and ^NIP pLNOk-oriP to obtain pLNOH2 ^-Hexon hIgG1-WT-oriP and ^Hexon pLNOk-oriP encoding the chimeric human H chain and L chain, respectively. The H chain encoding vector, pLNOH2- ^Hexon hIgG1-WT-oriP, was then used to generate h9C12 variants by replacing the CH ₂ and _CH 3 gene fragments with fragments containing the desired mutations. _CH 2 fragments were replaced using unique restriction sites recognized by AgeI and SfiI endonucleases, while CH3 fragments were replaced using SfiI and BamHI (all obtained from New England Biolabs). After co-transfection of H chain and L chain coding vectors into HEK293E cells using Lipofectamine 2000 (Life Technologies), h9C12 IgG1 variants were purified from the harvested supernatant using a CH1 specific CaptureSelect column (Life Technologies). Monomeric fractions were isolated by size exclusion chromatography using a Superdex 200 column (GE Healthcare). Protein integrity was confirmed by non-reducing SDS-PAGE gel electrophoresis (Life Technologies).

ELISA. 96-well plates (Nunc) were coated with recombinant AdV5 hexon (Abd Serotech) (diluted to 1 μg/ml in PBS) and incubated overnight at 4°C. The remaining surface area was blocked with PBS/4% skim milk (S) (Acumedia) before washing four times with PBS/0.005% tween-20 (T). Titrated amounts of h9C12 variants diluted in PBS/T/S were added to the wells and incubated for 1 hour at room temperature. After washing as described above, soluble GST-labeled human FcRn diluted in PBS/S/T was added to the wells and incubated for 1 hour at room temperature. After washing as described above, HRP-conjugated anti-GST antibody (1:8000) was diluted in added PBS/S/T (Rockland Immunochemicals, USA) followed by incubation for 1 h at room temperature. After washing as above, bound human FcRn was visualized by adding tetramethylbenzidine (TMB) solution (CalBiochem). The reaction was stopped by adding 100 μl of 1 M HCl and absorbance at 450 nm was recorded using a SUNRISE plate reader (TECAN). ELISA was performed using PBS with pH 6.0 or 7.4.

Surface plasmon resonance (SPR). A Biacore 3000 instrument (GE Healthcare) was used for all kinetic measurements. Humanized 9C12 IgG1 variants were immobilized by amine binding on CM5 chips according to the manufacturer's instructions. Binding was performed by injecting 1-2.5 μg/ml IgG1 variants dissolved in 10 mM sodium acetate, pH 4.5 (GE Healthcare). HBS-P buffer (0.01 M HEPES, 0.15 NaCl, 0.005% P20 surfactant, pH 7.4) was used as running and dilution buffer. Soluble monomeric human FcRn was then injected at various concentrations. Kinetic analysis was performed using BIAevaluation software and binding data were fitted using a simple first order (1:1) Langmuir biomolecular interaction model.

Results.

Fc-modified IgG antibodies with altered human FcRn binding are described herein as follows. The Q311R/N434W/M428E triple mutant variant exhibits improved pH-dependent binding, different from that of the published examples._______________________________________________________________

IgGl-Q311R/N434W/M428E IgGl-Q311R/N434W IgGl-Q311R IgGl-N434W IgG3(b)-Q311R/N434W/M428E IgG3(b)-Q311R/N434W/M438E/R435H IgGl-M252Y/S254T/T256E, also named YTE mutant/platform (Robbie, G.J., et al., Antimicrob Agents Chemother, 2013. 57(12): p. 6147-53; Dall'Acqua, W.F., P.A. Kiener, and H. Wu, J Biol Chern, 2006. 281(33): pp. 23514-24). Lead Candidate: AstraZeneca, included in as a standard.

- 25 042588

Table 1

Binding Kinetics of Human IgG1 Variants to Human FcRn Based on SPR Results

pH=6.0 IgGl variants Ka (10 ⁴ / ms) ^a Kd(10' ² /c) ^a KD (nM) ^a Change relatively wild type wild type 7.04±0.1 5.4±0.2 784.0 - M252Y/S254T/T256E 8.66±0.2 0.78±0.0 93.0 8.4 Q311R/N434W/M428E 12.2±0.2 0.50±0.0 38.5 20.3 Q311R/N434W 16.1±0.2 0.36±0.0 22.3 35.1 Q311R 8.3 NO, 1 5.72±0.2 686.9 1.1 N434W 21.7±0.2 0.32±0.1 14.8 52.9 pH=7.4 wild type No data No data No data - M252Y/S254T/T256E No data No data No data - Q311R/N434W/M428E No data No data No data - Q311R/N434W No data No data No data - Q311R No data No data No data - N434W No data No data 1980.0 -

Example 2

This example describes additional IgG1 and IgG3 variants with altered human FcRn binding.

Materials and methods.

Research under in vivo conditions. Mice of the Tg32-Alb ^-/ - line based on the genetic core C57BL/6J carry null FcRn HC (Fcgrt ^tm1Dcr ) and albumin (Alb ^em12Mvw ) alleles and express the hFcRn HC genomic transgene (FCGRT) under the control of the native hFcRn promoter. Mice line Tg32-Alb ^-/- (sex - females aged 7-9 weeks weighing from 17 to 27 g, 5 mice/group) received 5 mg/kg IgG1 variants by intraperitoneal injection. Blood samples (25 μl) were obtained from the retroorbital sinus 1, 3, 5, 7, 10, 12, 16, 19, 23, 30 and 37 days after injection. After sampling, blood was immediately mixed with 1 μl of 1% K3-EDTA to prevent coagulation and then centrifuged at 17,000xg for 5 min at 4°C. Plasma was isolated, diluted 1:10 in 50% glycerol/PBS and then stored at -20° C. until ELISA. In vivo studies were carried out at the Jackson Laboratory (Bar Harbor, Maine, USA).

Surface plasmon resonance. A Biacore T200 instrument (GE Healthcare) was used for all kinetic measurements. The h9C12 variants were immobilized by binding amines to CM5 chips according to the manufacturer's instructions. For all experiments, phosphate buffer (67 mM phosphate, 0.15 M NaCl, 0.005% Tween-20) at pH 6.0 or HBS-P buffer (0.1 M HEPES, 0. 15 NaCl, 0.005% surfactant P20) at pH 7.4 respectively. Recombinant human FcRn at various concentrations (1000.0-15.6 nM) was injected at a flow rate of 50 μl/min at 25°C. Kinetic analysis was performed using BIAevaluation software and binding data were fitted using a simple first order (1:1) Langmuir interaction model.

ELISA. Performed as described in example 1.

table 2

wild-type IgGl

IgGl-Q311R/N434W/M428E

IgGl-M252S/Q311R/N434W/M428E___________________________________________________

IgGl-Q311R/N434P/M428E

IgGl-Q311R/N434W/M428D_______________________________________________________________

IgGl-Q311R/N434W/M428E/H433K

IgGl-L309K/Q311R/N434W/M428E________________________________________________________

IgGl-L309R/Q311R/N434W/M428E

IgGl-L309S/Q311R/N434W/M428E________________________________________________________

IgG3(b)-Q311R/N434W/M428E/R435H___________________________________________________

Results.

The results are shown in FIG. 5-7 and in table. 3-5.

-

Claims (1)

Table 3 Binding of human h9C12 IgGl and IgG3 variants to human FcRn, as determined by ELISA h9C12 variants Change from wild type (ELISA) at pH=6.0 wild-type IgGl - IgGl-Q311R/N434W/M428E* 6 IgGl-M252S/Q311R/N434W/M428E 6 IgGl-Q311R/N434P/M428E 3 IgGl-Q311R/N434W/M428D 3 IgGl-Q311R/N434W/M428E/H433K 9 IgGl-L309K/Q311R/N434W/M428E 6 IgGl-L309R/Q311R/N434W/M428E 6 IgGl-L309S/Q311R/N434W/M428E 6 IgG3(b)-Q311R/N434W/M428E/R435H 3 Table 4 Binding Kinetics of Human IgG1 and IgG3 h9C12 Variants to Human FcRn Based on SPR Results pH=6.0 Variants h9C12 ka(10 ⁵ /ms) kd(10' ² /s) KD(hM) Change from wild type (KD) at pH=6.0 IgGl wild-type* 3.172 8.677 273 - IgGl-Q311R/N434W/M428E* 0.117 1.106 9.3 29.30 IgGl- M252S/Q311R/N434W/M428E He explored Not explored Not explored Not explored IgGl-Q311R/N434P/M428E 0.167 4.047 24.2 11.28 IgGl-Q311R/N434W/M428D 0.106 3.121 29.4 9.28 pH=7.4 IgGl wild-type* No data No data No data - IgGl-Q311R/N434W/M428E* No data No data No data - IgGl- M252S/Q311R/N434W/M428E Not explored Not explored Not explored - IgGl-Q311R/N434P/M428E No data No data No data - IgGl-Q311R/N434W/M428D No data No data No data - Table 5 Half-life (days) in mice. Tg32 line hemizygous for human FcRn IgGl variants IgGl specificity Path Dose (mg/kg) Half-life (days) (n=5) +/SD (days) mouse line wild-type IgGl h9C12 i/v 5 6.4 0.8 Hemizygous Tg32 mice IgGl- Q311R/N434W/M428E h9C12 i/v 5 7.5 2.0 Hemizygous Tg32 mice wild-type IgGl NIP i/v 5 8.4 2.5 Hemizygous Tg32 mice IgGl- NIP i/v 5 12.3 1.3 Hemizygous Q311R/N434W/M428E mice Tg32 All publications and patents mentioned in the description above are incorporated herein by reference. Various modifications and variations of the described method and system according to the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. While the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Accordingly, various modifications of the described methods for carrying out the present invention, which are obvious to those skilled in the art, are intended to be included within the scope of the following claims of the present invention. CLAIM 1. Composition for antibody-mediated therapy containing immunoglobulin in a therapeutically effective amount, where the specified immunoglobulin contains mutations Q311R, N434W and M428E, where the amino acid numbering corresponds to the EU numbering system, and the specified mutation increases the half-life of the specified immunoglobulin in blood serum compared to immunoglobulin that does not contain the specified mutation, and while the specified immunoglobulin is -