Engineered antibody fc variants for enhanced serum half life

Abstract

To provide new Fc domains that have an increased serum half life by enhanced pH-dependent binding to FcRn.SOLUTION: In some aspects of the present invention, Fc domain variants are provided which display enhanced pH-dependent affinity for FcRn. In some aspects of the present invention, methods are provided for isolating antibody Fc domains that display enhanced affinity at acidic pH and pH selectivity for FcRn. In another aspect of the present invention, specific mutations and combinations of mutations in IgG1 Fc domains are provided that can result in pH-selective binding and / or increased affinity to FcRn.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 544,622, filed Aug. 11, 2017, the entirety of which is incorporated herein by reference.

[0002] The present invention relates generally to the field of protein engineering. More specifically, the present invention relates to improved serum half-life of Fc antibody domains that confer enhanced pH-dependent binding to FcRn. [Background technology]

[0003] Currently, the top 25 commercially available recombinant therapeutic antibodies have sales of well over $43.5 billion / year, which is expected to increase to $62.7 billion / year by 2015, with an expected annual growth rate of 9.2% from 2010 to 2015 (Elvinet et al., 2013). Monoclonal antibodies (mAbs) make up the majority of recombinant proteins currently in the clinic, with 1064 products undergoing company-sponsored clinical trials in the US or EU, of which 164 are in Phase III (Elvinet et al., 2013). In terms of therapeutic focus, the mAb market has a large focus on oncology and inflammatory disorders, and products within these therapeutic areas will continue to be important growth drivers over the forecast period. Engineered mAbs, as a group, generally have a higher probability of successful FDA approval than small molecule drugs. At least 50 biotechnology companies and every major pharmaceutical company have active antibody discovery programs. The original method for isolation and production of mAbs was first reported by Milstein and Kohler in 1975 (Kohler and Milstein 1975) and involved the use of mouse lymphocytes and myeloma cells to give rise to mouse hybridomas. Therapeutic murine mAbs entered clinical studies in the early 1980s; however, problems with lack of efficacy and rapid clearance due to patient production of human anti-mouse antibodies (HAMA) became evident. These challenges, along with the time and expense associated with the technology, drove the evolution of mAb production technology. Polymerase chain reaction (PCR) facilitated the cloning of monoclonal antibody genes directly from lymphocytes of immunized animals and the expression of combinatorial libraries of antibody fragments in bacteria (Orlandiet al., 1989). More recent libraries were generated by entirely in vitro cloning techniques using naive genes with rearranged complementarity determining regions 3 (CDR3) (GriffthsandDuncan 1998; Hoogenboom et al., 1998). As a result, the isolation of antibody fragments with the desired specificity is no longer dependent on the immunogenicity of the corresponding antigen. These advantages allow for the detection of a large number of unique antigens, including small molecule compounds (haptens) (HoogenboomamdWinter, 1992), molecular complexes (Chames et al., 2000), labile compounds (Kjaer et al., 1998) and cell surface proteins (Desai et al., 1998). The development of antibody fragments has become easier.

[0004] One method for screening large combinatorial libraries of antibodies to identify clones that bind to ligands with desired affinity involves the expression and display of antibody fragments or full-length antibodies on the surface of bacterial cells, more specifically E. coli. Cells displaying antibodies or antibody fragments are incubated with a solution of fluorescently labeled ligand, and cells that bind to the ligand by the antibody displayed on their surface are isolated by flow cytometry. In particular, tethered periplasmic expression (APEx) is based on the tethering of antibody fragments to the periplasmic face of the inner membrane of E. coli, followed by disruption of the outer membrane, incubation with fluorescently labeled target, and sorting of spheroplasts (US Pat. No. 7,094,571, Harvey et al., 2004; Harvey et al., 2006). Among human serum proteins, albumin and immunoglobulin G (IgG) have a longer serum half-life (approximately 3 weeks) than other serum proteins in humans. It is known that the neonatal Fc receptor (FcRn) performs many important biological functions, such as recycling and transcytosis processes, leading to the unusually long serum persistence of IgG in humans for approximately 21 days (Challa et al., 2014). Such IgG intercellular transport is Delivery may regulate its homeostasis, resulting in reduced clearance and increased in vivo half-life by preventing IgG intracellular degradation (Ober et al., 2013). In almost all cell types, FcRn is mainly localized in intracellular vesicles such as early and recycling endosomes and sorting tubules, and the expression level of FcRn is restricted to the cell surface (Ober et al., 2004). Furthermore, IgG-FcRn interaction is optimal at acidic pH, but not at neutral pH. At pH 7.4, IgG is lost. Thus, the commonly accepted model of FcRn-mediated IgG recycling is that IgG is internalized by nonspecific pinocytosis, binds to FcRn in acidic endosomes (pH 6.0), and is salvaged back to the cell surface (physiological pH 7.4) so ​​that high serum levels of IgG are maintained in the blood (Ghetie et al., 1996; Ghetie and Ward, 2000; Israel et al., 1996; Kim et al., 1999; Martinet al., 2001; Roopenian and Akilesh, 2007; Roopenian et al., 2003). However, IgG that does not dissociate from FcRn at the cell surface is salvaged from transport to lysosomes or catabolized during receptor turnover, resulting in lysosomal degradation (Gane et al., 2009). Due to the physiological importance of Fc binding to FcRn and the importance of Fc binding to FcRn with therapeutic antibodies, there is a clear need for new Fc domains with increased serum half-life through enhanced pH-dependent binding to FcRn. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,094,571 [Non-patent literature]

[0006] [Non-Patent Document 1] Elvin et al., Int. J. Pharm. (2013) 440:83~98 [Non-Patent Document 2] Roopenian and Akilesh, Nat Rev Immunol., (August 17, 2007) 7(9):715-25 [Non-Patent Document 3] Roopenian et al., J Immunol. (2003) 170(7) 3528-3533 Summary of the Invention [Means for solving the problem]

[0007] In some embodiments, the present invention overcomes limitations in the prior art by providing Fc domain variants that exhibit enhanced pH-dependent affinity to FcRn. As shown in the examples below, engineered mutant IgG Fc domains are provided that both bind to FcRn at acidic pH with affinities far exceeding those of human IgG wild type, and in some embodiments, it has been further observed that the mutant Fc domains have very low to negligible or undetectable binding to FcRn at physiological pH (physiological pH 7.4). Such high pH selectivity of affinity to FcRn is highly desirable for increased serum half-life.

[0008] In some embodiments of the present invention, methods are provided for isolating antibody Fc domains that exhibit enhanced affinity and pH selectivity for FcRn at acidic pH. In another embodiment of the present invention, specific mutations and combinations of mutations in IgG1 Fc domains that can result in pH selective binding and / or increased affinity for FcRn are provided.

[0009] More specifically, in some embodiments, a mutant or variant human Fc domain is provided that exhibits (i) enhanced binding at pH 5.8 and (ii) reduced or no detectable binding at pH 7.4 to FcRn compared to the corresponding wild-type Fc domain. The mutant or variant Fc domain may be a mutant or variant IgG domain. The mutant or variant Fc domain may be comprised in a polypeptide such as an antibody. In some embodiments, the mutant or variant Fc domain may be comprised in a therapeutic antibody, such as an agonist antibody or an antagonist antibody. In some embodiments, there is a composition involving a polypeptide having a mutant or variant Fc domain derived from a human IgG1-4 antibody ("antibody Fc domain"). The mutant Fc domain may be a variant of the wild-type human IgG1 Fc domain (SEQ ID NO: 1), where the mutant or variant Fc domain can bind to FcRn with increased affinity at acidic pH but not at neutral pH. In some embodiments, the engineered Fc domain may exhibit, for example, about 5-fold increased affinity for FcRn over glycosylated wild-type Fc domains. In further embodiments, mutant human Fc domains of all other wild-type IgG subclasses (human IgG2, IgG3, and IgG4) are provided that can bind to FcRn with increased affinity at acidic pH, but not at neutral pH. To increase the binding of the mutant or variant Fc to FcRn at acidic pH (e.g., pH 5.8), but not at physiological pH (pH 7.4), the mutant or variant Fc domain may contain mutations (L / V309D, Q311H, N434S / Y), and optionally (V264E), compared to wild-type human IgG1 Fc (SEQ ID NO: 1), human IgG2 Fc (SEQ ID NO: 2), human IgG3 Fc (SEQ ID NO: 3), or human IgG4 Fc (SEQ ID NO: 4). For example, mutations (L / V309D, Q311H, N434S) were made in human IgG1, IgG2, IgG3, and IgG4, resulting in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.As noted above, these altered binding to FcRn at acidic pH can improve the pharmacokinetics and / or increase serum half-life of the protein. As observed in the example below, engineered Fc domains were observed to exhibit enhanced serum half-life in a human FcRn transgenic mouse model.

[0010] In some embodiments, methods are provided for isolating antibody Fc domains that exhibit increased affinity and pH selectivity for FcRn. The antibody Fc domains may, for example, comprise one or more of the specific substitution mutations or combinations of substitution mutations described herein to affect selective and increased affinity binding of the Fc domain to FcRn.

[0011] In some embodiments, there are compositions involving polypeptides having an Fc domain ("antibody Fc domain") derived from a human IgG1 antibody. In additional embodiments, the Fc domain is a variant or mutant of the human IgG1 Fc domain (SEQ ID NO: 1) that may exhibit enhanced or pH-selective binding to FcRn. In some embodiments, engineered amino acid substitutions of the Fc domain (mutations L / V309D, Q311H, and N434S / Y; and optionally V264E) when introduced into other IgG subclasses (IgG2, IgG3, and IgG4) result in both (i) pH-selective binding to FcRn and (ii) enhanced binding to FcRn at acidic pH. In some embodiments, the engineered Fc domains provided herein have a 3- to 5-fold or greater increased affinity for FcRn compared to a polypeptide having a wild-type Fc domain (e.g., a K for FcRn that is enhanced by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 5.5-fold). D ) may be observed.

[0012] The antibody Fc domain can be the Fc domain of an IgG antibody or a variant thereof.Furthermore, the antibody Fc domain can be defined as a human Fc domain.In certain embodiments, the Fc domain can be an IgG1 Fc domain, such as the Fc domain of an anti-HER2 antibody.It is also contemplated that the polypeptide can comprise a fusion of the engineered Fc domain disclosed herein fused to a polypeptide that is not derived from an antibody molecule.

[0013] In some embodiments, a polypeptide comprising an antibody Fc domain comprises specific amino acid substitutions. In some embodiments, there are multiple amino acid substitutions at more than one position from the following list: (264, 309, 311, and 434); (309, 311, and 434). In some embodiments, an engineered Fc domain may have substitution mutations at two, three, four, or all of these positions. The Fc domain may be glycosylated or non-glycosylated. For example, including a mutation at position 264 (e.g., V264E) may result in a non-glycosylated Fc domain. In some embodiments, an antibody Fc domain of IgG1 may have a substitution at amino acid 309 with aspartic acid (L309D), a substitution at amino acid 311 with histidine (Q311H), a substitution at amino acid 434 with serine or tyrosine (N434S / Y), or a combination of these substitutions. Optionally, the Fc domain IgG1 may further comprise a substitution at amino acid 264 with glutamic acid (V264E). In other preferred embodiments, the antibody Fc domain (such as an IgG2, IgG3 or IgG4 Fc domain) may comprise a substitution at amino acid 309 with aspartic acid (V309D), a substitution at amino acid 311 with histidine (Q311H), a substitution at amino acid 434 with serine or tyrosine (N434S / Y), or a combination of these substitutions. Optionally, the Fc domain IgG1 may further comprise a substitution at amino acid 264 with glutamic acid (V264E).

[0014] An aspect of the present invention relates to a polypeptide comprising a mutant or variant human IgG Fc domain capable of binding to human FcRn at acidic pH, the Fc domain having the following substitution mutations: (i) aspartic acid at position 309 (L / V309D); (ii) histidine at position 311 (Q311H); and (iii) substitution mutation at position 434 (N434). The substitution mutation at position 434 can be serine (N434S) or tyrosine (N434Y). The Fc domain may be glycosylated. In some embodiments, the Fc domain has substantially equal, essentially the same, nearly the same, or the same binding to FcγR as compared to wild type. In some embodiments, the Fc domain has substantially equal, essentially the same, nearly the same, or the same binding ability to one, two, or all of FcγRI, FcγRII, and FcγRIII as compared to wild type. The Fc domain may bind to FcRn with higher affinity at acidic pH than wild type. In some embodiments, the Fc domain does not detectably or selectively bind to FcRn at neutral pH, or shows no or essentially no binding to FcRn. In some embodiments, the Fc domain shows (i) enhanced binding at pH 5.8 and (ii) reduced binding or no detectable binding at pH 7.4 to FcRn compared to wild type. The Fc domain may be non-glycosylated. For example, in some embodiments, the Fc domain has a glutamic acid substitution mutation at position 264 (V264E). The IgG may be IgG1, IgG2, IgG3, or IgG4.In some embodiments, the Fc domain comprises or consists of EDHS (V264E, L309D, Q311H, and N434S; e.g., SEQ ID NO:5), EDHY (V264E, L309D, Q311H, and N434Y; e.g., SEQ ID NO:6), DHS (L309D, Q311H, and N434S; e.g., SEQ ID NO:7), DHY (L309D, Q311H, and N434Y; e.g., SEQ ID NO:8), IgG2-DHS (V309D, Q311H, and N434S; SEQ ID NO:9), IgG3-DHS (L309D, Q311H, and N434S; SEQ ID NO:10), or IgG4-DHS (L309D, Q311H, and N434S; SEQ ID NO:11). In some embodiments, the Fc domain comprises or consists of DHS (L309D, Q311H, and N434S; e.g., SEQ ID NO: 7) or DHY (L309D, Q311H, and N434Y; e.g., SEQ ID NO: 8). In some embodiments, the Fc domain has a K of less than about 550 nM, less than about 525 nM, less than about 500 nM, less than about 475 nM, less than about 450 nM, less than about 425 nM, less than about 400 nM, less than about 375 nM, less than about 350 nM, less than about 325 nM, less than about 300 nM, less than about 275 nM, less than about 250 nM, less than about 225 nM, less than about 200 nM, less than about 175 nM, less than about 150 nM, less than about 125 nM, or less than about 100 nM to FcRn. DThe polypeptide may further comprise a non-Fc receptor (non-FcR) binding domain. The non-FcR binding domain may be an Ig variable domain. The polypeptide may be a full-length antibody. In some embodiments, the antibody is an agonist or antagonist antibody. In some embodiments, the antibody selectively binds to Her2 / neu, CD20, CD40, IL-10, 4-1BB, PD-1, PD-L1, CTLA-4OX40, IL-1, IL-6, IL6R, TNFα, RANKL, EGFR, c-Met, CD11a, VEGF-A, VEGFR1, VEGFR2, C5, or Integrain-α4. The antibody may be chemically conjugated or covalently attached to a toxin. In some embodiments, the non-FcR binding region is not an antigen-binding site of the antibody. In some embodiments, the non-FcR binding region binds to a cell surface protein. In some embodiments, the non-FcR binding region binds to a soluble protein.

[0015] Another aspect of the present invention relates to a nucleic acid encoding any of the polypeptides of the present invention or above. The nucleic acid can be a DNA segment. In some embodiments, the nucleic acid is an expression vector.

[0016] Yet another aspect of the present invention relates to a host cell comprising a nucleic acid according to the invention or as described above. The cell is capable of expressing the nucleic acid.

[0017] Another aspect of the invention relates to a method for preparing a polypeptide comprising the steps of a) obtaining a host cell of the invention or as described above, b) incubating the host cell in culture under conditions to promote expression of a non-glycosylated polypeptide, and c) purifying the expressed polypeptide from the host cell, hi some embodiments, the host cell is a prokaryotic cell or a mammalian cell.

[0018] Yet another aspect of the invention relates to a pharmaceutical formulation comprising the inventive or above mentioned polypeptide or the inventive or above mentioned nucleic acid in a pharma- ceutically acceptable carrier.

[0019] Another aspect of the invention is a method of binding a protein in a subject comprising the step of providing to the subject an antibody, wherein the antibody binds to a protein and comprises an Fc domain of the invention or above. In some embodiments, the antibody has a K of less than about 550 nM, less than about 525 nM, less than about 500 nM, less than about 475 nM, less than about 450 nM, less than about 425 nM, less than about 400 nM, less than about 375 nM, less than about 350 nM, less than about 325 nM, less than about 300 nM, less than about 275 nM, less than about 250 nM, less than about 225 nM, less than about 200 nM, less than about 175 nM, less than about 150 nM, less than about 125 nM, or less than about 100 nM for human FcRn. D In some embodiments, the antibody is glycosylated and the Fc domain has nearly equal or equal binding to FcγRI, FcγRII, and FcγRIII compared to wild type. In some embodiments, the antibody is a glycosylated therapeutic antibody. In some embodiments, the antibody is an aglycosylated version of a therapeutic antibody.

[0020] Yet another aspect of the present invention relates to a method for treating a subject having a disease, comprising administering an effective amount of the present or above formulation to the subject. The method can induce antibody-dependent cellular cytotoxicity. In some embodiments, the disease is cancer, infection, or autoimmune disease (e.g., rheumatoid arthritis). The subject can be a human patient. The formulation can be administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion that directly bathes target cells, by catheter, or by lavage. In some embodiments, the disease is cancer, and the method further comprises administering at least a second anti-cancer therapy to the subject. The second anti-cancer therapy can be surgery, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy or cytokine therapy. The disease can include a tumor, such as a solid tumor or a hematological tumor.

[0021] In one embodiment, a composition is provided comprising a variant Fc domain of this embodiment or a nucleic acid encoding a variant Fc domain of this embodiment for use in treating a disease (e.g., cancer, infection, autoimmune disease, bacterial infection, or viral infection). Treating the disease may involve binding a select protein such that there is reduced immune activation or reduced complement dependent cytotoxicity while a therapeutic effect (e.g., resulting from binding of a toxin, or stimulation of a receptor with an agonist antibody, etc.) is achieved. In some aspects, the disease may be cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. In another embodiment, there is provided the use of a polypeptide according to this embodiment or a nucleic acid encoding a polypeptide according to this embodiment in the manufacture of a medicament for the treatment of a disease, such as cancer.

[0022] In some embodiments, a pharma- ceutically acceptable composition is provided comprising a polypeptide according to the present invention or above and a pharma- ceutically acceptable excipient. In some embodiments, a composition is provided for use in a method of treating a disease in a subject in need thereof, the composition comprising a polypeptide according to the present invention or above. The disease may be cancer, an infection, a bacterial infection, a viral infection, or an autoimmune disease.

[0023] A variant Fc domain polypeptide (also referred to as a mutant or engineered Fc domain) can be characterized as having a certain percentage of identity compared to an unmodified polypeptide (e.g., a wild-type Fc domain polypeptide such as a wild-type IgG Fc domain, or a human wild-type IgG Fc domain) or any polypeptide sequence disclosed herein. The percentage identity can be approximately at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% (or any range derivable therein) between the unmodified portion of the modified polypeptide (i.e., the sequence of the modified polypeptide excluding any specified substitutions) and the corresponding wild-type polypeptide. For example, a variant Fc domain may have, for example, at least 90%, at least about 95%, at least 99%, or 100% sequence identity compared to a wild-type Fc domain (e.g., a wild-type human IgG Fc domain, such as any one of SEQ ID NOs: 1-4) with respect to a region of the variant Fc domain excluding the specified substitution mutations (e.g., L / V309D, Q311H, and N434S / Y; and optionally V264E). A variant Fc domain may also include additional mutations in addition to the specified substitution mutations in the variant Fc domain compared to the wild-type Fc domain. It is also contemplated that the above percentages of identity may relate to the entirety of a variant Fc domain polypeptide compared to a wild-type Fc domain (e.g., a human IgG Fc domain). For example, a variant Fc domain polypeptide characterized as having at least 90% identity to a wild-type Fc domain means that at least 90% of the amino acids in the variant polypeptide are identical to those in the wild-type polypeptide.

[0024] The antibody Fc domain can be a mutant or variant Fc domain of a human IgG antibody (e.g., the Fc domain can be a human IgG1, IgG2, IgG3, or IgG4 Fc domain). It is also contemplated that the polypeptide can include a fusion of an engineered variant Fc domain disclosed herein fused to a polypeptide that is not derived from an antibody molecule. In some embodiments, the engineered Fc domain of the invention is included in an agonist antibody, such as an antibody that targets CD40, death receptor 5 (DR5), PD-1, or TNF receptor (TNFR) molecules.

[0025] The polypeptide comprising the variant Fc domain described herein may, in some embodiments, include a linker. In further embodiments, the linker is a conjugable linker. In some embodiments, the polypeptide contains an Fc domain derived from an antibody. It may contain other regions derived from an antibody, such as another binding domain. In some embodiments, the additional binding domain may not be an FcR binding domain. In some embodiments, the polypeptide may contain an antigen binding site or domain derived from an antibody, such as all or a portion of a variable region derived from an antibody. The polypeptide may contain an Fc domain derived from an antibody and another binding domain that is a non-FcR binding domain. In some embodiments, the non-Fc binding region specifically binds to a cell surface protein or a soluble protein, rather than the antigen binding site of an antibody. In some cases, the cell surface protein that the non-Fc binding region recognizes is a receptor, such as a receptor expressed on the cell surface.

[0026] Other polypeptides include polypeptides having an Fc domain variant (e.g., capable of enhanced binding to FcRn polypeptides at acidic pH but exhibiting reduced or no detectable binding to FcRn at physiological or neutral pH) and a second binding domain that is a non-Fc receptor binding domain, the second binding domain being capable of specifically binding to a cell surface molecule or a soluble protein. In some embodiments, the second binding domain is an antigen binding domain of an antibody ("Ig variable domain"). In some aspects, the polypeptide may be a full-length antibody. In some cases, the second binding domain is not an antibody antigen binding domain. In some embodiments, the second binding domain is capable of specifically binding to a cell surface molecule that is a protein or proteinaceous molecule. In some aspects, the second binding domain is capable of specifically binding to a soluble protein.

[0027] Some aspects relate to a nucleic acid encoding any of the polypeptides described herein. The nucleic acid can be isolated and / or recombinant. It can be an isolated and / or recombinant nucleic acid segment. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is a DNA segment. In some embodiments, the nucleic acid is an expression vector capable of expressing any of the polypeptides having an Fc binding domain with one or more substitutions that specifically bind to FcγRn. The nucleic acid can encode one or more of the polypeptides herein, which may or may not be glycosylated depending on the presence or absence of certain mutations and the manner in which the polypeptide is produced.

[0028] In some embodiments, the nucleic acid encodes a polypeptide comprising or consisting of a variant or mutant Fc domain capable of pH-selectively binding to FcRn as described herein. The nucleic acid can be placed (e.g., transfected or transformed) into a host cell capable of expressing the polypeptide, such as an unglycosylated version of the polypeptide. The host cell can be a prokaryotic cell, such as a bacterial cell. Alternatively, the host cell can be a eukaryotic cell, such as a mammalian cell. In some embodiments, the host cell contains a first expression vector, but may also contain a second expression vector. Since some antibodies are composed of multiple polypeptides, some embodiments may utilize a host cell that contains the necessary expression vector(s) to express the polypeptides. In some embodiments, the host cell expresses a first expression vector encoding a polypeptide comprising or consisting of an immunoglobulin heavy chain (e.g., containing a variant or mutant Fc domain that binds FcRn with enhanced affinity at acidic pH but not at neutral pH). In some embodiments, the host cell contains a second expression vector encoding a polypeptide comprising or consisting of an immunoglobulin light chain. The host cell may contain, for example, one or two expression vectors to allow expression of an antibody comprising a heavy and a light chain.

[0029] In some embodiments, a population of host cells is provided, the population comprising a plurality of host cells expressing polypeptides having different Fc domains. It is contemplated that the amino acid sequences of any two different Fc domains may differ by less than 20%, less than 15%, less than 10%, less than 5%, or less than identity.

[0030] In some aspects, methods of making the polypeptides described herein (e.g., polypeptides having an Fc region that binds to FcRn with enhanced affinity at acidic pH but not at neutral pH) as well as methods of using these polypeptides are provided. It is anticipated that the methods described herein or known to those of skill in the art can be for making or using any of the polypeptides described herein.

[0031] In some embodiments, the method involves purifying the polypeptide from the supernatant. This can involve subjecting the polypeptide from the supernatant to filtration, HPLC, anion or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof. In some embodiments, the method involves affinity chromatography using Staphylococcus aureus protein A, which binds to the IgG Fc region. Other purification methods are well known to those skilled in the art.

[0032] In some embodiments, a pharmaceutical formulation is provided that includes the polypeptide or nucleic acid of the embodiments in a pharma- ceutically acceptable carrier, or a pharmaceutical preparation that includes an excipient.

[0033] In some embodiments, an immune response can be induced in a subject by a method comprising the step of providing or administering (e.g., intravenously, etc.) an antibody to a subject, where the antibody comprises an Fc domain that binds with enhanced affinity to FcRn at acidic pH as described herein, but not at neutral or physiological pH.

[0034] In further embodiments, cancer, infection, autoimmune or inflammatory diseases can be treated by administering therapeutic polypeptides comprising a variant or mutant Fc domain described herein that binds to FcRn with enhanced affinity at acidic pH but not at neutral or physiological pH. It is envisioned that polypeptides comprising a mutant or variant Fc domain described herein may exhibit similar complement dependent cytotoxicity (CDC) as compared to that induced by a polypeptide comprising a wild-type human IgG Fc region. In yet further embodiments, the polypeptides of the present invention may exhibit similar ADCC or ADCP as compared to wild-type human IgG antibodies.

[0035] As used herein, "pH-selective binding to FcRn" or "pH-selective binding to FcRn" refers to the property of a polypeptide, such as an Fc domain (e.g., a mutant or variant IgG Fc domain), having the ability to bind to FcRn at acidic pH (e.g., pH 5.8), and preferably the polypeptide or Fc domain has the ability to exhibit increased binding to FcRn at acidic pH compared to a wild-type Fc domain (e.g., a wild-type Fc IgG domain). In some embodiments, an Fc domain or polypeptide that pH-selectively binds to FcRn also exhibits reduced binding to FcRn at physiological pH compared to a wild-type (e.g., a wild-type IgG Fc domain) or no detectable binding to FcRn at physiological pH.

[0036] As used herein, "essentially free of" is used herein to mean, with respect to a named component, that none of the named components are intentionally formulated in the composition and / or that it is present merely as a contaminant or in trace amounts. The total amount of the named components resulting from unintentional contamination of any of the compositions is therefore well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the amount of the named components cannot be detected by standard analytical methods.

[0037] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, K D The affinity of a binding domain for its target can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM); alternatively, it can be between 100 nM and 1 nM, or between 0.1 nM and 10 nM. Furthermore, it is contemplated that two agents specifically bind when there is an affinity between them within the affinity ranges noted above.

[0038] As used herein, the terms "encode" or "encoding" in reference to nucleic acids are used to enable one of skill in the art to readily understand the invention; however, these terms can be used interchangeably with "comprise" or "comprising," respectively.

[0039] As used herein, "a" or "an" can mean one or more. As used in the claim(s) herein, when used in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one.

[0040] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, the present disclosure supports the provision to refer to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0041] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device employed to measure the variable, the method used to determine the value, or the variation that exists among the study subjects. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention may become apparent to those skilled in the art from this detailed description. The following figures form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. In certain embodiments, for example, the following items are provided: (Item 1) A polypeptide comprising a mutant or variant human IgG Fc domain capable of binding to human FcRn at acidic pH, said Fc domain comprising: (i) aspartic acid at position 309 (L / V309D); (ii) histidine at position 311 (Q311H); and (iii) a substitution mutation at position 434 (N434) A polypeptide having a substitution mutation of (Item 2) 2. The polypeptide according to item 1, wherein the substitution mutation at position 434 is serine (N434S) or tyrosine (N434Y). (Item 3) 3. The polypeptide according to item 2, wherein the substitution mutation at position 434 is serine (N434S). (Item 4) 3. The polypeptide according to item 2, wherein the substitution mutation at position 434 is tyrosine (N434Y). (Item 5) 5. The polypeptide according to any one of items 1 to 4, wherein the Fc domain is glycosylated. (Item 6) 6. The polypeptide of item 5, wherein the Fc domain has binding to FcγR that is substantially equal, essentially the same, about the same, or the same as compared to wild-type. (Item 7) The polypeptide of item 5, wherein the Fc domain has substantially equal, essentially the same, nearly the same, the same, or equal binding ability to one, two, or all of FcγRI, FcγRII, and FcγRIII compared to the wild type. (Item 8) 8. The polypeptide according to any one of items 1 to 7, wherein the Fc domain binds to FcRn with higher affinity at acidic pH than a wild-type polypeptide. (Item 9) 9. The polypeptide of item 8, wherein the Fc domain does not detectably or selectively bind to FcRn at neutral pH, or exhibits no or essentially no binding to FcRn. (Item 10) 10. The polypeptide of item 9, wherein the Fc domain exhibits (i) enhanced binding at pH 5.8 and (ii) reduced or no detectable binding to FcRn at pH 7.4, compared to wild-type. (Item 11) 5. The polypeptide according to any one of items 1 to 4, wherein the Fc domain is non-glycosylated. (Item 12) 12. The polypeptide of item 11, wherein the Fc domain has a glutamic acid substitution mutation at position 264 (V264E). (Item 13) The polypeptide according to any one of items 1 to 12, wherein the IgG is IgG1. The polypeptide according to any one of items 1 to 12, wherein the IgG is IgG2. The polypeptide according to any one of items 1 to 12, wherein the IgG is IgG3. The polypeptide according to any one of items 1 to 12, wherein the IgG is IgG4. The Fc domain comprises: EDHS (SEQ ID NO:5; V264E; L309D; Q311H; N434S), EDHY (SEQ ID NO: 6; V264E; L309D; Q311H; N434Y), DHS (SEQ ID NO: 7; L309D; Q311H; N434S), DHY (SEQ ID NO: 8; L309D; Q311H; N434Y), IgG2-DHS (SEQ ID NO: 9; V309D; Q311H; N434S), IgG3-DHS (SEQ ID NO: 10; L309D; Q311H; N434S), or IgG4-DHS (SEQ ID NO: 11; L309D; Q311H; N434S) 13. The polypeptide according to any one of items 1 to 12, comprising or consisting of: (Item 18) 18. The polypeptide of item 17, wherein the Fc domain comprises or consists of DHS (SEQ ID NO: 7; L309D; Q311H; N434S) or DHY (SEQ ID NO: 8; L309D; Q311H; N434Y). (Item 19) The Fc domain has a K D 19. The polypeptide according to any one of items 1 to 18, wherein the polypeptide binds at a value of (Item 20) The Fc domain has a K D 20. The polypeptide according to item 19, which binds at a value (Item 21) The Fc domain has a K D 20. The polypeptide according to item 19, which binds at a value (Item 22) 22. The polypeptide of any one of items 1 to 21, further comprising a non-Fc receptor (non-FcR) binding domain. (Item 23) 23. The polypeptide of item 22, wherein the non-FcR binding domain is an Ig variable domain. (Item 24) 24. The polypeptide according to item 23, which is a full-length antibody. (Item 25) 25. The polypeptide of claim 24, wherein the antibody is an agonistic antibody. (Item 26) 25. The polypeptide according to item 24, wherein the antibody is an antagonist antibody. (Item 27) 25. The polypeptide of claim 24, wherein the antibody selectively binds to Her2 / neu, CD20, CD40, IL-10, 4-1BB, PD-1, PD-L1, CTLA-4OX40, IL-1, IL-6, IL6R, TNFα, RANKL, EGFR, c-Met, CD11a, VEGF-A, VEGFR1, VEGFR2, C5, or integrin-α4. (Item 28) 28. The polypeptide according to any one of items 1 to 27, wherein the antibody is chemically conjugated or covalently attached to a toxin. (Item 29) 23. The polypeptide of item 22, wherein the non-FcR binding region is not an antigen-binding site of an antibody. (Item 30) 23. The polypeptide of item 22, wherein the non-FcR binding region binds to a cell surface protein. (Item 31) 23. The polypeptide of item 22, wherein the non-FcR binding region binds to a soluble protein. (Item 32) A nucleic acid encoding any one of the polypeptides according to items 1 to 31. (Item 33) 33. The nucleic acid according to item 32, which is a DNA segment. (Item 34) 33. The nucleic acid according to item 32, which is an expression vector. (Item 35) A host cell comprising the nucleic acid according to any one of items 32 to 34. (Item 36) 36. The host cell of item 35, which expresses the nucleic acid. (Item 37) 1. A method for preparing a polypeptide comprising the steps of: a) obtaining a host cell according to item 36; b) incubating said host cell in culture under conditions to promote expression of a non-glycosylated polypeptide; c) purifying the expressed polypeptide from the host cell. A method comprising: (Item 38) 38. The method of claim 37, wherein the host cell is a prokaryotic cell or a mammalian cell. (Item 39) 35. A pharmaceutical formulation comprising the polypeptide according to any one of items 1 to 31 or the nucleic acid according to any one of items 32 to 34 in a pharma- ceutically acceptable carrier. (Item 40) 32. A method for binding a protein in a subject, the method comprising the step of providing an antibody to the subject, wherein the antibody binds to the protein and comprises an Fc domain according to any one of items 1 to 31. (Item 41) The antibody has a K D 41. The method according to item 40, wherein the antibody can specifically bind to the antibody at (Item 42) The antibody has a K D 42. The method according to item 41, wherein the antibody can specifically bind to the antibody at (Item 43) The antibody has a K D 43. The method according to item 42, wherein the antibody can specifically bind to the antibody at (Item 44) 44. The method of any one of items 40 to 43, wherein the antibody is glycosylated and the Fc domain has nearly equivalent or equivalent binding to FcγRI, FcγRII, and FcγRIII compared to wild type. (Item 45) 45. The method according to any one of items 40 to 44, wherein the antibody is a glycosylated therapeutic antibody. (Item 46) 45. The method according to any one of items 40 to 44, wherein the antibody is a non-glycosylated version of a therapeutic antibody. (Item 47) 40. A method for treating a subject having a disease, comprising administering to the subject an effective amount of the formulation of item 39. (Item 48) 48. The method according to item 47, which induces antibody-dependent cellular cytotoxicity. (Item 49) 48. The method of claim 47, wherein the disease is cancer, an infection, or an autoimmune disease. (Item 50) 48. The method of claim 47, wherein the subject is a human patient. (Item 51) 48. The method of item 47, wherein the formulation is administered intratumorally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intraocularly, intranasally, intravitreally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, or by lavage. (Item 52) 52. The method according to any one of items 47 to 51, wherein the disease is cancer, and further comprising the step of administering at least a second anti-cancer therapy to the subject. (Item 53) 53. The method of item 52, wherein the second anticancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. (Item 54) 32. The polypeptide according to any one of items 1 to 31 for use in the treatment of a disease. (Item 55) 55. The polypeptide according to item 54, wherein the disease is cancer, an infection, or an autoimmune disease. (Item 56) 55. The polypeptide according to item 54, wherein the disease is a bacterial infection or a viral infection. (Item 57) 32. Use of a polypeptide according to any one of items 1 to 31 in the preparation of a medicament for the treatment of a disease, such as cancer, an infection, a bacterial infection, a viral infection, or an autoimmune disease. (Item 58) 32. A pharma- ceutically acceptable composition comprising the polypeptide according to any one of items 1 to 31 and a pharma- ceutically acceptable excipient. (Item 59) 32. A composition for use in a method of treating a disease in a subject in need thereof, the composition comprising a polypeptide according to any one of items 1 to 31. (Item 60) 60. The composition of item 59, wherein the disease is cancer, an infection, a bacterial infection, a viral infection, or an autoimmune disease. [Brief description of the drawings]

[0042] [Figure 1] 1A-B are simplified diagrams of a two-plasmid system for bacterial periplasmic display of trastuzumab light chain (FIG. 1A) and trastuzumab heavy chain (FIG. 1B). [Diagram 2] Figure 2 shows the complex structure of the Fc domain of IgG1 with FcRn-β2m. Adjacent residues of the Fc domain near FcRn (<7 Å) are highlighted based on the FcRn-Fc complex structure (PDB ID: 4N0U) (Oganesyan V. et al., 2014). The side chains of adjacent residues in the Fc domain relative to FcRn are shown in the highlighted structure. Adjacent residues in the Fc domain relative to FcRn are randomized using primers containing degenerate codons. IgG1-Fc domain (left), FcRn-β2m (right). [Figure 3-1] Figure 3A-B shows the screening strategy and results of the Fc library. Figure 3A, IgG-displaying spheroplasts were labeled with single chain FcRn (scFcRn)-APC at pH 5.8 and then labeled with dimeric FcRn-FITC at pH 7.4. Figure 3B, the Fc library was screened with FcRn for five rounds and the resulting FcRn binding activity is shown. [Figure 3-2] Figure 3A-B shows the screening strategy and results of the Fc library. Figure 3A, IgG-displaying spheroplasts were labeled with single chain FcRn (scFcRn)-APC at pH 5.8 and then labeled with dimeric FcRn-FITC at pH 7.4. Figure 3B, the Fc library was screened with FcRn for five rounds and the resulting FcRn binding activity is shown. [Figure 3-3] Figure 3A-B shows the screening strategy and results of the Fc library. Figure 3A, IgG-displaying spheroplasts were labeled with single chain FcRn (scFcRn)-APC at pH 5.8 and then labeled with dimeric FcRn-FITC at pH 7.4. Figure 3B, the Fc library was screened with FcRn for five rounds and the resulting FcRn binding activity is shown. [Figure 4] Figure 4. FACS analysis of isolated Fc variants. E. coli spheroplasts expressing the Fc variants were labeled with 10 nM scFcRn-FITC at pH 5.8 or 50 nM dFcRn-APC at pH 7.4. [Figure 5-1] Figures 5A-F are surface plasmon resonance (SPR) sensorgrams of Herceptin (Figure 4A) and selected IgG variants, EDH (Figure 4B), EDHY (Figure 4C), EDHS (Figure 4D), Herceptin-LS (Figure 4E), and Herceptin-YTE (Figure 4F). Each antibody was immobilized on a CM5 chip, and serially diluted scFcRn proteins in PBS, pH 5.8, were injected onto the CM5 chip. [Figure 5-2] Figures 5A-F are surface plasmon resonance (SPR) sensorgrams of Herceptin (Figure 4A) and selected IgG variants, EDH (Figure 4B), EDHY (Figure 4C), EDHS (Figure 4D), Herceptin-LS (Figure 4E), and Herceptin-YTE (Figure 4F). Each antibody was immobilized on a CM5 chip, and serially diluted scFcRn proteins in PBS, pH 5.8, were injected onto the CM5 chip. [Figure 6]Figure 6 shows the binding activity of antibody variants to dFcRn at pH 7.4. Each antibody was coated onto a 96-well plate, and the binding activity of dFcRn was detected by anti-GST HRP. [Figure 7-1] Figures 7A-F show ELISA results of wild type Herceptin and selected IgG variant Herceptin-EDHS against FcγR; monomeric FcγRI (Figure 7A), dimeric FcγRIIAH131 (Figure 7B), dimeric FcγRIIAR131 (Figure 7C), dimeric FcγRIIB (Figure 7D) dimeric FcγRIIIAV157 (Figure 7E), and dimeric FcγRIIIAF157 (Figure 7F). [Figure 7-2] Figures 7A-F show ELISA results of wild type Herceptin and selected IgG variant Herceptin-EDHS against FcγR; monomeric FcγRI (Figure 7A), dimeric FcγRIIAH131 (Figure 7B), dimeric FcγRIIAR131 (Figure 7C), dimeric FcγRIIB (Figure 7D) dimeric FcγRIIIAV157 (Figure 7E), and dimeric FcγRIIIAF157 (Figure 7F). [Figure 8] 8 is a surface plasmon resonance (SPR) sensorgram of Herceptin-DHS. The antibody was immobilized on a CM5 chip, and serially diluted scFcRn protein in PBS pH 5.8 was injected onto the CM5 chip. [Figure 9] Figure 9 shows the binding activity of antibody variants to dimeric FcRn (dFcRn) at pH 7.4. Each antibody was coated onto a 96-well plate, and the binding activity of dFcRn was detected by anti-GST HRP. [Figure 10-1] Figures 10A-F show ELISA results of wild type Herceptin and selected IgG variant Herceptin-DHS against FcγR; monomeric FcγRI (Figure 10A), dimeric FcγRIIAH131 (Figure 10B), dimeric FcγRIIAR131 (Figure 10C), dimeric FcγRIIB (Figure 10D) dimeric FcγRIIIAV157 (Figure 10E), and dimeric FcγRIIIAF157 (Figure 10F). [Figure 10-2]Figures 10A-F show ELISA results of wild type Herceptin and selected IgG variant Herceptin-DHS against FcγR; monomeric FcγRI (Figure 10A), dimeric FcγRIIAH131 (Figure 10B), dimeric FcγRIIAR131 (Figure 10C), dimeric FcγRIIB (Figure 10D) dimeric FcγRIIIAV157 (Figure 10E), and dimeric FcγRIIIAF157 (Figure 10F). [Figure 10-3] Figures 10A-F show ELISA results of wild type Herceptin and selected IgG variant Herceptin-DHS against FcγR; monomeric FcγRI (Figure 10A), dimeric FcγRIIAH131 (Figure 10B), dimeric FcγRIIAR131 (Figure 10C), dimeric FcγRIIB (Figure 10D) dimeric FcγRIIIAV157 (Figure 10E), and dimeric FcγRIIIAF157 (Figure 10F). [Figure 11] 11A-B are Herceptin-DHS-mediated ADCC and ADCP assays. For each assay, antibodies were incubated with Her2-positive SK-BR3 cells and PBMCs or macrophages. [Figure 12] Figure 12 shows surface plasmon resonance (SPR) sensorgrams of Herceptin, Herceptin-DHS, Herceptin-LS, and Herceptin-YTE with three different densities of scFcRn. scFcRn was immobilized on a CM5 chip at three different levels, 500RU, 2000RU, and 4000RU, and serially diluted antibody proteins in PBS pH 7.4 were injected onto the CM5 chip. [Figure 13-1] 13A-C are surface plasmon resonance (SPR) sensorgrams of Herceptin, Herceptin-DHS, Herceptin-LS, and Herceptin-YTE with three different densities of scFcRn. scFcRn was immobilized on a CM5 chip at three different levels, 500 RU, 2000 RU, and 4000 RU, and 1 μM antibody protein at various pH values ​​was injected onto the CM5 chip. [Figure 13-2]13A-C are surface plasmon resonance (SPR) sensorgrams of Herceptin, Herceptin-DHS, Herceptin-LS, and Herceptin-YTE with three different densities of scFcRn. scFcRn was immobilized on a CM5 chip at three different levels, 500 RU, 2000 RU, and 4000 RU, and 1 μM antibody protein at various pH values ​​was injected onto the CM5 chip. [Figure 14] FIG. 14 shows the pharmacokinetics of antibodies in human FcRn transgenic mice. [Figure 15-1] Figures 15A-F are surface plasmon resonance (SPR) assays of IgG2 (Figure 15A), IgG2-DHS (Figure 15B), IgG3 (Figure 15C), IgG3-DHS (Figure 15D), IgG4 (Figure 15E), and IgG4-DHS (Figure 15F) against scFcRn. [Figure 15-2] Figures 15A-F are surface plasmon resonance (SPR) assays of IgG2 (Figure 15A), IgG2-DHS (Figure 15B), IgG3 (Figure 15C), IgG3-DHS (Figure 15D), IgG4 (Figure 15E), and IgG4-DHS (Figure 15F) against scFcRn. [Figure 15-3] Figures 15A-F are surface plasmon resonance (SPR) assays of IgG2 (Figure 15A), IgG2-DHS (Figure 15B), IgG3 (Figure 15C), IgG3-DHS (Figure 15D), IgG4 (Figure 15E), and IgG4-DHS (Figure 15F) against scFcRn. [Figure 16-1] Figures 16A-C are ELISA assays of IgG2-DHS, IgG3-DHS, and IgG4-DHS. FcγR binding activity of IgG variants compared to their wild type IgG. FcγR at 400nM, 80nM, 16nM, and 3.2nM were assayed with IgG variants. [Figure 16-2] Figures 16A-C are ELISA assays of IgG2-DHS, IgG3-DHS, and IgG4-DHS. FcγR binding activity of IgG variants compared to their wild type IgG. FcγR at 400nM, 80nM, 16nM, and 3.2nM were assayed with IgG variants. [Figure 17] FIG. 17 shows the pharmacokinetics of antibodies in human FcRn transgenic mice. [Figure 18-1] Figure 18 shows the SPR analysis of antibody variants. Each antibody was immobilized on a CM5 chip, and serially diluted FcγR or C1q was injected at 30 μl / min. All experiments were repeated three times independently, and the kinetic values ​​of each antibody are presented in Table 12. [Figure 18-2] Figure 18 shows the SPR analysis of antibody variants. Each antibody was immobilized on a CM5 chip, and serially diluted FcγR or C1q was injected at 30 μl / min. All experiments were repeated three times independently, and the kinetic values ​​of each antibody are presented in Table 12. [Figure 19] FIG. 19 shows the pharmacokinetics of antibodies in human FcRn transgenic mice. [Figure 20] FIG. 20. Rheumatoid factor binding assay for antibody variants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Provided herein are methods and compositions involving polypeptides having engineered antibody Fc domains that exhibit improved pH-dependent binding to FcRn. In some preferred aspects, the variant Fc domains may exhibit improved half-life or reduced degradation in vivo after administration to a mammalian subject. As shown in the examples below, these FcRn binding improvements have been observed in variant Fc domains that may be present in glycosylated or non-glycosylated antibodies of various antibody types (e.g., IgG1, IgG2, IgG3, IgG4). In some instances, non-glycosylated antibodies are associated with some risks (e.g., immunogenicity, poor in vivo stability, or unfavorable pharmacokinetics), so in some embodiments, it may be desirable to include variant Fc domains in glycosylated antibodies. Nevertheless, non-glycosylated antibodies can be used in a variety of useful or therapeutic approaches, and thus including variant Fc domains in non-glycosylated antibodies can also provide benefits, such as improved in vivo half-life of non-glycosylated antibodies. In some embodiments, the mutant Fc domain binds to FcRn at acidic pH and does not bind to FcRn at neutral pH, and has FcγR binding ability equivalent to the wild-type Fc domain. For example, a polypeptide may comprise a mutant or variant Fc domain that binds to FcRn in a pH-selective manner while at the same time having binding ability equivalent to the wild-type for binding to FcγRI, FcγRII, and / or FcγRIII.

[0044] I. Antibody Fc Domain The neonatal Fc receptor (FcRn) is well known to play a central role in regulating serum IgG levels in mammals (Ghetie, V., and Ward, ES 2000). Fc-binding FcRn is a heterodimer containing a membrane-tethered chain associated with β2-microglobulin (β2m) and a chain of major histocompatibility complex class I (MHC class I) molecules. The IgG-FcRn interaction shows a pronounced pH dependence, ranging from strong at slightly acidic pH to weak at neutral and basic pH (Rodewald, R. 1976 and Raghavan, M. et al., 1995). The generally accepted mechanism of FcRn-mediated IgG recycling involves the following steps: 1) internalization of IgG by pinocytosis, 2) acidification of endosomes (early endosomes: pH 6.0, late endosomes: pH 4.5) and interaction of IgG with FcRn, 3) exocytosis of IgG and dissociation from FcRn, 4) re-internalization of IgG that was not released by endocytosis, and 5) lysosomal fusion and degradation of IgG. As a result, recycled IgG shows a significantly extended serum half-life compared to other serum proteins (Ghetie, V. et al., 1996 and Borvak, J. et al., 1998). IgG that maintains significant binding to FcRn at neutral pH has a significantly reduced serum persistence (Dall'Acqua, WF et al., 2003).

[0045] Therapeutic antibodies with longer serum half-lives would be beneficial if they could increase efficacy due to sustained serum concentrations, less frequent dosing, and / or reduced costs. Although it seems logical to manipulate Fc-FcRn interactions to increase plasma half-life, the potential biological consequences of FcRn's role in transporting IgG are well known. In particular, this function includes transcytosis of IgG through the human placenta (Firanet al., 2001) and intestine (Israel et al., 1997 and Dickinson et al., 1999) and reabsorption of IgG in the human kidney (Hayman et al., 2000).

[0046] Based on FcRn-mediated homeostasis, Fc engineering to enhance serum half-life is usually focused on enhancing the affinity of Fc for FcRn at acidic pH and maintaining the original pH-dependent binding characteristics. Many engineered Fc variants against FcRn have been developed, such as Fc domains containing M428L / N434S (LS) or M252Y / S254T / T256E (YTE) amino acid substitutions, where LS and YTE show 10- to 20-fold enhanced affinity compared to wild-type IgG1 (WT) at endosomal pH and no significant affinity at pH 7.4, resulting in enhanced serum half-lives in transgenic human FcRn (hFcRn) mice, primates and / or humans (Acqua et al., 2002; Zalevsky et al., 2010; Ko et al., 2014; Deng et al., 2010; Hintone et al., 2004 and 2006; Yeung et al., 2009 and 2010; Borrok et al., 2015; Robbie et al., 2016). In particular, antibodies with the "YTE" variant have been evaluated in several clinical trials. Antibodies with the "YTE" mutation have been shown to have a serum half-life as long as 3 months, a property that allows for substantially reduced dosing, better patient compliance and better therapeutic efficacy. However, high FcRn affinity at acidic pH does not clearly correlate with enhanced serum half-life, and some engineered variants with similar or better improved FcRn binding characteristics at acidic pH have failed to show the predicted increased in vivo half-life (Borroket al., 2015; Datta-Mannan et al., 2007 and 2012, and Zheng et al., 2011). On the other hand, the affinity of antibodies at neutral pH is It has been shown that the PK of IgG at pH 7.4 correlates better with that of IgG at pH 7.4 based on the FcRn recycling mechanism (Cooper et al., 2014 and Wang et al., 2011). Affinity is a critical factor for antibody recycling. Accurate and precise measurement of antibody affinity at neutral pH is crucial. Recently, attempts to measure antibody affinity for FcRn at pH 7.4 by SPR using very high levels of immobilized FcRn have been reported. The YTE variant showed approximately 57-fold higher affinity than wild-type IgG1 at pH 7.4 by kinetic analysis using high-density FcRn, which was not shown in assays using low-density FcRn (Walter et al., 2016).

[0047] In certain embodiments, there are compositions comprising proteinaceous molecules that are modified relative to native or wild-type proteins. In some embodiments, the proteinaceous compounds have deleted amino acid residues; in other embodiments, the amino acid residues of the proteinaceous compounds have been replaced; while in still further embodiments, there is both a deletion and a replacement of amino acid residues in the proteinaceous compounds. Furthermore, the proteinaceous compounds can comprise amino acid molecules that comprise two or more polypeptide entities. As used herein, a "proteinaceous molecule," "proteinaceous composition," "proteinaceous compound," "proteinaceous chain," or "proteinaceous material" generally refers to, but is not limited to, a protein or full-length endogenous sequence translated from a gene of more than about 200 amino acids; a polypeptide of 100 amino acids or more; and / or a peptide of 3-100 amino acids. Any of the above "proteinaceous" terms may be used interchangeably herein; however, it is specifically contemplated that embodiments may be limited to certain types of proteinaceous compounds, such as polypeptides. Furthermore, these terms may be applied to fusion proteins or protein conjugates as well. A protein can comprise two or more polypeptides. For example, an IgG antibody has two heavy polypeptide chains and two light polypeptide chains held together by disulfide bonds.

[0048] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids, up to the full length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0049] As used herein, "amino acid residue" refers to any amino acid, amino acid derivative or amino acid mimic that would be known to those skilled in the art. In certain embodiments, the residues of a proteinaceous molecule are contiguous without any non-amino acid residues interrupting the sequence of amino acid residues. In other embodiments, the sequence can include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule can be interrupted by one or more non-amino acid moieties.

[0050] As used herein, a "distinct Fc domain" can be defined as a domain that differs from another Fc by only one amino acid. Methods for generating libraries of nucleic acids encoding distinct antibody Fc domains or antibodies are well known in the art. For example, in some cases, Fc domains can be amplified by error-prone PCR. Furthermore, in certain cases, multiple antibody Fc domains can contain a randomized stretch (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of amino acids. In certain cases, specific mutations can be engineered into the Fc domain. For example, in some embodiments, normally glycosylated residues in antibody Fc domains can be mutated. Furthermore, in certain embodiments, normally glycosylated residues (or adjacent residues) can be used as sites for insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0051] The polypeptide may comprise a mutant or variant antibody Fc domain capable of binding to an FcR polypeptide. In some embodiments, the Fc domain may be further defined as having a specific affinity for an FcR polypeptide under physiological conditions. For example, the Fc domain may have a specific affinity for an FcR polypeptide under physiological conditions of about 10 -6 M~about 10 -9M. Furthermore, in some embodiments, an aglycosylated Fc domain can be defined as comprising one or more amino acid substitutions or insertions relative to a wild-type sequence, such as a human wild-type sequence. The Fc domain can be glycosylated or aglycosylated.

[0052] Means for preparing such polypeptides include those described in PCT Publication WO2008 / 137475, which is incorporated herein by reference. Alternatively, such polypeptides can be prepared directly by genetic engineering techniques, for example by introducing selected amino acid substitutions or insertions into a known Fc background, which insertions or substitutions provide improved FcR binding ability to the non-glycosylated Fc region, as described above. In some embodiments, the Fc domain is engineered to bind to one or more specific Fc receptors. Additionally or alternatively, the Fc domain can be engineered to not specifically bind to one or more specific Fc receptors.

[0053] In some embodiments, the Fc domain comprises specific binding affinity for an FcR, such as human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI, or for C1q. In some embodiments, the antibody is a glycosylated antibody that exhibits FcR binding that is similar, essentially the same, or the same as that for a wild-type antibody, e.g., compared to a corresponding IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is glycosylated. In some embodiments, the antibody is an aglycosylated antibody. The binding affinity of an antibody Fc or other binding protein can be determined, for example, using the Scatter Binding Assay of Munson and Pollard (1980). The binding affinity can be determined by surface plasmon resonance or any other well-known method for determining the kinetics and equilibrium constants of protein:protein interactions. The isolated IgG variants are presented in Table 1 below. In various embodiments, mutations can be introduced into the IgG1 Fc domain (e.g., SEQ ID NOs: 5-8) or into the IgG2 Fc domain (e.g., SEQ ID NO: 9), IgG3 Fc domain (e.g., SEQ ID NO: 10), or IgG4 Fc domain (e.g., SEQ ID NO: 11), as desired. Corresponding mutations can be made in the Fc domain (eg, SEQ ID NO:11). Table 1: Isolated IgG variants (sequence numbering is based on Kabat, mutations are specified below) EDHS (V264E, L309D, Q311H, N434S; SEQ ID NO:5), EDHY (V264E, L309D, Q311H, N434Y; SEQ ID NO: 6), DHS (L309D, Q311H, N434S; SEQ ID NO: 7), DHY (L309D, Q311H, N434Y; SEQ ID NO: 8), IgG2-DHS (V309D, Q311H, N434S; SEQ ID NO: 9), IgG3-DHS (L309D, Q311H, N434S; SEQ ID NO: 10), IgG4-DHS (L309D, Q311H, N434S; SEQ ID NO: 11)

[0054] As used herein, "position" refers to a location in a protein sequence. Positions can be numbered consecutively or according to established formats, such as the EU index for antibody numbering.

[0055] For all positions described herein, numbering is according to the EU index. "EU index" or "EU index as in Kabat" or "EU numbering scheme" refers to the numbering of the EU antibody (Edelman et al., 1969; Kabat et al., 1991; both in their entirety). The entire disclosure of which is incorporated herein by reference.

[0056] In certain embodiments, the size of the at least one Fc polypeptide proteinaceous molecule is about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 850, 860, 870, 880, 89 The compounds may include, but are not limited to, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more amino acid residues, and any range derivable therein. The Fc polypeptide may comprise a sequence similar to that of SEQ ID NO: 1 or a sequence similar to that of SEQ ID NO: 5, or a number of contiguous amino acids from SEQ ID NO: 6, which may be further qualified as having a percent sequence identity or homology to a wild-type human IgG Fc domain (e.g., a percent sequence identity to any one of SEQ ID NOs: 1-4).

[0057] A. Modified Proteins and Polypeptides Some embodiments concern modified proteins and polypeptides, particularly modified proteins or polypeptides that exhibit at least one functional activity comparable to the unmodified version, and furthermore, the modified protein or polypeptide possesses additional advantages over the unmodified version, such as suppressing B-cell activation, being easier or cheaper to produce, inducing fewer side effects, and / or having better or longer efficacy or bioavailability. Thus, when the present application refers to a function or activity of a "modified protein" or "modified polypeptide", the skilled artisan will understand that this includes, for example, a protein or polypeptide that 1) performs at least one of the same activities or has at least one of the same specificities as the unmodified protein or polypeptide, but may have a different level of another activity or specificity; 2) possesses additional advantages over the unmodified protein or polypeptide. Determination of activity can be accomplished using assays well known to those skilled in the art, particularly for the activity of proteins, and can include, for comparison purposes, the use of, for example, native and / or recombinant versions of either the modified or unmodified protein or polypeptide. In particular, it is contemplated that embodiments pertaining to "modified proteins" can be implemented with respect to "modified polypeptides," and vice versa. In addition to the modified proteins and polypeptides described herein, embodiments can involve domains, polypeptides, and proteins described in PCT Publication WO2008 / 137475, specifically incorporated herein by reference.

[0058] Modified proteins can have amino acid deletions and / or substitutions; thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins.In some embodiments, these modified proteins can further include inserted or added amino acids, such as by fusion proteins or proteins with linkers.This can include inserting targeting peptides or polypeptides or simply inserting single residues.Terminal additions, called fusion proteins, are described below.

[0059] A "modified deleted protein" lacks one or more residues of a native protein, but retains the specificity and / or activity of the native protein. A "modified deleted protein" may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is a protein that has amino acid residues deleted from at least one antigenic region (i.e., a region of the protein that has been determined to be antigenic in a particular organism, such as the type of organism to which the modified protein may be administered).

[0060] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0061] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Thus, it includes sequences having between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids identical or functionally equivalent to those of a native polypeptide, provided that the biological activity of the protein is maintained. A modified protein may be biologically functionally equivalent to its native counterpart.

[0062] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including maintenance of biological protein activity to which protein expression pertains. The addition of terminal sequences applies particularly to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or may include various internal sequences, i.e., introns, which are known to occur within genes.

[0063] The following is a description based on the amino acid changes of proteins to create equivalent or even improved second generation molecules. For example, certain amino acids can be substituted for other amino acids in protein structures with or without appreciable loss of interactive binding ability with structures such as, for example, binding sites for substrate molecules. Because it is the interactive ability and properties of a protein that define its biological functional activity, certain amino acid substitutions can be made in protein sequences and their underlying DNA coding sequences, and still produce proteins with similar properties. Thus, as described below, it is contemplated that various changes can be made in the DNA sequence of a gene without appreciable loss of its biological usefulness or activity. A proteinaceous molecule is considered to have "homology" or be "homologous" to a second proteinaceous molecule if one of the following "homology criteria" is met: 1) at least 30% of the proteinaceous molecule has sequence identity at the same positions as the second proteinaceous molecule; 2) there is some sequence identity at the same positions as the second proteinaceous molecule, with at least 30% of the non-identical residues being conservative differences as described herein relative to the second proteinaceous molecule; or 3) at least 30% of the proteinaceous molecule has sequence identity with the second proteinaceous molecule, although there may be gaps of non-identical residues between the identical residues. As used herein, the term "homology" can equally apply to regions of a proteinaceous molecule instead of the entire molecule. When the term "homology" or "homology" is qualified by a number, e.g., "50% homology" or "50% homology," the homology criteria for 1), 2) and 3) are adjusted from "at least 30%" to "at least 50%." Thus, it is contemplated that there may be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homology between two proteinaceous molecules or portions of proteinaceous molecules.

[0064] Alternatively, a modified polypeptide can be characterized as having a certain percentage of identity to an unmodified polypeptide or any of the polypeptide sequences disclosed herein, including any of the mutant or variant Fc domains or sequences described herein (e.g., SEQ ID NOs: 5-11). The percentage identity can be at most or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) between two proteinaceous molecules or portions of proteinaceous molecules. It is contemplated that the above percentage of identity can relate to a particular region of a polypeptide as compared to an unmodified region of the polypeptide. For example, a polypeptide can include a modified or variant Fc domain that can be characterized based on the identity of the amino acid sequence of the modified or variant Fc domain to an unmodified or variant Fc domain from the same species. For example, a modified or variant human Fc domain characterized as having 90% identity to an unmodified Fc domain means that 90% of the amino acids in that domain are identical to the amino acids in the unmodified human Fc domain (e.g., SEQ ID NOs: 1-4).

[0065] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). The relative hydropathic character of amino acids can be used to determine the resulting It is accepted that molecular chemistry contributes to the secondary structure of a protein, which in turn determines the interactions of the protein with other molecules, e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0066] It is also understood in the art that substitutions of like amino acids can be usefully made on the basis of hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, describes that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that one amino acid can be substituted for another amino acid having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. Among such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0067] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various aforementioned characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0068] B. Antibodies and Proteinaceous Compounds Modified with Heterologous Regions Once the Fc domain is isolated, it may be desirable to link the molecule to at least one agent to form a conjugate to enhance the utility of the molecule. For example, to increase the effectiveness of the Fc domain or antibody molecule as a diagnostic or therapeutic agent, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, for example, cytotoxic activity. Non-limiting examples of effector molecules attached to antibodies include toxins, antitumor agents, therapeutic enzymes, radiolabeled nucleotides, antiviral agents, chelators, cytokines, growth factors, and oligo- or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include ligands such as enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or biotin. Another such example is the formation of conjugates comprising an antibody linked to a cytotoxic or anti-cellular agent, which may be termed "immunotoxins." Techniques for labeling such molecules are known to those of skill in the art and are described herein above.

[0069] Labeled proteins, such as Fc domains prepared according to the invention, can then also be used in immunodetection methods, for example, to bind, purify, remove, quantify and / or otherwise generally detect biological components, such as protein(s), polypeptide(s) or peptide(s). Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluoroimmunoassays, chemiluminescent assays, bioluminescent assays and Western blots, to name a few. Steps of various useful immunodetection methods are described, for example, in Doolittle and Ben-Zeev, 1999, each of which is incorporated herein by reference. 9; Gulbis and Galand, 1993; and De Jager et al., 1993, Scientific Literature It is stated in the dedication.

[0070] Fc domain molecules, including antibodies, can be used in conjunction with both fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for study by, for example, immunohistochemistry (IHC). Methods for preparing tissue blocks from such particulate specimens have been used successfully in previous IHC studies of various prognostic factors and / or are well known to those skilled in the art (Abbondanzo et al., 1990).

[0071] Some embodiments relate to Fc polypeptide proteinaceous compounds that can include amino acid sequences from two or more naturally occurring or native polypeptides or proteins. The embodiments described above are contemplated to apply to this section and vice versa. For example, the modified antibody is an antibody that contains a modified Fc domain with an antigen binding domain. Furthermore, the antibody can have two different antigen binding regions, such as different regions in each of the two heavy chains. Alternatively or additionally, in some embodiments, there are polypeptides that include multiple heterologous peptides and / or polypeptides ("heterologous" means that they are not derived from the same polypeptide). The proteinaceous compound or molecule can include, for example, a modified Fc domain with a protein binding region that is not derived from an antibody. In some embodiments, there are polypeptides that include a modified Fc domain with a protein binding region that binds to a cell surface receptor. These proteinaceous molecules that include multiple functional domains can be two or more domains chemically conjugated to each other, or can be fusion proteins of two or more polypeptides encoded by the same nucleic acid molecule. It is contemplated that a protein or polypeptide can include the whole or part of two or more heterologous polypeptides.

[0072] Thus, a multi-polypeptide proteinaceous compound may comprise all or part of a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, an eighth polypeptide, a It may consist of all or part of a ninth polypeptide, a tenth polypeptide or even higher polypeptides.

[0073] The proteinaceous moieties can be separated using amino acids, such as selectively cleavable linkers, synthetic linkers or other amino acid sequences.

[0074] Polypeptides or proteins (including antibodies) having an antigen binding domain or region of an antibody and a mutant or variant Fc domain can be used against any antigen or epitope, including but not limited to proteins, subunits, domains, motifs, and / or epitopes belonging to the following list of targets: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 Antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A , cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / ZIP, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2 , CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15 , CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay promoting factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA- A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein ( FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MM), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, insulin Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-1 0, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV)F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor activator), T cell receptor activator inhibitor (TCR-1 ... alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, PLAP-like alkaline phosphatase of testis, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcR1, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSF11B(OPGOCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY CROWN), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK polymer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg, TNFSF15(TL1A / VEGI), TNFSF18(GITR LINK, AITR TL6, TNFSF1A (TNF-a, DIF, TNFSF2), TNFSF1B (TNF-b). LTa、TNFSF1)、TNFSF3(LTbTNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor Tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (fit-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and hormone and growth factor receptors. In some embodiments, the polypeptide or protein has an antigen binding domain specific for one or more cell surface tumor antigens or B cell antigens. The methods and compositions can be used to target tumor cells or B cells.

[0075] Any antibody of sufficient selectivity, specificity, or affinity may be used as the basis for antibody conjugates. Such properties can be evaluated using conventional immunological screening methodologies known to those skilled in the art. Sites for binding to biologically active molecules in antibody molecules include, in addition to standard antigen binding sites, sites present in the variable domain that can bind to pathogens, B cell superantigens, T cell co-receptor CD4, and the HIV-1 envelope (Silverman, 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1981). In addition, the variable domain is involved in antibody self-binding (Kang et al., 1988) and can be used to identify epitopes (idiotopes) recognized by anti-antibodies. (Bhattacharya et al., 1989).

[0076] Although Fc domain can bind to FcR, it is contemplated that the regulation of immune response can be directed not only by antigen binding domain in polypeptide containing Fc domain, but also by some other protein binding domain. Thus, some embodiments may involve Fc domain and heterologous non-antigen binding domain. In certain embodiments, non-antigen binding domain binds to cell surface. Thus, these agents require either chemical conjugation or fusion with agents / proteins that can bind to specific target cells. The embodiments may further include connecting all or part of mutant or variant Fc domain to all or part of any of the proteins listed in Table 2. It is contemplated that embodiments include, but are not limited to, the examples provided in Table 2 and the description herein.

[0077] The ligand of the receptor can be used to target cells expressing the receptor of the ligand on its surface. Ligands include, for example, CD95 ligand, TRAIL, TNF (such as TNF-α or TNF-β), growth factors including those mentioned above such as VEGF, and cytokines such as interferons or interleukins, and variants thereof. Also contemplated are embodiments having multiple domains, such as a VEGF Trap fusion protein comprising the second extracellular domain of VEGF receptor 1 (Flt-1), the third domain of VEGF receptor 2 (KDR / FIK-1), and an IgG Fc region. [Table 2]

[0078] C. Antibody Fc Library Examples of techniques that can be used in conjunction with embodiments for the generation of diverse antibody Fc domains and / or antibodies comprising such domains include techniques similar to those for the expression of immunoglobulin heavy chain libraries described in U.S. Patent No. 5,824,520. Previously used Fc libraries are described in PCT Publication WO2008 / 137475, specifically incorporated herein by reference.

[0079] II. Antibody-Binding Polypeptides A variety of antibody binding domains (e.g., FcR polypeptides) are known in the art and can be used in the methods and compositions of the present invention. For example, in some aspects, an FcR can have specificity for a particular type or subtype of Ig, such as IgA, IgM, IgE, or IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). Thus, in some embodiments, an antibody binding domain can be defined as an IgG binding domain. An FcR polypeptide can include a eukaryotic, prokaryotic, or synthetic FcR domain. For example, an antibody Fc binding domain can be defined as a mammalian, bacterial, or synthetic binding domain. Some Fc binding domains for use in the present invention include, but are not limited to, a binding domain from one of the polypeptides in Table 3. For example, the Fc binding polypeptide may be encoded by FCGR2A, FCGR2B, FCGR2C, FCGR3A, FCGR3B, FCGR1A, Fcgr1, FCGR2, FCGR2, Fcgr2, Fcgr2, FCGR3, FCGR3, Fcgr3, FCGR3, Fcgr3, FCGRT, mrp4, spa or spg genes. Preferably, the FcR polypeptide for use according to the invention may be an Fc binding region from human FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcαRI or C1q. Various Fc receptors to which Fc domains bind are well known in the art and some examples of receptors are listed in Table 3 below. [Table 3-1] [Table 3-2]

[0080] III. Methods for Screening Antibody Fc Domains In certain embodiments, there are methods for identifying antibody Fc domains that have specific affinity for target ligands (e.g., antibody-binding polypeptides such as Fc receptors). Such methods are described herein, as well as in PCT Publication WO2008 / 137475, the entire contents of which are specifically incorporated herein by reference. For example, the methods can be used to identify additional mutations that can be further included in mutant or variant Fc domains or antibodies in addition to the Fc domain substitution mutations disclosed herein (e.g., in addition to L309D, Q311H, and N434S / Y in human IgG Fc domains). Various methods for identifying additional mutations are known in the art and can be used in the present invention (e.g., U.S. Patent Nos. 7,094,571, 7,419,783, 7,611,866, and U.S. Patent Publication No. 2003 / 0219870; Harvey et al., 2004; Harvey et al., 2006).

[0081] IV. Nucleic Acid-Based Expression Systems Nucleic acid-based expression systems may, in certain embodiments of the invention, find use in expressing recombinant proteins. For example, one embodiment of the invention involves the transformation of Gram-negative bacteria with a coding sequence for an antibody Fc domain, or preferably multiple separate Fc domains.

[0082] A. Nucleic acid delivery method Certain aspects of the invention can include delivery of nucleic acid to target cells (e.g., gram-negative bacteria). For example, bacterial host cells can be transformed with nucleic acids encoding candidate Fc domains that may bind to FcR. In certain embodiments of the invention, it may be desirable to target expression to the periplasm of bacteria. Transformation of eukaryotic host cells can similarly find use in expressing various candidate molecules that are identified as being capable of binding to target ligands.

[0083] Methods suitable for nucleic acid delivery for cell transformation are well known in the art. In some embodiments, the mutant or variant Fc domain is encoded by a nucleic acid expression vector, which can be used to transform cells, such as bacterial or eukaryotic cells.

[0084] The term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. An expression vector can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition, an expression vector can contain a promoter, an enhancer, an initiation signal, a multiple cloning site (MCS), a termination signal, one or more origins of replication sites (often named "ori"), a selectable and / or screenable marker (e.g., to confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol; or a protein such as GFP or chloramphenicol acetyltransferase (CAT)).

[0085] A "promoter" is a control sequence that is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It can contain genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct and functional location and / or orientation in relation to a nucleic acid sequence to control the transcription initiation and / or expression of this sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Those skilled in the art of molecular biology are generally familiar with the use of promoters, enhancers, and cell type combinations for protein expression, see, for example, Sambrook et al. (1989), which is incorporated herein by reference.

[0086] B. Host cells In the context of expression of heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic cell and includes any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be, and has been, used as a recipient for a vector. A host cell can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny.

[0087] In a particular embodiment of the invention, the host cell is a Gram-negative bacterial cell. These bacteria are suitable for use according to the invention in that they possess a periplasmic space between the inner and outer membranes, in particular the inner membrane being between the periplasm and the cytoplasm, also known as the cytoplasmic membrane. Thus, any other cell with such a periplasmic space can be used according to the invention. Examples of Gram-negative bacteria that can find use according to the invention include, but are not limited to, E. coli, Pseudomonas aeruginosa, Vibrio cholera, Salmonella typhimurium, Shigella flexneri, Haemophilus influenza, Bordotella pertussi, Erwinia amylovora, Rhizobium sp.

[0088] The appropriate host can be determined by those skilled in the art based on the vector backbone and the desired results. Plasmids or cosmids, for example, can be introduced into prokaryotic host cells for the replication of many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5α, JM109 and KC8, as well as many commercially available bacterial hosts such as SURE® competent cells and SOLOPACK™ Gold cells (Stratagene®, La Jolla). Alternatively, bacterial cells such as E. coli LE392 can be used as host cells for bacteriophages.

[0089] Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40 transformed monkey kidney cells (COS-1; ATCC CRL 1650) and mouse embryo cells (NIH-3T3; ATCC CRL 1658). The foregoing is intended to be illustrative, not limiting, of the many possible host organisms known in the art.

[0090] Mammalian host cells expressing the polypeptide are cultured under conditions typically used for culturing the parent cell line. Generally, the cells are cultured in standard media containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM or DMEM, typically supplemented with serum, such as 5%-10% fetal bovine serum. Culture conditions are also standard, e.g., cultures are incubated at 37°C in static or rotating culture until the desired level of protein is achieved.

[0091] Many host cells from various cell types and organisms can be utilized and will be known to those skilled in the art. Similarly, particularly viral vectors that are permissive for vector replication or expression can be used in conjunction with prokaryotic host cells. Some vectors can use control sequences that allow their replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions for incubating all of the above host cells to maintain them and allow vector replication. The techniques and conditions that will allow large-scale production of vectors and the production of the nucleic acid encoded by the vector and its cognate polypeptide, protein or peptide are also understood and known.

[0092] C. Expression Systems There are numerous expression systems that contain at least part or all of the compositions described above. Such systems can be used, for example, to produce a polypeptide product identified according to the present invention as capable of binding to a specific ligand. Prokaryotic-based systems can be used in accordance with the present invention to produce nucleic acid sequences or their cognate polypeptides, proteins and peptides. Many such systems are commercially available and widely available. Other examples of expression systems include vectors containing strong prokaryotic promoters such as T7, Tac, Trc, BAD, lambda pL, tetracycline or Lac promoters, the pET expression system and the E. coli expression system.

[0093] In certain aspects of the present invention, nucleic acid sequences encoding polypeptides are disclosed. Depending on which expression system is used, the nucleic acid sequence can be selected based on conventional methods. For example, if the polypeptide is derived from a human polypeptide and contains multiple codons that are rarely used in E. coli, this may interfere with the expression in E. coli. Therefore, each gene or its variant can be codon-optimized for E. coli expression. Various vectors can also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposon- or liposome-based vectors.

[0094] V. Protein Purification Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, homogenization and crude fractionation of cells, tissues, or organs into polypeptide and non-polypeptide fractions. Unless otherwise specified, the protein or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods that are particularly suitable for the preparation of pure peptides include ion exchange chromatography, size exclusion chromatography (SEC), reverse phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric focusing. A particularly efficient method for purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (also called high pressure liquid chromatography or HPLC). As is generally known in the art, it is contemplated that the order of carrying out the various purification steps, or the omission of certain steps, can still result in a method suitable for the preparation of substantially purified proteins or peptides.

[0095] A purified protein or peptide is intended to refer to a composition that can be isolated from other components, and the protein or peptide is purified to any degree compared to its naturally obtainable state. An isolated or purified protein or peptide therefore also refers to a protein or peptide that is free of the environment in which it may naturally occur. In general, "purified" refers to a protein or peptide composition that has been subjected to fractionation to remove various other components, and the composition will substantially retain its expressed biological activity. When the term "substantially purified" is used, this designation can refer to a composition in which the protein or peptide forms a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0096] Various methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art in light of the present disclosure. These methods include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides in a fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the degree of purity therein, as assessed by the "fold purification number". The actual units used to express the amount of activity may of course depend on the particular assay technique chosen to track purification, and whether the expressed protein or peptide exhibits detectable activity.

[0097] There is no general requirement that a protein or peptide always be provided in its most purified state. In fact, it is contemplated that in certain embodiments, products that are substantially less purified may have utility. Partial purification can be achieved by combining fewer purification steps or by utilizing different forms of the same general purification scheme. For example, it is recognized that cation exchange column chromatography performed using HPLC equipment generally results in a greater "fold" of purification than the same technique using a low pressure chromatography system. Methods that exhibit a lower degree of relative purification may have advantages in total recovery of protein product or in maintaining the activity of the expressed protein.

[0098] VI. Pharmaceutical Compositions When clinical application of pharmaceutical compositions containing polypeptides or antibodies is performed, it may generally be beneficial to prepare pharmaceutical or therapeutic compositions appropriate for the intended application. In general, pharmaceutical compositions may contain an effective amount of one or more polypeptides or additional agents dissolved or dispersed in a pharma-ceutically acceptable carrier. In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the polypeptide or antibody. In other embodiments, the polypeptide or antibody may contain, for example, between about 2% and about 75% of the weight unit or between about 25% and about 60%, and any range derivable therein. The amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage of the compound for any given unit dose is obtained. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimes may be desired.

[0099] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals, including humans, as appropriate. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, 1990, which is incorporated herein by reference. Furthermore, it is understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0100] Furthermore, in accordance with certain aspects of the present invention, compositions suitable for administration can be provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., paste, or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. As used herein, a "pharma- ceutically acceptable carrier" refers to any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solutions, parenteral vehicles, e.g., sodium chloride, Ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, e.g., ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonicity agents (e.g., sugars, sodium chloride), absorption retarders (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.), gels, binders, excipients, disintegrants, lubricants, fluid and nutrient replenishers, such materials and combinations thereof. Except insofar as any conventional vehicle, agent, diluent, or carrier is deleterious to the recipient or to the therapeutic effect of the composition in which it is contained, its use in administrable compositions for use in carrying out the present method is appropriate. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters. According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, pulverizing, etc. Such procedures are routine to those skilled in the art.

[0101] Certain embodiments of the present invention may include different types of carriers depending on whether they are to be administered in solid, liquid, or aerosol form and whether they need to be sterile for routes of administration such as injection. The compositions can be formulated for administration intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion directly bathing target cells, by catheter, by lavage, in lipid compositions (e.g., liposomes), or by other methods or combinations of any of the above, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference). Typically, such compositions include: It can be prepared either as a solution or suspension in liquid; solid forms suitable for use in preparing a solution or suspension by the addition of liquid prior to injection can also be prepared; preparations can also be emulsified. In some embodiments, a therapeutic protein comprising a mutant or variant Fc domain is administered to a mammalian subject (e.g., a human) by injection or intravenous administration.

[0102] Polypeptides can be formulated in the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, for example salts formed with free amino groups of proteinaceous compositions, or salts formed with inorganic acids, for example hydrochloric acid or phosphoric acid, or organic acids, for example acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can be derived from inorganic bases, for example sodium, potassium, ammonium, calcium or ferric hydroxide; or organic bases, for example isopropylamine, trimethylamine, histidine or procaine.

[0103] In further embodiments, the present invention may involve the use of pharmaceutical lipid vehicle compositions comprising a polypeptide, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" may be defined to include any of a wide range of substances that are characteristically insoluble in water and can be extracted with an organic solvent. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and their derivatives. Lipids may be of natural origin or synthetic (i.e., designed or produced by the hand of man). However, lipids are usually biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, ethers, and ethers. These include lipids having ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds that are understood by those of skill in the art as lipids, other than those specifically described herein, are also encompassed by the present compositions and methods.

[0104] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid vehicles.For example, the polypeptide or its fusion protein can be dispersed in a solution that contains lipid, dissolved in lipid, emulsified in lipid, mixed with lipid, combined with lipid, covalently bound to lipid, contained in lipid as a suspension, contained or complexed with micelles or liposomes, or otherwise associated with lipid or lipid structure by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.In some embodiments, the antibody that comprises mutant or variant Fc domain described herein can be included in antibody-targeted liposomes.

[0105] The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response as described above in conjunction with its administration, i.e., the appropriate route and treatment regimen. The amount to be administered according to both the number of treatments and the unit dose depends on the desired effect. The actual dosage of the composition of the present embodiment administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health and sex, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the idiopathic nature of the patient, the route of administration, and the potency, stability and toxicity of the particular therapeutic agent. In other non-limiting examples, dosages can include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight to about 1000 milligrams / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges that can be derived from the numbers listed herein, ranges from about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc. can be administered based on the above numbers. The physician responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0106] The present invention is not intended to be limited by the specific nature of the therapeutic preparation. For example, such compositions can be provided in a formulation together with physiologically acceptable liquid, gel or solid carriers, diluents and excipients. These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a similar manner to other therapeutic agents. In general, the dosage required for therapeutic efficacy can vary depending on the type of use and administration mechanism, as well as the specialized requirements of the individual subject. The actual dosage of the composition administered to an animal patient can be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic nature of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or frequency of administration of an effective amount can vary depending on the subject's response. The physician responsible for administration can, in any case, determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

[0107] VII. Treatment Methods Certain aspects of the invention provide polypeptides for treating diseases such as tumors, e.g., with therapeutic proteins or antibodies comprising mutant or variant Fc domains as described herein. In particular, the polypeptides may have human polypeptide sequences, thus preventing allergic reactions in human patients, allowing for repeated administration, and increasing therapeutic efficacy.

[0108] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject, or the performance of a procedure or modality in a subject, for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include the administration of a pharmacologic effective amount of an antibody that targets CDC to cancer cells without inducing cancer cell proliferation.

[0109] "Subject" and "patient" refer to either human or non-human animals, including primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0110] The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer may involve, for example, reducing tumor size, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis. Treating cancer may also refer to prolonging the survival of a subject with cancer.

[0111] The tumors for which the present treatment method is useful include any malignant cell type, such as those found in solid tumors or hematological tumors.Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast.Exemplary hematological tumors include tumors of bone marrow, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma , acral lentiginous melanoma, nodular melanoma and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's hypergammaglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML) and chronic myeloblastic leukemia.

[0112] The cancer may be of the following histological types, in particular, but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; bronchiolo-alveolar Adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophil carcinoma; eosinophil adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; ductal carcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease of the breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma w / squamous dysplasia;thymoma, malignant;ovarian stromal tumor, malignant;theca cell tumor, malignant;granulosa cell tumor, malignant;androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;lipid cell tumor, malignant;paraganglion Rioma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stroma sarcoma ;mixed tumor, malignant;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonic carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;hemangiopericytoma, malignant;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;chondroblastoma, malignant;mesenchymal chondrosarcoma;giant cell of bone Tumors;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroblastoma;Primitive neuroectodermal;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma ;retinoblastoma;olfactory neurogenic tumor;meningioma, malignant;neurofibrosarcoma;schwannoma, malignant;granular cell tumor, malignant;malignant lymphoma;Hodgkin's disease;Hodgkin type;lateral granuloma;malignant lymphoma, small lymphocytic;malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphoid leukemia;plasma cell leukemia;erythroleukemia;lymphosarcoma cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.

[0113] The polypeptides herein can be used as anti-tumor agents in various modalities to induce complement activation in tumor tissue or where this is considered desirable. In certain embodiments, the present invention contemplates a method of using the polypeptides as anti-tumor agents, thus comprising contacting a population of tumor cells with a therapeutically effective amount of the polypeptide for a period of time sufficient to inhibit tumor cell growth.

[0114] In one embodiment, in vivo contacting is accomplished by administering to the patient a therapeutically effective amount of a physiologically acceptable composition comprising a polypeptide of the invention by intravenous, intraperitoneal or intratumoral injection. The polypeptide can be administered parenterally by injection or by gradual infusion over time. The polypeptide can be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, dermally, can be delivered by peristaltic means, or can be injected directly into tissue containing tumor cells.

[0115] Therapeutic compositions containing polypeptides are conventionally administered intravenously, for example, by injection of a unit dose. The term "unit dose," when used in reference to a therapeutic composition, refers to physically discrete units suited as unitary administration to subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

[0116] The composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The exact amount of active ingredient required to be administered depends on the physician's judgment and is peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial and booster administration are also contemplated, typically represented by an initial administration followed by repeated doses at one or more time intervals by subsequent injections or other administrations. Exemplary multiple administrations are described herein and are particularly preferred for maintaining continuously high serum and tissue levels of the polypeptide. Alternatively, continuous intravenous infusion sufficient to maintain blood levels within the range specified for in vivo therapy is contemplated.

[0117] It is contemplated that in cancer patients with locally advanced or metastatic cancer, the polypeptide of the present invention can be administered systemically or locally to treat the disease, such as inhibiting tumor cell growth or killing cancer cells. It can be administered intravenously, intrathecally and / or intraperitoneally. It can be administered alone or in combination with an antiproliferative drug. In one embodiment, it can be administered to reduce the cancer burden in the patient prior to surgery or other procedures. Alternatively, it can be administered after surgery to ensure that any remaining cancer (e.g., cancer that surgery could not eliminate) does not survive.

[0118] The therapeutically effective amount of the polypeptide is a predetermined amount calculated to achieve the desired effect, i.e., to induce CDC in tumor tissue, thereby mediating tumor resection proinflammatory response. Thus, the dosage range of the administration of the polypeptide of the present invention is a dosage range that is large enough to produce the desired effect of reducing symptoms of tumor cell division and cell cycle progression. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, neurological effects, etc. In general, the dosage varies with the age, condition, sex and degree of disease in the patient, and can be determined by those skilled in the art. In case of any complication, the dosage can be adjusted by the individual physician.

[0119] VIII. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment involve the administration of polypeptide or antibody in combination with a second or additional therapeutic method.Such therapeutic methods can be applied in the treatment of any disease that is responsive to CDC.For example, the disease can be cancer.

[0120] Methods and compositions that include combination therapy enhance the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve a desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. This process can involve administration of a polypeptide or antibody and a second therapy. The second therapy may or may not have a direct cytotoxic effect. For example, the second therapy can be an agent that upregulates the immune system without a direct cytotoxic effect. The tissue, tumor, or cells can be exposed to one or more compositions or pharmacological formulations (single or multiple) that include one or more of the agents (e.g., polypeptides or anti-cancer agents), or the tissue, tumor, and / or cells can be exposed to two or more separate compositions or formulations, where one composition provides 1) a polypeptide or antibody, 2) an anti-cancer agent, or 3) both a polypeptide or antibody and an anti-cancer agent. It is also contemplated that such combination therapy may be used in conjunction with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0121] The terms "contacted" and "exposed," as applied to a cell, are used herein to describe the process by which a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell death, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0122] The polypeptide or antibody can be administered prior to, during, or after the anti-cancer treatment, or in various combinations. Administration can be at intervals ranging from concurrent to minutes to days to weeks. In embodiments where the polypeptide or antibody is given to the patient separately from the anti-cancer drug, one will generally ensure that a significant period of time has elapsed between each delivery such that the two compounds can still exert their beneficially combined effect on the patient, and have not exceeded their expiration date. In such cases, it is contemplated that the polypeptide and anti-cancer therapy can be given to the patient within about 12-24 or 72 hours of each other, more particularly within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the duration of treatment, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between the respective administrations.

[0123] In certain embodiments, the course of treatment may last for 1-90 days or more (including intervening days). It is contemplated that one agent may be given on any day from day 1 to day 90 (including intervening days), or any combination thereof, and another agent may be given on any day from day 1 to day 90 (including intervening days), or any combination thereof. Within a day (24 hour period), the patient may be given one or more doses of the agent(s). It is further contemplated that the course of treatment may be followed by a period during which no anti-cancer treatment is administered. This period may last for 1-7 days and / or 1-5 weeks and / or 1-12 months or more (including intervening days), depending on the patient's condition, such as their prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0124] Various combinations can be used. For the example below, the polypeptide or antibody is "A" and the anti-cancer therapy is "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0125] The administration of any polypeptide or therapeutic method of this embodiment to patient follows the general protocol of administration of such compounds, taking into account the toxicity of the agent, if any.Therefore, in some embodiments, there is a step of monitoring the toxicity caused by the combination therapy.In some embodiments involving the treatment of cancer in subject, the second therapeutic method can be, for example, chemotherapy, radiation therapy, immunotherapy, gene therapy, or surgery.

[0126] A. Chemotherapy A wide variety of chemotherapeutic agents can be used according to this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are categorized according to their mechanism of action in cells, for example, whether or not they affect the cell cycle and at what stage they do so. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or affect nucleic acid synthesis to induce chromosomal and mitotic abnormalities.

[0127] Examples of chemotherapy agents include thiotepa and cyclosphosphamide. alkylating agents, such as busulfan, improsulfan, and piposulfan; alkyl sulfonates, such as benzodopa, carboquone, meturedopa, and uredopa; aziridines, such as altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine. ethylenimine and methylolmelamine; acetogenins (especially , bullatacin and bullatacinone); camptothecin (synthetic analogs including topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and biceresin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); erytherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, tro Nitrogen mustards, such as phosphamide and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I); dynemicins, including dynemicin A; bisphosphatides, such as clodronate; phonates; esperamicin; and neocarzinostatin chromophore and related chromoproteins enediyne antibiotic chromophores, aclacinomysin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diaminobutyricin, Zo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, and other mitomycins mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ube antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; ancitabine, azacitidine, 6-azaurate, pyrimidine analogues such as lysine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; calusterone, Androgens such as dromostanolone propionate, epithiostanol, mepitiostane and testolactone; anti-adrenals such as mitotane and trilostane; Folic acid supplements such as folic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bessic acid; Trabucil (bestrabucil); Bisantrene (bisantrene); Edatrexate (edatraxate); Defofamine (defofamine); Demecolcine; Diaziquone (diaziquone); E Elformithine; Elliptinium acetate; Epothilone ;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidainine;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidanmol;Nitraerine;Pentostatin;Phenamet;Pirarubicin;Losoxantrone;Podophyllinic acid;2-Ethylhydrazine do;procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizophyllan;spirogermanium;tenuazonic acid;triazicon;2,2',2"-trichlorotriethylamine;trichothecines (especially T-2 toxin, verracurin A, roridin A and anguidine);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("Ara-C");cyclophosphamide;taxoids, e.g. paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;cisplatin, oxaliplatin platinum coordination complexes such as vincristine and carboplatin;vinblastine;platinum;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;vinorelbine;novantrone;teniposide;edatrexate;daunomycin;aminopterin;xeloda;ibandronate;irinotecan (e.g., CPT-11);topoisomerase inhibitors RFS2000;difluoromethylornithine (DMFO);retinoids such as retinoic acid;capecitabine;carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein trans tansferase inhibitors, transplatinum, and the above Any pharma- ceutically acceptable salt, acid or derivative is included.

[0128] B. Radiation therapy Other agents that have been widely used to produce DNA damage include what are commonly known as gamma radiation, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam radiation (U.S. Pat. Nos. 5,760,395 and 4,870,287) and UV radiation. All of these agents most likely affect widespread damage in DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens over extended periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0129] C. Immunotherapy Those skilled in the art will understand that immunotherapy can be used in combination or in conjunction with the method of the present embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules that target and suppress immune cells. Blinatumomab (Blincyto®) is one such example. Checkpoint inhibitors, such as ipilimumab, are another such example. The immune effector can be, for example, an antibody specific for a marker on the surface of tumor cells. The antibody can function alone as an effector of therapy, or can recruit other cells to actually affect cell killing. The antibody can be conjugated with a drug or toxin (chemotherapeutic drug, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as a targeting agent. Alternatively, the effector can be a lymphocyte with a surface molecule that interacts with tumor cell targets, either directly or indirectly. Various effector cells include cytotoxic T cells and NK cells.

[0130] In one aspect of immunotherapy, tumor cells must have a specific marker that is amenable to targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including: cytokines, e.g., IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, e.g., MIP-1, MCP-1, IL-8, and growth factors, e.g., FLT3 ligand.

[0131] Examples of immunotherapies currently under investigation or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1999). 8; cytokine therapy, e.g., interferon α, β and γ, IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2 and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent No. 5,830, and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2 and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in conjunction with the antibody therapies described herein.

[0132] D. Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised and / or destroyed, and can be used in conjunction with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs surgery).

[0133] When a part or the whole of a cancerous cell, tissue or tumor is excised, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection or local application of the area with additional anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also be of varying dosages.

[0134] E. Other Agents It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increased signaling between cells due to an increase in the number of GAP junctions may increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that inhibitors of cell adhesion improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. Additionally, it is contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment.

[0135] IX. Kit Certain aspects of the present invention can provide kits, such as therapeutic kits. For example, the kits can include one or more pharmaceutical compositions described herein and, optionally, instructions for their use. The kits can also include one or more devices for achieving administration of such compositions. For example, the subject kits can include a pharmaceutical composition and a catheter for achieving direct intravenous injection of the composition into a cancerous tumor. In other embodiments, the subject kits can include pre-filled ampoules of the polypeptide, optionally pharma- ceutical formulated or lyophilized, for use with the delivery device.

[0136] The kit may include a labeled container. Suitable containers include, for example, bottles, vials, and test tubes. The container may be made of a variety of materials, such as glass or plastic. The container may hold a composition that includes a polypeptide that is effective for a therapeutic or non-therapeutic application, such as those described above. The label on the container may indicate that the composition is to be used for a specific therapeutic or non-therapeutic application, and may also indicate instructions for either in vivo or in vitro use, such as those described above. The kits of the present invention may typically include the containers described above and one or more other containers that contain materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. EXAMPLES

[0137] X. Example The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples are techniques discovered by the inventors to work well in the practice of the invention and therefore can be considered to constitute preferred modes for practicing it. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0138] Example 1 Library construction strategy for isolating IgG1 Fc domains E. coli does not encode a protein glycosylation machinery, and thus the Fc domain of IgG expressed in the periplasm of E. coli is not glycosylated and lacks the glycan that is normally added to N297 of the Fc domain. The non-glycosylated Fc domain has a high degree of conformational flexibility, resulting in greatly attenuated or no detectable binding to effector FcγRs (FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIB) and C1q (Jefferise et al., 2005; Borrok et al., 2012), but the loss of the glycan does not affect the pH-dependent FcRn binding properties of IgG. To isolate Fc domain variants containing mutations that allow enhanced binding to FcRn over wild-type IgG, the binding site of the Fc domain to FcRn was randomized. Random amino acid substitutions were introduced at Asp249, Thr250, Leu251, Met252, Ile253, Ser254, Arg255, Val308, Leu309, Gln311, Asp312, Leu314, Glu430, Leu432, Tyr436, and Gln438. Eight primers (SEQ ID NOs: 12-19) were designed and used for mutagenesis as described in Example 2 below (Table 4). [Table 4]

[0139] Example 2 Construction of libraries for engineering Fc domains All plasmids and primers are listed in Tables 4, 10 and 11. All primers were synthesized by Integrated DNA Technologies. IgG polypeptides were expressed and displayed on the inner membrane of E. coli using vectors: pBAD30-PelB-VL-Ck-NlpA-VL-Ck-His-cMyc and pMopac12-pelB-IgG-VH-CH1-CH2-CH3-FLAG (Jung et al., 2012 and Lee et al., 2017) (Figures 1-2). Eight primers (SEQ ID NOs: 12-19) were designed to randomize the FcRn binding site of the Fc domain (Table 4 and Figure 2). Four fragments of the IgG1 heavy chain gene were amplified using the eight primers and bundled together by overlap extension with PCHT01 (SEQ ID NO: 12) and PCHT08 (SEQ ID NO: 19). Fc library genes were amplified using the following thermocycling program: 1 cycle at 94°C for 5 min; 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min; 1 cycle at 72°C for 5 min. The amplified heavy chain library genes were ligated in frame into SfiI-digested pMopac12-pelB-IgG-VH-CH1-CH2-CH3-FLAG vector. The resulting plasmid was transformed into E. coli JUDE-1 cells containing the plasmid pBAD30-PelB-VL-Ck-NlpA-VL-Ck-His-cMyc (Jung et al., 2010; Jung et al., 2012; Lee et al., 2017). The library size was 1 × 10 8 It was.

[0140] Example 3 Preparation of human FcRn-β2m-GST Plasmids for mammalian expression of FcRn were constructed as previously described (Lee et al., 2017). FcRn-β2m-GST was generated by transient transfection of HEK293F cells (Invitrogen) using pcDNA3.4 (Thermofisher). Transfected HEK293F cells were cultured for 5 days at 37°C in a 5% CO2 incubator. Supernatants were harvested by centrifugation at 4,000×g for 10 min and filtered using a 0.22 μm polyethersulfone (PES) membrane filter (PALL). FcRn-β2m-GST was purified using a glutathione sepharose (GE Healthcare) affinity column according to the manufacturer's instructions. To remove lipopolysaccharide (LPS) and non-specifically bound proteins, the FcRn-β2m-GST-bound resin was washed with 50 mL of PBS containing 0.1% Triton® X-114 (Sigma-Aldrich) and 50 mL of PBS. FcRn-β2m-GST was eluted with PBS containing 10 mM reduced L-glutathione. The buffer of the eluted FcRn-β2m-GST was exchanged with PBS using an Amicon Ultra-4 (Millipore) unit. Single chain FcRn-β2m-his (scFcRn) was also purchased from Acro Biosystems (Cat. No. FCM-H5286). Human scFcRn was labeled with FITC using the FITC Fast Conjugation Kit (Abcam, Cat. No. ab188285) according to the manufacturer's instructions. Furthermore, human FcRn-β2m-GST was labeled with R-phycoerythrin (R-PE) using the EasyLink R-PE Conjugation Kit (Abcam) according to the manufacturer's instructions.

[0141] Plasmids for mammalian expression of FcγR were constructed as previously described ( Jung et al., 2012 and Lee et al., 2017 ). FcγRI-His, FcγRIIa- H131 -GST, FcγRIIa- R131-GST, FcγRIIb-GST, FcγRIIIa- V158 -GST, and FcγRIIIa- F158 FcγRI-GST was produced by transient transfection of HEK293F cells (Invitrogen) using expression vectors derived from pMAZ-IgH (US Pat. No. 8,043,621) as described in Table 11. The detailed procedure is described in the above paragraph. FcγRI-His was purified using Ni-NTA (GE Healthcare) affinity column according to the manufacturer's instructions. FcγRI-His was eluted with PBS containing 250 mM imidazole. FcγR-GST was purified in the same way as FcRn-β2m-GST. The buffer of all eluted FcγR was exchanged with PBS by Amicon Ultra-4 (Millipore).

[0142] Example 4 Screening of Fc libraries against FcRn E. coli JUDE-1 cells were cultured overnight at 37°C and 250 rpm in Terrific Broth (TB) with chloramphenicol (40 μg / mL) and kanamycin (50 μg / mL). After overnight growth, the cells were diluted 1:50 into 100 mL of fresh TB medium with the two antibiotics. E. coli JUDE-1 cells were cultured at OD at 37°C and 250 rpm. 600The cells were cultured until the β-D-thiogalactopyranoside (IPTG, Sigma Aldrich) and 2% L-arabinose (Sigma-Aldrich) were then added to the E. coli JUDE-1 cells to facilitate protein expression, and the cells were then further incubated at 25°C for 20 h. The cultures (8 mL culture volume) were harvested by centrifugation and washed twice with 1 mL of ice-cold 10 mM Tris-HCl (pH 8.0). The washed cells were resuspended in 1 mL of ice-cold STE solution (0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA, pH 8.0) and incubated at 37°C for 30 min. Cells were centrifuged at 13,000 rpm for 1 min and washed with 1 mL of solution A (0.5 M sucrose, 20 mM MgCl2, 10 mM MOPS, pH 6.8). Washed cells were incubated with 1 mg / mL hen egg lysozyme (Sigma-Aldrich) in 1 mL of solution A for 15 min at 37 °C. After centrifugation at 13,000 rpm for 1 min, pelleted spheroplasts were resuspended in 1 mL of cold PBS (Jung et al., 2010; Jung et al., 2012; Lee et al., 2017).

[0143] To isolate enhanced FcRn-binding IgG1 variants, cells expressing the libraries described in Examples 1 and 2 were labeled with 100 nM FcRn-β2m-FITC and screened by FACSAria™ (BD Biosciences). For each of the four rounds of FACS screening, the top 3% of the population showing the highest fluorescence was collected, and these sorted spheroplasts were re-sorted to immediately remove false positives. The heavy chain genes in the sorted spheroplasts were rescued by PCR using two primers (PCHT01 and PCHT08) after boiling for 5 min, and ligated into the pMopac12 vector cut by SfiI. The ligated plasmids were transformed into E. coli JUDE-1 cells. Transformants were selected in chloramphenicol- and kanamycin-containing medium, and spheroplasts were prepared for the next round of screening using 100 nM FcRn-β2m-FITC (Figures 3A-B). To isolate pH-dependent binding IgG variants to FcRn, spheroplasts were labeled with 100 nM single chain FcRn-β2m-FITC in PBS pH 5.8 and then incubated with FcRn-β2m-GST-APC in PBS pH 7.4 (Figure 3A). Spheroplasts with FITC positive and APC negative signals are then sorted (Figure 3B).

[0144] Example 5 FACS analysis of IgG variants Four IgG variants were identified: EDH (V264E, L309D, and Q311H), EDHS (SEQ ID NO:5; V264E, L309D, Q311H, and N434S), EDHY (SEQ ID NO:6; V264E, L309D, Q311H, and N434Y), and TEDHY (A231T, V264E, L309D, Q311H, and N434Y). Each gene was transformed into E. coli JUDE-1, and the cells were spheroplasted and analyzed by FACS using 10 nM FcRn-β2m-FITC in PBS pH 5.8 or 50 nM FcRn-β2m-GST-APC in PBS pH 7.4. As shown in Figure 4, the four IgG variants exhibited mean fluorescence intensity (MFI) values ​​for FcRn-β2m-FITC that ranged from 2.6 to 5.1 times higher than wild-type nonglycosylated IgG. For FcRn-β2m-GST-APC in PBS pH 7.4, EDHY and TEDHY exhibited significantly higher binding activity than wild-type IgG1, whereas EDH and EDHS exhibited similar or slightly increased binding activity compared to wild-type IgG1.

[0145] Example 6 Expression and purification of selected mutant IgG variants All plasmids and primers are listed in Tables 4, 10 and 11. The four mutant Fc genes were amplified from pMopac12-pelB-IgG-VH-CH1-CH2-CH3-FLAG using two specific primers (TH083 (SEQ ID NO: 20) and TH084 (SEQ ID NO: 21)). The pcDNA3.4-IgH plasmid was amplified using two specific primers (TH081 (SEQ ID NO: 22) and TH082 (SEQ ID NO: 23)). The four Fc genes were cloned into pcDNA3.4 using the Gibson Assembly® Cloning Kit (NEB) according to the manufacturer's instructions (Leee et al., 2017). The Gibson assembled mixture was transformed into E. coli JUDE-1 cells and their sequences were confirmed. The Fab of trastuzumab was also used. The heavy chain genes of the four Trastuzumab-Fc variants were transiently transfected into HEK293F cells (Invitrogen) with an equal mass of the light chain plasmid. After 6 days of incubation at 37°C in a 5% CO2 incubator, the supernatant was harvested by centrifugation at 4,000 x g for 10 min and filtered using a 0.22 μm PES membrane filter (PALL). The filtered supernatant was passed three times through Protein A high-capacity agarose resin (Thermo Scientific). To remove LPS and nonspecifically bound proteins, the IgG-bound resin was washed with 50 mL of PBS containing 0.1% Triton® X-114 (Sigma-Aldrich) and 50 mL of PBS. All IgG variants were eluted with 100 mM glycine buffer (pH 3.0) and immediately neutralized with 1 M Tris-HCl buffer (pH 8.0). All buffers of the eluted trastuzumab-Fc antibody variants were exchanged with PBS by Amicon® Ultra-4 (Millipore). The reduced or non-reduced protein purity of the trastuzumab-Fc antibody variants and authentic (wt) trastuzumab expressed in HEK293 cells as described above was assessed by 4%-20% gradient SDS-PAGE gels (NuSep) under reducing and non-reducing conditions.

[0146] Example 7 Binding properties of selected IgG variants to human FcRn and FcγR The binding affinities of three IgG variants, EDH, EDHS and EDHY, to human FcRn were assessed using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

[0147] SPR measurements: SPR measurements were performed on a Biacore® 3000 (GE Healthcare) instrument. The reference channel of the CM5 sensor chip was closed without any protein immobilization to subtract the buffer effect and nonspecific binding signal. Clinical grade Herceptin, EDH, EDHY, and EDHS were immobilized on the CM5 sensor chip by amine coupling method at pH 5.0. Serially diluted FcRn (400-25 nM) proteins in phosphate buffered saline pH 5.8 (PBS, 1.8 mM KH2PO4, 10 mM Na2HPO4, 2.7 mM KCl, and 137 mM NaCl) were injected onto the CM5 chip at 30 μL / min for 1 min. The chip was regenerated after each binding event with 10 mM Tris (pH 8.0) with a contact time of 1 min. The obtained sensorgrams were fitted with a 1:1 Langmuir model using Biaevaluation 3.0 software (Figure 5; Table 5). As controls, previously reported Herceptin-LS (M428L / N434S, Xencor) and Herceptin-YTE (M252Y / S254T / T256E, Medimmune) were immobilized on a CM5 sensor chip by the same method and analyzed. Herceptin-EDH showed similar affinity compared to wild-type Herceptin (Figure 5A-B; Table 5). On the other hand, Herceptin-EDHY and Herceptin-EDHS showed enhanced affinity of 19.6-fold and 5.9-fold over Herceptin, respectively (Figure 5C-D; Table 5). Herceptin-LS and Herceptin-YTE showed 10-fold and 23.9-fold enhanced affinity over Herceptin, respectively, results consistent with previous reports (FIGS. 5E-F; Table 5). [Table 5]

[0148] ELISA measurement of IgG variants with hFcRn: 1 μg each of EDH, EDHY, EDHS, or Herceptin was coated on a 96-well EIA / RIA plate (Qiagen) overnight at 4° C., and the plate was washed three times with PBS containing 0.05% Tween® 20 (PBST). The plate was blocked with 1× fish gelatin blocking solution (Biotium) in PBS for 1 h at room temperature and washed three times with PBST. Then, serially diluted dimeric FcRn-β2m-GST (100 nM to 0.8125 nM) was added to the plate. After incubation at room temperature for 1 h, the plate was washed with PBST, and then it was incubated with 50 μL of PBS containing 1:5000 goat anti-GST HRP (GE Healthcare) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1M H2SO4, and absorbance at 450 nm was recorded. Wild-type Herceptin, Herceptin-EDH, and Herceptin-EDHS did not show any binding activity to dimeric FcRn-β2m-GST at physiological pH 7.4. On the other hand, Herceptin-LS and Herceptin-EDHY showed significant binding activity to dimeric FcRn-β2m-GST at physiological pH 7.4.

[0149] In particular, EDHS exhibited a 5.9-fold enhanced K D and no binding signal to FcRn at pH 7.4.

[0150] ELISA measurement of IgG variants with hFcγR: The binding properties of EDHS to human FcγR were assayed. Briefly, 1 μg each of wild-type Herceptin or Herceptin-EDHS was coated onto a 96-well EIA / RIA plate (Qiagen) overnight at 4°C, and the plate was washed three times with PBST. The plate was blocked with 1% BSA in PBS for 1 h at room temperature and washed three times with PBST. Serial dilutions of monomeric His-FcγRI, dimeric GST-FcγRIIa, and dimeric GST-FcγRIIa were then applied to the plate. R131 , dimeric GST-FcγRIIa H131 , dimeric GST-FcγRIIb, dimeric GST-FcγRIIIa V158 , and dimeric GST-FcγRIIIa F158 was added to the plate. After 1 h incubation at room temperature, the plate was washed with PBST and incubated with 50 μL of PBS containing 1:5000 goat anti-His or anti-GST HRP (GE Healthcare) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1 M H2SO4, and absorbance at 450 nm was recorded. Herceptin-EDHS showed a significant reduction in binding signal compared to wild-type Herceptin for all FcγRs (Figure 7A-F).

[0151] Example 8 Construction and characterization of DHS-Fc variants Effector function by FcγR is an antibody-mediated immune response that is essential for eliminating pathogens. Herceptin-EDHS was unable to bind to effector FcγR. Herceptin-EDHS contains four amino acid substitutions, V264E, L309D, Q311H, and N434S. V264E impairs the glycosylation of IgG, which is crucial for binding to effector FcR (XiaojieYu et al., 2013). Therefore, Herceptin-DHS (SEQ ID NO: 7), which does not have the V264E mutation, was not able to bind to effector FcγR. The binding affinity of DHS to human FcRn and FcγR was evaluated by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

[0152] SPR measurements: SPR measurements were performed on a Biacore® 3000 (GE Healthcare) instrument. The reference channel of the CM5 sensor chip was closed without any protein immobilization to subtract the buffer effect and nonspecific binding signal. Herceptin-DHS was immobilized on the CM5 sensor chip at pH 5.0 by the amine coupling method (Leee et al., 2017). Serially diluted FcRn (400-25 nM) proteins in PBS pH 5.8 were injected onto the CM5 chip at 30 μL / min for 1 min. The chip was regenerated after each binding event with 10 mM Tris (pH 8.0) with a contact time of 1 min. The obtained sensorgrams were fitted with a 1:1 Langmuir model using Biaevaluation 3.0 software (Figure 8). Herceptin-DHS showed similar affinity, K of 111 ± 20 nM, compared to Herceptin-EDHS. D (Figures 5 and 8; Table 5). Removal of V264E from Herceptin-EDHS does not affect FcRn binding ability.

[0153] ELISA measurement of IgG variants with hFcRn: 1 μg of each Herceptin-DHS was coated on a 96-well EIA / RIA plate (Qiagen) overnight at 4°C, and the plate was washed three times with PBST. The plate was blocked with 1× fish gelatin blocking solution (Biotium) in PBS for 1 h at room temperature and washed three times with PBST. Then, serially diluted dimeric FcRn-β2m-GST (100 nM to 0.8125 nM) was added to the plate. After incubation at room temperature for 1 h, the plate was washed with PBST, which was then incubated with 50 μL of PBS containing 1:5000 goat anti-GST HRP (GE Healthcare) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1 M H2SO4, and the absorbance at 450 nm was recorded. Herceptin-DHS did not show any binding activity to dimeric FcRn-β2m-GST at physiological pH 7.4 compared to wild-type Herceptin. As controls, Herceptin-LS and Herceptin-N434S were tested simultaneously.

[0154] ELISA measurement of IgG variants with hFcγR: The binding properties of EDHS to human FcγR were assayed as described above. Briefly, 1 μg each of wild-type Herceptin or Herceptin-EDHS was coated onto a 96-well EIA / RIA plate (Qiagen) overnight at 4° C., and the plate was washed three times with PBST. The plate was blocked with 1% BSA in PBS for 1 h at room temperature and washed three times with PBST. Serial dilutions of monomeric His-FcγRI, dimeric GST-FcγRIIa, and dimeric GST-FcγRIIa were then applied to the plate. R131 , dimeric GST-FcγRIIa H131 , dimeric GST-FcγRIIb, dimeric GST-FcγRIIIa V158 , and dimeric GST-FcγRIIIa F158was added to the plate. After incubation for 1 h at room temperature, the plate was washed with PBST and incubated with 50 μL of PBS containing 1:5000 goat anti-His or anti-GST HRP (GE Healthcare) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1 M H2SO4, and absorbance at 450 nm was recorded. Herceptin-DHS showed the same binding profile to all FcγRs compared to wild-type Herceptin (Figure 10A-F).

[0155] SPR measurements: SPR measurements were performed on a Biacore® 3000 (GE Healthcare) instrument. The reference channel of the CM5 sensor chip was closed without any protein immobilization to subtract the buffer effect and nonspecific binding signal. Wild-type Herceptin, Herceptin-DHS, Herceptin-LS, and Herceptin-YTE were immobilized on the CM5 sensor chip by amine coupling method at pH 5.0. Serially diluted FcγR or C1q (400-25 nM) proteins in HBS-EP at pH 7.4 were injected onto the CM5 chip at 30 μL / min for 1 min. The chip was regenerated after each binding event with 10 mM glycine (pH 5.0) with a contact time of 1 min. The obtained sensorgrams were fitted using the 1:1 Langmuir model or the equal binding model using Biaevaluation 3.0 software (Figure 18 and Table 12). The results of the kinetic analysis for Fc receptors were consistent with the ELISA results. Herceptin-DHS showed comparable kinetic values ​​for FcγR and C1q compared to wild-type Herceptin, whereas Herceptin-LS and Herceptin-YTE significantly lost FcγR binding capacity, especially FcγRIIIa binding capacity, a receptor important in ADCC (Figure 18 and Table 12).

[0156] In conclusion, removal of V264E does not affect the pH-dependent FcRn-binding ability of Herceptin-EDHS to FcRn, but restores its binding ability to FcγR. [Table 12-1] [Table 12-2]

[0157] Example 9 Effector functions of Herceptin-DHS ADCC assay: Human peripheral blood mononuclear cells (PBMCs) were isolated from human blood from healthy donors the day before the ADCC assay. 50 mL of human blood was collected in a heparinized vial (BD biosciences) and mixed thoroughly by gently inverting the tube several times. 25 mL of blood was layered on 25 mL of room temperature Ficoll Histopaque (Invitrogen) in a 50 mL conical tube. The tube was centrifuged at 2,500 rpm for 30 minutes in a swinging bucket rotor without brake. Human PBMCs were aspirated at the interface between the histopaque and the medium. Human PBMCs were resuspended in red blood cell (RBC) lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA) and washed twice with PBS. Isolated human PBMCs were mixed with calcein AM-labeled SK-BR3 Her2-positive cancer cells and various concentrations of IgG variants in a 96-well plate. The tumor to effector cell ratio was 1:10 and the plates were incubated at 37°C, 5% CO2 for 4 hours. The percentage of tumor cell lysis was calculated according to the following formula: 100 x (ES) / (MS), where E is the fluorescence of the experimental wells, S is the fluorescence in the absence of antibody (tumor cells were incubated with medium and effector cells), and M is the fluorescence of tumor cells with lysis buffer. As described in Example 8, Herceptin-DHS showed comparable ADCC activity compared to Herceptin in PBMCs, because Herceptin-DHS has the same affinity for FcγR compared to Herceptin (Figures 10A-F and 11A).

[0158] ADCP assay: CD14 positive monocytes were purified from PBMCs using a CD14 MicroBead Kit (Miltenyi Biotec). Monocytes were then differentiated into macrophages by culturing in RPMI medium containing 15% FBS and 50 ng / ml GM-CSF for 7 days and then combined with calcein-labeled SK-BR3 cells at an effector:tumor cell ratio of 10:1. For ADCP assay, SK-BR3 cells were incubated with various concentrations of antibodies and macrophages. After 2 hours at 37°C, cells were labeled with anti-CD11b-APC and anti-CD14-APC. Phagocytosis was assessed by FACS on an LSRFortessa (BD Bioscience) and reported as the fraction of double positive cells over the total number of tumor cells in the sample. Herceptin-DHS showed comparable ADCC activity compared to Herceptin in PBMCs. This is because Herceptin-DHS has the same affinity for FcγR as Herceptin (FIGS. 10A-F and 11B).

[0159] Example 10 Binding properties of Herceptin-DHS to FcRn To investigate the detailed pH-dependent binding activity of Herceptin-DHS, the antibody was evaluated by SPR with three different densities of scFcRn or under different pH conditions.

[0160] SPR measurements: SPR measurements were performed on a Biacore® 3000 (GE Healthcare) instrument. The reference channel of the CM5 sensor chip was closed without any protein immobilized to subtract the buffer effect and nonspecific binding signal. scFcRn was immobilized at three different immobilization levels (500RU, 2000RU, and 4000RU) on the CM5 sensor chip by the amine coupling method at pH 5.0. Serially diluted antibody (400-25 nM) proteins in PBS pH 7.4 were injected over the CM5 chip at 30 μL / min for 1 min. The chip was regenerated after each binding event with 10 mM Tris (pH 8.0) with a contact time of 1 min. The resulting sensorgrams were fitted with a 1:1 Langmuir model using Biaevaluation 3.0 software (Figure 12 and Table 6). Herceptin-DHS and wild-type Herceptin did not show any binding signals with low, medium and high density scFcRn. Herceptin-LS and Herceptin-YTE did not show any binding signals with low density scFcRn, but showed significant binding to scFcRn immobilized at medium and high density (Table 6). The K D The K of Herceptin-YTE is 58.7 nM for medium density FcRn and 22.7 nM for high density FcRn. D is 4.73 μM for medium density FcRn and 4.29 μM for high density FcRn. [Table 6] Next, to investigate the detailed pH-dependent binding activity of the antibodies, the antibodies were assayed under various pH conditions. 1 μM wild-type Herceptin, Herceptin-DHS, Herceptin-LS, and Herceptin-YTE were injected at 30 μL / min for 1 min on a CM5 chip in PBS pH 4.5-8.5. The chip was regenerated after each binding event with 10 mM Tris (pH 8.0) for a contact time of 1 min. As shown in Figure 13A-C, Herceptin-DHS showed better binding activity than wild-type Herceptin, but Herceptin-DHS significantly lost FcRn binding activity under three different FcRn densities, pH 6.2 to pH 6.5 conditions. Herceptin-DHS also completely lost FcRn binding activity at pH 6.8.

[0161] Example 11 Pharmacokinetics of Herceptin-DHS in human FcRn transgenic mice The pharmacokinetics of the modified IgG was carried out in hemizygous hFcRn transgenic mice (strain 276) generated by F1 mating of mouse FcRn-deficient B6.129X1-Fcgrttm1Dcr / DcrJ with the hFcRn cDNA transgenic strain B6.Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276 Dcr / DcrJ (Roopenian et al., 2003 and Chaudhury et al., 2003). FcRn mice were given a bolus intravenous (IV) dose of 2 mg / kg of antibody. Eleven mice were used per antibody. Blood samples were obtained from the tail vein using a capillary pipette at different time points throughout the 2-3 week study. A quantitative ELISA was used to monitor serum concentrations of the tested antibodies. Briefly, 96-well plates were coated with 2 μg / ml goat anti-human F(ab')2 fragment specific F(ab')2 (Jackson Immunoresearch). Plates were blocked with 1% fish gelatin (AMRESCO-inc) in PBS for 1 h and then incubated with appropriately diluted serum samples (1:200 for earlier time points and 1:50 or 1:100 for later time points). Human antibodies were detected using anti-kappa light chain antibody-HRP (Abcam) (dilution 1:5,000). Absorbance at 450 nm was measured after development with TMB substrate (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. Standard curves were generated for each antibody variant diluted in 1:100 pre-bled mouse serum. Human IgG in serum samples was then quantified using the linear portion of the standard curve generated in Prism (GraphPad Software). AUCinf (area under the curve to infinity) was calculated using the log-linear trapezoidal method. Elimination half-life (T1 / 2) was calculated using log-linear regression of concentration data including at least the last three sampling time points with measurable concentrations. Serum clearance was estimated as CL=dose / AUCinf. Descriptive statistics for the main PK parameters were then calculated. As a result, Herceptin-DHS exhibited a 6.8-fold increase in beta-phase serum half-life (β-phase T 1 / 2 =336±24.8 hours) (Figure 14 and Table 7). Herceptin-DHS also demonstrated a 9.5-fold reduced clearance rate (0.061±0.003 ml / day / kg), a 7.9-fold enhanced AUCinf (326.6±17.9 μg*day / ml), and a 3.96-fold reduced tissue distribution (0.28±0.01 ml / kg) compared to wild-type Herceptin. [Table 7]

[0162] Example 12 DHS mutations in IgG subclasses There are four subclass members of IgG: IgG1, IgG2, IgG3, and IgG4. The FcRn-binding site in IgG subclasses is highly conserved, and thus IgG2 and IgG4 exhibit serum half-lives comparable to that of IgG1. There are two single nucleotide polymorphisms at residue 435 of IgG: IgG3-R435 has a short serum half-life (approximately 8 days), whereas IgG3-H435 has a similar half-life (approximately 21 days) compared to other IgG subclasses. To investigate whether DHS mutations (L309D, Q311H, and N434S mutations) can enhance the FcRn-binding activity of other IgG subclasses, DHS mutations were introduced into the heavy chain genes of IgG2, IgG3-H435, and IgG4. The Fc genes of IgG2-DHS, IgG3-DHS, and IgG4-DHS were synthesized by IDT, and the Fc genes were cloned into pcDNA3.4 using the Gibson Assembly® Cloning Kit (NEB) according to the manufacturer's instructions (Leee et al., 2017). The Gibson assembled mixtures were transformed into E. coli JUDE-1 cells, and their sequences were confirmed. The Fab of trastuzumab was also used. The heavy chain genes of the four trastuzumab-Fc variants were transiently transfected into HEK293F cells (Invitrogen) with an equal mass of the light chain plasmid. After incubation at 37°C in a 5% CO2 incubator for 6 days, the supernatant was collected by centrifugation at 4,000 × g for 10 min and filtered using a 0.22 μm PES membrane filter (PALL). The filtered supernatant was passed three times through Protein A high-capacity agarose resin (Thermo Scientific). To remove LPS and non-specifically bound proteins, the IgG-bound resin was washed with 50 mL of PBS containing 0.1% Triton® X-114 (Sigma-Aldrich) and 50 mL of PBS. All IgG variants were eluted with 100 mM glycine buffer (pH 3.0) and immediately neutralized with 1 M Tris-HCl buffer (pH 8.0).All buffers of the eluted trastuzumab-Fc antibody variants were exchanged with PBS by Amicon® Ultra-4 (Millipore). Reduced or non-reduced protein purity for trastuzumab-Fc antibody variants and authentic (wt) trastuzumab expressed in HEK293 cells as described above was assessed by 4%-20% gradient SDS-PAGE gels (NuSep) under reducing and non-reducing conditions.

[0163] SPR measurements: SPR measurements were performed on a Biacore® 3000 (GE Healthcare) instrument. The reference channel of the CM5 sensor chip was closed without any protein immobilization to subtract the buffer effect and nonspecific binding signal. Wild-type IgG2, wild-type IgG3, wild-type IgG4, IgG2-DHS, IgG3-DHS and IgG4-DHS were immobilized on the CM5 sensor chip by amine coupling method at pH 5.0. Serially diluted FcRn (400-25 nM) proteins in PBS pH 5.8 were injected onto the CM5 chip at 30 μL / min for 1 min. The chip was regenerated after each binding event with 10 mM Tris (pH 8.0) with a contact time of 1 min. The obtained sensorgrams were fitted with a 1:1 Langmuir model using Biaevaluation 3.0 software (Figure 15). IgG2-DHS showed a 5.7-fold enhanced K for scFcRn compared to wild-type IgG2. D (377±26 nM) (FIGS. 15A-B; Table 8). IgG3-DHS showed a 5.5-fold enhanced K D (739 ± 75 nM) (Figures 15C-D; Table 8). IgG4-DHS showed a 3.0-fold enhanced K D (832±16 nM) (FIGS. 15E-F; Table 8). [Table 8]

[0164] ELISA measurement of IgG variants with hFcγR: The binding properties of EDHS to human FcγR were assayed. The detailed procedure was the same as in the above paragraph. Briefly, 1 μg each of wild-type IgG2, IgG2-DHS, wild-type IgG3, IgG3-DHS, wild-type IgG4, and IgG4-DHS were coated onto a 96-well EIA / RIA plate (Qiagen) overnight at 4°C, and the plate was washed three times with PBST. The plate was blocked with 1% BSA in PBS for 1 h at room temperature and washed three times with PBST. Then, serially diluted monomeric His-FcγRI, dimeric GST-FcγRIIa, and dimeric GST-FcγRIIa were coated onto a 96-well EIA / RIA plate (Qiagen) overnight at 4°C, and the plate was washed three times with PBST. R131 , dimeric GST-FcγRIIa H131 , dimeric GST-FcγRIIb, dimeric GST-FcγRIIIa V158 , and dimeric GST-FcγRIIIa F158 was added to the plate. After incubation at room temperature for 1 h, the plate was washed with PBST and incubated with 50 μL of PBS containing 1:5000 goat anti-His or anti-GST HRP (GE Healthcare) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1 M H2SO4, and absorbance at 450 nm was recorded. IgG2-DHS showed the same binding activity to all FcγRs compared to wild-type IgG2 (Figure 16A). IgG3-DHS showed the same binding activity to all FcγRs compared to wild-type IgG3 (Figure 16B). IgG4-DHS showed the same binding activity to all FcγRs compared to wild-type IgG4 (Figure 16C). In conclusion, the three amino acid substitutions do not affect the FcγR binding activity of IgG subclasses.

[0165] PK Study: The pharmacokinetics of IgG2-DHS and IgG4-DHS were evaluated using the same method as in Example 11. As a result, IgG2-DHS showed a 2.9-fold increased beta-phase serum half-life (β-phase T 1 / 2=199.8±4.9 hours) (Figure 17 and Table 9). IgG2-DHS also showed a 1.9-fold reduced clearance rate (0.067±0.015 ml / day / kg), a 1.9-fold enhanced AUCinf (148.3±7.5 μg*day / ml), and a 1.5-fold reduced tissue distribution (0.57±0.02 ml / kg) compared to wild-type IgG2. IgG4-DHS also showed a 3.6-fold increased beta-phase serum half-life (β-phase T 1 / 2 =277.4±14.9 hours) (Figure 16 and Table 9). IgG4-DHS also showed a 2.4-fold reduced clearance rate (0.067±0.048 ml / day / kg), a 2.4-fold enhanced AUCinf (149.2±6.3 μg*day / ml), and a 1.7-fold reduced tissue distribution (0.58±0.03 ml / kg) compared to wild-type IgG4. IgG3-DHS showed a 4.4-fold increased beta-phase serum half-life (β-phase T 1 / 2 =286.3±14.6 hours) (Figure 19 and Table 13). IgG3-DHS also showed a 3.8-fold reduced clearance rate (0.076±0.007 ml / day / kg), a 3.9-fold enhanced AUCinf (269.0±22.4 μg*day / ml), and a 2.1-fold reduced tissue distribution (0.41±0.05 ml / kg) compared to wild-type IgG3 (Figure 19 and Table 13). [Table 9] [Table 13]

[0166] Example 13 Rheumatoid factor binding properties In patients with autoimmune diseases, including rheumatoid arthritis (RA), rheumatoid factor (RF) is often observed, which may reduce the therapeutic efficacy of therapeutic antibodies by clearing serum antibodies from the blood (Newkirk2002, Ingegnoli et al., 2013, Maeda et al., 2017). Therefore, the RF-binding ability of antibody variants was tested by ELISA.

[0167] ELISA measurement of IgG variants with rheumatoid factor: The binding properties of DHS to RF were assayed. Briefly, 50 μl of rheumatoid factor positive serum (Lee Biosolutions) was coated onto 96-well EIA / RIA plates (Qiagen) overnight at 4°C, and the plates were washed three times with PBST. The plates were blocked with 1% BSA in PBS for 1 h at room temperature and washed three times with PBST. Serially diluted biotinylated antibody variants were then added to the plates. After incubation at room temperature for 1 h, the plates were washed with PBST and incubated with 50 μL of PBS containing 1:5000 streptavidin-HRP (Thermo Fisher Scientific) for 1 h. After washing three times with PBST, 50 μL of TMB substrate was added per well (Thermo Scientific), neutralized by adding 50 μL of 1 M H2SO4, and the absorbance at 450 nm was recorded. Herceptin-DHS showed a comparable level of binding response to wild-type Herceptin, while Herceptin-YTE and Herceptin-LS showed stronger binding activity than wild-type Herceptin (FIG. 20). [Table 10] [Table 11-1] [Table 11-2]

[0168] All of the methods disclosed and claimed herein can be prepared and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain specific agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. U.S. Patent No. 4,870,287 U.S. Patent No. 5,739,169 U.S. Patent No. 5,760,395 U.S. Patent No. 5,801,005 U.S. Patent No. 5,824,311 U.S. Patent No. 5,830,880 U.S. Patent No. 5,846,945 U.S. Patent No. 7,094,571 U.S. Patent No. 7,419,783 U.S. Patent No. 7,611,866 U.S. Patent Publication No. 2003 / 0219870 PCT Publication No. WO2008 / 137475 [ka] [ka] [ka]

Claims

[Claim 1] The invention as depicted in the drawings.

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