RU2412200C2 - Fc-VERSIONS WITH CHANGED BINDING WITH FcRn - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed are versions of polypeptides with Fc-segment from IgG, which possess increased binding FcRn due to introduction of mutation 308C, 308F, 308W, 308Y or modified binding with introduction of mutation 252Y/308F, 257L/308F, 257L/308Y, 257N/308Y, 279Q/308F, 279Y/308F, ^281S/308F, ^A281S/308Y, 284E/308F, 298A/308F/333A/334A, 308F/332E, 308F/311V, 308F/G385H, 308F/428L, and 308F/434Y. Described is antibody with said mutations in Fc region, as well as application of said Fc regions for obtaining fused protein. Application of the invention provides novel Fc-versions with modified FcRn binding.

EFFECT: possibility of application in medicine for obtaining various constructions, with prolonged time of preservation in blood serum in vivo or, vice versa, in case of therapy with application of radioactive medications, with reduced time of preservation in blood serum in vivo.

14 cl, 44 dwg, 4 ex

Description

This application claims rights under 35 U.S.C. §119 (e) for all the benefits of applications US 60/627,763, registered November 12, 2004; US 60 / 642,886, registered January 11, 2006; US 60 / 649,508, registered February 2, 2005; US 60 / 662,468 registered March 15, 2005; US 60 / 669,311, registered April 6, 2005; US 60 / 681,607, registered May 16, 2005, US 60 / 690,200, registered June 13, 2005; US 60 / 696,609, registered July 5, 2005; US 60 / 703,018, registered July 27, 2005 and US 60 / 726,453, registered August 12, 2005, which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to optimized variants of IgG immunoglobulins, to design methods for their preparation and to their use, especially for therapeutic purposes.

State of the art

Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. Each chain is assembled from individual immunoglobulin (Ig) domains, and thus the generic term immunoglobulin is used for such proteins. Each chain is assembled from two different sections related to the variable and constant regions. The variable regions of the light and heavy chains show a significant difference in sequence between the antibodies and are responsible for binding to the target antigen. Constant regions exhibit smaller differences in sequence and are responsible for binding to a large number of natural proteins to achieve significant biochemical effects. In humans, there are five different classes of antibodies, including IgA (which include subclasses of IgA1 and IgA2), IgD, IgE, IgG (which include subclasses of IgG1, IgG2, IgG3 and IgG4) and IgM. A characteristic feature of these classes of antibodies are their constant regions, although more subtle differences may exist in the V region. Figure 1 shows an IgG1 antibody used as an example here to describe the main structural features of immunoglobulins. IgG antibodies are tetrameric proteins consisting of two heavy chains and two light chains. The IgG heavy chain consists of four immunoglobulin domains joined from the N-terminus to the C-terminus in the order of VH-CH1-CH2-CH3, referring to the variable domain of the heavy chain, constant domain 1 of the heavy chain, constant domain 2 of the heavy chain and constant domain 3 the heavy chain, respectively (also called VH-Cγ1-Cγ2-Cγ3, referring to the variable domain of the heavy chain, the constant domain of gamma-1, the constant domain of gamma-2 and the constant domain of gamma-3, respectively). The IgG light chain consists of two immunoglobulin domains joined from the N-terminus to the C-terminus in the VL-CL order, relating to the variable domain of the light chain and the constant domain of the light chain, respectively.

The variable region of an antibody contains antigen-binding determinants of the molecule and thus determines the specificity of the antibody for the target antigen. The variable region is so named because it differs most strongly in sequence from other antibodies within the same class. The greatest differences in sequence are found in the hypervariable regions (CDR). There are 6 CDR sections in total, three for each heavy and light chain, designated as VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. The variable region outside the CDR regions refers to the framework region (FR). Among the various antibodies, sequence differences are observed in the FR regions, although not as significant as in the CDR regions. In general, this characteristic antibody structure provides a stable base (FR region) on which a significant diversity of antigen binding (CDRs) can be used by the immune system to achieve specificity for a wide variety of antigens. For many fragments of the variable regions of various organisms, a number of structures are available that are defined with high resolution, some of them are in free form, and some are in complex with antigens. The sequence and structural features of the variable regions of antibodies are well characterized (Morea et al., 1997, Biophys Chem 68: 9-16; Morea et al., 2000, Methods 20: 267-279, works included by reference in their entirety), conservative the properties of antibodies have allowed the development of many engineering technologies for the production of antibodies (Maynard et al., 2000, Annu Rev Biomed Eng 2: 339-376, the work is incorporated by reference in its entirety). For example, it is possible to transfer CDRs from one antibody, for example a murine antibody, to the frame region of another antibody, for example a human antibody. This process, called “humanization” in the art, allows the production of less immunogenic therapeutic antibodies from antibodies not related to human ones. Fragments comprising the variable region may exist in the absence of other regions of the antibody, such fragments include, for example, an antigen binding fragment (Fab) comprising VH-Cγ1 and VH-CL; variable fragment (Fv), including VH and VL; a single chain variable fragment (scFv), including VH and VL linked together in the same chain; as well as many other fragments of the variable region (Little et al., 2000, Immunol Today 21: 364-370, work incorporated by reference in its entirety).

An Fc antibody region interacts with various Fc receptors and ligands that give it many important functionalities, the so-called effector functions. In IgG immunoglobulins, the Fc region, as shown in Figures 1 and 2, includes the Cγ2 and Cγ3 immunoglobulin domain and the N-terminal hinge leading to Cγ2. An important family of IgG class Fc receptors includes Fc gamma receptors (FcγRs). These receptors bind antibodies and cellular mechanisms of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ravetch et al., 2001, Annu Rev Immunol 19: 275-290, all works included by references in its entirety). In humans, this protein family includes FcγRI (CD64), including the isoforms FcγRIa, FcγRIb and FcγRIc; FcγRII (CD32) comprising isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including the isoforms FcγRIIIa (including the allotypes V158 and F158) and FcγRIIIb (including the allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82: 57-65, reference is made by reference in its entirety). These receptors typically have an extracellular domain that mediates Fc binding, a membrane-bound region, and an intracellular domain that can mediate some signaling events within the cell. These receptors are expressed in various immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK) and γγ T cells. The formation of the Fc / FcγR complex directs these effector cells to antigen binding sites, which usually leads to signaling in the cells and important subsequent immune responses, such as the release of inflammatory mediators, activation of B cells, endocytosis, phagocytosis and cytotoxic attack. The ability to mediate cytotoxic and phagotoxic effector function is a potential mechanism by which antibodies destroy target cells. A cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognizes an antibody bound to the surface of the target cell and then causes lysis of the target cell is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181-220; Gheitie et al., 2000, Annu Rev Immunol 18: 739-766; Ravetch et al., 2001, Annu Rev Immunol 19: 275-290, all works are incorporated by reference throughout its fullness). A cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognizes an antibody bound on the surface of the target cell and then causes phagocytosis of the target cell is called antibody-dependent cell-mediated phagocytosis of target cells (ADCP). A number of extracellular structures of human FcγRs have been determined, including FcγRsIIa (access code in the pdb 1H9V database is included by reference in its entirety) (Sonderman et al., 2001, J Mol Biol 309: 737-749, work included by reference in its entirety) (access code in the pdb 1FCG database, included by reference in its entirety) (Maxwell et al., 1999, Nat Struc Biol 6: 437-442, work included by sending in its entirety), FcγRsIIb (access code in the pdb 2FCB database, incorporated by reference in its entirety) (Sondermann et al., 1999, Embo J 18: 1095-1103, operation enabled by sending trees in their entirety); and FcγRsIIIb (access code for the pdb 1E4J database, incorporated by reference in its entirety) (Sondermann et al., 2000, Nature 406: 267-273, operation incorporated by reference in its entirety). All FcγRs bind the same Fc region in the N-terminal region of the Cγ2 domain and the previous hinge region shown in Figure 1. This interaction is well characterized structurally (Sondermann et al., 2001, J Mol Biol 309: 737-749, work included by reference in its entirety), and several human Fc structures have been identified that bind to the extracellular domain of human FcγRsIIIb (access code in the pdb 1E4K database, included by reference in its entirety) (Sondermann et al., 2000, Nature 406 : 267-273, the work is enabled by sending in its entirety) (access code in the database pdb data 1IIS and 1IIX, included by reference in its entirety) (Radaev et al., 2001, J Biol Chem 276: 16469-16477, work included by reference in its entirety), as well as the structure of the Fc / FcєRIα human IgE complex (access code in the pdb 1F6A database, included by reference in its entirety) (Garman et al., 2000, Nature 406: 259-266, work included by sending in its entirety).

Different IgG subclasses have different affinities for FcγRs, usually IgG1 and IgG3 bind to receptors much better than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett. 82: 57-65, work included by reference in its entirety). All FcγRs bind to the same IgG Fc region, but with different affinities: the highest affinity FcγRI binds with an IgG1 Kd of 10 ^-8 M, while the low affinity receptors, FcγRII and FcγRIII, bind at 10 ^-6 M and 10 ^-5 M, respectively. The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical, however, FcγRIIIb does not have an intracellular signal domain. In addition, since FcγRI, FcγRIIa / c and FcγRIIIa are positive regulators of activation triggered by the immune complex, they are characterized by the presence of an intracellular domain, which includes an immunoreceptor tyrosine-dependent activation motif (ITAM), FcγRIIb incorporates an immunoreceptor tyrosine-dependent motive (ITIM) and therefore is an inhibitor. Thus, the first receptors relate to activating receptors, while FcγRIIb refers to inhibitory receptors. Receptors also vary in the nature and levels of expression on different immune cells. Another level of complexity is the existence of FcγR polymorphism in the human proteome. Especially important polymorphism with clinical significance are forms V158 / F158 FcγRIIIa. Human IgG1 binds with a higher affinity for the V158 allotype compared to the F158 allotype. The difference in affinity and the supposed effect of this difference on ADCC and / or ADCP have been shown to be a significant indicator of the effectiveness of the anti-rituximab anti-CD20 antibody (Rituxan®, BiogenIdec). Patients with the V158 allotype respond favorably to rituximab treatment, however, patients with a lower affinity F158 allotype respond poorly to such treatment (Cartron et al., 2002, Blood 99: 754-758, work included by reference in its entirety) . About 10-20% of people are V158 / V158 homozygotes, 45% are V158 / F158 heterozygotes and 35-45% of people are F158 / F158 homozygotes (Lehmbecher et al., 1999, Blood 94: 4220-4232; Cartron et al., 2002, Blood 99: 754-758, works incorporated by reference in their entirety). Thus, 80-90% of people are poorly responsive, as they have at least one F158 FcγRIIIa allele.

The overlapping but individual Fc region shown in Figure 1 serves as a contact surface for the C1q complement protein. In the same way that Fc / FcγR binding mediates ADCC, Fc / C1q binding mediates complement dependent cytotoxicity (CDC). To form the C1 complex, C1q forms a complex with the serine proteases C1r and C1s. C1q is capable of binding to six antibodies, although binding to two IgGs is sufficient to activate the complement cascade. Similar to the interaction of Fc with FcγRs, different IgG subclasses have different affinities for C1q, usually IgG1 and IgG3 bind better to FcγRs than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett. 82: 57-65, work included by reference in its entirety.

In IgG, the Fc region between the Cγ2 and Cγ3 domains (Figure 1) mediates an interaction with the neonatal FcRn receptor, the binding of which leads to the recirculation of the endocytotic antibody from the endosome back into the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12: 181 -220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766, both of which are incorporated by reference in their entirety). This process, associated with the elimination of renal filtration due to the large size of the full-sized molecule, leads to the fact that the half-life of suitable antibodies in the blood serum ranges from one to three weeks. Binding of Fc to FcRn also plays a key role in antibody transport. The binding site for FcRn on Fc is also the site in which bacterial proteins A and G bind. Strong binding to these proteins is usually used as a method for antibody purification using Protein A or Protein G affinity chromatography during protein purification. Thus, the exact reproduction of this region on Fc is very important both for the clinical properties of antibodies and for their purification. Available structures of the rat Fc / FcRn complex (Burmeister et al., 1994, Nature, 372: 379-383; Martin et al., 2001, Mol Cell 7: 867-877, both of which are incorporated by reference in their entirety) and complexes with proteins A and G (Deisenhofer et al., 1981, Biochemistry 20: 2361-2370; Sauer-Eriksson et al., 1995, Structure 3: 265-278; Tashiro et al., 1995, Curr Opin Struct Biol 5 : 471-481, all works are incorporated by reference in their entirety) provide an understanding of how Fc interacts with these proteins. The FcRn receptor is also responsible for the transfer of IgG into the gastrointestinal tract of newborns and into the lumen of the intestinal epithelium in adults (Ghetie and Ward, Annu. Rev. Immunol., 2000, 18: 739-766; Yoshida et al., Immunity, 2004, 20 (6): 769-783, both works are incorporated by reference in their entirety).

Studies on rat and human Fcγ domains have demonstrated the importance of certain Fc residues in FcRn binding. The sequences in the Fc regions of rats and humans are approximately 65% identical (residues 237-443, numbered according to Kabat et al.). See figures 3, 4, and 5 for alignment results of rat / human Fc, FcRn heavy chain, and FcRn (beta-2-microglobulin) light chain. Based on the existing structure of the rat Fc / FcRn complex, a model of the human Fc / FcRn complex was constructed (Martin et al., 2001, Mol. Cell 7: 867-877, the work is incorporated by reference in its entirety). The rat and human sequences contain some common residues that are critical for the binding of FcRn, for example, H310 and H435 (Medesan et al., 1997, J. Immunol. 158 (5): 221-7; Shields et al., 2001, J. Biol. Chem. 276 (9): 6591-6604, both of which are incorporated by reference in their entirety). In many positions, however, the proteins in rats and humans have different amino acids, providing a different environment and possibly different features for residues in the sequence in humans compared to sequences in rats. This variability limits the ability to transfer the characteristics of one homolog to another homolog.

In mouse Fcγ, random mutation and selection using phage display in the T252, T254, and T256 regions leads to the T252L / T254S / T256F triple mutant, in which the affinity for FcRn increases by 3.5 times and the half-life increases by 1.5 times (Ghetie et al., 1997, Nat. Biotech. 15 (7): 637-640, work incorporated by reference in its entirety).

The crystal structures of the rat Fc / FcRn complex have identified Fc residues important for FcRn binding (Burmeister et al., Nature, 372: 379-383 (1994); Martin et al. Molecular Cell. 7: 867-877 (2001), both incorporated by reference in its entirety). The initial structure of the Fc / FcRn complex was obtained in 1994 with a resolution of 6 E (table 2a, Burmeister et al., Nature, 372: 379-383 (1994), the work is incorporated by reference in its entirety). The higher-resolution structure performed in 2001 by Martin et al. Showed a more detailed view of the location of the side chains (Martin et al. Molecular Cell. 7: 867-877 (2001), the work is incorporated by reference in its entirety). This crystal structure of rat Fc bound to rat FcRn was resolved using an Fc dimer with one monomer containing the T252G / I253G / T254G / H310E / H433E / H435E mutation, which has broken binding to FcRn, and one monomer containing the wild Fc monomer type.

Mutation studies of human Fcγ have been performed on some residues that are important for binding to FcRn and which have been shown to have increased serum half-lives. Hinton et al. three residues separately from 19 common amino acids mutated in human Fcγ. Hinton et al. found that some point mutations of the double mutant increased affinity for FcRn binding (Hinton et al., 2004, J. Biol. Chem. 279 (8): 6213-6216, work incorporated by reference in its entirety). Two mutations increased the half-life of monkeys. Shields et al. the residues mutated, almost exclusively on Ala, and studied their binding to FcRn and FcγR's (Shields et al., 2001, J Biol Chem 276: 6591-6604, work incorporated by reference in its entirety).

To select Fc mutants that bind FcRn with increased affinity, Dall'Acqua et al. used a phage display (Dall'Acqua et al., 2002, J. Immunol. 169: 5171-5180, work included by reference in its entirety). The selected DNA sequences were double and triple mutants. Proteins encoded by many selected sequences are presented in reference, and some proteins are found that bind to FcRn more strongly than wild-type Fc.

In order to administer antibodies and proteins fused to Fc as therapeutic agents, injections with a prescribed frequency depending on the excretion characteristics and half-life of the proteins are necessary. A longer in vivo half-life makes infrequent injections or reduced dosages possible, which is undoubtedly beneficial. Although mutations made earlier in the Fc domain resulted in some proteins with increased affinity for binding to FcRn and increased in vivo half-lives, these mutations did not elicit optimal mutations and increased in-vivo half-lives.

A key feature of the Fc region is the conserved N-glycosylation shown in Figure 1, which is found on residue N297. This carbohydrate or oligosaccharide, as it is sometimes called, plays a special structural and functional role in the antibody, which is one of the principal reasons why antibodies must be produced using mammalian expression systems (Umana et al., 1999, Nat Biotechnol 17: 176- 180; Davies et al., 2001, Biotechnol Bioeng 74: 288-294; Mimura et al., 2001, J Biol Chem 276: 45539-45547; Radaev et al., 2001, J Biol Chem 276: 16478-16483; Shields et al., 2001, J Biol Chem 276: 6591-6604; Shields et al., 2002, J Biol Chem 277: 26773-26740; Simmons et al., 2002, J Immunol Methods 263: 133-147). However, the carbohydrate effect is negligible if there is any specific contact with FcγR (Radaev et al., 2001, J Biol Chem 276: 16469-16477; Radaev et al., 2001, J Biol Chem 276: 16469-16477 and Krapp et al., 2003, J. Mol. Biol. 325: 979-989, works incorporated by reference in its entirety).

Antibodies have been developed for therapeutic use. The most typical publications relevant to such treatment include Chamow et al., 1996, Trends Biotechnol 14: 52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9: 195-200; Cragg et al., 1999, Curr. Opin. Immunol. 11: 541-547; Glennie et al., 2000, Immunol. Today 21: 403-410; McLaughlin et al., 1998, J. Clin. Oncol. 16: 2855-2833 and Cobleigh et. al., 1999, J. Clin. Oncol. 17: 2639-2648, all works are incorporated by reference in their entirety. For anticancer therapy, any minor improvement in mortality rates is currently defined as success. Certain IgG variants disclosed herein, at least in part, increase the effectiveness of antibodies in limiting the further growth or destruction of target cancer cells.

Antitumor efficacy of antibodies is realized by increasing their ability to mediate cytotoxic effector functions such as ADCC, ADCP and CDC. Examples include the work of Clynes et al., 1998, Proc Natl Acad Sci USA 95: 652-656; Clynes et al., 2000, Nat Med 6: 443-446 and Cartron et al., 2002, Blood 99: 754-758, all works are incorporated by reference in their entirety.

Human IgG1 is the antibody most commonly used for therapeutic purposes, and most design studies have been performed in this context. Various IgG class isotypes, including IgG1, IgG2, IgG3, and IgG4, however, have unique physical, biological, and clinical properties. There is a need in the art to develop such options to improve binding to FcRn and / or to increase the in vivo half-life compared to native IgG polypeptides. This application meets all these and other requirements.

Disclosure of invention

The present invention discloses the creation of new variants of Fc domains, including those found in antibodies fused with Fc and immunoadhesins that have increased binding to the FcRn receptor and increased retention in serum in vivo. An additional aspect of the invention is an increase in binding to FcRn compared to the wild type, especially at low pH values of about 6.0, in order to ensure binding of Fc / FcRn in endosomes. An additional aspect of the present invention is the preferred binding of the constructed variants at a pH of about 6 compared to their binding at a pH of about 7.4 in order to facilitate the release of Fc into the blood after cell recycling.

Another aspect of the present invention relates to the construction of Fc variants with reduced binding to FcRn and reduced in vivo half-life. Such proteins, including mutations to reduce the affinity for FcRn and / or in vivo half-life, are useful for many treatment and diagnostic options, including the delivery and monitoring of radioactive drugs, where, ideally, the half-life of the radioactive label is approximately equal to the in vivo half-life of its protein conjugate.

Another aspect of the present invention relates to a change in the binding of an Fc domain to FcR's, for example, to FcgRI, FcgRIIa, FcgRIIb, FcgRIIIa in humans. These receptors are responsible for the induction of various effector functions of antibodies. Therefore, another aspect of the invention relates to a change in the effector functions of the Fc domain, such as dependent antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP).

Another aspect of the present invention relates to Fc variants that possess both altered binding to FcRn and altered binding to Fcg to affect both the in vivo half-life and the effector functions of a protein including Fc. For example, these options may have an increased in vivo half-life, as well as improved ADCC. Variants, for example, may have an increased in vivo half-life and reduced CDC.

In yet another aspect, the invention provides recombinant nucleic acids encoding variant Fc proteins, expression vectors, and host cells.

In an additional aspect, the invention provides methods for producing a protein comprising Fc, comprising culturing the host cells of the invention under conditions suitable for expression of the protein.

In yet another aspect, the invention provides pharmaceutical compositions comprising a variant Fc protein of the invention and a pharmaceutical carrier.

In yet another aspect, the invention provides methods for treating disorders, comprising administering to a patient a protein comprising the variant Fc of the invention.

In an additional aspect, the invention provides an Fc region of a variant in a parental Fc polypeptide comprising at least one modification in the Fc region of a parental polypeptide, wherein said variant protein exhibits altered binding to FcRn compared to the parent polypeptide, and where said Fc the option includes at least one modification selected from the group consisting of:

where the numbering corresponds to the EU index and the symbol “^” means insertion after the indicated position, and the symbol “#” means the deletion at this indicated position.

In yet another aspect, the invention provides Fc variants comprising at least one modification selected from the group consisting of:

In an additional aspect, the invention provides Fc variants comprising at least one modification selected from the group consisting of:

Brief Description of the Drawings

Figure 1: Structure and function of antibodies. A model of a full-sized human IgG1 antibody is shown, modeled using the structure of a humanized Fab fragment with access code 1CE1 in the pdb database (James et al., 1999, J Mol Biol 289: 293-301, the work is incorporated by reference in its entirety) and structure Fc fragment of human IgG1 with access code 1DN2 in the pdb database (DeLano et al., 2000, Science 287: 1279-1283, the work is incorporated by reference in its entirety). A movable hinge that links the Fab and Fc portions is not shown. IgG1 is a homodimer consisting of heterodimers composed of two light chains and two heavy chains. The Ig domains that make up the antibody are marked and include V _L and C _L in the light chain and V _H , C-gamma-1 (Cγ1), C-gamma-2 (Cγ2) and C-gamma-3 (Cγ3) for severe chains. The Fc region is marked. The binding sites of important proteins are marked and include the binding site of the antigen in the variable region and the binding sites of FcγRs, FcRn, C1q and proteins A and G in the Fc region.

Figure 2: Human IgG sequences used in the present invention, numbered in accordance with the EU index of the database Kabat et al.

Figure 3: An example of human and rodent IgG sequences used in the present invention, numbered according to the EU index of the Kabat et al. Database.

Figure 4: Example of human and rodent FcRn heavy chain sequences used in the present invention.

Figure 5: Example sequences of human beta-2-microglobulin and rodents used in the present invention.

Figure 6: Model of the human Fc / FcRn complex based on rat structures (Burmeister et al., 1994, Nature, 372: 379-383; Martin et al., 2001, Mol Cell 7: 867-877, both included by references in its entirety). Some histidine residues are shown as space-filling atoms in the FcRn (light gray) and Fc polypeptide (dark gray) chains.

Figure 7: Illustration of some of the concepts used in constructing options, including insertions and deletions.

Figure 8: Variants of the present invention.

Figure 9: Variants of the present invention.

Figure 10: Variants of the present invention.

Figure 11: Scheme of the pcDNA3.1 Zeo + vector, which can be used in the construction of Fc variants.

Figure 12: Data on competition of binding of wild-type Fc and Fc variants of the present invention with FcRn. In each panel, the Fc variants of the present invention are shown as a left curve (red or dark gray), and wild-type trastuzumab is shown as a right curve (blue or light gray).

Figure 13: Generalized results of the FcRn binding properties of Fc variants. Columns from right to left show FcRn binding modifications, immunoglobulin used, other modifications, relative FcRn affinity measured using AlphaScreen ™ competitive assay versus wild type (median value), number of assays performed and protein identification number. The relative affinity of FcRn, which is higher than 1.0, shows an improvement in binding compared to the wild type.

Figure 14: Data on the binding of FcRn Fc-variants of the present invention. Fc variants of the present invention are alemtuzumab and trastuzumab. Shows a multiple increase in binding compared with the wild type.

Figure 15: Generalized experiments carried out by surface plasma resonance with Fc variants having improved binding to FcRn. The histogram shows the increase in affinity for binding to each variant FcRn with respect to the wild-type Fc domain.

Figure 16: Experiments conducted by surface plasma resonance with wild-type antibodies and variants of the present invention. The curves shown represent the association and dissociation of an Fc variant antibody with FcRn at pH 6.0.

Figure 17: Assays for binding of Fc variants of the present invention to FcRn. AlphaScreen ™ direct binding assay results are shown at pH 6.0 (a and b) and at pH 7.0 (s).

Figure 18: Assays for binding of Fc variants of the present invention to FcRn. The surface plasmon resonance units obtained by binding the Fc variant with the FcRn attached to the surface are given.

The implementation of the invention

The present invention discloses the creation of new variants of Fc domains, including those found in antibodies fused with Fc and immunoadhesins that have enhanced binding to the FcRn receptor. As indicated here, binding to FcRn leads to their longer retention in serum in vivo.

To increase the retention of Fc proteins in vivo, the increase in binding affinity should be at approximately pH 6.0 without a concomitant increase in affinity at approximately pH 7.4. Although this is still being clarified, it is believed that Fc regions have a longer half-life in vivo, since binding to FcRn at pH 6 in the endosome leads to capture of Fc (Ghetie and Ward, 1997, Immunol. Today, 18 (12): 592 -598, work included by reference in its entirety). The endosomal compartment then returns Fc to the cell surface again. After the compartment opens into the extracellular space, higher pH values of about 7.4 induce the release of Fc back into the bloodstream. Dall 'Acqua et al. showed that Fc mutants with enhanced FcRn binding at pH 6 and 7.4 actually have reduced serum concentrations and the same half-life as wild-type Fc (Dall 'Acqua et al., 2002, J. Immunol ., 169: 5171-5180, work included by reference in its entirety). It is believed that the increased Fc affinity for FcRn at pH 7.4 does not allow Fc to be released back into the blood. Thus, Fc mutations that will increase the in vivo half-life of Fc will ideally increase FcRn binding at low pH values, while still providing Fc release at higher pH values. The amino acid histidine changes its charge in the pH range from 6.0 to 7.4. Therefore, it is not surprising to detect His residues at important positions in the Fc / FcRn complex (Figure 6).

In Figures 8-10 and 17-19, additional data is presented on the options disclosed in the originally filed description. In Figures 20, 27 and 30 show data obtained in vivo.

The Figure 20 presents a summary of the results of measuring the binding affinity of the Fc variants of the present invention with human, macaque, and mouse FcRn obtained by surface plasma resonance (SPR). Values greater than one indicate an increase in the binding of the Fc variant of the present invention to FcRn. The measurements were carried out using approximation of the curves obtained by the SPR method in accordance with the Langmuir binding model with a 1: 1 binding stoichiometry.

The Figure 27 presents the results obtained in vivo. Shown are the concentrations of wild-type antibodies (WT) and variants in the serum of mice with human FcRn nokin. The following anti-VEGF antibodies were used: wild-type antibodies (WT) - open squares, V308F - closed squares, P257L - closed triangles, and P257N - crosses.

The Figure 30 presents data on the binding of wild-type antibodies and variants with FcRn on the surface of 293T cells.

An additional aspect of the invention is the increase in binding to FcRn compared to the wild type, especially at low pH values of about 6.0, in order to facilitate the binding of Fc / FcRn in the endosome. The application also discloses Fc variants with altered binding to FcRn and altered binding to another class of Fc receptors, FcγR's, since it has been shown that different binding to FcγRs, in particular increased binding to FcγRIIIb and reduced binding to FcγRIIb, leads to an increase effectiveness.

Definitions

So that the application could be more fully understood, below are some definitions. Of course, such definitions include grammatical equivalents.

The term “ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein means a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize antibodies bound to the target cell and subsequently cause lysis of the cell - the target.

The term “ADCP” or “antibody-dependent cell-mediated phagocytosis” as used herein means a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγRs recognize antibodies bound to the target cell and subsequently cause phagocytosis of the target cell.

The term "modification of amino acids" means here a replacement, insertion and / or deletion in the polypeptide sequence.

The terms "replacement of amino acids" or "replacement" means here the substitution of an amino acid at a certain position in the original polypeptide sequence with another amino acid. For example, the substitution E272Y refers to a variant polypeptide, in this case the Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine.

The term “amino acid insertion” or “insertion” means here the addition of an amino acid at a specific position in the parent polypeptide chain. For example, -233E or ^ 233E denotes the insertion of glutamic acid after position 233 and to position 234. In addition, -233ADE or ^ 233ADE denotes the insertion of AlaAspGlu after position 233 and before position 234.

The term "deletion of an amino acid" or "deletion", as used here, means the removal of an amino acid, in particular, at a certain position in the original polypeptide sequence. For example, E233- or E233 # denotes a deletion of glutamic acid at position 233. In addition, an EDA233- or EDA233 # deletion deletes the GluAspAla sequence that starts from position 233.

The term “variant protein” or “variant protein” or “variant” as used herein means a protein that differs from the original protein in at least one amino acid modification. A variant of the protein may refer to the protein itself, to a composition that includes a protein, or to a sequence of amino acids that encodes it. Preferably, when the variant protein has at least one amino acid modification compared to the original protein, for example from about one to about ten amino acid modifications and preferably from about one to about five amino acid modifications compared to the original protein. The sequence of the variant protein here will preferably have at least 80% homology with the sequence of the original protein, most preferably at least about 90% homology, more preferably at least about 95% homology. A variant protein may refer to the variant protein per se, to a composition that includes a variant of the protein, or to an amino acid sequence that encodes it. Accordingly, the term “antibody variant” or “variant antibody” as used herein means an antibody that differs from the parent antibody in at least one amino acid modification, the term “IgG variant” or variant “IgG” as used herein means an antibody which differs from the original IgG by at least one amino acid modification, and the term “immunoglobulin variant” or “variant immunoglobulin” as used herein means an immunoglobulin sequence that differs are derived from the original immunoglobulin sequence of at least one amino acid modification. Options may include unnatural amino acids. Examples include US 6,586,207; WO 98/48032; WO 03/073238; US 2004-021988 A1; WO 05 / 35727A2; WO 05 / 74524A2; J. W. Chin et al., (2002) Journal of the American Chemical Society 124: 9026-9027; J. W. Chin & P. G. Schultz, (2002) ChemBioChem 11: 1135-1137; J. W. Chin et al., (2002), PICAS United States of America 99: 11020-11024; and L. Wang & P. G. Schultz, (2002), Chem. 1-10, all works are incorporated by reference in their entirety).

The term "protein" here means at least two covalently linked amino acids, the term includes proteins, polypeptides, oligopeptides and peptides. The peptidyl group may include naturally occurring amino acids and peptide bonds or synthetic peptide-like structures, that is, “analogs” such as peptoids (see Simon et al., PNAS USA 89 (20): 9367 (1992), the work is incorporated by reference in its entirety its fullness). Amino acids can be either natural or non-naturally occurring, as is well known in the art. For example, homophenylalanine, citrulline and norleucine are considered as amino acids used for the purposes of the present invention, and can be used as D- and L- (R or S) amino acid configurations. Embodiments of the present invention may include modifications that include the use of unsaturated amino acids, including the use, for example, of technologies developed by Schultz et al., Including but not limited to the methods described by Cropp & Schultz, 2004, Trends Genet. 20 (12): 625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. USA 101 (2): 7566-71, Zhang et al., 2003, 303 (5656): 371-3 and Chin et al., 2003, Science, 301 (5635): 964-7, all works are incorporated by reference in its entirety). In addition, polypeptides may include the preparation of synthetic derivatives of one or more side or main chains, glycosylation, PEGylation, cyclic rearrangement, cyclization, linkers to other molecules, fusion with proteins or protein domains, and attachment of protein tags or tags.

The term “residue,” as used herein, means a position in a protein and its associated amino acid identity. For example, asparagine-297 (also called Asn297, also called N297) is a residue of human IgG1 antibody.

The term “Fab” or “Fab region” as used herein means polypeptides that include the VH, CH1, VL, and CL domains of immunoglobulins. A Fab may be related to this region in isolation or this region in the context of a full length antibody or antibody fragment.

The term “IgG subclass modification” as used herein means an amino acid modification that converts the amino acid of one IgG isotype to the corresponding amino acid in another, sequence aligned IgG isotype. For example, since IgG1 at EU position 296 includes tyrosine and IgG2 phenylalanine, the replacement of F296Y with IgG2 means a modification of the IgG subclass.

The term “non-naturally occurring modification” as used herein means an amino acid modification that is not isotypic. For example, since none of the IgG contains glutamic acid at position 332, the replacement of I332E with IgG1, IgG2, IgG3 or IgG4 is considered as a non-naturally occurring modification.

The terms “amino acid” or “amino acid identity” are used herein to mean one of the 20 naturally occurring amino acids or any non-naturally occurring analogs that may be present in a specific position.

The term "effector function" here means a biochemical phenomenon that occurs as a result of the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC.

An “effector cell” as used herein means an immune system cell that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK) and γδT cells that can come from any organism, including but not limited to humans, mice, rats, rabbits and monkeys.

The term “IgG Fc ligand” as used herein means a molecule, preferably a polypeptide from any organism, that binds the Fc region of an IgG antibody to form the Fc / Fc ligand complex. Fc ligands include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcγRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G and viral FcγR. Fc ligands also include homologs of the Fc receptor (FcRH), which belong to the family of Fc receptors homologous to FcγRs (Davis et al., 2002, Immunological Reviews 190: 123-136, work incorporated by reference in its entirety). Fc ligands may include hitherto not open molecules that bind to Fc. Special IgG Fc ligands are FcRn and Fc gamma receptors. The term “Fc ligand” as used herein means a molecule, preferably a polypeptide from any organism, that binds the Fc region of an antibody to form an Fc / Fc ligand complex.

The terms “Fc-gamma receptor” or “FcγR” as used herein mean any member of a family of proteins that bind the Fc fragment of an IgG antibody and which are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms of FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), including isoforms of FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82: 57-65, included by reference in its entirety), as well as any still not open human FcγRs or isoforms or allotypes of FcγR. FcγR may be derived from, but not limited to, humans, mice, rats, rabbits, and monkeys. Murine FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD 16-2), as well as any still not open murine FcγRs or isoforms or allotypes FcγR.

The term “FcRn” or “neonatal Fc receptor” as used herein means a protein that binds the Fc region of an IgG antibody and is encoded at least in part by the FcRn gene. FcRn can be derived from any organism, including, but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein comprises two polypeptides, often called a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless specifically stated in the text, FcRn or FcRn protein refers to the complex of the FcRn heavy chain with beta-2-microglobulin. FcRn sequences of particular interest, especially human sequences, are shown in the drawings.

The term "source polypeptide", which is used in the text, means a polypeptide that is significantly modified to create a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered species of a naturally occurring polypeptide. The parent polypeptide may refer to either the polypeptide itself, a composition that includes the parent polypeptide, or an amino acid sequence that encodes it. Accordingly, the term “parent immunoglobulin”, which is used here, means an unmodified immunoglobulin polypeptide, which is then modified to create a variant, and the term “parent antibody”, which is used here, means an unmodified antibody, which is then modified to create a variant antibody. It should be noted that the “parent antibody” includes known commercially available antibodies, recombinantly produced antibodies, as noted below.

The term “position,” as used herein, means a specific place in a protein sequence. Provisions can be numbered sequentially or in accordance with the established format, for example, in accordance with the EU index in the Kabat database. For example, position 297 is a position in the human IgG1 antibody.

The term “target antigen,” as used herein, means a molecule that specifically binds to the variable region of a specific antibody. The target antigen may be a protein, carbohydrate, lipid, or other chemical substance.

The term “target cell,” as used herein, means a cell that expresses a target antigen.

The term “variable region” as used herein means a portion of an immunoglobulin that includes one or more Ig domains, mainly encoded by any of the Vκ, Vλ and / or V _H genes, which contain the genetic loci of kappa, lambda and the heavy locus immunoglobulin chains, respectively.

The term “wild type or WT,” as used herein, means an amino acid sequence or nucleotide sequence that is found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that is not intentionally modified.

The present invention is directed to antibodies that exhibit modulated binding to FcRn (modulation, including increased or decreased binding). For example, in some cases, increased binding leads to cellular recycling of the antibody and thus to an increased half-life, for example, in the case of therapeutic antibodies. Alternatively, reduced binding to FcRn is required, for example, in the case of diagnostic antibodies or therapeutic antibodies that contain a radioactive label. In addition, antibodies exhibiting increased binding to FcRn and altered binding to other Fc receptors, for example FcγRs, find use in the present invention.

Antibodies

The present invention is directed to antibodies that include amino acid modifications that modulate binding to FcRw. Of particular interest are antibodies that minimally include the Fc region, or a functional variant thereof, which exhibit an increased binding agent for FcRn at lower pH and do not show a significantly altered binding at higher pH.

Conventional antibody structural units typically include a tetramer. Each tetramer usually consists of two identical pairs of polypeptide chains, each pair has one “light” (usually with a molecular weight of about 25 kDa) and one “heavy” chain (usually with a molecular weight of about 50-70 kDa). In humans, light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon chains and antibody isotypes such as IgM, IgD, IgG, IgA and IgE are determined, respectively. IgG immunoglobulin has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM immunoglobulin has subclasses including but not limited to IgM1 and IgM2. Thus, the term "isotype", which is used here, means any of the subclasses of immunoglobulins with specific chemical and antigenic characteristics of their constant regions. Known isotypes of human immunoglobulins are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE.

The N-terminal portion of each chain includes a variable region of approximately 100-110 or more amino acids, which is initially responsible for antigen recognition. In the variable region, in each V-domain of the heavy chain and light chain, three loops are assembled to form an antigen-binding site. Each loop refers to a hypervariable region (hereinafter referred to as “CDR”), in which the difference in amino acid sequence is most significant.

The carboxy-terminal part of each chain defines a constant region, initially responsible for effector function. Kabat et al. collected many primary sequences of the variable regions of the heavy chains and light chains. Based on the degree of sequence conservatism, they classified the individual primary sequences into CDR and framework and created a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 ™ edition, NIH publication. No. 91-3242, Kabat et al., Incorporated by reference in its entirety).

In the IgG subclass of immunoglobulins in the heavy chain there are several immunoglobulin domains. The term “immunoglobulin (Ig) domain” as used herein means an immunoglobulin site having an isolated tertiary structure. In the present invention, domains of heavy chains, including constant heavy (CH) domains, and hinge region domains are of interest. In the context of IgG antibodies, each of the IgG isotypes has three CH sites. Accordingly, “CH” domains in the context of IgG are: “CH1” referring to positions 118-220 in accordance with the EU index in the Kabat database. “CH2” refers to provisions 237-340 according to the EU index in the Kabat database and “CH3” refers to provisions 341-447 according to the EU index in the Kabat database.

Another type of heavy chain Ig domain relates to the hinge region. The terms “hinge” or “hinge portion” or hinge portion of an antibody, as used herein, refer to a mobile polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at the EU position 220, and the IgG CH2 domain begins at the residue at the EU position 237. Thus, in the IgG antibody hinge is defined here as a domain including positions from 221 (D221 in IgG1) to 236 ( G236 in IgG1), where the numbering corresponds to the EU index in the Kabat database. In some embodiments, for example in the context of the Fc region, a lower hinge is included, typically relating to positions from 226 or 230.

Of particular interest in the present invention are Fc regions. The terms “Fc” or “Fc region” are used herein to mean a polypeptide comprising the constant region of an antibody except for the first constant region of the immunoglobulin domain and, in some classes, a hinge part. Thus, Fc refers to the last two constant regions of the IgA, IgD and IgG immunoglobulin domains, and to the last three constant regions of the IgE and IgM immunoglobulin domains and to the N-terminal hinge of these domains. IgA and IgM Fc may include chain J. As shown in Figure 1 for IgG, Fc includes the C-gamma-2 and C-gamma-3 immunoglobulin domains (Cg2 and Cg3) and the lower hinge region between C-gamma-1 (Cg1) and C-gamma-2 (Cg2). Although the interfaces between the Fc region may vary, the Fc region of the heavy chain of human IgG typically includes C226 or P230 residues at the C-terminus, where the numbering corresponds to the EU index of the Kabat database. Fc may be related to this site in isolation or to the site in the context of the Fc polypeptide, as described below. The term “Fc polypeptide” as used herein means a polypeptide that includes all or part of the Fc region. Fc polypeptides include antibodies, Fc fusion proteins, isolated Fc and Fc fragments.

In some embodiments, the antibodies are full length. By “full-length antibody” is meant a structure that creates a natural biological form of the antibody, including variable and constant regions, including one or more modifications, as noted here.

Alternatively, antibodies can be many structures including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, fused antibodies (sometimes called “antibody conjugates”) and fragments thereof, respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particular interest are antibodies that include Fc regions, Fc fusions, and a heavy chain constant region (CH1-hinge-CH2-CH3), again including fused constant heavy regions.

Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains, (ii) an Fd fragment consisting of VH CH1 domains, (iii A) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragment (Ward et al., 1989, Nature 341: 544-546, the work is incorporated by reference in its entirety), which consists of a separate variable region, (v) isolated sections of the CDR, (vi) F ( a b ') 2 fragments, a bivalent fragment comprising two linked Fab fragments, (vii) single chain Fv molecules (scFv), where the VH and VL domains are linked through a peptide linker, which allows two domains to associate with the formation of an antigen binding site (Bird et al., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883, works incorporated by reference in their entirety), (viii) bispecific single chain Fv (WO 03/11161, incorporated herein by reference) and (ix) dimers and trimers of small antibody fragments, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326: 461-479 ; WO94 / 13804; Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448, works incorporated by reference in their entirety). Antibody fragments may be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature. Biotech. 14: 1239-1245, work incorporated by reference in its entirety).

Chimeric and humanized antibodies

In some embodiments, the components of the skeleton may be a mixture of various species. Actually, if the antibody is any antibody, such an antibody may be a chimeric antibody and / or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species. For example, “chimeric antibodies” typically include a variable region (s) from a mouse (or, in some cases, a rat) and a constant region (s) from a human. “Humanized antibodies” generally refer to non-human antibodies that include framework regions of the variable domain with substitution for sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody, with the exception of CDR's, is encoded by a human polynucleotide or is identical to such an antibody, with the exception of its CDR's. CDR's, some or all of which are encoded by nucleic acids originating from non-human organisms, are transplanted into the beta-folded frame region of the variable region of a human antibody to create an antibody whose specificity is determined by transplanted CDR's. The creation of such antibodies is described, for example, in WO 92/11018, Jones et al., 1986, Nature 321: 522-525; Verhoeyen et al., 1988, Science, 239: 1534-1536, works incorporated by reference in their entirety. The "reverse mutation" of the selected amino acid residues of the acceptor framework site to the corresponding donor amino acid residues is often necessary to restore the affinity that is lost in the original structure during transplantation (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 6054297; US 6407213, all works are incorporated by reference in their entirety). An optimally humanized antibody will also include at least a portion of the constant region of immunoglobulin, usually belonging to a human immunoglobulin, and therefore will include a human Fc region. Humanized antibodies can also be obtained using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20: 639-654, work included by reference in its entirety. Many techniques are well known in the art for humanizing and restoring non-human antibodies to their original form (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited there, all works included by reference in its entirety). Humanization methods include, but are not limited to, the methods described in Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988; Nature 332: 323-329; Verhoeyen et al., 1988, science, 239: 1534-1536; Queen et al., 1989, Proc. Natl. Acad. Sci., USA 86: 10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89: 4285-9, Presta et al., 1997, Cancer Res. 57 (20): 4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88: 4181-4185; O'Connor et al., 1998, Protein Eng 11: 321-8, all works are incorporated by reference in their entirety. Humanization or other methods of reducing the immunogenicity of the variable regions of non-human antibodies may include surface repair methods, as described, for example, in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969-973, incorporated by reference in its entirety. In one embodiment, the parent antibody is affinity matured as is known in the art. Structural methods can be used for humanization and affinity maturation, as, for example, described in US 11 / 004,590. For humanization and affinity maturation of the variable regions of antibodies, selection methods can be used that include, but are not limited to, the methods described by Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272 (16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271 (37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16 (10): 753-759, incorporated by reference in its entirety. Other methods of humanization may include transplanting only parts of the CDR, these methods include, but are not limited to, the methods described in US 091810,502; Tan et al., 2002, J. Immunol. 169: 1119-1125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084, incorporated herein by reference in its entirety.

Bispecific Antibodies

In one embodiment, the antibodies of the invention are a multispecific antibody and an exclusively bispecific antibody, also sometimes referred to as "diabodies." These are antibodies that bind two (or more) different antigens. Diabodies can be made by a variety of methods known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449, work included by reference in its entirety), for example, obtained by chemical means or from hybrid hybrids.

Mini-antibodies

In one embodiment, the antibody is a miniantibody. Miniantibodies are minimized antibody-like proteins including scFv attached to the CH1 domain. Hu et al., 1996, Cancer Res. 56: 30553061, work included by reference in its entirety. In some cases, scFv may be attached to the Fc region and may include some or all of the hinge region.

Human antibodies

In one embodiment, the antibody is a fully human antibody with at least one modification, as already indicated. “Entirely human antibody” or “absolutely human antibody” refers to a human antibody having the sequence of an antibody derived from the human chromosome with the modifications specified in the text of the application.

Fused Antibodies

In one embodiment, the antibodies of the invention are antibody fusion proteins (sometimes referred to herein as an “antibody conjugate”). One type of fusion antibody includes Fc fusion in which the Fc region is linked to a fusion partner. The term "fused Fc". as used herein, means a protein in which one or more polypeptides are efficiently attached to the Fc region. The Fc fusion here is synonymous with the terms immunoadhesin, Ig fusion, Ig chimera and receptor globulin (sometimes spelled with a dash) as previously used (Chamow et al., 1996, Trends Biotechnol 14: 52-60; Ashkenazi et al., 1997 Curr Opin Immunol 9: 195-200). A Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which basically can be any protein or small molecule. In fact, any protein or small molecule can be attached to the Fc to create a fused Fc. Protein fusion partners are not limited to these examples, but may include a variable region of an antibody that binds a portion of a target receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. A small molecule as fusion partners may include any therapeutic agent that directs the fused Fc to a therapeutic target. Such targets can be any molecule, preferably an extracellular receptor that is involved in the disease. Thus, IgG variants can be attached to one or more fusion partners. In one alternative embodiment, the IgG variant is conjugated or efficiently attached to another therapeutic compound. The therapeutic compound may be a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, cytokine or other therapeutically active agent. IgG may be attached to one of a variety of non-protein polymers, for example, polyethylene glycol, propylene glycol, polyalkylene or copolymers of polyethylene glycol and propylene glycol.

In addition to Fc fusion, fusion antibodies include fusion of the constant region of the heavy chain with one or more fusion partners (in addition, including the variable region of any antibody), while other fusion antibodies are substantially complete antibodies or full length antibodies with fusion partners merge. In one embodiment, the role of the fusion partner is to mediate binding to the target and, thus, it is a functional analogue of the antibody variable regions (and may actually be). In fact, any protein or small molecule can be attached to an Fc to create a Fc fusion (or fusion antibody). Protein fusion partners are not limited to these examples, but may include a target receptor binding site, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. A small molecule as fusion partners may include any therapeutic agent that directs the fused Fc to a therapeutic target. Such targets can be any molecule, preferably an extracellular receptor that is involved in the disease.

A merger partner can be protein and non-protein; the latter is usually created using functional groups located on the antibody and fusion partner. For example, linkers are known in the art; for example, homo- or hetero-bifunctional linkers are well known (see the 1994 catalog of the Pierce Chemical Company technical section relating to cross-linking agents, pp. 155-200, incorporated herein by reference).

Suitable conjugates include, but are not limited to, labels, as described below, drugs and cytotoxic agents, including but not limited to cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria chain A, chain A of exotoxin, chain A of ricin, chain A of abrin, curcin, crotin, phenomycin, enomycin, and the like. Cytotoxic agents also include radioactive chemicals made by fusing radioisotopes with antibodies or by binding radionuclides to chelating agents that have been covalently attached to an antibody. Additional embodiments use calicheamicin, auristatins, geldanamycin, maytansine and duocarmycins and analogues (the latter are described in US 2003/0050331 A1, incorporated by reference in its entirety).

Covalent Antibody Modifications

Covalent modifications of antibodies are included in the scope of this invention and are mainly, but not always, made post-translationally. For example, several types of covalent modifications of an antibody are introduced into a molecule by reacting specific amino acid residues of an antibody with an organic derivatizing agent that is capable of reacting with selectable side chains or N- or C-terminal residues.

Cysteine residues most often react with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to form carboxymethyl or carboxyamidomethyl derivatives. Cysteine residues can also be converted to derivatives by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidazoyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, para-chloromethane , 2-chloromercuri-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

The histidine residues are converted into derivatives by reaction with diethyl pyrocarbonate at a pH of 5.5-7.0, since this compound is relatively specific for the histidine side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodilate at pH 6.0.

Lysine residues and terminal amino groups are reacted with succinic and other carboxylic acid anhydrides. Derivative production using these agents has the effect of changing the charge of lysine residues. Other suitable reagents for derivatizing residues containing alpha amino groups include imido esters such as picolinimidate methyl ester; pyridoxalphosphate; pyridoxal; chloroborohydride; trinitrobenzosulfonic acid; O-methylisourea; 2,4-pentanedione and a reaction with glyoxylate catalyzed by transaminase.

Residues of arginine are modified by reaction with one or more common reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. To convert arginine residues to their derivatives, it is necessary that the reaction is carried out under alkaline conditions due to the high pKa of the guanidine functional group. In addition, these reagents can react with lysine groups, as well as with the epsilon-amino group of arginine.

Specific modifications of tyrosine residues can be made, with the introduction of spectral labels into tyrosine residues by reaction with aromatic compounds of diazonium or tetranitromethane of particular interest. Most often, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosine derivatives and 3-nitro derivatives, respectively. Tyrosine residues are iodinated using ¹²⁵ I or ¹³¹ I to produce labeled proteins for use in radioimmunoassay, a method using chloramine T described above is suitable.

Lateral carboxyl groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N = C = N-R '), where R and R' are optionally different alkyl groups such as 1-cyclohexyl-3- (2 morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, residues of aspartic and glutamic acids are converted to residues of asparagine and glutamine by reaction with ammonium ions.

Derivatization using bifunctional agents is useful for crosslinking antibodies with a water-insoluble support matrix or surface for use in many methods, in addition to the methods described below. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example 4-azidosalicylic acid esters, homofunctional imido esters, including disuccinimidyl esters, such as 3.3 ' dithiobis (succinimidyl propionate) and bifunctional maleimides, such as bis-N-maleimido-1,8-octane. Derivatizing agents, such as methyl 3 - [(para-azidophenyl) dithio] propioimidate, provide photoactivated intermediates that are capable of crosslinking in the presence of light. Alternatively, reactive water-insoluble matrices, such as cyanogen bromide activated carbohydrates and reactive substrates, are described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440, incorporated by reference in their entirety, are used to immobilize proteins.

Glutamine and asparagine residues are often deamidated to produce the corresponding glutamic and aspartic acid residues. Alternatively, these residues can be deamidated under mild acidic conditions. Both methods of forming these residues are included in the scope of the present invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine and threonine residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (i.e., Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 [1983], the work is incorporated by reference in its entirety), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Glycosylation

Another type of covalent modification is glycosylation. In another embodiment, the IgG variants disclosed herein may be modified to include one or more engineered glycoforms. The term “engineered glycoforms” as used herein means a carbohydrate mixture that is covalently attached to IgG, wherein said carbohydrates are chemically different from the carbohydrates of the original IgG. Engineered glycoforms can be useful for many purposes, including but not limited to increasing or decreasing effector function. Designed glycoforms can be created using a variety of methods known in the art (Umaňa et al., 1999, Nat Biotechnol 17: 176-180; Davies et al., 2001, Biotechnol Bioeng 74: 288-294; Shields et al. , 2002, J Biol Chem 277: 26733-26740; Shinkawa et al., 2003, J Biol Chem 278: 3466-3473; US 6,602,684; US 10 / 277,370; US 10 / 113,929; PCT WO 00 / 61739A1; PCT WO 01 / 29246A1; PCT WO 02 / 31140A1; PCT WO 02 / 30954A1, all sources are incorporated by reference in their entirety (Potelligent ^TM technology [Biowa, Inc., Princeton, NJ]; GlycoMAb ™ glycosylation engineering technology [GLYCART biotechnology AG, Zűrich , Switzerland]). Many of these methods are based on the regulation of the level of fucosylated and / or branching oligosaccharides, which which are covalently attached to the Fc region, for example, by expressing IgG in various organisms or cell lines constructed or otherwise made (for example, in Lec-13 CHO cells or rat hybrid YB2 / 0 cells) by regulating the enzymes involved to the glycosylation pathway (eg, FUT8 [α1,6-fucosyltransferase] and / or β1-4-N-acetylglucosaminyltransferase III [GnTIII]) or by modifying the carbohydrate (s) after IgG expression. The engineered glycoform usually relates to various carbohydrates or oligosaccharides; therefore, the IgG variant, for example, an antibody or Fc fusion, may include engineered glycoform. Alternatively, the engineered glycoform may be related to an IgG variant that includes a different carbohydrate or oligosaccharide. As is known in the art, glycosylation methods can depend both on the protein sequence (for example, on the presence or absence of particular amino acid residues suitable for glycosylation), and on the host cell or organism in which the protein is produced. Individual expression systems are discussed below.

Glycosylation of polypeptides usually occurs either through an N-bond or through an O-bond. N-glycosylation refers to the attachment of a carbohydrate molecule to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a carbohydrate molecule to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-glycosylation refers to the addition of one of the sugars: N-acetylgalactosamine, galactose or xylose, to hydroxyamino acid, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

The inclusion of glycosylation sites in an antibody is conveniently carried out by changing the amino acid sequence so that it contains one or more tripeptide sequences from those described above (for N-linked glycosylation sites). The change can be made by adding or replacing one or more residues of serine or threonine to start the sequence (for glycosylation sites linked to the O atom). For convenience, the amino acid sequence of an antibody is preferably modified by changing at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases so that codons are formed that will translate into the desired amino acids.

Other ways to increase the number of carbohydrate molecules in an antibody is by chemical or enzymatic attachment of glycosides to a protein. These procedures are useful in those methods that do not require protein production in the host cell, which is capable of glycosylation at the N - and O-bonds. Depending on the attachment method used, the sugar (s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., Pp. 259-306, incorporated herein by reference in its entirety.

Removing the carbohydrate moiety present in the parent antibody can be carried out chemically or enzymatically. Chemical deglycosylation requires exposure to a protein of a trifluoromethanesulfonic acid compound or equivalent compound. This treatment results in cleavage of most or all of the sugars, with the exception of linked sugars (N-acetylglucosamine or N-acetylgalactosamine), while the leaving protein remains intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52 and Edge et al., 1981, Anal. Biochem. 118: 131, both works are incorporated by reference in their entirety. Enzymatic cleavage of the carbohydrate moiety of the polypeptide can be achieved using a variety of endo- and exoglycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138: 350, incorporated by reference in its entirety. Glycosylation at potential glycosylation sites can be prevented by using the tunicamycin compound as described by Duskin et al., 1982, J. Biol. Chem. 257: 3105, incorporated herein by reference in its entirety. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

Another type of covalent modification of an antibody involves attaching the antibody to various non-protein polymers, including but not limited to various polyols, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, as described, for example, in 2005-2006 PEG Catalog from Nektar Therapeutics (available on the website Nektar), US Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, all of which are incorporated by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions within the antibody to facilitate the attachment of polymers such as PEG. See, for example, US Publication No. 2005 / 011037A1, incorporated by reference in its entirety.

Labeled antibodies

In some embodiments, the covalent modification of an antibody of the invention includes the incorporation of one or more labels. In some cases, they are considered as fused antibodies. The term “labeling group” means any detectable label. In some embodiments, the labeled group is attached to the antibody through spacer legs of varying lengths to reduce potential steric hindrance. Various methods for producing labeled proteins are known in the art and can be used in the practice of the present invention.

Basically, labels come in three classes, depending on the analysis in which these labels are detected: a) isotopic labels, which can be radioactive or heavy isotopes; b) magnetic marks (e.g. magnetic particles); c) redox active substances; d) optical paints; enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by secondary reporters (for example, a pair of “leucine zipper sequences”, binding sites for secondary antibodies, metal-binding domains, epitope tags, etc.). In some embodiments, the labeled group is attached to the antibody through spacer legs of various lengths to reduce potential steric mismatch. Various methods for producing labeled proteins are known in the art and can be used in the practice of the present invention.

Specific labels include optical inks, including, but not limited to, chromophores, phosphors, and fluorophores, the latter in many cases being specific. Fluorophores can be either fluorescent "small molecules" or fluorescent tags of protein nature.

The term “fluorescent label” means any molecule that can be detected using its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red Red, LC Red LC 640, Su 5, Su 5.5, LC Red 705, Oregon green, Alexa-Fluor paints (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 ( Amersham Life Science, Pittsburgh, PA). Suitable optical dyes including fluorophores are described in Molecular Probes Handbook by Richard P. Haugland, incorporated herein by reference in its entirety.

Suitable protein fluorescent labels include, but are not limited to, green fluorescent protein, including GFP from the species Renilla, Ptilosarcus or Aequorea (Chalfie et al., 1994, Science 263: 802-805), EGFP (Clontech Laboratories, Inc., code Genbank U55762), a blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24: 462-471; Helm et al. , 1996, Cur. Biol. 6: 178-182), enhanced fluorescence yellow protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150: 5408-5417), β -galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85: 2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98/26277, WO 99 / 49 019, US Patent Nos. 5292658, 5418155, 5683888, 5741668, 5777079, 5804387, 5874304, 5876995, 5925558). All of the above sources in this section are expressly incorporated by reference.

IgG variants

In one embodiment, the invention provides variant IgG proteins. At a minimum, IgG variants include an antibody fragment comprising the CH2-CH3 region of the heavy chain. In addition, suitable IgG variants include Fc domains (for example, including a lower hinge region), as well as IgG variants including a constant region of the heavy chain (CH1-hinge-CH2-CH3) are also useful in the present invention, all of which can be joined to merger partners.

The IgG variant includes one or more amino acid modifications related to the parent IgG polypeptide, in some cases related to the wild type IgG. The IgG variant may have one or more optimized properties. The IgG variant differs in amino acid sequence from its original IgG in at least one amino acid modification. Thus, IgG variants have at least one amino acid modification compared to the original protein. Alternatively, IgG variants may have more than one, for example, from about one to fifty amino acid modifications, preferably from about one to ten amino acid modifications, and most preferably from about one to about five amino acid modifications compared to the parent protein.

Thus, the sequences of the IgG variants and the starting Fc polypeptide are substantially homologous. For example, variant IgG variant sequences here will have approximately 80% homology with the sequence of the original IgG variant, preferably at least about 90% homology, and more preferably at least about 95% homology. Modifications can be made genetically using molecular biology, or can be made enzymatically or chemically.

Those skilled in the art will recognize that the above list of targets applies not only to specific proteins and biomolecules, but also to the biochemical pathway or pathways that include them. For example, reference to CTLA-4 as a target antigen implies that the ligands and receptors that make up the T cell costimulatory pathway, including CTLA-4, B7-1, B7-2, CD28 and any other currently not discovered ligands and the receptors that bind these proteins are also targets. Thus, the term “target”, which is used here, refers not only to specific biomolecules, but also to a number of proteins that interact with the specified target and participants in the biochemical pathway to which the specified target belongs. Those skilled in the art will further understand that any of the above target antigens, ligands or receptors that bind them, or other participants in the corresponding biochemical pathway can be efficiently attached to the Fc variants of the present invention to form a fused Fc. For example, a Fc fusion that targets EGFR can be constructed by efficiently attaching the Fc variant to EGF, TGF-b, or any other ligand currently explored or not discovered that binds to EGFR. Accordingly, an Fc variant of the present invention can be efficiently attached to an EGFR to form an Fc fusion that binds EGFR, TGF-b, or any other ligand that is currently or not currently discovered that binds to EGFR. Thus, virtually any polypeptide, or ligand, or receptor, or some other protein or protein domain, including but not limited to the above targets and proteins that make up the corresponding biochemical pathways, can be effectively attached to the Fc variants of the present invention to develop merged Fc.

The selection of a suitable antigen depends on the desired practical application. For anticancer therapy, it is desirable to have a target whose expression will be limited in cancer cells. Some targets for which it has been proven that they respond particularly well to antibody treatment are targets with signaling functions. Other therapeutic antibodies exert their effects by blocking receptor signaling by inhibiting the binding of the receptor and the ligand recognized by this receptor. Another mechanism of action of therapeutic antibodies is that they cause suppressive regulation of the receptor. Other antibodies do not function by signaling through their target antigen. In some cases, antibodies are used that are directed against agents that cause infectious diseases.

In one embodiment, the Fc variants of the present invention are introduced into the antibody along with a cytokine. Alternatively, Fc variants are fused or conjugated to a cytokine. The term “cytokine” as used herein means a generic term for proteins released by one cell population that act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional peptide hormones. For example, as described by Penichet et al., 2001, J Immunol Methods 248: 91-101, incorporated herein by reference, cytokines can be fused to an antibody to provide a set of desired properties. Examples of such cytokines are lymphokines, monokines, and the usual polypeptide hormones. Cytokines include growth hormone, for example, human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, for example follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; anti-muller hormone; murine gonadotropin-associated peptide; inhibin; activin; endothelial vascular growth factor; integrin; thrombopoietin (TPO), nerve growth factors, such as NGF-beta; platelet growth factor; transforming growth factors (TGFs), for example TGF-alpha and TGF-beta; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons, for example interferon alpha, beta and gamma; colony stimulating factors (CSFs), for example macrophage-colony stimulating factor (N-CSF), granulocyte-macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (ILs), e.g. IL-1, IL-1alpha, 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, for example TNF-alpha or TNF-beta; 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 cultures and biologically active equivalents of cytokines with natural sequences.

Cytokines and soluble targets, for example, members of the TNF superfamily, are preferred targets when using variants of the present invention. For example, anti-VEGF antibodies and anti-CTLA-4 antibodies, and anti-TNF antibodies, or fragments thereof, are particularly good antibodies for the use of Fc variants with enhanced FcRn binding. Therapeutic agents aimed at these targets are often used in the treatment of autoimmune diseases and this requires multiple injections over a long period of time. Therefore, increased serum half-lives and less frequent exposures provided by embodiments of the present invention are particularly preferred.

For some antibodies and Fc fusions that are approved for use that are in clinical trials or development, Fc variants of the present invention may be useful. These antibodies and Fc fusions are referred to herein as "clinical products and candidates." Thus, in a preferred embodiment, the Fc polypeptides of the present invention can be used in a number of clinical products and candidates. For example, for some antibodies targeted by CD20, the Fc polypeptides of the present invention may be useful. For example, the Fc polypeptides of the present invention may find use in antibodies that are substantially similar to rituximab (Rituxan®, IDEC / Genentech / Roche) (see, for example, US 5,736,137), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin lymphoma; HuMax-CD20, an anti-CD20 antibody currently being developed by Genmab, an anti-CD20 antibody described in US Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedic, Inc.), HumaLYM (Intracel) and PRO70769 (PCT / US2003 / 040426 entitled "Immunoglobulin Variants and Uses Thereof"). For some antibodies targeted by members of the epidermal growth factor receptor family, including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (Erb-4), Fc- may be useful polypeptides of the present invention. For example, the Fc polypeptides of the present invention may find use in an antibody that is substantially similar to trastuzumab (Herceptin®, Genentech) (see, for example, US 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; Pertuzumab (rhuMab-2C4, Omnitarg ™), currently under development by Genentech; anti-Neg2 antibody described in US 4,753,894; cetuximab (Erbitux®, Imclone) (US 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody undergoing clinical trials for use in many types of cancer; ABX-EFG (US 6,235,883), currently under development by Abgenix / Immunex / Amgen; HuMax-EGFr (US 10 / 172.317), currently under development by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (US 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 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J Cell Biophys. 1993, 22 (1-3): 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 5,891,996; US 6,506,883; Mateo et al., 1997, Immunotechnology, 3 (1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, 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 Institute) (PCT WO 0162931A2) 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. Fc polypeptides of the present invention may find multiple antibodies or Fc fusions that are substantially similar to other clinical products and candidates, including, but not limited to, muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson , ibritumumab tiuksetan (Zevalin®), anti-CD20 antibody developed by IDEC / Schering AG, gemtuzumab ozogamicin (Mylotarg®), anti-CD33 antibody (protein p67) developed by Celltech / Wyeth, alefasept (Amevive®), anti-LFA-3 antibody fused to Fc developed by Biogen, abciximab (ReoPro®) developed by Centocor / Lilly, Basiliximab (Simultec®), developed by Novartis, palivizumab (Sinagis®), developed by MedImmune, infliximab (Remicade®), TNF-alpha antibody developed by Centocor, adalimumab (Humira®) , Anti-TNF-alpha antibody developed by Abbott, Humicade ™, Anti-TNF-alpha antibody developed by Celltech, Etanercept (Enbrel®), Anti-TNF-alpha antibody fused to Fc, developed by Immunex / Amgen ", ABX-CBL, anti-CD 147 antibody developed by Abgenix, ABX-IL8, anti-IL8 antibody developed by Abgenix, ABX-MA1, anti-MUC18 antibody developed by paniey «Abgenix», pemtumomab (R1549, ⁹⁰ Y-muHMFG1), antibody to MUC1 in the formulation now «Antisoma», Therex (R1550) , an antibody to MUC1, developed by the company «Antisoma», AngioMab (AS1405) , being developed by «Antisoma» , HuBC-1, developed by Antisoma, Thioplatin (AS1407), developed by Antisoma, Antegren® (natalizumab), anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 developed by Biogen, VLA-1 mAb, integrin antibody VLA-1, developed by Biogen, LTBR mAb, anti-lymphotoxin beta receptor (LTBR), developed by Biogen, CAT-152, anti TGFβ2, developed developed by Cambridge Antibody Technology, J695, an anti-IL-12 antibody developed by Cambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1 antibody developed by Cambridge Antibody Technology and Genzyme, CAT- 213, anti-eotaxin-1 antibody developed by Cambridge Antibody Technology, LymphoStat-B ™ anti-Blys antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc., Avastin ™ (bevacizumab, rhuMAb-VEGF), anti-VEGF antibody developed by Genentech, anti-rat family HER fluids developed by Genentech, Anti-Tissue Factor (ATF), tissue factor antibody developed by Genentech, Xolair ™ (omalizumab), IgE immunoglobulin antibody developed by Genentech, Raptiva ™ (efalizumab) the anti-CD-11 antibody developed by Genentech and Xoma, the MLN-02 antibody (formerly called LDP-02) developed by Genentech and Millenium Pharmaceuticals, HuMax CD4, the anti-CD4 antibody developed by Genmab ”, HuMax-IL15, anti-IL15 antibody developed by Genmab and Amgen; HuMax-Inflam developed by Genmab and Medarex , HuMax-Cancer, anti-heparanase I antibody developed by Genmab and Medarex and Oxford GcoSciences; HuMax-Lymphoma drug developed by Genmab and Amgen; HuMax-TAC developed by Genmab, IDEC- 131 and an anti-CD40L antibody developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody developed by IDEC Pharmaceuticals, IDEC-152, anti-CD23 antibody developed by IDEC Pharmaceuticals, anti-macrophage migration inhibiting factor (MIF) antibodies developed by IDEC Pharmaceuti cals ", BEC2, anti-idiotypic antibody developed by Imclone, IMC-1C11, anti-KDR antibody developed by Imclone, DC101, anti-flk-1 developed by Imclone, anti-VE cadherin antibodies developed by Imclone, CEA-Cide ™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics, LymphoCide ™ (epratuzumab), anti-CD22 antibody developed by Immunomedics, AFP-Cide, developed by Immunomed ", MyelomaCide, developed by Immunomedics, LkoCide, developed by Immunomedics, P rostaCide, 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 Immuno-Designed Moleculas, HuMax ™ -CD4, anti-CD-4 antibody developed by Medarex and Genmab ", HuMax-IL15, anti-IL15 antibody developed by Medarex and Genmab, CNTO 148, anti-TNFα antibody developed by Medarex and Centocor / J & J, CNTO 1275, cytokine antibody, developed developed by Centocor / J & J, MOR101 and MOR102, antibodies to the intracellular adhesion molecule-1 (ICAM-1) (CD54), developed by MorphoSys, MOR201, an antibody to fibroblast growth factor receptor 3 (FGFR-3), developed MorphoSys, Nuvion® (visilizumab), anti-CD-3 antibody developed by Protein Design Labs, HuZaF ™, anti-interferon-gamma antibody developed by Protein Design Labs, integrin antibody α5β1, developed by Protein Design Labs ", an anti-IL-12 antibody developed by Protein Design Labs, ING-1, an EP-CAM antibody developed by Xoma and MLN01, an anti-beta2 integrin antibody developed by Xoma, all of the above sources in this section are expressly incorporated by reference.

Fc polypeptides of the present invention can be included in the above clinical candidates and products, or in antibodies and Fc fusions that are substantially similar to them. Fc polypeptides of the present invention can be included in varieties of the above clinical candidates and products, which are humanized, affinity-matured, engineered or modified in any way varieties.

In one embodiment, the Fc polypeptides of the present invention are used to treat autoimmune, inflammatory diseases, or as indicated by transplantation. Target antigens and clinical products and candidates that are important in such diseases include, but are not limited to, antibodies to α4β7 integrin, such as LDP-02, antibodies to beta-2 integrin, such as LDP-01, antibodies to complement (C5), such as 5G1.1, anti-CD2 antibodies, such as BTI-322, MEDI-507, anti-CD3 antibodies, such as OKT3, SMART anti-CD3, anti-CD4 antibodies, such as IDEC-151, MDX- CD4, OCT4A, antibodies to CD11a, antibodies to CD14, such as IC14, antibodies to CD18, antibodies to CD23, such as IDEC 152, antibodies to CD25, such as zenapax, antibodies to CD40L, such as 5c8, Antova, IDEC-131 Antibodies to CD64, such as MDX-33, anti-CD80 antibodies such as IDEC-114; anti-CD147 antibodies such as ABX-CBL; anti-E-selectin antibodies such as CDP850; anti-gpIIb / IIIa antibodies such as ReoPro / Abcixima; anti-ICAM-3 antibodies such as ICM3, antibodies to ICE, such as VX-740, antibodies to FcR1, such as MDX-33, antibodies to IgE, such as rhuMab-E25, antibodies to IL-4, such as SB-240683, antibodies to IL-5, such as SB-240563, SCH55700, antibodies to IL-8, such as ABX-IL8, antibodies to interferon-gamma, antibodies to TNF (TNF, TNFa, TNFa, TNF-alpha), such as CDP571, CDP870, D2E7, infliximab , MAK-195F, and anti-VLA-4 antibodies, such as integrin.

Fc variants of the present invention with increased binding to FcRn can be used in TNF inhibitor molecules to provide improved properties. Useful TNF-inhibitory molecules include any molecule that inhibits the action of TNF-alpha in mammals. Suitable examples include Enbrel® Fc protein (etanercept) and Humira® antibodies (adalimumab) and Remicade® (infliximab). Monoclonal antibodies (such as Remicad and Humira) engineered using the Fc variants of the present invention, to improve binding to FcRn, can be converted to antibodies with better efficiency by increasing half-life.

In some embodiments, antibodies against infectious diseases are used. Antibodies against eukaryotic cells include antibodies directed to yeast cells, including but not limited to Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, Plasmodium liparipia liparipia farciparia farciparium falciparia liparipia falciparium falciparium falciparium.

Antibodies to other fungal cells including target antigens associated with Candida strains including Candida glabrata, Candida albicans, C.krusei, C. lusitaniae and C.maltosa, as well as representatives of the genus Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces are also useful. and Penicillium et al.

Antibodies directed against target antigens associated with protozoa include, but are not limited to antibodies associated with Trypanosoma, representatives of the Leishmania genus, including Leishmania donovanii, Plasmodium spp., Pneumocystis carinii, Cryptosporidium parvum, Giardia lamblia, Entamoebaospoisolytispoi.

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

In some aspects, the antibodies are directed against viral infections; these 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 (e.g., smallpox virus, vaccinia virus), enteroviruses (e.g. poliovirus, Coxsackie virus), hepatitis viruses (including A, B and C), herpes viruses (e.g. herpes simplex virus, chickenpox virus, cytomegalovirus , Epstein-Barr virus), otavirusy, Norfolk viruses, hantavirus, arenaviruses, rhabdovirus (e.g., rabies virus), retroviruses (including HIV, HTLV-I and II), papovaviruses (e.g. papillomavirus), polyomaviruses, and picornaviruses, and the like

Properties of optimized IgG variants

The present invention also provides IgG variants that are optimized for a wide range of therapeutically important properties. An IgG variant designed or predicted to demonstrate one or more optimized properties is referred to herein as an “optimized IgG variant”. The most preferred properties that can be optimized include, but are not limited to increasing or decreasing the affinity for FcRn and increasing or decreasing the half-life in vivo. Suitable embodiments include antibodies that exhibit improved affinity for binding to FcRn at reduced pH values, such as those associated with endosomes, for example at pH 6.0, and at the same time do not exhibit corresponding improved affinity for binding at higher values pH, for example, at a pH of 7.4, which allows an increase in uptake into endosomes at normal release rates. Similarly, these FcRn modulated binding antibodies do not necessarily have other desired properties, such as FcγR modulated binding, such as described in US 11 / 174,287, 11 / 124,640, 10 / 822,231, 10 / 672,280, 10 / 379,392 and the patent application entitled “IgG Immunoglobulin variants with optimized effector function” and filed October 21, 2005 under ________. Thus, optimized properties also include, but are not limited to, increasing or decreasing the affinity for FcγR. In one optional embodiment, the IgG variants are optimized in order to increase their affinity for human activating FcγR, preferably FcγRIIIa, in addition to the FcRn binding profile. In yet another optional alternative preferred embodiment, the IgG variants are optimized to reduce their affinity for the human FcγRIIb inhibitor receptor. Hence, particular embodiments include the use of antibodies that exhibit increased binding to FcRn and increased binding to FcγRIIIa. In other embodiments, antibodies are used that exhibit increased binding to FcRn and increased binding to FcγRIIIa. These embodiments provide IgG polypeptides with enhanced therapeutic properties for humans, for example, with enhanced effector function and higher anti-cancer efficacy. In an alternative embodiment, the IgG variants are optimized so that they have increased or decreased affinity for FcRn and increased or decreased affinity for human FcγR, including but not limited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb, including variations thereof. These embodiments provide IgG polypeptides with enhanced therapeutic properties for humans, for example, with increased serum half-lives and reduced effector function. In other embodiments, IgG variants provide increased affinity for FcRn and increased affinity for one or more FcγRs, and, in addition, reduced affinity for one or more FcγRs. For example, the IgG variant may have improved binding to FcRn and FcγRIIIa and, in addition, reduced binding to FcγRIIb. Alternatively, the IgG variant may have reduced binding to FcRn and FcγR's. In another embodiment, the IgG variant may have reduced affinity for FcRn and increased affinity for FcγRIIb, and, in addition, reduced affinity for one or more activator FcγRs. In yet another embodiment, the IgG variant may have increased serum half-life and impaired effector functions.

Preferred embodiments include optimizing binding to human FcRn and FcγR, however, in alternative embodiments, the IgG variants have increased or decreased affinity for FcRn and FcγRs from non-human organisms, including but not limited to non-human rodents and primates. IgG variants that have optimized binding to non-human FcRn can be used in research. For example, mouse models are suitable for the study of many diseases and allow testing properties such as efficacy, toxicity, and pharmacokinetics of a particular drug candidate. As is known to those skilled in the art, cancer cells can be inoculated or introduced into a mouse to mimic human cancer, a process known as xenograft. Testing IgG variants, which include IgG variants optimized for FcRn, can provide useful information regarding protein clearance characteristics, its clearance mechanism, and the like. IgG variants can also be optimized to enhance functionality and / or increase solubility in aglycosylated form. Fc ligands include, but are not limited to, FcRn, FcγRs, C1q, and proteins A and G, and may be from any source including, but not limited to, a mouse, rat, rabbit, or monkey, but the preferred source is human. In an alternative preferred embodiment, the IgG variants are optimized to become more stable and / or more soluble than the aglycosylated form of the parent IgG variant.

IgG variants may include modifications that modulate interaction with Fc ligands that are not related to FcRn and FcγRs, which include, but are not limited to, complement proteins and Fc receptor homologs (FcRHs). FcRHs include, but are not limited to, FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190: 123-136, work incorporated by reference in its entirety).

Preferably, the specificity of the IgG variant for the Fc ligand will determine its therapeutic use. The applicability of a particular IgG variant for therapeutic purposes will depend on the epitope or form of the target antigen and the disease or symptom being treated. For most targets and indications, enhanced FcRn binding may be preferred, since enhanced FcRn binding can lead to an increase in serum half-life. The increased half-life in blood serum allows you to use a lower frequency of dosing or a low dosage of drugs. This may be particularly preferred if the therapeutic agent is used in response to indications that require repeated administration of the agent. For some targets and indications, a reduced affinity for FcRn may be preferred. This may be particularly preferred if it is desirable to have an Fc variant with increased clearance or reduced half-life in blood serum, for example, in an Fc polypeptide used as a visiographic agent or as a radioactive therapeutic agent.

You can apply IgG variants, which include IgG variants, providing increased affinity for FcRn with enhanced activator FcγR and / or reduced affinity for inhibitory FcγR. For some targets and indications, in addition, it may be useful to use IgG variants that provide different selectivity for different activator FcγR; for example, in some cases, enhanced binding to FcγRIIa and FcγRIIIa, but not to FcγRI, may be desirable, while in other cases, enhanced binding to only FcγRIIa may be preferred. For some targets and indications, it may be preferable to use IgG variants that alter FcRn binding and enhance effector functions, both FcγR mediated and complement mediated effector functions, whereas in other cases it may be useful to use IgG variants that enhance binding FcRn or increase the serum half-life and enhance effector functions, both FcγR-mediated and complement-mediated effector functions. For some targets or cancer indications, it may be useful to reduce or completely eliminate one or more effector functions, for example by knocking out C1q binding, one or more FcγR, FcRn, one or more other Fc ligands. For other targets and indications, it may be preferable to use IgG variants that provide increased binding to inhibitory FcγRIIb and, in addition, provide binding to activator FcγR at the WT level, at a reduced level, or do not bind to activator FcγR at all. This can be especially useful, for example, if the goal of the IgG variant is to inhibit inflammation or an autoimmune disease or modulate the immune system in some way. Since autoimmune diseases generally continue for a long period of time and re-dosing is used for treatment, it is particularly preferable to treat these diseases with Fc variants with increased half-life due to increased FcRn.

To improve the stability, solubility, functioning or clinical use of IgG, a modification can be made. In a preferred embodiment, the IgG variants may include modifications to reduce the immunogenicity in humans. In a most preferred embodiment, the immunogenicity of the IgG variant is reduced by the methods described in US Pat. No. 11 / 004,590, incorporated by reference in its entirety. In alternative embodiments, the IgG variants are humanized (Clark, 2000, Immunol Today 21: 397-402, work incorporated by reference in its entirety).

IgG variants may include modifications that reduce immunogenicity. Modifications designed to reduce immunogenicity may include modifications that impair the binding of processed peptides derived from the precursor sequence to proteins of the major histocompatibility complex (MHC). For example, amino acid modifications can be designed in such a way that they would not have at all or would have a minimal number of immune epitopes, presumably associated with high affinity for any prevailing MHC alleles. Several methods are known in the art for identifying the protein sequences of epitopes that bind to MHCs, and they can be used to determine the sum of scores for epitopes in an IgG variant. See, for example, WO 98/52976; WO 02/079232; WO 00/13317; US09 / 903.378; US 10 / 039,170; US 60 / 222,697; US 10 / 754,296; PCT WO 01/21823 and PCT WO 02/00165; Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: 942-948; Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol. 154: 5927-5933 and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358, all works are incorporated by reference in their entirety. Sequence-based information can be used to determine the amount of scoring for binding in the interaction of a particular peptide and MHC (see, for example, Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et. al., 1999, Nature Biotech. 17: 555-561, all works are incorporated by reference in their entirety).

Construction of IgG variants

Embodiments of the present invention can be constructed in various ways. Variants, as described herein, may be insertions, deletions, substitutions, other modifications, or combinations thereof and other changes. In particular, the latest embodiment of the present invention is the construction of inserts and deletions that both improve and impair the binding of the Fc polypeptide to the Fc ligand. As disclosed in this application, insertions and deletions can be made that increase or decrease the affinity of the Fc polypeptide for FcRn. Insertions and deletions can be constructed using rationalized approaches or using approaches that include the use of random components, such as the creation of libraries of random or semi-random options or screening. In an alternative embodiment, substitutions are disclosed that increase or decrease the affinity of the Fc polypeptide for FcRn.

Insertions and Deletions

Variant Fc polypeptides can be created by replacing the parent amino acid at any position of the Fc polypeptide with a variant amino acid. By replacing one or more amino acids in the Fc polypeptide with variant amino acids, side chains are changed at these positions. The most useful substitutions modify the properties of Fc by changing the side chains of Fc. The substituted side chains can interact directly or indirectly with a partner that binds to the Fc associated with the function or property of Fc. At least one substitution changes the covalent structure of one or more side chains of the original Fc polypeptide.

Alternatively, variant Fc polypeptides can be created that alter the covalent structure of the backbone of the parent Fc polypeptide. Protein atoms that make up the backbone of a molecule include a peptide nitrogen atom, an alpha-carbon atom, a carbonyl or peptide carbon atom, and carbonyl oxygen. Changing the covalent structure of the main chain provides additional methods for changing the properties of Fc polypeptides. The covalent structure of the Fc backbone can be changed by adding atoms to the backbone of the molecule, for example by inserting one or more amino acids, or by removing atoms from the backbone, for example by deletion of one or more amino acids. The covalent structure of the main chain can also be modified by changing individual atoms of the main chain to other atoms (Deechongkit et al., J. Am. Che. Soc. 2004, 126 (51): 16762-71, the work is incorporated by reference in its entirety ) As is known in the art and illustrated herein, insertions and deletions of amino acids in Fc polypeptides can be made using insertions or deletions of the corresponding nucleotides in DNA encoding an Fc polypeptide. Alternatively, as is known in the art, insertions and deletions can be made during the synthesis of Fc polypeptides.

The construction of insertions and deletions of amino acids that alter the interaction of the Fc polypeptide with one or more binding partners (e.g., Fc-gamma R's, FcRn, C1q) can be made based on the structure of the complex of the Fc polypeptide and its binding partner. In a less preferred embodiment, the construction can be carried out based on the structure of the Fc polypeptide and information about the Fc region involved in binding to the binding partner. This information can be obtained using experiments on mutagenesis, experiments using phage display, by comparison by homology, using computer modeling and other methods.

Preferred amino acid positions for insertions and deletions that affect Fc binding interactions but do not affect the overall structure, stability, expression or use of the Fc polypeptide are in the Fc / Fc binding partner interaction sites. To modify the binding of FcRn to the Fc polypeptide, the preferred loop positions for insertions and deletions are positions 244-257, 279-284, 307-317, 383-390 and 428-435 (numbering according to the EU index in the Kabat database et al., Burmeister et al., 1994, Nature, 372: 379-383; Martin et al., 2001, Mol Cell 7: 867-877, all works are incorporated by reference in their entirety). To modify the binding of the Fc gamma receptor to the Fc polypeptide, the preferred loop positions for insertions and deletions are positions 229-239, 266-273, 294-299 and 324-331 (numbering according to the EU index in the Kabat database et al., pdb code 1E4K.pdb Sondermann et al., 2000, Nature 406: 267, all works are incorporated by reference in their entirety). Loops are regions of the polypeptide that are not involved in the structure of alpha helices and beta folds (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp2-67, the work is incorporated by reference in its entirety ) Loop positions are preferred since the backbone atoms of a molecule are usually more mobile and less likely to be involved in hydrogen bonding compared to backbone atoms of alpha helices and beta folds. Therefore, lengthening or shortening of loops due to the insertion or deletion of one or more amino acids is less likely to lead to large, disruptive changes in the Fc polypeptide, including its stability, expression and other problems.

Insertions and deletions can be used to change the length of the polypeptide. For example, in the loop region, changing the loop length will lead to altered mobility and conformational entropy of the loop. Loop inserts will generally increase the conformational entropy of the loop, which can be defined as the Boltzmann constant multiplied by the natural logarithm of the number of possible conformations (van Holde, Johnson and Ho. Principles of Physical Biochemistry. Prentice Hall, New Jersey 1998, Chapter 1 pp78, work included by reference in its entirety). When at least one amino acid is inserted into a polypeptide, the total number of conformations possible in the polypeptide increases. These additional conformations may be useful for generating favorable binding Fc / Fc partner interactions, since the Fc polypeptide may use one of the additional conformations in binding to the Fc binding protein. In this case, the insertion can lead to stronger Fc / Fc binding partner interactions. If additional conformations are not used on the binding surface, then the insertion may lead to weaker interactions of the Fc / Fc binding partner, since the additional conformations must compete with the conformation competent for binding. Similarly, deletion of the polypeptide segment can also result in both stronger and less strong Fc / Fc binding partner interactions. If a segment deletion that reduces the possible number of conformations of the backbone of a molecule eliminates a binding competent conformation, then a deletion can lead to weaker Fc / Fc binding partner interactions. If a segment deletion does not eliminate the binding competent conformation, then the deletion can lead to stronger Fc / Fc binding partner interactions, since the deletion can eliminate the conformations competing with the binding competent conformation.

Insertions and deletions can be used to alter the positions and orientations of amino acids in the Fc polypeptide. Since insertions and deletions can cause changes in the covalent structure of the main chain, they will necessarily cause a change in the positions of the atoms of the main chain. Figure 7 compares the positions of the main chain in some loop segments labeled L1-L4 in three different main chains. The original main structure contains four loop segments, while a deletion in the main chain leads to the loss of the L1 segment, and the insertion of the segment includes an additional segment located in front of the L1 segment, for example, in front of the N-terminus. Deletions and insertions cause a large-scale change in the structure of the main chain near the site of insertion or deletion. Due to the removal of the segment located near the N-terminal part of the loop, for example the L1 segment, the loop is shortened, and the remaining segments shift their position closer to the N-end of the loop. This has an effect on the movement of the L2 segment towards the previous location of the L1 segment and towards the N-end of the loop. This change in the position of the L2 segment relative to the L1 segment can enhance the binding of the Fc complex / Fc binding partner and is preferable if there is preliminary information suggesting that the amino acid or amino acids located in L2 make favorable interactions with the Fc binding partner when located in L1 . For example, if L2 contains alanine and tyrosine and replacing the two amino acids in L1 with alanine and tyrosine previously led to an Fc variant with improved binding, then a deletion of L1 can create an Fc variant with increased affinity for the Fc binding partner.

Similarly, inserting a polypeptide segment into an Fc polypeptide at the N-terminal portion of the loop will cause the position of the segments of the loop to shift toward the C-terminal portion of the loop. In figure 7, the insertion of one or more amino acids in front of the L1 segment, for example at the N-terminus, changes the conformation of the main chain, including the movement of the L1 segment towards the C-end of the loop. This type of insertion is preferred if it is known that the amino acids located in the L1 segment favor interaction when they are in the L2 position, since the insertion can lead to stronger Fc / Fc binding partner interactions. If weaker interactions of the Fc / Fc binding partner are desired, an insert can be used to shift the unfavorable amino acid to a new position. The inserted, deleted, and parent segments (L1-L4 in FIG. 7) may be one or more amino acids in the Fc polypeptide.

Alternatively, like inserts and deletions at the N-terminal portion of the loops, inserts and deletions at the C-terminal portion of the loops can be used. The inserts at the C-terminus of the loops can cause the positions of the N-terminal portion of the insert to move toward the N-terminus of the loop. Deletions at the C-terminus of the loops can lead to the movement of the positions of the N-terminal portion of the deletion towards the C-end of the loop. The choice when using insertion or deletion at the N-terminal or C-terminal portion of the loop is based on the amino acids located in the loop, the need to increase or decrease the affinity of the Fc / Fc binding partner complex and the desired positional shift.

Insertions and deletions can be used in any region of the Fc polypeptide, including regions of loops, alpha helices and beta folds. Preferred sites for insertions and deletions include loops that are not alpha helix and beta fold sites. Loops are preferred because they usually perceive changes in the main chain better than alpha helices and beta folds. Particularly preferred locations for insertions or deletions that result in stronger protein / protein interactions are the N-terminal and C-terminal edges of the loop. If the side chains of the loop are involved in the Fc / Fc binding partner interactions, it is less likely that insertions or deletions at the edges will lead to very harmful changes in the binding interactions. It is more likely that deletions at the exact center of the loop eliminate important residues on the binding surface of the Fc / Fc binding partner, and inserts at the exact center of the loop are more likely to create adverse interactions at the binding surface of the Fc / Fc binding partner. The number of residues to be removed or inserted can be determined by the volume of the desired main chain change, preferably about 15 inserts or deletions or less, more preferably about 10 inserts or deletions or less, and most preferably about 5 inserts or deletions or less.

After the location and size of the deletion Fc variant have been designed, the complete sequence of the polypeptide is finally determined, and the polypeptide can be created using methods known in the art.

Insertion Fc variants, however, have additional steps of constructing a sequence of at least one inserted amino acid. Inserts of polar residues, including Ser, Thr, Asn, Gln, Ala, Gly, His, are preferred at the positions that are expected to be exposed in the Fc polypeptide. Particularly preferred are smaller amino acids, including Ser, Thr and Ala, as it is less likely that the small size will sterically hinder the interactions of the Fc / Fc binding partner. Ser and Thr also have the ability to form hydrogen bonds with the atoms of the Fc binding partner.

The inserts may also have additional flexibility that can be constructed in the insertion polypeptide to create favorable interactions with the Fc binding partner, as would be necessary if stronger binding of the Fc / Fc binding partner is desired. The length of the insertion into the main chain can be determined by modeling a variant main chain using the simple base sequence used for insertion. For example, polyserine, polyglycine or polyalanine inserts of various lengths can be constructed and modeled. Modeling can be carried out using a variety of methods, including homologous modeling, based on known three-dimensional structures of homologues, including inserts, and using computer modeling, including MODELLER (MA Marti-Renom et al., Annu. Rev. Biophys. Biomol. Struct. 29 , 291-325, 2000) and ROSETTA (Kuhlman et al. (2003). Science 302, 1364-8), both of which are incorporated by reference in their entirety. Usually, different conformations of the main chain are first created, and the final structure of the main chain can be determined after the side chain identities are established. Side chains can be designed using PDA® algorithms (US 6,188,965; 6,269,312; 6,403,312; 6,801,861; 6,804,611; 6,792,356, 6,950,754 and US 09 / 782,004; 09 / 927,790; 10 / 101,499; 10 / 666,307; 10 / 216,102; 10 , all are incorporated by reference in their entirety).

Insertions and deletions can be designed in any polypeptides other than Fc polypeptides using the methods described here. For example, insertions or deletions in a member of the TNF superfamily, APRIL, can be designed using its three-dimensional structure (PDB code IXU1.pdb, Hymowitz, et al. (2005) J.Biol.Chem. 280: 7218, incorporated by reference in their entirety completeness). Insertions or deletions can be designed to enhance the binding of APRIL to its receptor, TACI. Preferred loop residues as insertion or deletion sites are Ser118-Val124, Asp164-Phe167, Pro192-Ala198, Pro221-Lys226 residues. These loops interact with TACI in the APRIL / TACI complex and mediate binding.

Polypeptides Including Variants

IgG variants can be based on human IgG sequences, and thus, human IgG sequences are used as “main” sequences, with which other sequences are compared, including but not limited to sequences from other organisms, such as rodent and primate sequences. IgG variants may also include sequences from other classes of immunoglobulins, such as IgA, IgE, IgD, IgM, and the like. It is believed that although IgG variants are constructed in the context of one source IgG, variants can be designed and “transferred” to the context of another, second source IgG. This is done by determining the "equivalent" or "corresponding" residues and substitutions between the first and second IgG, which is usually based on the sequence or structural homology between sequences of IgGs. To establish homology, the amino acid sequence of the first IgG indicated here is directly compared with the sequence of the second IgG. After the sequence alignment procedure using one or more homology programs known to specialists in this field (for example, using residues conservative in various species), allowing insertions and deletions to exist, if necessary, in order to maintain alignment (that is, to avoid the exclusion of conservative residues from due to random insertions and deletions), amino acid residues are established that are equivalent to certain amino acids in the primary sequence of the IgG variant. The alignment of conservative residues should preferably retain 100% of such residues. However, alignment of more than 75% or at least 50% of the conserved residues is also sufficient to determine equivalent residues. Equivalent residues can also be established by determining the homology between the first and second IgG at the level of the tertiary structure for IgGs for which a tertiary structure has already been established. In this case, residues are called equivalent for which the atomic coordinates of two or more atoms of the main chain of a particular amino acid residue in the original polypeptide or its precursor (N to N, CA to CA, C to C, O to O) during alignment are within 0.13 nm and preferably within 0.1 nm. Alignment is achieved after the model of best fit is oriented and positioned in such a way as to ensure maximum overlap of atomic coordinates related to protein atoms that are not hydrogen atoms. Regardless of how many equivalent or corresponding residues are determined, and regardless of the particularity of the initial IgG, on the basis of which the IgGs are created, it is a matter of the fact that known IgG variants can be designed into any second initial IgG that has significant homology in the sequence and structure with the IgG variant. Thus, for example, if you receive a variant antibody in which the original antibody is human IgG1, using the methods described above or other methods for determining equivalent residues, the variant body can be designed into another source IgG1 antibody that binds another antigen into the original antibody Human IgG2, parental human IgA antibody, parental mouse IgG2a or IgG2b antibody and the like. In addition, as described above, the context of the original IgG variant does not affect the ability to transfer the IgG variants to other original IgGs.

Methods are provided for creating, producing and screening IgG variants. This does not mean that the described methods are limited by the scope of any practical application or mechanism of action. Rather, the methods provided are intended to illustrate broadly that one or more IgG variants can be constructed, produced, and experimentally selected in order to obtain IgG variants with optimized effector function. Many methods for the construction, production and testing of antibodies and parent variants are described in US 10 / 754,296 and US 10 / 672,280, both incorporated by reference in their entirety.

For the design of IgG variants with optimized effector function, many methods for constructing proteins can be used. In one embodiment, a design method based on a known structure can be applied in which available structure information is used to manage substitutions, where substitutions are designed based on their energy compliance in computer calculations. See, for example, US 10 / 754,296 and US 10 / 672,280 and the references cited therein, all of which are incorporated by reference in their entirety.

Sequence alignment can be used to control substitutions at set positions. Those skilled in the art will understand that the use of sequence information can inhibit the introduction of substitutions that are potentially harmful to the protein structure. Sources of sequences can vary greatly and include one or more well-known databases, including, but not limited to, the Kabat database (Northwestern University); Johnson & Wu, 2001, Nucleic Acids Res 29: 205-206; Johnson & Wu, 2000, Nucleic Acids Res 28: 214-218), IMGT database (IMGT, ImMunoGeneTics information system®; Lefranc et al., 1999, Nucleic Acids Res 27: 209-212; Ruiz et al. , 2000 Nucleic Acids Res 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res 29: 207-209; Lefranc et al., 2003, Nucleic Acids Res 31: 307-310) and VBASE, all incorporated by reference in its entirety. Information about antibody sequences can be obtained, collected, and / or created from sequence alignments of the germ line of cells or naturally occurring antibodies from any organism, including but not limited to mammals. It will be clear to those skilled in the art that the use of sequences that are human or mainly human can also benefit from their lower immunogenicity when administered to humans. Other databases, which are typically nucleic acid or protein databases, that is, not only related to antibodies, include, but are not limited to SwissProt, GenBank, Entrez, and EMBL Nucleotide Sequence Database. Aligned sequences may include the sequences VH, VL, CH and / or CL. There are a large number of sequence-based alignment programs and methods known in the art, and all of them find application for creating sequence alignments.

Alternatively, random or semi-random mutagenesis techniques can be used to obtain amino acid modifications at desired positions. In these cases, positions are chosen randomly or amino acid substitutions are made using simplified techniques. For example, all residues can be mutated into alanine; this process is called alanine scanning. Such methods can be coupled with more complex engineering approaches in which selection methods are used to select higher levels of sequence varieties. As is known in the art, there are a variety of breeding technologies that can be applied in such approaches and which include, for example, display technologies, for example, phage display, ribosomal display, cell surface display, and the like, as described below.

Methods for the preparation and selection of IgG variants are well known in the art. The main methods of molecular biology of antibodies, expression, purification, and screening are described in Antibody Engineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5: 683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2: 339-76. Also see methods described in US 10 / 754,296; US 10/672,280 and US 10/822,231 and 11/124,620 are all incorporated by reference in their entirety.

Preferred embodiments of the present invention include those shown in Figure 8. Alternatively preferred embodiments of the present invention include those shown in Figure 9. In addition, alternatively preferred embodiments of the present invention include those shown in Figure 10. Particularly preferred embodiments of the present invention include G358H and N434Y. Most preferred embodiments of the present invention include 257C, 257M, 257L, 257N, 257Y, 279Q, 279Y, 308F, and 308Y.

Production of IgG variants

IgG variants can be made by any methods known in the art. In one embodiment, to generate nucleic acids that encode sequences of a representative and which can then be cloned into host cells, expressed and, if necessary, analyzed, IgG variants are used. These methods are performed using well-known procedures, and a variety of methods that may find application are described in Molecular Cloning - A Laboratory Manual, 3 ^nd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001) and Current Protocols in Molecular Biology (John Wiley & Sons), both incorporated by reference in their entirety. For protein expression, nucleic acids that encode IgG variants may be included in the expression vector. Expression vectors usually include a protein that is effectively connected, that is, in functional relationships with control or regulatory sequences, selective markers and fusion partners and / or additional elements. IgG variants can be created by culturing a host cell transformed with a nucleic acid, preferably an expression vector comprising a nucleic acid encoding the IgG variants, under conditions suitable for inducing or inducing protein expression. A huge variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. For example, many cell lines that may find use in the present invention are described in the ATCC cell line catalog, available from the American Type Culture Collection and incorporated by reference in its entirety. Methods for introducing exogenous nucleic acid into host cells are well known in the art and vary depending on which host cell is used.

In a preferred embodiment, the IgG variants are purified or isolated after expression. Proteins can be isolated or purified using numerous methods known to those skilled in the art. Standard purification methods include chromatographic methods, electrophoresis, immunological methods, sedimentation, dialysis and isoelectric focusing. As is well known in the art, a variety of naturally occurring proteins bind antibodies, such as bacterial proteins A, G, and L, and these proteins can be used in purification. Cleaning can often be done using a specific merger partner. For example, proteins can be purified using glutathione resin if fused GST is used; by N ^{+ 2} affinity chromatography, if His-tag is used, or by using an immobilized anti-flag antibody using a flag tag. For an introduction to suitable cleaning methods, see Antibody Purification: Principles and Practice, 3 ^rd Ed., Scopes, Springer-Verlag, NY, 1994, incorporated by reference in its entirety.

IgG variants can be screened using a variety of methods, including, but not limited to, methods that use in vitro assays, in vivo assays, cell assays, and selection techniques. Screening procedures can use automatic and high-performance screening technologies. A fusion partner or label, such as an immune label, an isotopic label, or a label representing a small molecule, such as a fluorescent or colorimetric dye, may be used in the screening.

In a preferred embodiment, the in vitro assay selects the functional and / or biophysical properties of the IgG variants. In a preferred embodiment, the protein is selected based on its functionality, for example, its ability to catalyze a reaction or affinity for binding to its target. Binding assays can be performed using a variety of methods known in the art, including, but not limited to, assays based on FRET (resonance fluorescence energy transfer) and BRET (bioluminescence resonance energy transfer), AlphaScreen ™ (homogeneous proximity analysis using enhanced luminescence), proximity scintillation analysis, ELISA (enzyme immunoassay), SPR (surface plasma resonance, also known as BIACORE®), isothermal titration calorimetry, differential Scanning calorimetry, gel electrophoresis, and chromatography, including gel filtration. These and other methods may take advantage of some merge partners or tags. A variety of detection methods can be used in the analyzes, including, but not limited to, chromogenic, fluorescent, luminescent and isotopic labels. Biophysical properties of proteins, such as stability or solubility, can be selected using a variety of methods known in the art. Protein stability can be determined by measuring the thermodynamic equilibrium between folded and unfolded states. For example, IgG variants can be deployed using a chemical denaturing compound, heating or pH, and this transition can be monitored using methods including, but not limited to, circular dichroism spectroscopy, fluorescence spectroscopy, absorption spectroscopy, NMR spectroscopy, calorimetry and proteolysis. As recognized by experts in this field of technology, the kinetic parameters of transitions of the rolled-up and expanded states can also be traced using these and other technologies. The solubility and overall structural integrity of the IgG variants can be characterized qualitatively and quantitatively using a wide range of methods known in the art. Methods that can be used to characterize the biophysical properties of IgG variants include gel electrophoresis, chromatography such as size exclusion chromatography and reverse phase high performance liquid chromatography, mass spectrometry, ultraviolet absorption spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, calorimetry, calorimetry titration, differential scanning calorimetry, analytical ultracentrifugation, dynamic light scattering, proteols and intermolecular cross-linking, turbidity measurement, filter delay analysis, immunoassays, binding assays fluorescent dye, analysis of protein staining, microscopy, and detection of aggregates via ELISA analysis or other binding assay. Structural analysis using crystallographic methods using x-rays and NMR spectroscopy are also possible.

As is known in the art, a group of screening methods allows the selection of useful library members. The methods described herein are referred to as “selection methods”, and these methods find use in screening for IgG variants. If protein libraries are screened using a selection method, then only those library members who are useful, that is, who meet certain selection criteria, are propagated, isolated and / or marked. It is believed that in connection with obtaining only the most suitable options, such methods allow screening of libraries that are larger in comparison with libraries that are selected using methods that analyze the individual suitability of library members. Selection can be carried out by any method, technique or using a fusion partner that covalently or non-covalently combines the phenotype of a protein with its genotype, that is, the function of the protein with a nucleic acid that encodes it. For example, the use of the phage display method as a selection method becomes possible when fusing library members with gene III protein. In this case, the selection or production of IgG variants that meet certain criteria, for example, the affinity for binding to protein targets, is also used to select or obtain the nucleic acid that encodes them. After isolation, the gene or genes encoding the IgG variants can then be amplified. This process of isolation and amplification, called panning, can be repeated and enriches the library with useful IgG variants. Finally, sequencing of the attached nucleic acid allows the gene to be identified.

In this area of technology there are many selection methods that may find application for screening protein libraries. These include, but are not limited to, the phage display method (Phage display of peptides and proteins: a laboratory manual, Kay et al., 1996 Academic Press, San Diego, CA, 1996; Lowman et al., 1991, Biochemistry 30: 10832-10838; Smith, 1985, Science 228: 1315-1317) and its modifications, such as the method of selective infection with phage (Malmborg et al., 1997, J Mol Biol 273: 544-551), the method of selectively infectious phage (Krebber et al., 1997, J Mol Biol 268: 619-630) and panning for delayed infectivity (Benhar et al., 200, J Mol Biol 301: 893-904), cell surface display (Wittrup, 2001, Curr Opin Biotechnol, 12 : 395-399), e.g. bacteria display (Georgiou et al., 1997, Nat Biotechnol 15: 29-34; Geourgiou et al., 1993 , Trends Biotechnol 11: 6-10; Lee et al., 2000, Nat Biotechnol 18: 645-648; Jim et al., 1998, Nat Biotechnol 16: 576-80), yeast (Boder & Wittrup, 2000, Methods Enzymol 328: 430-44; Boder & Wittrup, 1997, Nat Biotechnol 15: 553-557) and on mammalian cells (Whitehom et al., 1995, Biotechnology 13: 1215-1219), as well as in vitro display technologies (Amstutz et al., 2001, Curr Opin Biotechnol 12: 400-405), such as a polysome display (Matheakis et al., 1994, Proc Natl Acad Sci USA 91: 9022-9026), a ribosome display (Hanes et al., 1997 Proc Natl Acad Sci USA 94: 4937-4942), mRNA display (Roberts & Szostak, 1997, Proc Natl Acad Sci USA 94: 12297-12302; Nemoto et al., 1997, FEBS Lett 414: 405-408) and a ribosome inactivation display system (Zhow et al., 2002, J Am Chem Soc 124: 538-543). All sources are included in this paragraph by reference in its entirety.

Other selection methods that may find application include methods that are not related to display, for example, in vivo selection methods including, but not limited to, periplasm expression and cytometric screening (Chen et al., 2001, Nat Biotechnol 19 : 537-542, work included by reference in its entirety), complement analysis of protein fragments (Johnsson & Varshavsky, 1994, Proc Natl Acad Sci USA 91: 10340-10344; Pelletier et al., Proc Natl Acad Sci USA 95: 12141 -12146, all works are incorporated by reference in their entirety) and yeast double-hybrid screening system (Field s & Song, 1989, Nature 340: 245-246, work included by reference in its entirety), used in selective mode (Visintin et al., 1999, Proc Natl Acad Sci USA 96: 11723-11728, work included by reference in its entirety). In an alternative embodiment, the selection can be carried out using a fusion partner that binds to a specific sequence of the expression vector, thus covalently or non-covalently linking the fusion partner and its associated member of the Fc variants library with the nucleic acid that encodes them. For example, patents US 09 / 642,574; US 10 / 080,376; US09 / 792,630; US 10 / 023,208; US09 / 792,626; US 10 / 082,671; US09 / 953.351; US 10 / 097,100; US 60 / 366,658; PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466, all incorporated herein by reference in their entirety, describe such a merger partner and a technique that may find application. In an alternative embodiment, in vivo selection can occur if protein expression confers on the cells some of the benefits of growth, reproduction or survival.

A subset of selection methods related to “directed evolution” are methods that include combining or propagating useful sequences during selection, sometimes by incorporating new mutations. As recognized by those skilled in the art, directional evolution techniques can help identify the most useful library sequences and can increase the diversity of the selected sequences. Many directed evolutionary methods are known in the art that can be used to screen libraries of IgG variants and which include, but are not limited to, DNA rearrangement (PCT WO 00/42561 A3; PCT WO 01/70347 A3), exon rearrangement ( US 6,365,377; Kolkman & Stemmer, 2001, Nat Biotechnol 19: 423-428), family rearrangement (Crameri et al., 1998, Nature 391: 288-291; US 6,376,246), RACHITT ™ (Coco et al., 2001, Nat Biotechnol 19: 354-359; PCT WO 02/06469), STEP and random in vitro recombination priming (Zhao et al., 1998, Nat Biotechnol 16: 258-261; Shao et al., 1998, Nucleic Acids Res 26: 681 -683), exonuclease-dependent gene assembly (US 6,352,842; US 6,361, 974), Gene Site Saturation Mutagenesis ™ method (US 6,358,709), Gene Reassembly ™ method (US 6,358,709), SCRATCHY (Lutz et al., 2001, Proc Natl Acad Sci USA 98: 11248-11253), DNA fragmentation methods (Kikuchi et al., Gene 236: 159-167), rearrangement of single-stranded DNA (Kikuchi et al., 2000, Gene 243: 133-137) and AMEsystem ™ (applied molecular evolution) protein engineering for directed evolution (US 5,824,514; US 5,817,483; US 5,814,476; US 5,763,192; US 5,723,323). All sources cited in the paragraph are incorporated by reference in their entirety.

In a preferred embodiment, the IgG variants are screened using one or more cell or in vivo assays. For such assays, exogenously purified or untreated proteins are usually added so that the cells are exposed to certain variants or a pool of variants belonging to the library. These analyzes are usually, but not always, based on IgG function, i.e., the ability of IgG to bind its target antigen and mediate some biochemical events, such as effector function, inhibition of ligand / receptor binding, apoptosis, etc. Such assays often include monitoring the response of cells to IgG, for example, such as cell survival, cell death, a change in cell morphology or activation of transcription, for example, cell expression of a natural gene or reporter gene. For example, in such assays, the ability of IgG variants to induce ADCC, ADCP or CDC can be measured. For some analyzes, in addition to the target cells, it may be necessary to add additional cells or components, for example, serum complement or effector cells, such as peripheral blood monocytes (PBMC), NK cells, macrophages, etc. Such additional cells can come from any organism, but it is preferable that they be cells of humans, mice, rats, rabbits and monkeys. Antibodies can induce apoptosis of certain cell lines expressing the target, or they can mediate an attack on target cells using immune cells that have been added for analysis. Methods for monitoring cell death or viability are known in the art and include the use of dyes, immunochemical, cytochemical, and radioactive reagents. For example, a caspase staining assay provides an opportunity to measure apoptosis, and uptake and release of radioactive substrates or fluorescent dyes such as Alamar blue makes it possible to observe cell growth or cell activation. In a preferred embodiment, a DELFIA® EuTDA-based cytotoxicity assay cytotoxicity assay (Perkin Elmer, MA) is used. Alternatively, dead or damaged target cells can be monitored by the release of one or more naturally occurring intracellular proteins, such as lactate dehydrogenase. Transcription activation can also serve as a way to analyze function in cellular measurements. In this case, the response can be monitored using natural genes or proteins that can be regulated by activation, for example, the release of certain interleukins can be measured, or, alternatively, the response can be mediated by the reporter construct. Cellular assays may also include measuring morphological changes in cells in response to the presence of protein. Cell types in such assays can be prokaryotic or eukaryotic, and a variety of cell lines that are known in the art can be used. Alternatively, selection using cells is performed using cells that have been transformed or transfected with a nucleic acid encoding the variants. That is, IgG variants are not exogenously added to the cells. For example, in one of the embodiments in the selection based on the use of cells, the method of presentation on the cell surface is used. A fusion partner can be used that makes it possible to present IgG variants on the cell surface (Witrrup, 2001, Curr Opin Biotechnol, 12: 395-399, work included by reference in its entirety).

In a preferred embodiment, the immunogenicity of the IgG variants is determined experimentally using one or more cell assays. Several methods can be used for experimental confirmation of epitopes. In a preferred embodiment, ex vivo T cell activation is used to experimentally quantify immunogenicity. In this method, antigen-presenting cells and native T cells from suitable donors are provoked one or more times by the peptide or whole protein of interest. T cell activation is then detected using a number of methods, for example, by monitoring the production of cytokines or by measuring the uptake of thymidine labeled with tritium. In a most preferred embodiment, gamma-interferon production is monitored using an Elispot assay (Schmittel et al., 2000, J. Immunol. Meth. 24: 17-24, work incorporated by reference in its entirety).

The biological properties of IgG variants can be characterized in experiments on cells, tissues or the whole body. As is known in the art, drugs are often tested on animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs and monkeys, in order to determine the effectiveness of drugs for treating diseases or in disease models; or to measure the pharmacokinetics, toxicity, and other properties of drugs. Animals can be classified as disease models. Therapeutic agents are often tested on mice, including, but not limited to, hairless mice, SCID mice, xenograft mice and transgenic mice (including knockouts and knockouts). Such an experiment can provide very important information for determining the potential of a protein used as a therapeutic agent. Any organism, preferably a mammal, can be used in trials. For example, since monkeys are genetically similar to humans, they can be suitable therapeutic models, and thus can be used to test the efficacy, toxicity, pharmacokinetics, or other properties of IgG. Ultimately, human testing is required to test them as medicines, and, of course, therefore, such experiments are provided. Thus, IgG can be tested in humans to determine therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics and / or other clinical properties.

Methods of using IgG variants

IgG variants can be used in a wide range of products. In one embodiment, the IgG variant is a therapeutic, diagnostic or research reagent, preferably a therapeutic agent. The IgG variant may find use in the composition of antibodies, which may be monoclonal or polyclonal. In a preferred embodiment, IgG variants are used to kill target cells that carry a target antigen, such as cancer cells. In an alternative embodiment, IgG variants are used to block the target antigen, to be antagonists or agonists of the target antigen, for example, to be antagonists for a cytokine or cytokine receptor. In an alternative preferred embodiment, IgG variants are used to block the target antigen, to be antagonists or agonists of the target antigen, and to destroy target cells carrying the target antigen.

IgG variants can be used for various therapeutic purposes. In a preferred embodiment, antibodies comprising the IgG variant are administered to a patient for the treatment of an antibody dependent disorder. The term "patient" for the purposes of the present invention includes both humans and other animals, preferably mammals and most preferably humans. The terms “antibody-dependent disorder” or “antibody-sensitive disorder” or “condition” or “disease” as used herein mean a disorder that can be improved by administering a pharmaceutical composition comprising an IgG variant. Antibody-dependent disorders include, but are not limited to, autoimmune diseases, immunological diseases, infectious diseases, inflammatory diseases, neurological diseases, and oncological and neoplastic diseases, including cancer. The terms "cancer" and "cancerous" refer to or describe the physiological state in mammals, which is usually characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

In one embodiment, the IgG variant is the only therapeutically active agent administered to a patient. Alternatively, the IgG variant is administered together with one or more therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, antihormonal agents, kinase inhibitors, antiangiogenic agents, cardioprotectors or other therapeutic agents. IgG variants can be introduced in addition to one or more other therapeutic regimens. For example, an IgG variant may be administered to a patient along with chemotherapy, radiotherapy, or simultaneously with chemotherapy and radiotherapy. In one embodiment, the IgG variant may be introduced in combination with one or more antibodies, which may or may not be an IgG variant. In accordance with another embodiment, an IgG variant and one or more types of anti-cancer therapy are used ex vivo to effect cancer cells. It is believed that such an ex vivo effect may be useful in bone marrow transplantation, and especially in autologous bone marrow transplantation. Of course, it is believed that IgG variants can also be used in combination with other therapeutic methods, such as surgery.

For administration together with IgG variants, many other therapeutic agents may find use. In one embodiment, IgG is administered together with an antiangiogenic agent. The term "anti-angiogenic agent", which is used here, means a compound that blocks or to some extent interferes with the development of blood vessels. The anti-angiogenic agent, for example, can be a small molecule or protein, for example, an antibody fused with Fc or a cytokine that binds to a growth factor or growth factor receptor involved in stimulating angiogenesis. A preferred anti-angiogenic factor is an antibody that binds to endothelial vascular growth factor (VEGF). In an alternative embodiment, IgG is administered together with a therapeutic agent that includes or enhances an adaptive immune response, for example, with an antibody targeted by CTLA-4. In an alternative embodiment, IgG is administered together with a tyrosine kinase inhibitor. The term "tyrosine kinase inhibitor." which is used here means a molecule that to some extent inhibits the tyrosine kinase activity of tyrosine kinase. In an alternative embodiment, IgG variants are administered together with a cytokine.

Pharmaceutical compositions are provided that comprise an IgG variant and one or more therapeutically active agents. Compositions IgG-variants are prepared for storage by mixing the IgG, having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16 ^{th edition, Osol, A. Ed.} , 1980, work incorporated by reference in its entirety ) in the form of lyophilized compositions or aqueous solutions. The compositions used for in vivo administration are preferably sterile. This is easily done by filtration through sterile filtration membranes or other methods. IgG variants and other therapeutically active agents disclosed herein can be used in the composition as immunoliposomes and / or placed in microcapsules.

The concentration of the therapeutically active IgG variant in the composition may vary from about 0.1 to 100% by weight. In a preferred embodiment, the concentration of IgG is in the range of 0.003 to 1.0 M. A therapeutically effective dose of the IgG variant can be administered to treat a patient. The term "therapeutically effective dose" here means a dose that causes the effects for which it is administered. The exact dose will depend on the purpose of the treatment and is set by a specialist using known techniques. Dosages may be from 0.01 to 100 mg / kg body weight or more, for example 0.01, 0.1, 1.0, 10 or 50 mg / kg body weight, preferably from 1 to 10 mg / kg. As is known in the art, adjustments may be necessary that take into account protein degradation, the ratio of systemic delivery to localized delivery, and the level of synthesis of new proteases, as well as age, body weight, general health, gender, nutrition, time of administration, drug interaction, and severity state, and this will be clarified by specialists in the process of routine experimental work.

The introduction of a pharmaceutical composition comprising the IgG variant, preferably in the form of a sterile aqueous solution, can be carried out in many ways, including, but not limited to, oral, subcutaneous, intravenous, parenteral, intranasal, intraocular, intraocular, rectal, vaginal, transdermal, superficial (for example, when using gels, ointments, lotions, creams, etc.), intraperitoneal, intramuscular, intrapulmonary (for example, using the AERx® inhalation method commercially available from Aradigm, or using the Inhance ™ Lung Delivery System, commercially available from Nektar Terapeutics), administration methods, etc. The therapeutic agents described herein can be additionally administered together with other therapeutic agents, that is, the therapeutic agents described here can be administered together with other therapies or together with therapeutic agents that include, for example, small molecules, other biological compounds , radiation therapy, surgery, etc.

Examples

The following are examples illustrating the present invention. This does not mean that the examples given limit the scope of the present invention to any particular methods of application or theories of action. In all the provisions discussed in the present invention, the numbering corresponds to the EU index in the Kabat database (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Intitules of Health, Bethesda, work included by reference in its entirety). Those skilled in the art will recognize that this arrangement includes inconsistent numbering of immunoglobulin sequences in specific sections of the sequence, which permits normal reference to conservative positions in immunoglobulin families. Thus, the provisions of any immunoglobulin provided, determined using the EU index, do not necessarily coincide with its sequential numbering.

Example 1: DNA Construction, Expression and Purification of Fc Variants

Fc variants were constructed using the Fc domain of human IgG1 and the variable region of trastuzumab (Herceptin®, Genentech). Fc polypeptides were part of alemtuzumab, trastuzumab, or AC10. Alemtuzumab (Campath®, a trademark of Millenium) is a humanized monoclonal antibody currently approved for the treatment of chronic B-cell lymphocytic leukemia (Hale et al., 1990. Tissue Antigens 35: 118-127, work included by reference in its entirety). Trastuzumab (Herceptin®, a Genentech trademark) is an anti-Her2 / neu antibody for the treatment of metastatic breast cancer. AC10 is a monoclonal antibody to CD30. The variable region of herceptin was composed using the recursive PCR method. This variable region was then cloned together with IgG1 into the pcDNA3.1 / Zeo (+) vector (Invitrogen) shown in Figure 11. Plasmids were expanded in E. coli One Shot TOP10 cells (Invitrogen) and purified using the Hi-Speed Plasmid Maxi Kit Kit (Qiagen). Plasmids were sequenced to check for the presence of cloned inserts.

Site-directed mutagenesis was performed using the Quikchange ™ method (Stratagene). Plasmids containing the desired substitutions, insertions, and deletions were expanded in E. coli One Shot TOP10 cells (Invitrogen) and purified using the Hi-Speed Plasmid Maxi Kit (Qiagen). DNA was sequenced to confirm sequence accuracy.

Plasmids containing the heavy chain gene (VH-Cγ1-Cγ2-Cγ3) (wild type or variants) were co-transfected with a plasmid containing the light chain gene (VL-Cκ) to 293T cells. The medium was collected 5 days after transfection and the antibodies from the supernatant were purified by protein A affinity chromatography (Pierce). The concentration of antibodies was determined using analysis with bicinchoninic acid (ICA) (Pierce).

Example 2: Measurement of binding affinity

The binding of Fc polypeptides to Fc ligands was analyzed by measuring surface plasma resonance. Surface plasma resonance (SPR) measurements were performed using a BIAcore 3000 instrument (BIAcore AB). The wild-type antibody or variant antibody was fixed with immobilized protein L (Pierce Biotechnology, Rockford, IL) and binding to the receptor analyte was measured. Protein L was covalently coupled to a CM5 sensor chip at a concentration of 1 μM in 10 mM sodium acetate, pH 4.5 on a CM5 sensor chip using N-hydroxysuccinimide / N-ethyl-N '- (- 3-dimethylamino-propyl) carbodiimide (NHS / EDC) with a flow rate of 5 μl / min. Flow cell 1 of each sensor chip was treated with NHS / EDC as a negative control for binding. The working buffer was 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% w / w surfactant P20 (HBS-EP, Biacore, Upsala, Sweden), and the chip regeneration buffer was 10 mM glycine HC1 pH 1.5. A 125 nM trastuzumab antibody, wild-type or variant, was coupled to the protein L-CM5 chip in HBS-EP at a speed of 1 μl / min for 5 minutes. FcRn-His-GST, FcRn, fused to His-tag and glutathione-S-transferase, in serial dilution in the range from 1 to 250 nM, was injected for association for 20 minutes, for dissociation for 10 minutes in HBS- EP at a rate of 10 μl / min. The response, measured in resonance units (RU) and reflecting close to stationary binding, was detected 1200 seconds after the injection of the receptor. A cycle conducted only with antibody and buffer provided a baseline signal level. We plotted the dependence of RU on 1 / log concentration and approximated the sigmoidal dose-response curve using the nonlinear regression method using the GraphPad Prism program.

The binding of Fc polypeptides to Fc ligands was also performed using an AlphaScreen ™ assay (homogeneous proximity assay using enhanced luminescence). AlphaScreen ™ - Non-radioactive luminescent proximity measurement analysis. Laser excitation of donor beads excites oxygen, which in the case of closely located acceptor beads will cause a cascade of chemiluminescent phenomena, ultimately leading to fluorescence emission at 520-620 nm. The principal advantage of AlphaScreen ™ analysis is its sensitivity. Since one donor bead emits up to 60,000 excited oxygen molecules per second, the amplification of the signal becomes extremely high, which makes it possible to detect attomolar levels (10 ^-18 ). The wild-type antibody was biotinylated using standard methods to attach to streptavidin donor beads and labeled Fc ligand, for example FcRn, was attached to glutathione chelating beads. AlphaScreen ™ was used as a direct binding assay in which the interaction of the Fc / Fc ligand resulted in the association of donor and acceptor beads. In addition, AlphaScreen ™ was used in a competitive assay to screen engineered Fc polypeptides. In the absence of competing Fc polypeptides, the wild-type antibody and FcRn interacted and gave a signal at 520-620 nm. Unlabeled Fc domains competed with the wild-type / FcRn interaction, quantitatively reducing fluorescence, which makes it possible to determine the relative binding affinity.

Example 3: FcRn-binding properties of Fc variants.

The binding affinity of Fc IgG1 to FcRn was measured with variant antibodies using AlphaScreen ™. Fc polypeptides were part of alemtuzumab or trastuzumab. Alemtuzumab (Campath®, Ilex) is a humanized monoclonal antibody currently approved for the treatment of chronic B-cell lymphocytic leukemia (Hale et al., 1990. Tissue Antigens 35: 118-127, reference is hereby incorporated by reference in its entirety) . Trastuzumab (Herceptin®, "Genentech") is an anti-Her2 / neu antibody for the treatment of metastatic breast cancer.

AlphaScreen ™ competitive assay data was collected to measure the relative binding of Fc variants compared to wild-type antibody at pH 6.0. Examples of the AlphaScreen ™ signal as a function of a competitive antibody are shown in Figure 12. The curves for 12 variants: P257L, P257N, V279E, V279Q, V279Y, ^ 281S, E283F, V284E, L306Y, T307V, V308F and Q311V, show an increased affinity since In the graph in the corresponding window, the curve for each variant shifts to the left of the curve for the wild type. AlphaScreen ™ competitive assay data for the Fc variants of the present invention are summarized in Figures 13 and 14. A table is presented of the relative binding of FcRn variants compared to the wild type. Values greater than 1 demonstrate improved binding of the Fc variant to FcRn compared to the wild type. For example, variants E283L and V284E show binding that is 9.5 and 26 times stronger than that of the wild type, respectively. Surface plasma resonance measurements of many variants also show increased binding to FcRn, as shown in figures 15 and 16.

At position 257, all variants that have removed the proline amino acid and replaced an amino acid that does not have an N atom in the main chain with a covalent bond of the side chain provide greater main chain mobility, which provides greater freedom of the Fc domain for better FcRn binding. In particular, variants with L and N at position 257 possess strong binding of FcRn at pH 6, which demonstrates that the 4-atom side chain and the gamma-branched side chain element helps the Fc domain to produce, i.e., stronger, interaction with FcRn . Position 308 interacts with position 257. Both of these positions, in turn, interact with H310, which is directly involved in Fc / FcRn interactions (Table 2, Burmeister et al., 1994, Nature, 372: 379-383, work included by reference in its entirety). The affinity of the Fc variants of V308F and V308Y to FcRn is 2.9 times and 4.3 times higher compared to the wild type (Figure 13). Positions 279 and 385 interact with FcRn since all variants of V279E, V279Q and V279Y, and G385H, and G385N are more strongly associated with FcRn. All of these options are related to amino acids that are capable of forming hydrogen bonds.

The Fc variant of N434Y has particularly strong binding to FcRn at pH 6.0, as shown in Figure 13. A single variant of N434Y has 16-fold increased binding. Combinations of these options with other modifications result in even stronger binding. For example, P257L / N434Y, ^ 281S / N434Y, and V308F / N434Y demonstrate 830,180 and 350 fold enhancement of FcRn binding.

Example 4: Options Including Insertions and Deletions

Insertions and deletions were constructed that alter the strength of the Fc / FcRn interaction and their binding properties for various Fc ligands were measured. To enhance the FcRn binding properties of the Fc domain, an Fc variant was designed with an inserted Ser residue between 281 and 282 residues, according to the EU index according to Kabat et al. This option is referred to as ^ 281S, where the symbol “^” means insert immediately after the specified position. The inserted sequence, which may include more than one residue, is assigned the following position number. This Fc variant was constructed in the Kappa-IgG1 antibody trastuzumab (Herceptin®, "Genentech") using the methods disclosed in this application. The insert in the area between residues 281 and 282 shifts the remains of the Fc loop to the C-terminal portion of residue 281 towards the C-terminal portion of the loop and changes the location of the side chain. It is believed that the offset at the C-terminal portion of the loop in the Fc variants, including substitutions at positions 282, 283 and 284, was advantageous (see figure 14). Another variant with an N286 deletion, sometimes called N286 #, was also designed to move the position of the FcRn binding loop. Both of these variants show enhanced affinity for FcRn at pH 6.0.

AlphaScreen ™ analysis results show binding of the ^ 281S variant and other variants to FcRn. These AlphaScreen ™ results were collected as a direct binding assay. Higher levels of the chemiluminescent signal demonstrate stronger binding. With increasing concentrations of options in the analysis receive more powerful signals. These data at pH 6.0 shown in FIGS. 17a and 17b show an increased affinity for the ^ 281S, P257L, P257L / ^ 281S variants (combination of substitution and insertion variants) and other variants compared to wild-type Fc. Also shown is a double substitution, T250Q / M428L, which, as previously shown, has an increased serum half-life in monkeys (Hinton et al., 2004, J. Biol. Chem. 279 (8): 6213-6216, work included by reference in its entirety). The ^ 281S insert itself enhances Fc / FcRn binding. In addition, ^ 281S enhances P257L binding if two modifications are combined in the P257L / ^ 281S variant, as shown in the results for the point of ~ 40 nM. The results shown in figure 17C demonstrate that these options do not have increased FcRn binding at pH 7.0. A reduced affinity at pH 7.0 is desirable to increase the in vivo half-life, as this allows the release of Fc polypeptides from FcRn into the extracellular space, which is an important step in Fc recycling.

Surface plasma resonance experiments also show improved binding of ^ 281S to FcRn. Figure 18 shows the signal units arising from the binding of various Fc variants to FcRn on the surface of the chips. After the option is fully associated with the chip, the signal units are displayed, which are shown on the ordinate. The ^ 281S insert demonstrates binding properties comparable to the other variants shown here, which have an increased affinity for FcRn compared to the wild type (see, for example, Figures 13, 14 and 15).

The deletion variant of N286 #, including the deletion of N286, also shows an increased affinity for FcRn compared to the wild type. This variant has a 2-fold increase in FcRn affinity, as shown in FIG. 13, and also presents AlphaScreen ™ assay data on competition studies at pH 6.0. Various options have been used to inhibit the binding of wild-type Fc attached to donor beads to FcRn attached to acceptor beads. To inhibit the binding of donor and acceptor beads through the Fc / FcRn complex, half the free N286 # was required compared to wild-type free Fc. This shows a 2-fold increase in N286 # binding compared to the wild type.

Other Fc variants, including insertion and division, had a reduced affinity for FcRn. The insertion variant ^ 254N has significantly reduced FcRn binding, as expected from the nature of the variant and its location. In this embodiment, the Asn insert is located in the middle of the FcRn binding loop. This insert has only 1.1% binding affinity compared to the wild type (Figure 13).

Although particular embodiments of the present invention have been described above for purposes of illustration, it will be apparent to those skilled in the art that many modifications to the details can be made without departing from the invention, as described in the appended claims at the end of the text. All publications cited herein are incorporated by reference in their entirety.

Claims (14)

1. The polypeptide showing altered binding to FcRn, including the Fc variant of the original IgG Fc polypeptide, containing the modification in the Fc region selected from the group consisting of 308C, 308F, 308W, 308Y, where the specified Fc variant shows increased binding to FcRn compared to the original Fc polypeptide, where the numbering corresponds to the EU index. 2. The polypeptide according to claim 1, where the specified Fc-variant contains at least one substitution selected from the group consisting of: 308F, 308W and 308Y. 3. The polypeptide according to claim 1 or 2, where the specified Fc-variant contains 308F. 4. The polypeptide according to any one of claims 1 to 3, where the specified Fc-variant shows an altered binding to FcγR compared to the specified source Fc-polypeptide. 5. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant shows increased binding to FcγR. 6. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant shows reduced binding to FcγR. 7. The polypeptide according to any one of claims 1 to 4, where the specified Fc-variant shows an altered binding to FcRn compared to the original Fc-polypeptide, and where the specified polypeptide is further characterized in that the polypeptide has specificity for the target molecule, which selected from the group consisting of a cytokine, a soluble protein factor, and a protein expressed on cancer cells. 8. A polypeptide showing altered binding to FcRn, comprising the Fc variant of the original IgG Fc polypeptide, wherein said Fc variant shows increased binding to FcRn compared to the original Fc polypeptide, where the specified Fc variant includes modifications in the Fc region of the specified the source polypeptide selected from the group consisting of 252Y / 308F, 257L / 308F, 257L / 308Y, 257N / 308Y, 279Q / 308F, 279Y / 308F, ^ 281S / 308F, ^ 281S / 308Y, 284E / 308F, 298A / 308F / 333A / 334A, 308F / 332E, 308F / 311V, 308F / G385H, 308F / 428L and 308F / 434Y, where ^ means insertion, where the numbering corresponds to the EU index. 9. The polypeptide of claim 8, where the specified Fc-variant shows an altered binding to FcγR compared to the specified source Fc-polypeptide. 10. The polypeptide according to any one of claims 8 and 9, wherein said Fc variant exhibits increased binding to FcγR. 11. The polypeptide according to any one of claims 8 and 9, wherein said Fc variant exhibits reduced binding to FcγR. 12. The polypeptide according to any one of claims 8 and 9, wherein said Fc variant exhibits altered binding to FcRn compared to the original Fc polypeptide, and wherein said polypeptide is further characterized in that the polypeptide has specificity for the target molecule, which selected from the group consisting of a cytokine, a soluble protein factor, and a protein expressed on cancer cells. 13. An antibody showing altered binding to FcRn containing the Fc variant according to any one of claims 1 to 12. 14. The use of the Fc variant, characterized in any one of claims 1 to 13, to obtain fused to Fc protein, showing increased binding to FcRn.

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