Engineered FcRIIb-selective IgG1 Fc variants and their use

Abstract

In some aspects, mutant Fc domains or variant Fc domains are provided that can exhibit increased affinity or selectivity for FcγRIIB. The variant Fc domain can be a mutant Fc domain of IgG1. In some embodiments, the mutant Fc domain or variant Fc domain can be present in a therapeutic antibody, for example, in an agonist antibody. Further methods for using and identifying mutant Fc domains are also provided. TIFF2025519556000040.tif104170

Description

Technical Field

[0001] Priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 351,282, filed on June 10, 2022, and U.S. Provisional Patent Application No. 63 / 385,877, filed on December 2, 2022, the entire contents of both provisional applications being incorporated herein by reference.

[0002] Incorporation of Sequence Listing This application includes a Sequence Listing XML, which is submitted electronically and is incorporated herein by reference in its entirety. The XML Sequence Listing was created on June 6, 2023, has the name "CLFRP0495WO.xml", and is 15,515 bytes in size.

Background Art

[0003] Background of the Invention 1. Field of the Invention The present invention generally relates to the field of protein engineering. More specifically, the present invention relates to an improved composition, which is a composition of the Fc domain of an antibody that provides high binding to FcγRIIB and altered effector function.

[0004] 2. Description of Related Art A variety of antibodies are being developed for therapeutic purposes. The top 25 marketed recombinant therapeutic antibodies currently have sales well in excess of $43.5 billion per year. Given an expected annual growth rate of 9.2% from 2010 to 2015, this is expected to increase to $62.7 billion per year by 2015 (Elvin et al., 2013). Monoclonal antibodies (mAbs) currently account for the majority of recombinant proteins in clinical use, and in the US or EU, 1,064 products are in industry-sponsored clinical trials, 164 of which are in Phase III (Elvin et al., 2013). The mAb market has received significant attention with respect to oncology and inflammatory disorders, and products within these therapeutic areas are likely to remain important drivers well beyond the period considered. As a group, genetically engineered mAbs generally have a higher likelihood of successful FDA approval than small molecule drugs. At least 50 biotechnology companies and all of the major pharmaceutical companies are actively pursuing antibody drug discovery programs. The first method for isolating and generating mAbs was first reported in 1975 by Milstein and Kohler (Kohler and Milstein, 1975). This involved fusing mouse lymphocytes with myeloma cells to generate mouse hybridomas. Therapeutic mouse mAbs were introduced into clinical trials in the early 1980s; however, problems with lack of efficacy and rapid disappearance due to patients producing human anti-mouse antibodies (HAMA) became apparent. These points were a driving force for evolving the technology for making mAbs, as well as the time and cost consumed in relation to that technology. The polymerase chain reaction (PCR) facilitated the direct cloning of monoclonal antibody genes from the lymphocytes of immunized animals and the expression of combinatorial libraries of antibody fragments in bacteria (Orlandi et al., 1989).Subsequently, a library was fully generated by in vitro cloning techniques using naive genes with the reconstituted complementarity determining region 3 (CDR3) (Griffiths and Duncan, 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 have facilitated the development of antibody fragments against some unique antigens, including small molecule compounds (haptens) (Hoogenboom and Winter, 1992), molecular complexes (Chames et al., 2000), labile compounds (Kjaer et al., 1998), and cell surface proteins (Desai et al., 1998).

[0005] For the purpose of identifying clones that bind to a ligand with the desired affinity, one method for screening a large combinatorial library of antibodies involves expressing and presenting antibody fragments or full-length antibodies on the surface of bacterial cells, more specifically on the surface of Escherichia coli (E. coli). Cells presenting the antibody or antibody fragment are incubated with a solution of fluorescently labeled ligand, and the cells that bind to the ligand due to the antibody presented on their surface are isolated by flow cytometry. In particular, Anchored Periplasmic Expression (APEx) is based on immobilizing antibody fragments on the periplasmic side of the inner membrane of E. coli, subsequently disrupting the outer membrane, incubating with a fluorescently labeled target, and sorting the spheroplasts (U.S. Patent No. 7,094,571, Harvey et al., 2004; Harvey et al., 2006).

[0006] Receptors for the Fc domain of antibodies are expressed on a variety of immune cells and are important for both promoting and regulating the immunological response to antibody-antigen complexes (referred to as immune complexes). Binding of the Fc region of an antibody that has formed an immune complex with a pathogenic target cell to various Fc receptors expressed on the surface of leukocytes induces antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), or complement-mediated reactions including complement-dependent cytotoxicity (CDC).

[0007] In humans, there are two major classes of FcγRs for IgG antibodies: activating receptors are characterized by the presence of an immunoreceptor tyrosine-based activation motif (ITAM) sequence on the cytoplasmic side linked to the receptor, and inhibitory receptors are characterized by the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence (Daeron, 1997 and Bolland et al., 1999). Notably, the activating FcγRs, FcγRI, FcγRIIA, FcγRIIIA, and FcγRIIIB, induce activating or pro-inflammatory responses, while the inhibitory FcγRIIB induces anti-inflammatory or inhibitory responses. Among the activating FcγRs, FcγRIIA and FcγRIIIA have natural allotypes that can affect their binding ability to IgG. FcγRIIA H131 shows higher binding affinity for IgG than FcγRIIA R131 and FcγRIIIA V158 shows higher binding affinity for IgG than FcγRIIIA F158 . All naturally produced antibodies and glycosylated recombinant antibodies produced by tissue culture contain an Fc domain that binds to both activating and inhibitory FcγRs (Boruchov et al. 2005; Kalergis et al., 2002).

[0008] As described above, non-glycosylated antibodies do not show any detectable binding to FcγRIIB. Due to the physiological importance of Fc binding to FcγRIIB and the importance of Fc binding to FcγRIIB when using therapeutic antibodies (e.g., agonist antibodies), a novel Fc domain, for example, a non-glycosylated Fc domain that can selectively bind to FcγRIIB, is definitely needed. SUMMARY OF THE INVENTION

[0009] The present disclosure overcomes the limitations in the prior art by providing an Fc domain variant that selectively binds to FcγRIIB while not inducing activating FcγRs and does not cause inflammatory effector functions, such as antibody-mediated cell phagocytosis or antibody-dependent cell cytotoxicity (ADCP and ADCC, respectively), and does not cause such effector functions. In contrast to previously engineered Fc mutants, in some embodiments, the engineered Fc provided herein confers highly selective binding to FcγRIIB but does not bind to activating receptors (FcγRI, FcγRIIa H131 、FcγRIIa R131 、FcγRIIIa F158 、FcγRIIIa V158) and has no binding ability to C1q or only has significantly impaired binding ability. In addition, the variants specific to FcγRIIB disclosed herein bind to the fetal receptor FcRn in a pH-dependent manner, similar to the wild-type Fc domain. The variant Fc domain may or may not be glycosylated. The ability of the variant Fc that selectively binds to FcγRIIB and does not induce activating FcγR or cause antibody-mediated phagocytosis can be particularly beneficial for various therapies, including, for example, therapeutic applications where it is necessary to minimize or suppress inflammatory effects mediated by antibodies due to binding to activating FcγR or complement. The disclosed variant Fc that selectively binds to FcγRIIB includes SEQ ID NOs: 2 to 5, which is preferably SEQ ID NO: 5. Also provided are hexameric constructs (such as those shown in Figure 11; SEQ ID NOs: 7 to 8 and 12 to 13, preferably SEQ ID NO: 7 or SEQ ID NO: 8) containing Fc domain variants that selectively bind to FcγRIIB, and related methods of using such constructs.

[0010] Some aspects of the present disclosure relate to polypeptides comprising a human IgG variant Fc domain or variant Fc domain that can bind to human FcγRIIb, wherein the human IgG variant Fc domain or variant Fc domain comprises substitution mutations that are valine at position 233 (E233V), leucine at position 239 (S239L), proline at position 238 (H268P), leucine at position 327 (A327L), alanine at position 328 (L328A), and substitution mutations at positions 234 (L234) and 235 (L235); the amino acid numbering follows the Kabat system. The human IgG variant Fc domain or variant Fc domain may further preferably comprise substitution mutations that are glycine at position 298 (S298G) and alanine at position 299 (T299A). The substitution mutation at position 234 may be proline at position 234 (L234P) or may be aspartic acid at position 234 (L234D). In some embodiments, the substitution mutation at position 234 is aspartic acid at position 234 (L234D). The substitution mutation at position 235 may be threonine at position 235 (L235T) or may be phenylalanine at position 235 (L235F). In some embodiments, the substitution mutation at position 235 is phenylalanine at position 235 (L235F). In some embodiments, the substitution mutation at position 234 is proline at position 234 (L234P) and the substitution mutation at position 235 is threonine at position 235 (L235T). The human IgG variant Fc domain or variant Fc domain may further comprise a substitution mutation that is aspartic acid at position 237 (S267D). In some embodiments, the human IgG variant Fc domain or variant Fc domain further comprises substitution mutations that are glutamine at position 332 (I332Q) and / or valine at position 334 (K334V). In some embodiments, the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc V8.2 (SEQ ID NO:2).In some embodiments, the substitution mutation at position 234 is aspartic acid at position 234 (L234D), and the substitution mutation at position 235 is phenylalanine at position 235 (L235F). The human IgG variant Fc domain or variant Fc domain may further include one, two, three, or all of the substitution mutations that are arginine at position 236 (G236R), aspartic acid at position 237 (G237D), histidine at position 330 (A330H), and / or isoleucine at position 333 (E333I). The human IgG variant Fc domain or variant Fc domain may further include a substitution mutation that is aspartic acid at position 267 (S267D). In some embodiments, the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc 2B18K (SEQ ID NO:3). The human IgG variant Fc domain or variant Fc domain may further include a substitution mutation that is glutamine at position 292 (R292Q). The human IgG variant Fc domain or variant Fc domain may include Fc 2B18KQ (SEQ ID NO:4), or may consist of it. The human IgG variant Fc domain or variant Fc domain may further include a substitution mutation that is glutamine at position 292 (R292Q). The human IgG variant Fc domain or variant Fc domain may include Fc 2B18KQS (SEQ ID NO:5), or may consist of it. In some embodiments, the human IgG variant Fc domain or variant Fc domain is not glycosylated. In some embodiments, the human IgG variant Fc domain or variant Fc domain is glycosylated. In some embodiments, the Fc domain does not selectively or detectably bind to one, two, three, four, or all of human FcγRI, FcγRIIa H131, FcγRIIIa F158, FcγRIIIa V158, and / or C1q polypeptide. In some embodiments, the Fc domain does not selectively or detectably bind to human FcγRI.In some embodiments, the Fc domain does not bind selectively or detectably to any of human FcγRIIa H131, FcγRIIIa F158, and FcγRIIIa V158. In some embodiments, the Fc domain has a binding affinity for FcγRIIa that is at most 1 / 30 or 1 / 40 as high as that of wild-type Fc. R131 The Fc domain may further include a substitution mutation at position 299 (e.g., leucine at amino acid position 299 (T299L), etc.) and / or a substitution mutation at position 298. In some embodiments, the Fc domain includes serine at position 298 and threonine at position 299. The polypeptide may further include a domain that binds other than the Fc receptor (FcR). The domain that binds other than the FcR may be an Ig variable domain, an antibody variable domain, or an antibody heavy chain variable domain. In some embodiments, the polypeptide is a full-length antibody or an agonistic antibody (e.g., an anti-CD40 agonistic antibody). The Ig variable domain may include the antibody heavy chain variable domain. The Ig variable domain may be included in a single-domain antibody. The Ig variable domain may constitute a ScFv. The human IgG variant Fc domain or variant Fc domain may be included in a multimeric oligomer (e.g., a hexameric Fc fusion protein). The hexameric Fc fusion protein may include TIFF2025519556000002.tif17147. As shown in the following examples, these peptides can be used to facilitate the formation of hexameric Fc fusion proteins. In some preferred embodiments, the hexameric Fc fusion protein It includes TIFF2025519556000003.tif4128. The human IgG variant Fc domain or variant Fc domain may include a substitution mutation that is a Leu residue at position 309, where the amino acid position numbering follows the Kabat system. The hexameric Fc fusion protein may or may not be glycosylated. In some embodiments, the human IgG variant Fc domain or variant Fc domain includes a Gly residue at position 298 and / or an Ala residue at position 299. In some embodiments, the human IgG variant Fc domain or variant Fc domain includes a Ser residue at position 298 and / or a Thr residue at position 299. In some preferred embodiments, the hexameric Fc fusion protein includes SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, or SEQ ID NO:13, and more preferably, the hexameric Fc fusion protein may include SEQ ID NO:7 or SEQ ID NO:8. The polypeptide or antibody may be chemically conjugated or covalently bound to a toxin. The region that binds other than to FcR may bind to a cell surface protein. In some embodiments, the region that binds other than to FcR binds to a soluble protein. The domain that binds other than to FcR may constitute a single domain antibody, scFv, or nanobody. The polypeptide may or may not be glycosylated. In some embodiments, the Fc domain does not cause or essentially does not cause antibody-mediated phagocytosis. In some embodiments, the Fc domain does not cause or essentially does not cause antibody-dependent cell-mediated cytotoxicity. In some embodiments, the Fc domain does not cause or essentially does not cause the induction of activating FcγR.

[0011] Another aspect of the present disclosure relates to a nucleic acid encoding any of the polypeptides described above or described herein. The nucleic acid may be a DNA segment. In some embodiments, the nucleic acid is an expression vector.

[0012] Yet another aspect of the present disclosure relates to a host cell comprising the nucleic acid described hereinabove. The host cell can express the nucleic acid. The host cell may be a eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, or a yeast cell.

[0013] Another aspect of the present disclosure relates to a method for preparing a non-glycosylated polypeptide, the method comprising the steps of: a) obtaining a host cell as described above or described herein; b) incubating the host cell in culture under conditions that promote the expression of the non-glycosylated polypeptide; and c) purifying the expressed polypeptide from the host cell. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, or a yeast cell).

[0014] Yet another aspect of the present disclosure relates to a pharmaceutical formulation comprising, in a pharmaceutically acceptable carrier, a polypeptide as described above or described herein, or a nucleic acid as described above or described herein.

[0015] Another aspect of the disclosure relates to a method of binding to a protein in a mammalian subject, the method comprising the steps of: administering an antibody to the subject, wherein the antibody binds to the protein and the antibody comprises an Fc domain as described above or described herein. In some embodiments, the antibody is capable of specifically binding to human FcγRIIb and the antibody has a reduced binding affinity for one or more activating Fcγ receptors as compared to the wild-type human IgG Fc domain. The antibody may or may not be glycosylated. In some embodiments, after the step of administering, the antibody does not cause or essentially does not cause antibody-mediated phagocytosis in the subject. In some embodiments, the mammalian subject is human. In some embodiments, the antibody binds to FcγRIIa in the subject with an affinity that is higher by about 1 / 30 or about 1 / 40, even if higher, compared to wild-type Fc. R131 to the receptor. In some embodiments, the antibody does not selectively or detectably bind to one or more activating human Fcγ receptor polypeptides in the subject. The activating human Fcγ receptor polypeptide may be FcγRI, FcγRIIa H131, FcγRIIIa F158, and / or FcγRIIIa V158. In some embodiments, the antibody does not specifically or detectably bind to one or more activating human C1q. The antibody may be a non-glycosylated version of a therapeutic antibody.

[0016] Yet another aspect of the disclosure relates to a method of treating a subject having a disease, the method comprising administering to the subject an effective amount of a formulation as described above or described herein. In some embodiments, the method does not induce antibody-dependent cell cytotoxicity. The disease can be cancer, an infectious disease, or an autoimmune disease. In some embodiments, the subject is a human patient.

[0017] In some embodiments, variant Fc domains or mutant Fc domains are provided, which exhibit selective binding to FcγRIIB compared to the corresponding wild-type Fc domain, and (ii) show reduced binding or no detectable binding to all of the activating human Fcγ receptors, namely FcγRI, FcγIIa H131, FcγIIa R131, FcγIIIa F158, or FcγIIIa V158, and to C1q. In some preferred embodiments, the variant Fc domain or mutant Fc domain is a human variant Fc domain or mutant Fc domain. The variant Fc domain or mutant Fc domain may be contained or incorporated in a polypeptide, such as in an antibody or a fusion protein. In some embodiments, the variant Fc domain or mutant Fc domain may be contained in a therapeutic antibody, such as an agonistic antibody. In some embodiments, there are compositions related to a polypeptide having an Fc domain derived from a human IgG1 antibody that is not glycosylated (the "Fc domain of the antibody" that is not glycosylated). In some embodiments, the Fc domain is a variant of the Fc domain of human IgG1 (SEQ ID NO: 1) that allows for highly selective binding to only FcγRIIB, but not for such binding to any of the effector Fc receptors, namely FcγRI, FcγIIA, and FcγIIIA. The Fc domain can bind highly selectively to FcγRIIB both when expressed in a glycosylated form and when expressed in a non-glycosylated form, but shows little or no binding to effector Fc receptors.

[0018] Another aspect of the invention relates to a pharmaceutically acceptable composition comprising a polypeptide of the invention and a pharmaceutically acceptable excipient.

[0019] Another further aspect of the present invention relates to a composition for use in a method of treating a disease in a subject in need thereof, the composition comprising a polypeptide of the present disclosure. In some embodiments, the disease is cancer, an infectious disease, a bacterial infection, a viral infection, or an autoimmune disease.

[0020] The Fc domain of the antibody may be the Fc domain of an IgG antibody or a variant thereof. Further, the Fc domain of the antibody may be that defined as a human Fc domain. In one aspect, the Fc domain may be the Fc domain of IgG1, which may be, for example, the Fc domain of an anti-HER2 antibody, more specifically the Fc domain of trastuzumab, and also the Fc domain of an anti-CD20 antibody, more specifically the Fc domain of rituximab. It is also contemplated that the polypeptide may form a fusion, wherein in the fusion, the engineered Fc domain disclosed herein is fused to a polypeptide not derived from an antibody molecule.

[0021] In some examples, the antibody may also have a substitution at amino acid position 297 or 299, such that when the antibody is expressed in mammalian cells that recognize the glycosylation motif in the Fc domain of the antibody, N-linked glycosylation does not occur. Any mutation at amino acid position 297 or 299, known to render glycosylation impossible, can be used (e.g., WO2005018572A2), and it is contemplated that any such mutation, including replacing 299T with a leucine residue, can be utilized.

[0022] In some embodiments, there are multiple amino acid substitutions at one or more positions among the following lists: (264, 328, 329, 330, 332, 333), (336; 234, 235, 236, 238), and / or (351 and 311); in some embodiments, the engineered Fc domain may have substitution mutations at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of these positions. The non-glycosylated Fc domain of the antibody described herein may include a substitution to alanine at amino acid position 264 (V264A), a substitution with serine at amino acid position 328 (L328S), a substitution to cysteine at amino acid position 329 (P329C), a substitution to tryptophan at amino acid position 330 (A330W), a substitution to asparagine at amino acid position 332 (I332N), a substitution to glycine at amino acid position 333 (E333G), a substitution to valine at amino acid position 336 (I336V), or a combination of those substitutions. The engineered Fc domain may further include one or more of the substitutions at amino acid positions 234, 235, 236, 238, and 351; and in some embodiments, the substitution at amino acid position 234 is arginine (L234R), the substitution at amino acid position 235 is glutamic acid (L235E), the substitution at amino acid position 236 is glutamic acid (G236E), the substitution at amino acid position 238 is arginine (P238R), and the substitution at amino acid position 351 is glutamine (L351Q). In some embodiments, the engineered Fc domain includes an additional amino acid substitution at residue position 311, which is, for example, lysine (i.e., Q311K) in some preferred embodiments. Combinations of substitution mutations may be present in the variant Fc domain or the variant Fc domain of the present disclosure.The human IgG variant Fc domain or variant Fc domain may contain the following substitution mutations: one, two, three, or four of alanine at amino acid position 264 (V264A), cysteine at amino acid position 329 (P329C), glycine at position 333 (E333G), and valine at amino acid position 336 (I336V); optionally, one, two, or three of tryptophan at amino acid position 330 (A330W), asparagine at amino acid position 332 (I332N), and serine at amino acid position 328 (L328S), in combination with the above; optionally, threonine at position 299 (T299L) in combination with the above. In some embodiments, the variant Fc domain may contain one, two, three, four, five, six, or all of the mutations that are V264A, L328S, P329C, A330W, I332N, E333G, I336V, which may optionally be combined with a mutation at 299, such as T299L. In some embodiments, the variant Fc domain may contain one, two, three, four, five, or all of the mutations that are L234R, L235E, G236E, P238R, T299L, and / or L351Q. In some embodiments, the variant Fc domain contains the Q311K mutation, which may optionally be combined with the T299L mutation. The engineered Fc domain of an antibody described herein may further contain one or more of the amino acid substitutions disclosed in U.S. Patent No. 10,526,408.

[0023] A variant Fc domain polypeptide (also referred to as a variant Fc domain or an engineered Fc domain) may be characterized as having a certain percentage identity when 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 by way of example), or when compared to any polypeptide sequence disclosed herein. The percentage identity may be at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100% (or any range derivable therein) between the unmodified portion of the modified polypeptide (i.e., the sequence of the modified polypeptide excluding all specified substitutions) and the corresponding wild-type polypeptide. For example, a variant Fc domain may have at least 90% (or at least about 95%, etc.) sequence identity with a wild-type Fc domain (e.g., a human wild-type Fc domain) with respect to the region of the variant Fc domain excluding the specified substitution mutations (e.g., excluding the substitution mutation at position 299 (e.g., T299L) in addition to any other specified substitution mutations). A variant Fc domain may include additional mutations compared to the wild-type Fc domain in addition to the substitution mutations specified in the variant Fc domain. It is also intended that the percentage of identity discussed above may relate to the entirety of the variant Fc domain polypeptide when compared to the 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 the amino acids in the wild-type polypeptide.

[0024] The Fc domain of the antibody may be the Fc domain of a human IgG antibody or a variant thereof. In certain aspects, the Fc domain may be the Fc domain of IgG1. It is also contemplated that the polypeptide can form a fusion, wherein in the fusion, the engineered variant Fc domain disclosed herein is fused to a polypeptide 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 targeting a CD40, cell death receptor 5 (DR5), or TNF receptor (TNFR) molecule.

[0025] The polypeptide comprising the variant Fc domain described herein may, in some embodiments, include a linker. In further embodiments, the linker is a conjugatable linker. In some embodiments, the polypeptide comprises an Fc domain derived from an antibody. The polypeptide may include other regions derived from the antibody, such as, for example, another binding domain. The additional binding domain may not be a domain that binds to an FcR. In some embodiments, the polypeptide may include an antigen-binding site or domain derived from an antibody, such as, for example, all or a portion of a variable region derived from an antibody. The polypeptide may include an Fc domain derived from an antibody and another binding domain that is a domain that binds other than to an FcR. In some embodiments, the binding region that is not Fc is not the antigen-binding site of the antibody, but specifically binds to a cell surface protein or a soluble protein. In some examples, the cell surface protein recognized by the binding region that is not Fc is a receptor, such as, for example, a receptor expressed on the cell surface.

[0026] Other polypeptides include those having a non-glycosylated variant Fc domain (e.g., one that can bind to an FcγRIIb polypeptide but exhibits a reduced or abolished binding to activating FcRs) and a second binding domain that is a domain that binds to something other than an Fc receptor, where the second binding domain can specifically bind to a cell surface molecule or a soluble protein. In some embodiments, the second binding domain is an antigen-binding domain of an antibody (an "Ig variable domain"). In some aspects, the polypeptide may be a full-length antibody. In some examples, the second binding domain is not an antigen-binding domain of an antibody. In some embodiments, the second binding domain can specifically bind to a cell surface molecule, which is a protein or a proteinaceous molecule. In some aspects, the second binding domain can specifically bind to a soluble protein.

[0027] Some aspects relate to nucleic acids encoding any of the polypeptides discussed herein. The nucleic acid may be isolated and / or recombinant. The nucleic acid may be an isolated and / or recombinant nucleic acid segment. In some embodiments, the nucleic acid is DNA, while in other cases, the nucleic acid is 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 a binding domain that is an Fc, where the binding domain that is an Fc has one or more substitutions and specifically binds to FcγRIIb. The nucleic acid may encode one or more of the polypeptides herein, and the polypeptide may be glycosylated or not glycosylated depending on the presence or absence of a particular mutation and on how the polypeptide is made.

[0028] In some embodiments, the nucleic acid encodes a polypeptide comprising or consisting of a variant Fc domain or mutant Fc domain that can selectively bind to FcγRIIb as described herein. The nucleic acid may 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 may be a prokaryotic cell, such as a bacterial cell. Alternatively, the host cell may be a eukaryotic cell, such as a mammalian cell. In some embodiments, the host cell contains a first expression vector, although the host cell may also contain a second expression vector as well. Since some antibodies are composed of multiple polypeptides, in some embodiments, host cells containing the expression vectors necessary to express such polypeptides may be utilized. For example, in some embodiments, the host cell contains a second expression vector that encodes a polypeptide comprising or consisting of an immunoglobulin light chain. In some embodiments, the host cell expresses a first expression vector that encodes a polypeptide comprising or consisting of an immunoglobulin heavy chain (e.g., one that contains a variant Fc domain or mutant Fc domain that selectively binds to FcγRIIb). The host cell may contain one or two expression vectors that enable the expression of an antibody, for example, one that contains both a heavy chain and a light chain.

[0029] In some aspects, a population of host cells is provided, where the population comprises a plurality of host cells each expressing a polypeptide having a different Fc domain. It is contemplated that the amino acid sequences of any two distinct Fc domains may differ by less than 20%, less than 15%, less than 10%, less than 5%, or less in their identity.

[0030] In some aspects, provided are methods of making the polypeptides described herein (e.g., polypeptides having an unglycosylated Fc region that can selectively bind to FcγRIIb), and methods of using those polypeptides. It is contemplated that any of the polypeptides described herein can be made or used by the methods described herein or methods known to those of skill in the art.

[0031] In some embodiments, there are methods for preparing an unglycosylated polypeptide, the method comprising the following steps: a) obtaining a host cell capable of expressing an unglycosylated polypeptide comprising an Fc domain that can selectively bind to FcγRIIb as described herein; b) incubating the host cell in culture under conditions that promote the expression of the unglycosylated polypeptide; and c) purifying the expressed polypeptide from the host cell. In some embodiments, the host cell is a prokaryotic cell, such as, for example, a bacterial cell. In other embodiments, the host cell is a eukaryotic cell and the polypeptide comprises a substitution mutation (e.g., T299L) at position 299 of a variant or mutant IgG Fc domain. In a further embodiment, the method may involve recovering the expressed variant polypeptide (e.g., from the supernatant), which may be done prior to purification.

[0032] In some embodiments, the method involves purifying the polypeptide from the supernatant. This may involve subjecting the polypeptide derived from the supernatant to filtration, HPLC, anion exchange chromatography or cation exchange chromatography, high performance liquid chromatography (HPLC), affinity chromatography, or combinations thereof. In some embodiments, the method involves affinity chromatography using Staphylococcus protein A that binds to the IgG Fc region. Other purification methods are well known to those of skill in the art.

[0033] In some embodiments, pharmaceutical formulations comprising the polypeptides or nucleic acids of the present disclosure are provided in a pharmaceutically acceptable carrier or in a pharmaceutical preparation comprising an excipient.

[0034] In some embodiments, an immune response may be induced in a subject by a method comprising the step of providing or administering an antibody to the subject (e.g., intravenously, etc.), wherein the antibody is not glycosylated and comprises an Fc domain that selectively binds to FcγRIIb, as described herein. In some aspects, the non-glycosylated antibody may be able to specifically bind to human FcγRIIb. In some aspects, the non-glycosylated antibody may be able to specifically bind to any of the activating FcγR polypeptides at a level that is at most 1 / 30 or 1 / 40 as high as compared to the wild-type human IgG1 antibody. In some embodiments, the antibody may comprise a variant Fc domain provided herein, which variant Fc domain does not exhibit specific or detectable binding to the FcγRI polypeptide, does not stimulate antibody-mediated phagocytosis, and / or does not elicit effector functions in a mammalian host. In some aspects, the antibody may be a glycosylated version of a therapeutic antibody or its non-glycosylated version.

[0035] In some aspects, cancer, an infectious disease, an autoimmune disease, or an inflammatory disease may be treated by administering a therapeutic polypeptide comprising a variant Fc domain or a variant Fc domain that selectively binds to FcγRIIb, as described herein. The polypeptide comprising the variant Fc domain or variant Fc domain described herein may exhibit reduced CDC as compared to the CDC induced by a polypeptide comprising the human wild-type IgG Fc region. The polypeptide comprising the variant Fc domain provided herein may exhibit reduced ADCC or ADCP as compared to the human wild-type IgG antibody.

[0036] In yet another aspect, inhibition of a protein target for therapeutic purposes can be achieved by an antibody comprising a variant Fc polypeptide as contemplated herein. In some aspects regarding polypeptides comprising a variant Fc domain or mutant Fc domain that are capable of selectively binding to inhibitory FcγRIIb but exhibit reduced binding to activating Fc, the polypeptide may exhibit reduced CDC as compared to CDC induced by a polypeptide comprising a human wild-type IgG Fc region.

[0037] A method for treating a subject having a disease is provided, the method comprising administering to the subject an effective amount of a pharmaceutical formulation of the present disclosure. In some aspects, the tumor is a solid tumor or a hematologic tumor. In one aspect, the subject can be a human patient. In some aspects, the pharmaceutical formulation can be administered into the tumor, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intravitreally, intranasally, intravaginally, rectally, intramuscularly, subcutaneously, subconjunctivally, intravesically, mucosally, intrapericardially, intraumbilically, orally, by inhalation, by injection, by infusion, by continuous infusion, by directly locally perfusing the target cells in a bath, via a catheter, or via perfusion. In some aspects, the method may further comprise administering to the subject at least a second anti-cancer therapy, which can be, for example, surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy.

[0038] In one aspect, a composition comprising the variant Fc domain of this aspect or a nucleic acid encoding the variant Fc domain of this aspect is provided for use in the treatment of a disease. The treatment of the disease may be related to achieving a therapeutic effect (e.g., due to the binding of a toxin or due to the stimulation of a receptor by an agonistic antibody, etc.) while causing a decrease in immune activation or a decrease in complement-dependent cytotoxicity by binding a selected protein. In some aspects, the disease can be cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. In another aspect, the use of the polypeptide of this aspect or the use of a nucleic acid encoding the polypeptide of this aspect in the manufacture of a medicament for treating a disease, such as cancer, etc., is provided.

[0039] As used herein, "selective binding to FcγRIIb" or "selectively binds to FcγRIIb" refers to the property of a polypeptide having the ability to bind to FcγRIIb, such as the property of an Fc domain (e.g., a mutant or variant IgG Fc domain), and preferably, the polypeptide or Fc domain has the ability to bind to FcγRIIb as compared to the wild-type Fc domain (e.g., wild-type IgG Fc domain). The binding to FcγRIIb may be equivalent or decreased (e.g., 1 / 4) compared to the wild-type binding, but in all cases, the mutant Fc domain needs to show detectable and selective binding to FcγRIIb. In some aspects, the Fc domain or polypeptide that selectively binds to FcγRIIb also shows significantly decreased binding or no detectable binding to all human activating (inflammation-inducing) Fcγ receptors as compared to the wild-type (e.g., wild-type IgG Fc domain). In some aspects, the mutant Fc domain provided herein binds to FcRn with an affinity that is the same as or not significantly different from wild-type Fc.

[0040] In some embodiments, the variant Fc domain or variant Fc domains provided above or provided herein are contained within an antibody or covalently attached to an antibody fragment (e.g., the heavy chain variable domain of an antibody, scFv, etc.). Generally, the term "antibody" includes, but is not limited to: polyclonal antibodies, monoclonal antibodies, single-chain antibodies (including one heavy chain variable region, also called VHH), humanized antibodies, deimmunized antibodies, minibodies, dibodies, tribodies, and antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies, Fv, or single-chain Fv (scFv) antibodies, single domain antibodies, etc., and antibody mimetics such as anticalins, etc., and any mixtures thereof. In some embodiments, single domain antibodies (sdAb) can offer several advantages over full-length antibodies, such as, for example, being smaller in size, having more accessible epitopes, and lower production costs (e.g., Hoey et al., 2019). In some embodiments, variants of the variant Fc domain are covalently attached to a single-chain antibody (scFv) or single domain antibody or expressed as a fusion protein with them. In one related aspect, the domain targeting the cell may be an avimer polypeptide.

[0041] As used herein, "essentially free of" with respect to a particular component means that the particular component is not intentionally formulated in the composition and / or is present only as an impurity or in trace amounts. Thus, the total amount of the particular component due to any unintentional contamination in the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the particular component cannot be detected as an amount therein using standard analytical methods.

[0042] As used herein, the term "affinity" refers to the equilibrium constant when two types of agents reversibly bind, and this is represented as K D and is expressed as such. The affinity of a binding domain for its target may 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 may be from 100 nM to 1 nM or 0.1 nM to 10 nM, or any range derivable therefrom. Further, when the affinity between two types of agents is within the range of affinity discussed above, the agents are intended to specifically bind.

[0043] As used herein, the terms "encoding" or "codes for" with respect to nucleic acids are used to enable those skilled in the art to readily understand the present invention; however, these terms may also be used interchangeably with "comprising" or "comprises" respectively in some cases.

[0044] As used in the specification text of this specification, "a" or "an" may mean one or more. As used in the claims of this specification, the words "a" or "an" used with the word "comprising" may mean one or more than one.

[0045] The use of the term "or" in the claims is used to mean "and / or" unless it is clearly specified to refer only to alternatives or the alternatives are mutually exclusive, but this disclosure demonstrates both a definition that refers only to alternatives and a definition that refers to "and / or". As used herein, "another" may mean at least a second or more.

[0046] Throughout this application, the term "about" is used to indicate that a value includes inherent variations due to manufacturing tolerances of the device, variations inherent in the method used to determine the value, or variations that exist between the test subjects.

[0047] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0048] This patent or the patent application file contains at least one drawing created in color. Copies of this patent with color drawings or copies of the patent application publication will be provided by the authorities upon request and payment of the necessary fees.

[0049] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. In combination with the detailed description of specific embodiments presented herein, the invention can be more fully understood by referring to one or more of these drawings.

[0050]

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Mode for Carrying Out the Invention

[0051] Description of Exemplary Embodiments Provided herein are methods and compositions related to polypeptides having engineered Fc domains of antibodies, wherein the Fc domains exhibit selective binding to FcγRIIB while showing reduced or abolished activity (e.g., reduced or abolished antibody-mediated phagocytosis, reduced or abolished effector function) towards activating FcγRs. Such polypeptides may include Fc domains containing one or more substitution mutations compared to the wild-type Fc domain (SEQ ID NO: 1). Additionally, some Fc domains may selectively bind to FcγRIIB but not to activating FcγRs (e.g., not detectably bind to FcγRIIaH131, FcγRIIIa, or FcγRIIIb). For example, the polypeptide may include a non-glycosylated Fc domain that selectively binds to FcγRIIB but not detectably to activating FcγRs. In some embodiments, the Fc domain shows reduced binding to FcγRIIaR131 (e.g., 1 / 40 binding compared to WT Fc). The fact that mutant Fc induces reduced or abolished effector function can provide significant advantages for the treatment of diseases where such immune responses (e.g., phagocytosis stimulated by antibodies) may be undesirable.

[0052] I. Fc Domain of Antibodies The Fc domain of IgG that binds to FcγRIIB has been shown in a variety of separate assays to suppress the activation of various immune cells (Sidman, C. L. and Unanue, E. R. 1976; Phillips, N. E. and Parker, D. C. 1984). FcγRIIB is the only FcγR expressed by B cells, and when it cross-links with the B cell receptor (BCR), the threshold of B cell activation increases, and B cell differentiation and subsequent antibody production decrease. In other immune cells including dendritic cells (DCs), macrophages, activated neutrophils, mast cells, and basophils, FcγRIIB inhibits functions mediated by activating FcγRs, including phagocytosis and the release of pro-inflammatory cytokines. When expressed by follicular DCs (FDCs), FcγRIIB is important for capturing immune complexes containing antigens, which are thought to be essential for driving the germinal center response (Qin et al. 2000; Barrington 2002). The diversity of FcγRIIB expression and function underlies its importance in regulating defense against infection and susceptibility to autoimmune diseases.

[0053] Importantly, binding to FcγRIIB in effector cells and stromal cells has been shown to be essential for the agonistic function of therapeutic antibodies against TNFRS (agonistic antibodies targeting important TNF receptor (TNFR) molecules). Many agonistic antibodies against TNFRS, including those against CD40 or anti-cell death receptor 5 (DR5), have been shown to play extremely important roles in immune regulation and activation. For example, signaling by an agonistic antibody targeting CD40 has been shown to require ligation of the Fc domain of the antibody by FcγRIIB expressed on adjacent cells in the microenvironment (Nimmerjahn et al. 2005; Wilson et al. 2011).

[0054] The site on IgG1 that binds to FcγR is determined by the co-crystal structure of the Fc fragment and the extracellular domain of FcγR. This binding site is generally located in the CH2 domain. In IgG1, the more distal hinge region (Leu234 - Ser239) and the Asp265 - Ser267 segment of the CH2 domain play important roles in the interaction with all FcγRs (Christine Gaboriaud et al., 2003 and Jenny M. Woof et al., 2004).

[0055] The CH2 domain has one N - glycosylation site at Asn297, and the N - linked glycosylation at this Asn297 bridges the gap between the two CH2 domains. This bridging maintains the proper conformation of the CH2 domain for binding to FcγR. On the other hand, when the glycan is removed at Asn297, there is a significant increase in the conformation of the CH2 domain such that the non - glycosylated Fc binds to FcγR with significantly reduced affinity or does not bind at all, and thus ADCC, ADCP, and other biological effects mediated by the Fc:FcγR interaction are significantly reduced (Borrok et al., 2012).

[0056] Given the importance of FcγRIIB binding for the biological function of antibodies, extensive efforts have been made to engineer the Fc domain of IgG1 to bind this FcγRIIB with increased affinity and / or selectivity compared to other Fcγ receptors. All of these efforts have been related to engineering glycosylated IgG1 to bind with higher affinity to FcγRIIB, because antibodies lacking the glycan at position 297 and thus not glycosylated do not show any binding to FcγRIIB. Two Fc variants of IgG1 with significantly improved binding to FcγRIIB have been reported: the so-called "EF-Fc" variant developed by Xencor and the so-called "V12-Fc" variant developed by Chugai (Chu et al., 2008; Mimoto et al., 2013; WO 2012 / 115241 A1). The EF-Fc variant contains two mutations, S267E and L328F. The V12-Fc variant has five mutations, E233D, G237D, H268D, P271G, and A330R. The EF variant was reported to have a KD (equilibrium dissociation constant) of 1 / 430 for FcγRIIB, while the V-12 variant showed 64-fold higher affinity. However, the Fc domain was FcγRIIB-selective. Specifically, the Fc domain of EF showed similar affinity for FcγRI and FcγRIIA H131 compared to the native (wild-type) human IgG1 Fc domain, and showed significantly enhanced affinity for FcγRIIA R131 . The V12-Fc variant was reported to have similar affinity for FcγRIIA R131 compared to the Fc domain of native IgG1 and decreased affinity for FcγRI and FcγRIIA H131 .

[0057] In certain embodiments, there are compositions comprising a proteinaceous molecule that is modified compared to a native or wild-type protein. In some embodiments, the proteinaceous compound lacks certain amino acid residues; in other embodiments, certain amino acid residues of the proteinaceous compound are substituted; while in yet further embodiments, both a deletion of certain amino acid residues and a substitution of certain amino acid residues are made in the proteinaceous compound. Further, the proteinaceous compound may comprise amino acid molecules that include multiple polypeptide entities. As used herein, the terms “proteinaceous molecule,” “proteinaceous composition,” “proteinaceous compound,” “proteinaceous chain,” or “proteinaceous substance” generally refer to, but are not limited to: proteins that are longer than about 200 amino acids or full-length endogenous sequences translated from genes; polypeptides of 100 amino acids or more; and / or peptides of 3 to 100 amino acids. All of the above terms with “proteinaceous” can be used interchangeably herein; however, it is specifically intended that there may be embodiments limited to certain types of proteinaceous compounds, such as polypeptides. Further, these terms may equally apply to fusion proteins or protein conjugates. A protein may comprise multiple polypeptides. For example, an IgG antibody has two heavy-chain polypeptides and two light-chain polypeptides, which are linked to each other via disulfide bonds.

[0058] As used herein, the terms protein or peptide generally refer to, but are not limited to: proteins that are longer than about 200 amino acids but at most up to the full-length sequence translated from a gene; polypeptides longer than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein.

[0059] As used herein, "amino acid residue" refers to any amino acid, any amino acid derivative, or any amino acid mimetic, as known to those of skill in the art. In certain embodiments, the residues of a proteinaceous molecule are contiguous and have no non-amino acid residues interrupting the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule may be interrupted by one or more non-amino acid moieties.

[0060] As used herein, "distinct Fc domain" may be defined as a domain that differs by only one amino acid when compared to another Fc. Methods for generating libraries of distinct Fc domains of antibodies, or libraries of nucleic acids encoding antibodies, are well known in the art. For example, in some instances, the Fc domain may be amplified by error-prone PCR. Further, in some instances, multiple Fc domains of an antibody may include stretches of randomized amino acids (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In one example, specific mutations may be incorporated into the Fc domain. For example, in some aspects, residues that are normally glycosylated in the Fc domain of an antibody may be mutated. Further, in some aspects, residues that are normally glycosylated (or adjacent residues) may be used as sites for insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0061] The polypeptide may be a non-glycosylated Fc domain of an antibody, which may include an Fc domain capable of binding to an FcR polypeptide. In some aspects, the non-glycosylated Fc domain may be further defined as having a specific affinity for the FcR polypeptide under physiological conditions. For example, the Fc domain may have an equilibrium dissociation constant of about 10-6 M to about 10-9 M under physiological conditions. Further, in some aspects, the non-glycosylated Fc domain may be defined as including one or more amino acid substitutions or insertions as compared to a wild-type sequence, such as a human wild-type sequence.

[0062] Means for preparing such polypeptides include those discussed below: PCT International Publication No. WO 2008 / 137475, which is incorporated herein by reference. Alternatively, such polypeptides can be prepared directly by genetic engineering techniques, such as by introducing selected amino acid substitutions or insertions into a known Fc background, where the insertion or substitution provides an improvement in its ability to bind to FcR, as discussed above. In some embodiments, the Fc domain is engineered to bind to one or more specific types of Fc receptors. In addition to or instead of the above, the Fc domain may be engineered to not selectively bind to one or more specific types of Fc receptors.

[0063] In some embodiments, the non-glycosylated Fc domain has a specific binding affinity for FcR or C1q, and FcRs include, for example, human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI, and the like. Thus, in some aspects, the non-glycosylated Fc domain of the present invention is defined as an Fc domain having a specific affinity for FcγRIIB. The binding affinity of the Fc of an antibody, or of another binding protein, can be determined, for example, by the following Scatchard analysis: Munson and Pollard (1980). Alternatively, the binding affinity can be determined by surface plasmon resonance, or by any other well-known method for determining the kinetics and equilibrium constants for protein:protein interactions.

[0064] The amino acid sequence showing the changes compared to wild-type Fc (SEQ ID NO: 1) of an isolated IgG variant having a specific affinity for FcγRIIB is shown below.

[0065] (Table 1) Isolated IgG variants having an affinity for FcγRIIB (numbering of the sequences is based on Kabat and the mutations are shown below) TIFF2025519556000004.tif47146

[0066] In Table 1 above, specific point mutations for the mutant Fc domain or variant Fc domain are listed; these mutations indicate the differences between the mutant Fc domain or variant Fc domain and the wild-type IgG Fc domain (SEQ ID NO:1). Some aspects of the present disclosure relate to a polypeptide having an Fc domain of IgG (e.g., a non-glycosylated Fc domain of IgG) or a nucleic acid encoding said domain, and said domain has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therefrom, sequence identity to the mutant Fc domain or variant Fc domain described in Table 1. In some embodiments, for example, substitution mutations at T299 (e.g., T299L) are also included in the Fc mutants described in Table 1, thereby enabling the production of a non-glycosylated Fc domain in mammalian cells.

[0067] In some embodiments, the variant of the variant Fc domain comprises or consists of the following. Fc 2B18K: TIFF2025519556000005.tif30145 or a polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therefrom, sequence identity.

[0068] In some embodiments, the variant of the variant Fc domain comprises or consists of the following. Fc 2B18KQ: TIFF2025519556000006.tif31146 or a polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therefrom, sequence identity.

[0069] In some embodiments, the variant of the variant Fc domain comprises or consists of the following. Fc 2b18KQS: A polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TIFF2025519556000007.tif30146, or any range derivable therefrom.

[0070] In some embodiments, the variant Fc or variant Fc is contained within a hexameric Fc polypeptide. For example, the variant Fc or variant Fc (e.g., SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5) may be contained within a polypeptide that includes the μ - tailpiece of human IgM (e.g., TIFF2025519556000008.tif4128), where the tailpiece promotes polymerization of the Fc domain to form a hexameric construct. For example, in some preferred embodiments, a variant of the variant Fc domain (e.g., any one of SEQ ID NOs: 3 - 6) is expressed as a fusion construct that is fused to the variant Fc domain at the C - terminus of the μ - tailpiece of human IgM (e.g., SEQ ID NO:6), or is fused to the tailpiece at the C - terminus of the polypeptide.

[0071] To generate multimeric oligomers, it is contemplated that various polypeptides can be included in the variant Fc or variant Fc provided herein. In some preferred embodiments, the μ-tailpiece of human IgM described below is included to form hexamers (Rowley et al., 2018; Spirig et al., 2018). By including a substitution mutation at position 309 which is a Leu residue according to Kabat numbering in the human IgG variant Fc domain or variant Fc domain, a disulfide bond is introduced between Fc domains, and the formation of multimeric oligomers is promoted. The multimeric oligomer or hexamer may or may not be glycosylated. Other polypeptides that can be used to generate multimeric oligomers (for example, when expressed using the variants of the variant Fc provided herein) include TIFF2025519556000009.tif17154, which may also be used to form hexamers, and two mutations V567I and A572G may help to stabilize the hexamer structure (Yuan, et al., 2022).

[0072] In some embodiments, the hexameric Fc polypeptide comprises or consists of the following. Hex 2b18KQS (non-glycosylated Fc): TIFF2025519556000010.tif31145 or a polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range of sequence identity derivable therebetween.

[0073] In some embodiments, the hexameric Fc polypeptide comprises or consists of the following. Hex 2b18KQS-ST (glycosylated): A polypeptide having an array identity of 31145 or at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therefrom, with TIFF2025519556000011.tif.

[0074] As used herein, "position" means a position in the sequence of a protein. Positions may be numbered consecutively or may be numbered according to an established format, for example, according to the EU index for numbering antibodies.

[0075] For all positions discussed herein, the numbering follows the EU index. "EU index" or "EU index in Kabat" or "Kabat numbering" or "EU numbering scheme" refers to the numbering of EU antibodies (Edelman et al., 1969; Kabat et al., 1991; both of which are incorporated herein by reference in their entirety).

[0076] In certain embodiments, the size of at least one Fc polypeptide, which is a proteinaceous molecule, can include, but is not limited to: about 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, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, 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, 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 therefrom. The compounds can include a number of contiguous amino acids as described above, derived from SEQ ID NO:1 (human IgG Fc polypeptide) or from a variant Fc domain listed in Table 1, and these can be further specified as having a percent identity or percent homology to SEQ ID NO: 1 (discussed herein).

[0077] A. Modified Proteins and Polypeptides Some embodiments relate to modified proteins and polypeptides, which are, in particular, modified proteins and polypeptides that exhibit at least one functional activity similar to that of their unmodified version, and furthermore, the modified proteins and polypeptides have additional advantages over their unmodified version, which are, for example, suppressing B cell activation, being easier or cheaper to produce, having fewer induced side effects, and / or having better or longer-term efficacy or bioavailability. Thus, when the present application refers to the function or activity of a "modified protein" or a "modified polypeptide", one of ordinary skill in the art will understand that they include, for example, the following proteins or polypeptides: 1) exhibiting at least one activity that is the same as that of the unmodified protein or polypeptide, or having at least one specificity that is the same as that of the unmodified protein or polypeptide, but may have different levels for other activities or specificities; and 2) having additional advantages over the unmodified protein or polypeptide. The determination of activity may be achieved using assays well known to those of ordinary skill in the art, particularly assays related to the activity of the protein, and this may include, for the purpose of comparison, using, for example, either the modified protein or polypeptide, or the unmodified protein or polypeptide, in their native and / or recombinant versions. Embodiments regarding "modified proteins" may apply to "modified polypeptides", and vice versa, is particularly intended. In addition to the modified proteins and polypeptides discussed herein, some embodiments may relate to the domains, polypeptides, and proteins described below: PCT International Publication No. WO 2008 / 137475, which is specifically incorporated herein by reference.

[0078] The modified protein may have amino acid deletions and / or substitutions; thus, a protein with a deletion, a protein with a substitution, and a protein with both a deletion and a substitution are modified proteins. These modified proteins may further contain an insertion or may further contain additional amino acids, such as, for example, fusion proteins or proteins with linkers. This may include inserting a targeting peptide or targeting polypeptide or simply inserting a single residue. Additions to the termini, referred to as fusion proteins, are discussed below.

[0079] A "deleted-modified protein" is one that lacks one or more residues of the native protein but has the specificity and / or activity of the native protein. A "deleted-modified protein" may also have reduced immunogenicity or antigenicity. An example of a deleted-modified protein is one in which amino acid residues are 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 in which the modified protein may be administered).

[0080] A substitution variant or replacement variant typically involves the exchange of one amino acid for another at one or more sites within a protein, and the variant may be designed to alter one or more of the properties of the polypeptide, particularly its effector function and / or bioavailability. The substitution may be conservative or non-conservative, where conservative means that an amino acid is replaced by an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following exchanges: 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.

[0081] The term "biologically functional equivalent" is well understood in the art and is further defined in more detail herein. Thus, sequences having about 70% to about 80% amino acids that are identical or functionally equivalent to the amino acids of the native polypeptide, or about 81% to about 90% amino acids, or even about 91% to about 99% amino acids are included, provided that the biological activity of the protein is maintained. The modified protein can be a biologically functional equivalent of its native counterpart.

[0082] Even if the amino acid sequence and nucleic acid sequence contain additional residues, such as additional amino acids at the N-terminus or C-terminus or 5' or 3' additional sequences, etc., it is still understood that, as long as the sequence meets the above criteria, including that the biological activity of the protein is maintained with respect to protein expression, it is essentially defined as one of the sequences disclosed herein. The addition of terminal sequences applies in particular to nucleic acid sequences that may contain various non-coding sequences adjacent to either the 5'-side or 3'-side portion of the coding region, or to various internal sequences, i.e., nucleic acid sequences that may contain introns known to occur within the gene.

[0083] The following is a consideration based on changing the amino acids of a protein to produce a second-generation molecule that is equivalent or even an improved second-generation molecule. For example, an amino acid can be substituted with another amino acid in a protein structure while significantly losing or without losing the interaction-binding ability related to the structure, such as the binding site with a substrate molecule. Since the interaction ability and properties of a protein define such biological functional activities of the protein, some amino acid substitutions may be made in the protein sequence and in the underlying DNA coding sequence, and even in such a case, it is possible to produce a protein with similar characteristics. Therefore, as discussed below, it is intended that various changes can be made in the DNA sequence of a gene without significantly losing its biological utility or activity. A proteinaceous molecule is considered to have "homology" to or be "homologous" to a second proteinaceous molecule if it meets one of the following "homology criteria": 1) at least 30% of a proteinaceous molecule has sequence identity at the same position relative to the second proteinaceous molecule; 2) there is a certain degree of sequence identity at the same position relative to the second proteinaceous molecule, and at least 30% of those residues that are not identical are conservative differences as described herein relative to the second proteinaceous molecule; or 3) at least 30% of a proteinaceous molecule has sequence identity relative to the second proteinaceous molecule, but there may be gaps of non-identical residues between the identical residues. As used herein, the term "homologous" can be equally applicable to a region of a proteinaceous molecule rather than the whole molecule. When the terms "homology" or "homologous" are modified by a numerical value, such as "50% homology" or "50% homologous", the homology criteria in 1), 2), and 3) are adjusted from "at least 30%" to "at least 50%".Accordingly, it is contemplated that there may be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher, homology or sequence identity between two proteinaceous molecules or between two portions of a proteinaceous molecule.

[0084] Alternatively, a modified polypeptide may be characterized as having a percentage identity to an unmodified polypeptide or to any polypeptide sequence disclosed herein, including variants of the variant Fc domains listed in Table 1. The percentage identity may 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 between two portions of a proteinaceous molecule. It is contemplated that the percentage of identity discussed above may relate to a particular region of the polypeptide, the region being compared to an unmodified region of the polypeptide. For example, a polypeptide may comprise an Fc domain that is modified or is a variant, where the modified or variant Fc domain may be characterized based on the amino acid sequence identity of the modified or variant Fc domain to an unmodified or non-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 the domain are identical to the amino acids in the unmodified human Fc domain (SEQ ID NO:1).

[0085] When making such changes, the hydrophobicity index of amino acids may be considered. The importance of the hydrophobicity index of amino acids in conferring interacting biological functions to proteins is widely understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophobicity characteristics of amino acids are involved in the resulting protein secondary structure and, accordingly, define the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0086] It is also understood in the art that substitutions with similar amino acids can be effectively carried out based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, describes that the maximum local average hydrophilicity of a protein, which is affected by the hydrophilicity of its adjacent amino acids, correlates with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values are 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 amino acids can be substituted with another amino acid having a similar hydrophilicity value and still produce a protein that is biologically and immunologically equivalent. In such changes, substitutions between amino acids with hydrophilicity values within ±2 are preferred, particularly preferred if within ±1, and even more particularly preferred if within ±0.5.

[0087] As outlined above, amino acid substitutions typically are based on the relative similarity of the amino acid side chain substituents, which is, for example, relative similarity in terms of their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various properties described above are well known to those skilled in the art and include the following: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0088] B. Modified Antibodies and Modified Proteinaceous Compounds Having Heterologous Regions Once the Fc domain is isolated, it may be desirable to conjugate at least one agent to the molecule to form a conjugate to enhance the utility of the molecule. For example, conjugation, covalent attachment, or complex formation with at least one desired molecule or moiety for the purpose of increasing the effectiveness of the Fc domain or antibody molecule as a diagnostic or therapeutic agent is conventional. Such a molecule or moiety may be, but is not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules having a desired activity, such as cytotoxic activity, etc. Non-limiting examples of effector molecules conjugated to an antibody include the following: toxins, anti-tumor agents, therapeutic enzymes, radiolabeled nucleotides, anti-viral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast to the above, a reporter molecule is defined as any molecule that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to an antibody include the following: enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin, etc. Another such example is the formation of a conjugate comprising an antibody conjugated to a cytotoxic or anti-cellular agent, and this may be referred to as an "immunotoxin." Techniques for labeling such molecules are known to those skilled in the art and are described above herein.

[0089] Labeled proteins, such as those prepared according to the present disclosure, and labeled Fc domains may also be utilized, which may be used, for example, in immunological detection methods for binding, purifying, removing, quantifying, and / or broadly detecting biological components such as proteins, polypeptides, or peptides. Some immunological detection methods include, by way of example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescence immunoassay, chemiluminescence assay, bioluminescence assay, and Western blot. The various stages of useful immunological detection methods are described in the scientific literature, which includes, for example: Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; and De Jager et al., 1993, each of which is incorporated herein by reference.

[0090] Fc domain molecules, including antibodies, may be used, for example, with tissue blocks prepared for testing by immunohistochemical analysis (IHC), both fresh frozen and paraffin-embedded tissue blocks, and / or formalin-fixed and paraffin-embedded tissue blocks. Methods for preparing tissue blocks from those particulate specimens have been successfully used in previous IHC tests for various prognostic factors and / or are well known to those skilled in the art (Abbondanzo et al., 1990).

[0091] Some embodiments relate to proteinaceous compounds that are Fc polypeptides that can include an amino acid sequence derived from one or more naturally occurring polypeptides or proteins or natural polypeptides or proteins. The embodiments discussed above are intended to apply to this section and vice versa. For example, an engineered antibody includes a modified Fc domain together with an antigen-binding domain. Further, an antibody may have two different antigen-binding regions, which are, for example, different regions on each of two heavy chains. Instead of or in addition to the above, in some embodiments, there are polypeptides that include multiple heterologous peptides and / or polypeptides (where "heterologous" means not derived from the same polypeptide). A proteinaceous compound or molecule may include, for example, a modified Fc domain together with a region that binds to a protein, a region not derived from an antibody. In some embodiments, there are polypeptides that include a modified Fc domain together with a region that binds to a protein and a region that binds to a cell surface receptor. These proteinaceous molecules that include multiple functional domains may be two or more domains that are chemically conjugated to each other, or these proteinaceous molecules may be fusion proteins of two or more polypeptides encoded by the same nucleic acid molecule. It is intended that a protein or polypeptide may include all or part of two or more heterologous polypeptides.

[0092] Accordingly, a multipolypeptide proteinaceous compound may be composed of all or part of a first polypeptide and all or part of a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, an eighth polypeptide, a ninth polypeptide, a tenth polypeptide, or a further polypeptide.

[0093] An amino acid, for example, a selectively cleavable linker, a synthetic linker, or another amino acid sequence may be used to separate a proteinaceous moiety.

[0094] A polypeptide or protein (including an antibody) having an antigen-binding domain or antigen-binding region of an antibody and a non-glycosylated Fc domain may be used against any antigen or epitope, including but not limited to proteins, subunits, domains, motifs, and / or epitopes belonging to the following target list: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, adenosine A1 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, α-1-antitrypsin, α-V / β-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-6, Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, 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), carcinoma-associatedantigen), 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, the tumor-associated antigen keratin, DAN, DCC, DcR3, DC-SIGN, disintegration 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-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV envelope glycoprotein gB, HCMV) envelope glycoprotein gH, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) glycoprotein gB, HSV glycoprotein gD, HGFA, high molecular weight melanoma associated antigen (HMW-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-α (INF-α), INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (integrin αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-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, lung surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4, or neurotrophin-6, neurulin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prolactin, 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, RSVFgp, 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 receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, pan-specific TGF-β, TGF-βRI (ALK-5), TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, 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 (OPG, OCIF, TR1), TNFRSF12 (TWEAK R, FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM, ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR, p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR, AITR), TNFRSF19 (TROY, TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI, CD120a, p55-60), TNFRSF1B (TNF RII, CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR, TNF RIII, TNFC R), TNFRSF4 (OX40, ACT35, TXGP1R), 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 ligand, TL2), TNFSF11 (TRANCE / RANK ligand, ODF, OPG ligand), TNFSF12 (TWEAK, Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL, TALL2), TNFSF13B (BAFF, BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT, HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand, AITR ligand, TL6), TNFSF1A (TNF-a, Conectin, DIF, TNFSF2), TNFSF1B (TNF-b, LTa, TNFSF1), TNFSF3 (LTb, TNFC, p33), TNFSF4 (OX40 ligand, gp34, TXGP1), TNFSF5 (CD40 ligand, CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, 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-associated antigen CA125. Tumor-associated antigens expressing Lewis Y-related sugar chains, 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 antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and receptors for growth factors. In some embodiments, the polypeptide or protein has an antigen-binding domain specific for one or more of the tumor antigens or B cell antigens on the cell surface. The methods and compositions may be utilized to target tumor cells or B cells.

[0095] Any antibody having sufficient selectivity, specificity, or affinity may be utilized as the basis of an antibody conjugate. Such properties can be evaluated using conventional immunological screening methods known in the art. Sites for binding biologically active molecules in antibody molecules, added separately from the canonical antigen-binding site, include sites present in variable domains that can bind to the following: pathogens, B cell superantigens, the co-receptor CD4 of T cells, and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991). In addition, the variable domains are involved in the self-binding of antibodies (Kang et al., 1988) and contain epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[0096] The Fc domain can bind to FcR, but it is intended that the regulation of the immune response can be controlled not only by the antigen-binding domain on the polypeptide containing the Fc domain, but also by a domain that binds to some other protein. Accordingly, some embodiments can relate to an Fc domain and a heterologous domain that binds to something other than an antigen. In one embodiment, the domain that binds to something other than an antigen binds to the cell surface. Thus, these agents require either being chemically conjugated to, or fused with, an agent / protein that can bind to a particular target cell. One embodiment can further include linking all or a portion of an unglycosylated Fc domain to all or a portion of any of the proteins listed in Table 2. One embodiment is intended to include, but not be limited to, the examples provided in Table 2 and the descriptions herein.

[0097] Ligands for receptors may be utilized to target cells expressing the receptor for the ligand on their surface. Ligands also include, by way of example, the following: CD95 ligand, TRAIL, TNF (e.g., TNF-α or TNF-β), growth factors including those discussed above such as VEGF, and cytokines such as interferon or interleukin, and variants thereof. Embodiments having multiple domains are also contemplated, for example, the VEGF-Trap fusion protein includes the second extracellular domain of VEGF receptor 1 (Flt-1) and the third domain of VEGF receptor 2 (KDR / FIK-1), and the Fc region of IgG.

[0098] (Table 2) Agents / proteins capable of binding to specific target cells TIFF2025519556000012.tif89146TIFF2025519556000013.tif155146

[0099] C. Library of antibody Fc Examples of techniques available for generating diverse Fc domains of antibodies and / or antibodies containing such domains may utilize techniques similar to those for expressing immunoglobulin heavy chain libraries described below: U.S. Patent No. 5,824,520. Fc libraries previously utilized are discussed below: PCT International Publication No. 2008 / 137475, specifically incorporated herein by reference. In some embodiments, yeast surface display libraries are used (e.g., Choi et al. 2015; Wozniak-Knopp et al., 2010).

[0100] II. Polypeptides that bind to antibodies Various domains that bind to antibodies (e.g., FcR polypeptides) are known in the art and may be used in the methods and compositions of the present invention. For example, in some aspects, the FcR may have specificity for a particular type or subtype of Ig, which may be, for example, specificity for IgA, IgM, IgE, or IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). Thus, in some embodiments, the domain that binds to the antibody may be defined as a domain that binds to IgG. The FcR polypeptide may comprise a eukaryotic, prokaryotic, or synthetic FcR domain. For example, the domain that binds to the Fc of an antibody may be defined as a mammalian, bacterial, or synthetic binding domain. Some of the domains that bind to Fc used in the present invention include, but are not limited to, binding domains derived from one of the polypeptides in Table 3. For example, the polypeptide that binds to Fc may be encoded by the following genes: FCGR2A, FCGR2B, FCGR2C, FCGR3A, FCGR3B, FCGR1A, Fcgr1, FCGR2, FCGR2, Fcgr2, Fcgr2, FCGR3, FCGR3, Fcgr3, FCGR3, Fcgr3, FCGRT, mrp4, spa, or spg. The FcR polypeptide may be an Fc binding region derived from: human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI, or C1q. Various Fc receptors that bind to the Fc domain are well known in the art, and some examples of the receptors are listed in Table 3 below.

[0101] (Table 3) Selected FcR polypeptides TIFF2025519556000014.tif95155TIFF2025519556000015.tif215155TIFF2025519556000016.tif136155

[0102] III. Methods for screening the Fc domain of an antibody In certain instances, there are methods for identifying an Fc domain of an antibody that has specific affinity for a target ligand (e.g., a polypeptide that binds to an antibody, such as an Fc receptor, by way of example). Such methods are described herein and also described below: PCT International Publication No. WO 2008 / 137475, which is hereby specifically incorporated by reference in its entirety. In some embodiments, screening methods using eukaryotic cells (e.g., yeast surface display libraries) can be used.

[0103] The screened polypeptides may comprise a large library of diverse candidate Fc domains or may comprise Fc domains of a particular class (e.g., engineered point mutations or amino acid insertions) selected in view of structural features thought to render them more likely to bind the target ligand. In one embodiment, the candidate polypeptide may be an intact antibody, or a fragment or portion thereof, that includes the Fc domain.

[0104] To identify candidate Fc domains capable of binding a target ligand, the following steps may be carried out: providing a population of Gram-negative bacterial cells, each expressing a distinct Fc domain of an antibody; mixing the bacteria with a target ligand (FcR polypeptide) that is either labeled with at least a first label or immobilized and capable of contacting the Fc domain of the antibody; and identifying at least a first bacterium that expresses a molecule capable of binding the target ligand.

[0105] In some aspects of the above method, binding between the Fc domain of the antibody and the labeled FcR polypeptide prevents diffusion outside the bacterial cell. In this manner, labeled ligand molecules can be retained in the periplasm of bacteria with a permeabilized outer membrane. Alternatively, since the Fc domain has been shown to bind to the inner membrane, the periplasm may be removed, whereby the Fc domain retains the bound candidate molecule. The label may then be used to isolate cells expressing a binding polypeptide that can bind to the FcR polypeptide, and a gene encoding the Fc domain polypeptide may also be isolated. Molecules capable of binding to the target ligand may then be produced in large quantities using in vivo or ex vivo expression methods and subsequently used for any desired application, such as diagnostic or therapeutic applications. Furthermore, it is understood that the Fc domain of the isolated and identified antibody can be used to construct an antibody fragment containing an antigen-binding domain or a full-length antibody containing an antigen-binding domain.

[0106] In a further aspect, the screening method may include at least two selection rounds, where a sub-population of bacterial cells obtained from a first selection round is subjected to at least a second selection round based on the binding of the Fc domain of a candidate antibody to an FcR. The sub-population of bacterial cells obtained in the first selection round may be grown under permissive conditions prior to the second selection (to increase the total number of cells). The method may include, for example, two, three, four, five, six, seven, eight, nine, ten, or more selection rounds. In some aspects, the sub-population of bacterial cells obtained from each selection round is grown under permissive conditions prior to the next selection round. Cells isolated after one or more such selection rounds may be subjected to additional mutagenesis rounds. In some examples, the selection is performed after removing FcR polypeptides that do not bind to the antibody. The stringency of the selection can be varied by adjusting the pH, salt concentration, or temperature of the solution containing the bacteria presenting the antibody. As an example, the bacterial cells may be grown at a temperature below physiological temperature, such as about 25°C.

[0107] A method of producing a bacterial cell is provided, and the bacterial cell may comprise a nucleic acid sequence encoding a variant Fc domain provided herein. The bacterial cell produced by the method provided herein may be used to clone a nucleic acid sequence encoding an Fc domain having a specific affinity for an FcR polypeptide. For example, methods for isolating and amplifying such nucleic acids from cells by PCR are well known in the art and are further described below. The nucleic acid sequence produced by the method described above constitutes an aspect of the present disclosure. It is possible to express the nucleic acid in a cell in order to produce the Fc domain provided herein or to produce a polypeptide comprising the Fc domain provided herein. In some embodiments, the Fc domain of an antibody provided herein may be included in a polypeptide having one or more variable regions of the antibody (e.g., a single domain antibody, scFv, etc.), wherein the variable region has an affinity for a particular target ligand.

[0108] B. Periplasmic expression of the Fc domain of an antibody In some embodiments, a polypeptide comprising the Fc domain of an antibody may be expressed in the periplasmic space of Gram-negative bacteria. Further, in some aspects, the Fc domain of the antibody may be immobilized on the periplasmic side of the inner membrane. Methods and compositions for immobilizing polypeptides to the inner membrane of Gram-negative bacteria have been previously described (U.S. Pat. Nos. 7,094,571, 7,419,783, 7,611,866, and U.S. Patent Application Publication No. 2003 / 0219870; Harvey et al., 2004; Harvey et al., 2006). For example, the Fc domain may be directly fused to a transmembrane polypeptide or a membrane-bound polypeptide, or may interact (e.g., via protein-protein interactions) with a transmembrane polypeptide or a membrane-bound polypeptide. This technique may be referred to as "immobilized periplasmic expression" or "APEx". In some examples, the Gram-negative bacterial cell may be defined as an E. coli cell. Further, in some aspects, the Gram-negative bacterial cell may be defined as a genetically engineered bacterial cell, e.g., as the Jude-1 strain of E. coli.

[0109] The fusion protein may include an N-terminal fusion or a C-terminal fusion having an Fc domain, and in some examples, the fusion protein may include additional linker amino acids between the membrane anchor polypeptide and the Fc domain. In certain examples, the membrane anchor polypeptide may be: the first 6 amino acids encoded by the NlpA gene of E. coli, one or more transmembrane α-helices from an inner membrane protein of E. coli, the gene III protein of filamentous phage or a fragment thereof, or an inner membrane lipoprotein or a fragment thereof. Thus, as an example, the membrane anchor polypeptide may be an inner membrane lipoprotein or a fragment thereof, which may be derived from, for example, AraH, MglC, MalF, MalG, MalC, MalD, RbsC, RbsC, ArtM, ArtQ, GlnP, ProW, HisM, HisQ, LivH, LivM, LivA, LivE, DppB, DppC, OppB, AmiC, AmiD, BtuC, ThuD, FecC, FecD, FecR, FepD, NikB, NikC, CysT, CysW, UgpA, UgpE, PstA, PstC, PotB, PotC, PotH, Pod, ModB, NosY, PhnM, LacY, SecY, TolC, DsbB, DsbD, TouB, TatC, CheY, TraB, ExbD, ExbB, or Aas.

[0110] In further examples, the population of Gram-negative bacteria in the present invention is at least about 1 x 10 3 species, 1 x 10 4 species, 1 x 10 5 species, 1 x 10 6 species, 1 x 10 7 species, 1 x 10 8 species, 1 x 10 9It may be defined as including distinct Fc domains of one or more types of antibodies. In some specific examples, a population of gram-negative bacterial cells may be produced by a method comprising the following steps: (a) preparing a plurality of nucleic acid sequences encoding distinct Fc domains of antibodies; and (b) transforming a population of gram-negative bacteria with the nucleic acids, wherein the gram-negative bacteria contain a plurality of Fc domains of antibodies expressed in the periplasm.

[0111] C. Outer Membrane Permeabilization Methods for disrupting, making permeable, or removing the outer membrane of bacteria are well known in the art; see, for example, U.S. Patent No. 7,094,571. For example, prior to contacting the bacterial cells with the FcR polypeptide, the outer membrane of the bacterial cells may be treated with high osmotic pressure conditions, physical stress, lysozyme, EDTA, digestive enzymes, chemicals that disrupt the outer membrane, may be treated by infecting the bacteria with phage, or may be treated by a combination of the above methods. Thus, in some examples, the outer membrane may be disrupted by treatment with lysozyme and EDTA. Further, in certain embodiments, the outer membrane of the bacteria may be completely removed.

[0112] Methods for increasing the permeability of the outer membrane to one or more labeled ligands may be utilized. This allows access during screening to labeled ligands that would otherwise not be able to cross the outer membrane. However, a class of molecules, such as hydrophobic antibiotics larger than the 650 Da exclusion limit, can diffuse through the outer membrane of bacteria on their own, independent of the membrane porins (Farmer et al., 1999). This process can make the membrane permeable (Jouenne and Junter, 1990). Also, certain long-chain phosphate polymers (100 Pi) appear to completely bypass the normal molecular sieving activity of the outer membrane (Rao and Torriani, 1988).

[0113] Conditions have been identified for permeating a ligand into the periplasm without the cell losing viability or releasing the proteins it expresses, and the present invention may be practiced without maintaining the outer membrane. For Fc domains expressed or immobilized in the periplasmic space, the need to maintain the outer membrane (as a barrier to prevent leakage of binding proteins from the cell) is eliminated in order to detect the labeled ligand that is bound. Thus, cells that either have a partially permeable membrane or have had the outer membrane almost completely removed can be easily labeled by incubating them with a solution of the labeled ligand, cells that express a binding protein immobilized on the outside (periplasmic side) of the cytoplasmic membrane.

[0114] For example, treatments such as high osmotic shock can significantly improve labeling. It is known that many agents, including calcium ions (Bukau et al., 1985) and even Tris buffer (Irvin et al., 1981), can change the permeability of the outer membrane. Furthermore, phage infection also stimulates the labeling process. Both pIII, an inner membrane protein of filamentous phage, and pIV, a large multimeric outer membrane protein, can change the membrane permeability (Boeke et al., 1982), and mutants of pIV are known to improve access to maltodextrin, which is normally excluded (Marciano et al., 1999). To achieve high permeability, it is possible to use combinations of strains, salts, and phages (Daugherty et al., 1999). Cells containing a polypeptide bound to a labeled ligand, whether immobilized or bound in the periplasm, can then be easily isolated from cells expressing a binding protein that does not have affinity for the labeled ligand using flow cytometry or other related techniques. However, in some cases, it is desirable to use less disruptive techniques for the purpose of maintaining cell viability. Treatment with EDTA and lysozyme can also be useful in this regard.

[0115] D. Labeled Target Ligand As described above, it is typically desirable to provide an FcR polypeptide labeled with one or more detectable agents. This can be accomplished, for example, by linking at least one detectable agent to a ligand to form a conjugate. For example, linkage, covalent bonding, or complex formation with at least one detectable molecule or moiety is commonly used. A "label" or "detectable label" is a compound and / or element that can be detected due to specific functional and / or chemical properties, and by using this, it becomes possible to detect the ligand to which it is bound and / or, if desired, to further quantify it. Examples of labels that can be used include, but are not limited to: enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin. In some embodiments, streptavidin-biotinylated FcγR tetramers may be used for screening and FcγR having an Avi-tag may be used for biotinylation.

[0116] In one aspect of the invention, a visually detectable marker is used such that automated screening of cells with respect to the label can be performed. By visualization, examples of agents that can be detected using appropriate equipment are known in the art, as is the case for methods for binding them to the desired ligand (see, for example: U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each of which is incorporated herein by reference). Such agents can include: paramagnetic ions; radioisotopes; fluorescent dyes; substances detectable by NMR; and substances for X-ray imaging. In particular, fluorescent labels are beneficial in that they enable the use of flow cytometry to isolate cells expressing the desired binding protein or antibody.

[0117] Another type of FcR conjugate is one in which the ligand is linked to a second binding molecule and / or an enzyme (enzyme tag) that produces a colored product when contacted with a chromogenic substrate. Examples of such enzymes include urease, alkaline phosphatase, horseradish peroxidase, or glucose oxidase. In such examples, it is desirable that the selected cells are viable. Preferred second binding ligands are biotin and / or avidin and streptavidin-based compounds. The use of such labels is well known to those skilled in the art and is described, for example, in: U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is incorporated herein by reference.

[0118] Molecules containing azide groups can be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2-azido and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes to identify proteins that bind nucleotides in crude cell extracts (Owens and Haley, 1987; Atherton et al., 1985). 2-Azidonucleotides and 8-azidonucleotides have also been used to map domains that bind nucleotides in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can also be used as agents that bind ligands.

[0119] The labeling can be carried out by any technique well-known to those skilled in the art. By way of example, the FcR polypeptide can be labeled by contacting a ligand with a desired label and a chemical oxidizing agent such as sodium hypochlorite, or with a desired label and an oxidizing agent by an enzyme such as lactoperoxidase. Similarly, a ligand exchange process can also be used. Alternatively, direct labeling techniques may be used, which is, for example, by incubating a label, a reducing agent such as SnCl2, a buffer solution such as a sodium potassium phthalate solution, and a ligand. Intermediate functional groups on the ligand may also be used, which can be used, for example, to bind a label to the ligand in the presence of diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0120] Other methods for conjugating a ligand to its conjugate moiety or for conjugation are also known in the art. Some conjugation methods involve the use of an organic chelating agent bound to the ligand, such as: diethylenetriaminepentaacetic acid (DTPA); ethylenetriamine tetraacetic anhydride or ethylenediamine tetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3 (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). The FcR polypeptide may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with a fluorescein marker can be prepared by reaction in the presence of a coupling agent or using isothiocyanate. In U.S. Pat. No. 4,938,948, breast tumor imaging has been achieved using monoclonal antibodies, and a detectable imaging moiety has been conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate. In a further aspect, the FcR polypeptide may be fused to a reporter protein, which may be, for example, an enzyme as described above, or a fluorescent protein.

[0121] E. Isolation of Cells Bound to a Labeled Target Ligand 1. Column-Based or Bead-Based Immobilization One of ordinary skill in the art understands that methods for selecting cells based on their interaction (binding) with FcRs are well known in the art. For example, FcRs may be immobilized on a column or beads (e.g., magnetic beads), and cells that bind to the FcR (e.g., bacterial cells or eukaryotic cells such as yeast) may be separated by repeatedly washing the beads (e.g., by magnetic separation) or by repeatedly washing the column. Further, the target ligand may be labeled (e.g., labeled with a fluorophore, radioisotope, or enzyme). Thus, in some instances, cells may be selected by detecting the label on the FcR to which they are bound. Further, in some aspects, cells may be selected based on their binding to two or more FcR polypeptides or based on the lack of such binding. For example, bacteria presenting antibodies that bind to two types of FcR polypeptides may be selected, where each FcR is used to sequentially select the bacteria. Conversely, in some aspects, bacteria presenting the Fc domain of an antibody that binds to one type of FcR (e.g., an FcR containing a first label) but not to a second FcR (e.g., one containing a second label) may be selected. The methods described above may be used, for example, to identify the Fc domain of an antibody that binds to one particular FcR but not to a second particular FcR.

[0122] 2. Flow Cytometry In one aspect of the invention, fluorescence-activated cell sorting (FACS), or other automated flow cytometry techniques, may be used to efficiently isolate bacterial cells containing a labeled ligand that binds to the Fc domain. Equipment for performing flow cytometry is known to those of skill in the art and is generally commercially available. Examples of such equipment include: FACS Star Plus, FACScan, and FACSort, instruments from Becton Dickinson (Foster City, CA); Epics C from the Epics division of Coulter Corporation (Hialeah, FL); and MOFLO™ from Cytomation (Colorado Springs, CO).

[0123] Flow cytometry techniques generally involve separating cells or other particles in a liquid sample. Typically, the purpose of flow cytometry is to analyze the separated particles with respect to one or more of their properties, e.g., the presence of a labeled ligand or other molecule. The basic steps of flow cytometry involve directing a fluid sample through an apparatus such that a stream of liquid passes through a sensor region. The particles need to pass through the sensor one at a time, and the particles are classified based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.

[0124] In flow cytometry, not only is the analysis of cells performed, but also the sorting of cells. U.S. Patent No. 3,826,364 discloses an apparatus for physically separating particles, such as functionally different cell types. In this apparatus, a laser is focused by an appropriate lens or lens system to irradiate a stream of particles, resulting in highly localized scattering from the particles therein. In addition, a high-intensity irradiation source is directed at the stream of particles to excite fluorescent particles in the stream. Some of the particles in the stream can be selectively charged and then separated by changing their paths to designated containers. The standard form of this separation is via antibodies tagged with fluorescence, which are used to label one or more cell types for the purpose of separation.

[0125] Other examples of methods related to flow cytometry include, but are not limited to, those described below: U.S. Patent Nos. 4,284,412; 4,989,977; 4,498,766; 4,857,451; 4,774,189; 4,767,206; 4,714,682; 5,160,974; 5,478,722; and 4,661,913, each of which is specifically incorporated herein by reference.

[0126] One useful aspect of flow cytometry is that multiple rounds of screening can be performed continuously. To improve the stringency of screening, cells can be isolated from the first round of sorting and then promptly reintroduced into the flow cytometer and screened again. Another advantage known to those skilled in the art is that it is possible to recover non-viable cells using flow cytometry. Since flow cytometry is essentially a technique for sorting particles, the ability of cells to grow or proliferate is not required. Techniques for recovering nucleic acids from such non-viable cells are well known in the art and include the use of template-dependent amplification techniques, such as PCR.

[0127] F. Cloning of the Coding Sequence of the Fc Domain After a bacterial cell has been identified as producing a molecule with the desired specificity, affinity, and / or activity, the corresponding coding sequence may be cloned. In this manner, it is possible to isolate and sequence the DNA encoding the molecule using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to a gene encoding an antibody or binding protein). Those skilled in the art understand that it is possible to clone nucleic acids from viable or non-viable cells. In the case of viable cells, for example, it may be desirable to use amplification of the cloned DNA, such as by PCR. This may also be carried out using viable cells, whether or not the cells have been further propagated.

[0128] Once isolated, the DNA of the Fc domain of the antibody may be placed into an expression vector, and then the vector may be used to transfect a host cell, such as a bacterium. The DNA may also be modified, for example, by adding the sequences of the variable domains of the human heavy and light chains, or by covalently attaching all or part of the coding sequence of a polypeptide other than an immunoglobulin to the coding sequence of the immunoglobulin. In this manner, a binding protein that is "chimeric" or "hybrid" is prepared to have the desired binding specificity. For example, the identified Fc domain of an antibody may be fused to a therapeutic polypeptide or toxin and used to direct cells expressing a particular FcR (in vitro or in vivo).

[0129] Chimeric or hybrid Fc domains may also be prepared in vitro using techniques known in protein synthesis chemistry, which include techniques involving cross-linking agents. For example, targeted toxins may be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0130] IV. Nucleic Acid-Based Expression Systems Nucleic acid-based expression systems can be utilized in certain aspects of the present invention to express recombinant proteins. For example, one aspect of the present invention involves transforming Gram-negative bacteria using the coding sequence of the Fc domain of an antibody, or preferably, the coding sequences of multiple distinct Fc domains.

[0131] A. Nucleic Acid Delivery Methods Certain aspects of the present invention may include the delivery of nucleic acids to target cells (e.g., Gram-negative bacteria). For example, host cells that are bacteria may be transformed using nucleic acids encoding candidate Fc domains that may be able to bind to FcRs. In certain embodiments of the present invention, it may be desirable to direct expression into the bacterial periplasm. Transformation of host cells that are eukaryotes can also be similarly utilized in the expression of various candidate molecules identified as being able to bind to target ligands.

[0132] Suitable methods for delivering nucleic acids for cell transformation are considered to include virtually any method by which it is possible to introduce nucleic acids (e.g., DNA) into cells or even into their organelles.Such methods include, but are not limited to, direct delivery of DNA such as the following: by injection (U.S. Pat. Nos. 5,994,624; 5,981,274; 5,945,100; 5,780,448; 5,736,524; 5,702,932; 5,656,610; 5,589,466; and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonoporation (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT International Publication Nos. 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each incorporated herein by reference); or by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); by uptake of DNA via desiccation / inhibition (Potrykus et al., 1985).By applying the techniques as described above, it is possible to stably or transiently transform cells.

[0133] B. Vector Vectors can be utilized in connection with the present invention, for example, in the transformation of cells using nucleic acid sequences encoding candidate Fc domains. In one aspect of the invention, a “library” of nucleic acid sequences encoding polypeptides, which may be a heterogeneous “library” in its entirety, may be introduced into a cell population, thereby enabling screening of the entire library. The term “vector” is used to refer to a nucleic acid molecule that is a carrier into which a nucleic acid sequence for introduction into a cell can be inserted, and in which the nucleic acid molecule can be replicated in the cell. The nucleic acid sequence can be “foreign” or “heterologous,” meaning that the nucleic acid sequence is foreign to the cell into which the vector is being introduced, or that the sequence is homologous to a sequence within the cell but is in a position within the nucleic acid sequence of the host cell where it is not normally found. Vectors include plasmids, cosmids, and viruses (such as bacteriophages). One of ordinary skill in the art can construct vectors using standard recombinant techniques, which are described below: Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference.

[0134] The term “expression vector” refers to a vector that contains a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some instances, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription, and in some cases translation, of a coding sequence that is operably linked in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.

[0135] 1. Promoters and Enhancers A "promoter" is a control sequence, which is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. This can include genetic elements to which regulatory proteins and regulatory molecules, such as RNA polymerase and other transcription factors, can bind. The expressions "functionally disposed", "functionally linked", "under control", and "under transcriptional control" mean that the promoter is in an accurate and functional position and / or orientation with respect to the nucleic acid sequence that controls the initiation of its transcription and / or its expression. A promoter may or may not be used together with an "enhancer", where an "enhancer" refers to a cis-acting regulatory sequence involved in the activation of transcription of a nucleic acid sequence. Generally, those skilled in the art of molecular biology are well acquainted with the use of combinations of promoters, enhancers, and cell types for expressing proteins. See, for example, Sambrook et al. (1989), which is incorporated herein by reference.

[0136] 2. Initiation Signals Specific initiation signals may also be required to efficiently translate a coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide foreign translation control signals, including the ATG initiation codon. Those skilled in the art will be able to readily determine such a need and provide the necessary signals. It is well known that the initiation codon needs to be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Foreign translation control signals and initiation codons can be either natural or synthetic. The expression efficiency can be improved by including appropriate transcriptional enhancer elements.

[0137] 3. Multiple Cloning Sites The vector may contain a multiple cloning site (MCS), which is a region on a nucleic acid that contains multiple restriction enzyme sites, any of which can be used with standard recombinant techniques for digesting the vector (see, e.g., Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, which are incorporated herein by reference). "Digestion with a restriction enzyme" refers to the catalytic cleavage of a nucleic acid molecule using an enzyme that functions only at a specific position on the nucleic acid molecule. Many of these restriction enzymes are commercially available. Vectors are often linearized or fragmented using restriction enzymes, which cut the vector within the scope of the MCS, enabling the ligation of foreign sequences to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques regarding restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant techniques.

[0138] 4. Termination Signal Vectors or constructs prepared according to the present disclosure generally contain at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of RNA transcription by RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. Terminators may be necessary to achieve the desired messenger level in vivo. Terminators contemplated for use in the present invention include, but are not limited to, any transcription terminator known to those skilled in the art, including ρ-dependent or ρ-independent terminators. In certain embodiments, the termination signal may be lacking in sequences that are transcribable or translatable, for example, due to sequence shortening.

[0139] 5. Origin of Replication For the purpose of increasing the vector in a host cell, the vector may contain one or more origins of replication (often referred to as "ori"), which are special nucleic acid sequences where replication begins.

[0140] 6. Selectable Markers and Screenable Markers In certain embodiments of the invention, cells containing the nucleic acid constructs of the disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers can confer an identifiable change to the cell, thereby allowing easy identification of cells containing the expression vector. Generally, selectable markers confer properties that allow selection. A marker that allows positive selection is one that enables its selection by its presence, while a marker that allows negative selection is one that prevents its selection by its presence. An example of a marker that allows positive selection is a drug resistance marker.

[0141] Generally, including a drug selection marker facilitates the cloning and identification of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer a phenotype that allows identification of transformants based on the implementation of certain conditions, other types of markers are also contemplated, including screenable markers whose principle is colorimetric analysis, such as GFP for example. Alternatively, screenable enzymes such as chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will also understand how to utilize immunological markers, optionally in conjunction with FACS analysis. The marker used is not considered a point of emphasis as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable markers and screenable markers are well known to those skilled in the art.

[0142] C. Host Cell In the context of expressing a heterologous nucleic acid sequence, a "host cell" refers to a prokaryotic or eukaryotic cell (e.g., a yeast cell, an insect cell, or a mammalian cell), and includes any organism that can be transformed and can replicate a vector and / or express a heterologous gene encoded by the vector. A host cell can be used as a recipient of a vector and has been used as a recipient of a vector. A host cell may be "transfected" or "transformed," which refers to the process by which foreign nucleic acid is introduced into or transferred into the host cell. Transformed cells include the subject primary cells and their progeny.

[0143] In certain embodiments of the invention, the host cell is a Gram-negative bacterial cell. Such bacteria have a periplasmic space between an inner membrane and an outer membrane and are particularly suitable for use in the present invention in that they have the inner membrane described above between the periplasm and the cytoplasm, where the inner membrane is also known as the cytoplasmic membrane. Thus, any other cell having such a periplasmic space can also be used in the present invention. Examples of Gram-negative bacteria that can be utilized in the present invention include, but are not limited to: Escherichia coli, Pseudomonas aeruginosa, Vibrio cholera, Salmonella typhimurium, Shigella flexneri, Haemophilus influenza, Bordotella pertussi, Erwinia amylovora, Rhizobium sp.

[0144] One skilled in the art can determine an appropriate host based on the vector backbone and the desired product. Plasmids or cosmids, for example, can be introduced into prokaryotic host cells to replicate many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5α, JM109, and KC8, as well as numerous 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.

[0145] Examples of mammalian host cells include the following: 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), monkey kidney cells transformed with SV40 (COS-1; ATCC CRL 1650), mouse fetal cells (NIH-3T3; ATCC CRL 1658), and human fetal kidney cells (e.g., EXPI293 cells). The above are examples of many available host organisms known in the art and are not intended to limit the host organisms.

[0146] Mammalian host cells expressing polypeptides are cultured under conditions typically utilized for culturing the parental cell line. Generally, the cells are cultured in a standard medium containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM, or DMEM, and typically 5% - 10% serum, such as fetal bovine serum for example, is added. The culture conditions are also standard, for example, the culture is incubated at 37°C as a static or rotating culture until the desired level of protein is achieved.

[0147] A variety of host cells derived from various cell types and various organisms are available, and such host cells will be known to those skilled in the art. Similarly, viral vectors may be used with prokaryotic host cells, which in particular enable replication or expression of the vector. Some vectors may utilize control sequences that enable their replication and / or expression in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the incubation conditions for maintaining the above-described host cells and for causing replication of the vector for all of the above-described host cells. Along with the techniques and conditions that may enable large-scale production of vectors, the techniques and conditions that may enable production of the nucleic acids encoded by the vectors and the polypeptides, proteins, or peptides related thereto are also understood and known.

[0148] D. Expression Systems Numerous expression systems exist that contain at least a portion or all of the compositions discussed above. Such systems may be used, for example, to produce polypeptide products identified in the present invention as being capable of binding to a particular ligand. Prokaryote-based systems may be used in conjunction with the present disclosure to produce nucleic acid sequences, or the polypeptides, proteins, and peptides related thereto. Many such systems are widely commercially available. Other examples of expression systems include expression systems of vectors containing strong prokaryotic promoters such as, for example, T7, Tac, Trc, BAD, λpL, tetracycline promoter, or Lac promoter, pET expression systems, and E. coli expression systems.

[0149] In one aspect of the present invention, a nucleic acid sequence encoding a polypeptide is disclosed. Depending on the expression system used, it is possible to select a nucleic acid sequence based on conventional techniques. For example, if a polypeptide is derived from a human polypeptide and contains multiple codons that are rarely used in Escherichia coli, they may interfere with expression in Escherichia coli. Therefore, each gene or its variant may be codon-optimized for expression in Escherichia coli. Various vectors may also be used to express the protein of interest. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0150] V. Protein Purification Protein purification techniques are well known to those skilled in the art. These techniques, at one level, involve homogenizing cells, tissues, or organs and then crudely fractionating them into polypeptide and non-polypeptide fractions. The protein or polypeptide of interest may be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity), unless otherwise specified. Analytical methods particularly suitable for preparing pure peptides are as follows: ion exchange chromatography, size exclusion chromatography, 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 more so, high-performance liquid chromatography (HPLC). As is widely known in the art, it is considered that changing the order in which the various purification steps are carried out or omitting some of the steps will still result in a suitable method for preparing a substantially purified protein or peptide.

[0151] A purified protein or peptide is intended to refer to a composition that can be isolated from other components, where the protein or peptide is purified to any extent compared to the state in which it can be obtained naturally. Thus, an isolated or purified protein or peptide also refers to a protein or peptide that is removed from the environment in which it may exist naturally. Generally, "purified" refers to a protein composition or peptide composition that has been subjected to fractionation to remove various other components and that substantially retains the biological activity it exhibits. When the term "substantially purified" is used, this expression refers to a composition in which the protein or peptide forms the main component of the composition, for example, constitutes about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0152] Various methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art from the perspective of the present disclosure. These include, for example, determining the specific activity of the active fraction or evaluating the amount of polypeptide in the fraction by SDS / PAGE analysis. A preferred method for evaluating the purity of a fraction is to calculate the specific activity of the fraction, compare it with the specific activity of the initial extract, and accordingly calculate the degree of purification in the fraction, which is evaluated by the "purification fold", but which calculates such a degree. The actual units used to represent the magnitude of the activity will, of course, vary depending on the specific assay technique selected following purification and also depending on whether the expressed protein or peptide exhibits a detectable activity.

[0153] Generally, proteins or peptides are not required to be provided always in their most purified state. In fact, it is contemplated that substantially less purified products may be useful in certain embodiments. Partial purification can be achieved by using fewer of the purification steps that are combined or by utilizing different formats within the same general purification scheme. For example, cation exchange chromatography performed using an HPLC apparatus generally provides a higher "magnitude" of purification compared to the same technique using a low-pressure chromatography system. Methods that exhibit a relatively low degree of purification may have advantages in terms of the overall recoverability of the protein product or in maintaining the activity of the expressed protein.

[0154] Affinity chromatography is a chromatographic technique that utilizes the specific affinity between the substance to be isolated and a molecule to which the substance can specifically bind. This is a receptor-ligand type of interaction. The column substrate is synthesized by covalently attaching one of the binding partners to an insoluble matrix. Therefore, it is possible to specifically adsorb the substance from the solution to the column substrate. Elution occurs by changing the conditions under which binding does not occur (e.g., changes in pH, ionic strength, temperature, etc.). The matrix should be a substance that does not adsorb molecules to any significant extent and has broad chemical, physical, and thermal stability. The ligand should bind in a manner that does not affect its binding properties. The ligand should also provide a relatively strong bond. It should be possible to elute the substance without destroying the sample or the ligand.

[0155] Size-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size, or more specifically, based on their hydrodynamic volume. This is generally applied to macromolecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to pass a sample through a column, the technique is known as gel filtration chromatography, while the name gel permeation chromatography is used when an organic solvent is used as the mobile phase. The principle underlying SEC is that particles of different sizes elute (are filtered) through the stationary phase at different speeds. This results in the separation of a particle solution based on size. If all the particles are loaded simultaneously or almost simultaneously, particles of the same size should elute together.

[0156] High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography that is frequently used in the fields of biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds a chromatographic packing material (stationary phase), a pump that moves the mobile phase through the column, and a detector that indicates the retention time of the molecules. The retention time varies depending on the interaction between the stationary phase, the molecule being analyzed, and the solvent used.

[0157] VI. Pharmaceutical Compositions When attempting to clinically apply a pharmaceutical composition containing a polypeptide or an antibody, it is generally beneficial to prepare a pharmaceutical composition or a therapeutic composition suitable for the intended use. Generally, a pharmaceutical composition may contain an effective amount of one or more of a polypeptide or additional active substances dissolved or dispersed in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% polypeptide or antibody. In other embodiments, the polypeptide or antibody may constitute, for example, from about 2% to about 75% by unit weight, or from about 25% to about 60%, and any range derivable therein. The amount of the active compound in each therapeutically useful composition may be prepared in a manner such that an appropriate dosage is obtained in any given unit dose of the compound. In preparing such pharmaceutical formulations, one of ordinary skill in the art will consider factors such as, for example, solubility, bioavailability, biological half-life, route of administration, shelf-life of the product, and other pharmacological considerations, and thus various dosages and various treatment regimens may be desirable.

[0158] The expression "pharmaceutical, or pharmaceutically acceptable" refers to molecular entities and compositions that do not cause adverse reactions, allergic reactions, or other inappropriate reactions when properly administered to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is known to those of ordinary skill in the art from the perspective of the present disclosure, as exemplified below: "Remington's Pharmaceutical Sciences", 18th Ed., 1990, which is incorporated herein by reference. Further, with respect to administration to animals (such as humans), it is understood that the preparation should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA's standards for biological agents.

[0159] Furthermore, in certain aspects of the present invention, a composition suitable for administration may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier needs to be capable of being taken up by the body and includes liquid carriers, semi-solids, i.e., carriers that are pastes, or solid carriers. Examples of carriers or diluents include fats, oils, water, physiological saline, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. As used herein, "pharmaceutically acceptable carrier" includes any of the following: aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, physiological saline, parenteral vehicles such as sodium chloride, dextrose plus Ringer's, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coating agents (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), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (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, sweeteners, flavoring agents, dyes, replenishing fluids and nutrient supplements, similar substances and combinations thereof as known to those skilled in the art. Except when any commonly used medium, agent, diluent, or carrier is harmful to the recipient or is harmful to the therapeutic effect of the composition containing them, it is appropriate to use such media, etc. in an administrable composition for use in the practice of the method. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.The composition may be combined with a carrier in any convenient and practical manner, namely, by solution, suspension, emulsification, mixing, encapsulation, absorption, grinding, etc. Those skilled in the art routinely perform such techniques.

[0160] Certain aspects of the present disclosure may include different types of carriers depending on whether it is administered in solid form, liquid form, or aerosol form, and depending on whether it needs to be sterilized in its route of administration, such as by injection. The composition can be formulated for: intravenous administration, intradermal administration, transdermal administration, intrathecal administration, intraarterial administration, intraperitoneal administration, intranasal administration, intravaginal administration, rectal administration, intramuscular administration, subcutaneous administration, mucosal administration, oral administration, topical administration, local administration, administration by inhalation (e.g., aerosol inhalation), administration by injection, administration by infusion, administration by continuous infusion, administration by directly locally perfusing the target cells in a bath, administration via a catheter, administration via perfusion, administration in a lipid composition (e.g., liposome), or administration by other methods known to those skilled in the art, or by any combination of the above (see, e.g., "Remington's Pharmaceutical Sciences", 18th Ed., 1990, incorporated herein by reference). Typically, such compositions may be prepared as either a liquid solution or a suspension; solid forms suitable for use in preparing a solution or suspension by adding a liquid prior to injection are also possible to prepare; and their preparations may also be emulsified.

[0161] The polypeptide may be formulated in a composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, which are formed, for example, with the free amino groups of a proteinaceous composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0162] In a further aspect, the disclosure may relate to the use of a pharmaceutical lipid vehicle composition, which comprises a polypeptide, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” is defined to include any of a wide range of substances that are characterized as being insoluble in water and extractable using an organic solvent. This broad class of compounds is well known to those skilled in the art, and when the term “lipid” is used herein, the term is not limited to any particular structure. Examples thereof include compounds containing long-chain aliphatic hydrocarbons, and compounds containing derivatives of long-chain aliphatic hydrocarbons. Lipids may be naturally occurring or synthetic (i.e., artificially designed or made). However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example: neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glycolipids, sulfatides, lipids having ether-linked fatty acids and lipids having ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by those skilled in the art as lipids are also included in the present composition and method.

[0163] One skilled in the art would be familiar with a series of techniques that can be utilized to disperse a composition in a lipid vehicle. For example, a polypeptide, or a fusion protein thereof, may be dispersed in a lipid-containing solution, dissolved using lipids, emulsified using lipids, mixed with lipids, combined with lipids, covalently bonded to lipids, included as a suspension in lipids, included in micelles or liposomes, or form a complex with micelles or liposomes, or be bound to a lipid or lipid structure, by any means known to those skilled in the art. The dispersion may or may not cause the formation of liposomes.

[0164] The terms "unit dose" or "dosage" refer to physically discrete units suitable for use in a subject, where each unit contains a predetermined amount of the therapeutic composition calculated to produce the desired response discussed above in conjunction with its mode of administration, i.e., the appropriate route and treatment regimen. The amount administered will vary depending on the desired effect, taking into account both the number of treatments and the unit dose. The actual dosage of the composition of this aspect administered to a patient or subject can be determined by physical and physiological factors such as, for example, the subject's weight, age, health status, and gender, the type of disease to be treated, the degree of penetration of the disease, past or current therapeutic interventions, the idiosyncrasies of the patient, the route of administration, and the efficacy, stability, and toxicity of the particular therapeutic substance. In another non-limiting example, the dosage may also include the following: from about 1 microgram / kg / body weight, from about 5 micrograms / kg / body weight, from about 10 micrograms / kg / body weight, from about 50 micrograms / kg / body weight, from about 100 micrograms / kg / body weight, from about 200 micrograms / kg / body weight, from about 350 micrograms / kg / body weight, from about 500 micrograms / kg / body weight, from about 1 milligram / kg / body weight, from about 5 milligrams / kg / body weight, from about 10 milligrams / kg / body weight, from about 50 milligrams / kg / body weight, from about 100 milligrams / kg / body weight, from about 200 milligrams / kg / body weight, from about 350 milligrams / kg / body weight, from about 500 milligrams / kg / body weight, from about 1000 milligrams / kg / body weight or more per administration, and any range derivable therefrom. In non-limiting examples of ranges derivable from the numerical values listed herein, based on the above numerical values, it is possible to administer ranges such as from about 5 milligrams / kg / body weight to about 100 milligrams / kg / body weight, from about 5 micrograms / kg / body weight to about 500 micrograms / kg / body weight, etc. In any case, it will be the physician administering the treatment who will determine the concentration of the active ingredient in the composition and the dosage appropriate for the individual subject.

[0165] The present invention is not intended to be limited by the specific properties of the therapeutic preparation. For example, such a composition may be provided as a formulation with a physiologically tolerable carrier, diluent, and excipient that is liquid, gel, or solid. The therapeutic preparation may be administered to mammals, such as domesticated animals, for veterinary use, and may be administered to humans for clinical use in a manner similar to other therapeutic agents. Generally, the dosage required for a therapeutic effect varies depending on the form of use and the mode of administration, and also varies according to the needs specified for each individual subject. The actual dosage of the composition administered to an affected animal can be determined by physical and physiological factors, such factors being, for example, body weight, severity of the condition, type of disease to be treated, past or current therapeutic interventions, idiosyncrasy of the affected animal, and route of administration. Depending on the dosage and the route of administration, the number of times of administering a preferred dosage and / or an effective amount may be varied while observing the response of the subject. In any case, it will be the physician administering the treatment who determines the concentration of the active ingredient in the composition and the dosage appropriate for each individual subject.

[0166] VII. Treatment Methods One aspect of the present disclosure provides a polypeptide for treating a disease, such as a tumor. In particular, the polypeptide may have a human polypeptide sequence, and thus, this may prevent an allergic reaction in a human patient, enable repeated administration, and increase the therapeutic effect.

[0167] "Treatment" and "treating" refer to administering or applying a therapeutic agent to a subject, or performing a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit against a disease or a health-related condition. For example, treatment may include administering a pharmaceutically effective amount of an antibody that directs CDC to cancer cells without causing the proliferation of cancer cells.

[0168] "Subject" and "patient" refer to either a human or a non-human, such as, for example, primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0169] As used throughout this application, the terms "therapeutic benefit" or "therapeutically effective" refer to anything that promotes or enhances the well-being of a subject with respect to the medical treatment of a condition. The terms include, but are not limited to, a decrease in the frequency or severity of the signs or symptoms of a disease. By way of example, the treatment of cancer can be related to, for example, a decrease in tumor size, a decrease in tumor invasiveness, a decrease in the cancer growth rate, or the prevention of metastasis. The treatment of cancer can also refer to extending the survival period of a subject having cancer.

[0170] In some aspects, the disease can be, for example, cancer (such as those using agonist antibodies, such as anti-CD40 agonist antibodies, as an example), an infectious disease, or an immune disease. The immune disease can be an autoimmune disease, such as, for example, lupus, rheumatoid arthritis, psoriasis, etc.

[0171] 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, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include: tumors of the bone marrow, T-cell cancer or B-cell cancer, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can be treated using the methods provided herein include, but are not limited to: carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or corpus cancer, salivary gland cancer, kidney cancer 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, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin 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 non-cleaved cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myelogenous leukemia.

[0172] Cancer can specifically be of the following histological types, but is not limited thereto: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; trichoblastoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma - cholangiocarcinoma; cord adenocarcinoma; adenoid cystic carcinoma; adenomatous polyp carcinoma; familial polyposis coli carcinoma; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; anaplastic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary - follicular adenocarcinoma; unencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; 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; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant mesothelioma; malignant granulosa cell tumor; malignant androblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipoid cell tumor; malignant paraganglioma; malignant extra - mammary paraganglioma; pheochromocytoma; glomus angiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epitheloid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated embryonal cell tumor; fetal carcinoma; malignant teratoma; malignant ovarian teratoma; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant angioendothelioma; Kaposi's sarcoma; malignant pericytic tumor; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontogenic sarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; anaplastic glioma; anaplastic astroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma;Hodgkin's disease; Hodgkin's granuloma; small lymphocyte malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblast leukemia; myelosarcoma; and hairy cell leukemia.;

[0173] The polypeptides herein may be used as anti-tumor agents in various modalities to cause complement activation in tumor tissue or at locations where it is considered desirable. In certain embodiments, the invention contemplates a method of using a polypeptide as an anti-tumor agent and thus includes contacting a population of tumor cells with a therapeutically effective amount of the polypeptide for a period sufficient to inhibit the growth of the tumor cells.

[0174] In one embodiment, in vivo contact is achieved by administering to a patient a therapeutically effective amount of a physiologically tolerable composition comprising the polypeptide of the invention by intravenous injection, intraperitoneal injection, or intratumoral injection. The polypeptide may be administered parenterally by injection or by slow infusion over time. The polypeptide may be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, to the skin, and may be delivered by a peristaltic method or may be directly injected into the tissue containing the tumor cells.

[0175] A therapeutic composition containing the polypeptide is administered intravenously as is conventional, for example by injecting unit doses. The term "unit dose" when used in relation to a therapeutic composition refers to physically discrete units suitable as unit dosage for the subject, where each unit contains a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.

[0176] The composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount administered will vary depending on the subject being treated, the capacity of the subject's system to utilize the active ingredient, and the degree of desired therapeutic effect. The exact amount of active ingredient required for administration will vary according to the judgment of the physician and is specific to each individual. However, ranges of suitable dosages for systemic application are disclosed herein and these vary according to the route of administration. Regimens suitable for initial administration and for booster administration are also contemplated and exemplary thereof is to administer an initial dose and then, at intervals of 1 hour or longer, to perform repeated administration by the next injection or other means of administration. Exemplary multiple administrations are described herein and it is particularly preferred that the serum and tissue levels of the polypeptide be continuously maintained high. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the range designated for in vivo therapy is also contemplated.

[0177] The polypeptide of the present invention is intended to be administered systemically or locally to inhibit the growth of tumor cells or to kill cancer cells, for example, in cancer patients having locally advanced cancer or cancer having distant metastases, for treating the disease. The polypeptide may be administered intravenously, intrathecally, and / or intraperitoneally. The polypeptide may be administered alone or in combination with an anti-proliferative agent. In one embodiment, the polypeptide is administered to reduce the amount of cancer in a patient prior to a surgical or other procedure. Alternatively, the polypeptide may be administered after a surgical procedure to ensure that any remaining cancer (for example, cancer that could not be removed by the surgical procedure) does not survive.

[0178] The therapeutically effective amount of the polypeptide is calculated to achieve the desired effect, i.e., a predetermined amount calculated to cause CDC in tumor tissue and thereby mediate an inflammatory response that eliminates the tumor. Thus, the dosage range for administering the polypeptide of the present invention is wide enough to produce the desired therapeutic effect (e.g., a decrease in cancer cell division or an increase in cancer cell death, other clinical benefits). The dosage should preferably not be so large as to significantly cause significantly harmful side effects, such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, neurological effects, etc. Generally, the dosage can vary depending on the patient's age, health status, gender, and the degree of the disease, and those skilled in the art can determine the dosage. In the case of any complications, an individual physician can adjust the dosage. In some embodiments, the dosage can be from about 0.1 mg / kg to about 10 mg / kg.

[0179] VIII. COMBINATION THERAPY In one aspect, the compositions and methods of this aspect relate to administering a polypeptide or antibody in combination with a second or additional therapy. Such therapies are applicable in the treatment of any disease that is responsive to CDC. For example, the disease can be cancer.

[0180] Methods and compositions, including combination therapies, enhance a therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-proliferative therapy. Therapeutic and prophylactic methods, as well as therapeutic and prophylactic compositions, may be provided in a combined total amount effective to achieve a desired effect, such as killing cancer cells and / or inhibiting cell overgrowth. This process may be related to administering 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 may be an agent that upregulates the immune system but does not have a direct cytotoxic effect. Tissues, tumors, or cells may be exposed to one or more compositions or pharmaceutical formulations containing one or more agents (such as polypeptides or anti-cancer agents, etc.), or this may be done by exposing the tissues, tumors, and / or cells to two or more separate compositions or pharmaceutical 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. Also, such combination therapies are intended to be used with chemotherapy, radiation therapy, surgical therapy, or immunotherapy.

[0181] When applied to cells, the terms "contact" and "exposure" are used herein to describe a process by which a therapeutic polypeptide or therapeutic antibody, and a chemotherapeutic or radiotherapeutic agent, are delivered to the target cells or placed in direct proximity to the target cells. To achieve killing of the cells, both agents are delivered to the cells in a combined total amount effective to, for example, kill the cells or prevent their division.

[0182] The polypeptide or antibody may be administered before, during, after, or in various combinations with the anti-cancer treatment. Their administrations may be made at intervals ranging from simultaneously to several minutes, several days, or several weeks. In the manner in which the polypeptide or antibody is provided to the patient separately from the anti-cancer agent, generally, it is advisable to ensure that a long period does not elapse between the respective delivery times so that the two compounds can continue to exhibit a beneficial combined effect on the patient. In such an example, it is contemplated that both the polypeptide and the anti-cancer therapy can be provided to the patient within about 12 to 24 hours or up to 72 hours, and more specifically, within about 6 to 12 hours. In some situations, it may be desirable to significantly extend the treatment period by taking a drug-free period of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration.

[0183] In one aspect, one course of treatment spans 1 to 90 days or longer (including interrupted days). One type of agent may be administered on any day within the first to 90th day (including interrupted days), or any combination thereof, and another agent may be administered on any day within the first to 90th day (including interrupted days), or any combination thereof. During one day (a 24-hour period), the patient may receive the administration of the agent once or multiple times. Further, after one course of treatment, a period of administration of a treatment other than the anti-cancer treatment is contemplated. Such a period may span 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or longer (including interrupted days), depending on the patient's condition, such as their prognosis, physical strength, health status, etc. The treatment cycle is expected to be repeated as necessary.

[0184] Various combinations are available. In the following examples, the polypeptide or antibody is "A" and the anti-cancer therapy is "B". TIFF2025519556000017.tif17130

[0185] Administration of any polypeptide or any therapy of this aspect to a patient is, in some cases, carried out in accordance with the general protocol for administering such a compound, taking into account the toxicity of the agent. Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy. In some embodiments related to the treatment of cancer in a subject, the second therapy can be, for example, chemotherapy, radiotherapy, immunotherapy (such as checkpoint inhibitors, for example anti-PD1 antibody or anti-PDL1 antibody), gene therapy, anti-inflammatory agents, antibiotics, or surgical procedures, etc.

[0186] A. Chemotherapy For this aspect, a wide range of chemotherapeutic agents may be used. The term "chemotherapy" refers to the use of drugs to treat cancer. The term "chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. The agent or drug is classified by the mode of its activity within the cell, which is, for example, whether they affect the cell cycle and at which stage of the cell cycle they have an impact, etc. Alternatively, the agent may be characterized based on its ability to directly crosslink DNA, based on its ability to intercalate into DNA, or based on its ability to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0187] Examples of chemotherapeutic agents include the following: alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bratasin and bratasinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); ellipticine; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, noburembycin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (such as calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1); dynemicin (including dynemicin A); bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore, and related chromoproteide engyin antibiotics chromophore, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardifilin, chromomycinis, daunorubicin, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;Defofamine; Demecolcine; Diazicon; Elformithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially, T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids such as paclitaxel and docetaxel; Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum complexes such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine; Carboplatin, Procarbazine, Plicomycin, Gemcitabien, Navelbine, Farnesyl protein transferase inhibitor, Transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.;

[0188] B. Radiation Therapy Other factors that cause DNA damage and are widely used include those well-known as γ-rays, as X-rays, and / or as the targeted delivery of radioisotopes to tumor cells. Other forms of factors that damage DNA are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these factors have a very high likelihood of causing extensive damage to DNA, to the precursors of DNA, to DNA replication and repair, and to the assembly and maintenance of chromosomes. The dose range of X-rays spans from a daily dose of 50 to 200 roentgens for a long period (3 - 4 weeks) to a single dose of 2,000 to 6,000 roentgens. The dose range of radioisotopes varies significantly and this varies according to the half-life of the isotope, according to the intensity and type of radiation emitted, and according to the uptake by neoplastic cells.

[0189] C. Immunotherapy One of ordinary skill in the art will understand that immunotherapy can be used in combination with, or together with, the methods of this aspect. With respect to the treatment of cancer, generally immunotherapeutic agents require the use of immune effector cells and immune effector molecules to target and suppress immune cells. Blincyto® is an example of this. Checkpoint inhibitors, such as ipilumimab, are another such example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can act alone as an effector of immunotherapy, or the antibody can recruit other cells that actually perform cell killing. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and used simply as a targeting agent. Alternatively, the effector can be a lymphocyte bearing surface molecules that interact with tumor cell targets either directly or indirectly. Various effector cells include cytotoxic T cells and NK cells.

[0190] In one aspect of immunotherapy, tumor cells need to carry some marker suitable for targeting, i.e., some marker that is not present in the majority of other cells. Many tumor markers exist, and any of them can be suitable for targeting according to this aspect. Common tumor markers include the following: CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy is to combine the anti-cancer effect with an immune-stimulating effect. Immune-stimulating molecules also exist and include the following: cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, etc., chemokines such as MIP-1, MCP-1, IL-8, etc., and growth factors such as FLT3 ligand.

[0191] Examples of immunotherapies currently under investigation or in use are as follows: immunoadjuvants 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., 1998); cytokine therapies such as interferon α, interferon β, and interferon γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be utilized in conjunction with the antibody therapies described herein.

[0192] D. Surgical procedures Approximately 60% of people with cancer undergo some type of surgical procedure, which includes surgical procedures for prevention, diagnosis or staging, treatment, and palliation. Surgical procedures for treatment include resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and the surgical procedure may be used in combination with other therapies, such as the procedures of the present aspect, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. The excision of a tumor refers to physically removing at least a part of the tumor. In addition to tumor excision, treatments by surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgery (Mohs surgery).

[0193] When part or all of a cancerous cell, tissue, or tumor is excised, a cavity may be formed in the body. Treatment may be achieved by performing perfusion, direct injection, or topical application to the area using additional anti-cancer therapies. Such treatments may be repeated, for example, daily, every two days, every three days, every four days, every five days, every six days, or every seven days, or every week, every two weeks, every three weeks, every four weeks, and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months, etc. Similarly, the dosage for these treatments can vary.

[0194] E. Other agents In order to improve the therapeutic effect of the treatment, it is contemplated that other agents may be used in combination with certain aspects of the present aspect. These additional agents include the following: agents that affect the upregulation of cell surface receptors and GAP binding, cell division inhibitors and differentiating agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. By increasing intracellular signaling by increasing the number of GAP bindings, the anti-hyperproliferative effect on adjacent hyperproliferative cell populations can be increased. In other aspects, a cell division inhibitor or differentiating agent may be used in combination with certain aspects of the present aspect to improve the anti-hyperproliferative effect of the treatment. A cell adhesion inhibitor is intended to improve the effect of the present aspect. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents, such as the c225 antibody, which increase the sensitivity of hyperproliferative cells to apoptosis, may also be used in combination with certain aspects of the present aspect to improve the effect of the treatment.

[0195] IX. Kit Certain aspects of the present invention may provide a kit, such as, for example, a therapeutic kit. For example, the kit may include one or more of the pharmaceutical compositions described herein and optionally include instructions for using them. The kit may also include one or more devices for achieving administration of such compositions. For example, a kit of interest may include a pharmaceutical composition and a catheter, the catheter being for achieving direct intravenous injection of the composition into a cancerous tumor. In other aspects, a kit of interest may include an ampoule pre-filled with a polypeptide, the polypeptide being formulated as a medicament or lyophilized, optionally for use with a delivery device.

[0196] The kit may include a labeled container. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed from various materials, such as glass or plastic. The container may hold a composition containing a polypeptide effective for therapeutic or non-therapeutic use, such as those described above. The label attached to the container may indicate that the composition is used for a specific therapy or a specific non-therapeutic use, and may also indicate instructions for either in vivo or in vitro use, such as those described above. The kit of the present invention typically includes the above-described container and also one or more other containers containing materials desirable from a commercial and user perspective, and the materials include buffers, diluents, filters, needles, syringes, and an accompanying document with instructions for use.

Examples

[0197] X. Examples To demonstrate the preferred embodiments of the present invention, the following examples are included. The techniques disclosed in the following examples are representative of those that the inventor has found to be fully functional for practicing the present invention and, therefore, can be considered to constitute a preferred mode for practicing the present invention. However, from the perspective of the present disclosure, it should be understood by those skilled in the art that many modifications can be made in the specific embodiments disclosed, and even in that case, similar or comparable results can be obtained without departing from the spirit and scope of the present invention.

[0198] Example 1 Library construction strategy for isolating the Fc domain of IgG1 that binds to FcγRIIB Mutagenesis was performed on the residues that bind to the Fc receptor in non-glycosylated V8.2 Fc, and additional random mutations in the Fc were introduced by error-prone PCR. The residues in the Fc that bind to FcγRIIb are ELLGG (positions 233 - 237 in EU numbering), SH (positions 267, 268), NST (positions 297 - 299), and ALPAPIE (positions 327 - 333), and these were used as residues for site-saturation mutagenesis for the FcγRIIb-selective Fc library. This library was displayed on the surface of yeast to screen for FcγRIIb-selective variants. Fc mutants with Aga2 linked to the N-terminus were expressed and linked to Aga1 by two disulfide bonds and displayed on the cell surface of yeast cells. Populations showing binding affinity for FcγRIIb were sorted using other tetrameric FcγRs coated with streptavidin as competitors. The first library and the second library were high concentrations of FcγRIIa as competitors during screening R131 Sorted for binding to the fluorescently labeled FcγRIIb tetramer in the presence of the tetramer. To sort only the FcγRIIb-selective population, a two-step sorting method was used for sorting the third library and the fourth library. First, the library was incubated with FcγRIIa R131 (the most competitive FcγR), and the negative population was sorted to remove those that bind to FcγRIIa R131 . Those that did not bind to FcγRIIa R131 were sorted at this stage. Next, these sorted cells were incubated with the FcγRIIb tetramer, and the population positive for 2b was sorted. After sorting the fourth library, 91 single clones were selected from the final library, and 8 variants were expressed in HEK293 cells to measure the affinity.

[0199] Example 2 Binding characteristics of the Fc2b variant Table 4 shows a list of engineered Fc variants used in the further experiments described below. V8.2 is an Fc variant made using the method described in Example 1. EF, V11, and V12 are previously reported Fc variants that show enhanced binding affinity for FcγRIIb (Teige et.al., 2019), V11 and V12: (Mimoto, et al., 2013), and these variants were evaluated for comparison purposes. The mutation G237D in the 2b18K variant is also present in V12 and V11. E233V is a different mutation at the same site in V12, and it is not present in V11. H268, A330 are different mutations at the same site in V12 and V11. L328 is a different mutation at the same site in EF. The substitution mutations in Table 4 are shown using the Kabat numbering.

[0200] (Table 4) Mutations in the Fc2b variant TIFF2025519556000018.tif20891

[0201] To verify the binding affinity for Fcγ receptors, a Biolayer Interference (BLI) assay was performed on an Octet RED96 system (ForteBio Inc., California, USA). A high-precision streptavidin (SAX) biosensor (Sartorius Inc., 18-5117) was used, and the assay was performed at 25 °C with shaking at 1,000 rpm. To measure KD, biotinylated FcgR was immobilized on the biosensor sufficiently up to a shift of 0.5 nm or 1.0 nm. Monoclonal antibodies were allowed to bind for 2 minutes and then dissociated for 2 minutes. KD was calculated using the baseline drift model in BIAevaluation software with a 1:1 binding. The results are shown in Table 5.

[0202] (Table 5) Binding characteristics of engineered antibody variants measured by BLI TIFF2025519556000019.tif22279a: Reported data, NA: No assay, NB: No conjugation

[0203] FcγRIIb-selective Fc variants candidates (2b18K, 2b18KQ, and 2b18KQS) showed binding affinity for FcγRIIb by BLI, but showed no binding affinity or only very low binding affinity for activating receptors (Table 5). In contrast, previously reported ones with enhanced binding to 2b (i.e., EF, V11, and V12) showed binding affinity for activating receptors, particularly FcγRIIa R131 showed binding affinity for (FcγRIIa R131 has 95% identity to the extracellular domain of FcγRIIb and only three residues located at the binding interface are different).

[0204] To more accurately measure the binding affinity of the Fc variant 2b18KQS, a surface plasmon resonance (SPR) assay was performed using the 2b18KQS variant. The results are shown in Table 6 below. The method used was the one previously described below: Lee et al., 2017.

[0205] (Table 6) Affinity of the 2b18KQS Fc variant for Fc receptors measured by SPR (by steady-state analysis) TIFF2025519556000020.tif22060

[0206] The Fc variant 2b18KQS showed no detectable binding affinity for activating receptors, and this was even the case for FcγRI, a high-affinity Fc receptor (Table X3). The variant showed a decrease in binding to FcγRIIb (1 / 4 compared to WT Fc) and FcγRIIa R131showed almost undetectable binding (1 / 40 compared to WT Fc). The binding affinity of 2b18KQS Fc for FcRn was similar to that of WT Fc, which may be related to the circulating half-life.

[0207] The binding characteristics of Fc variants by BLI do not necessarily accurately reflect the binding characteristics of immune complexes due to differences in avidity. For the purpose of measuring the binding profile under high avidity conditions, a binding assay of opsonized SK-BR-3 with beads coated with FcγR was performed using the 2b18K variant. To mimic the arrangement of Fc receptors on effector cells, biotinylated Fcγ receptors were coated on the beads, and the binding was measured by flow cytometry. The results are shown in Figure 1.

[0208] In Figure 1, the first column shows the results for cancer cells opsonized with the WT antibody, and the subsequent columns show the data for the Fc variants V8.2, 2b18K, V12, and EF. The values shown indicate the fold difference in binding affinity compared to wild-type Fc measured by BLI, and the values within the basket (within the parentheses in Figure 1) are the values published by other research institutions. WT shows strong binding to all Fcγ receptors. V8.2 shows strong binding to 2aR and 2b, although it shows 1 / 3 of the binding affinity compared to WT in BLI. 2b18K shows weak binding to 2aR and strong binding to 2b. V12 and EF show strong binding to 2a and 2b as well as 3aV.

[0209] A binding assay of opsonized SK-BR-3 with beads coated with FcγR was performed using the Fc variants 2b18KQ and 2b18KQS (Figure 2). As a negative control, the Fc variant of LALAPG was included (this is a well-known science Fc and has completely lost the effector function mediated by Fc). The 2b18K variant is FcγRIIaH131 showed no binding to FcγRIIIa and FcγRIIIb, but maintained its binding to FcγRIIb. 2b18KQS had the lowest binding affinity for FcγRIIa among the 2b18K variants. R131 showed the lowest binding affinity for FcγRIIa.

[0210] To confirm the binding to actual cells, an IC binding assay was performed using Raji cells (ATCC CCL-86), a Burkitt lymphoma. Raji cells express only FcγRIIb on the surface among FcγRs. Thus, the binding to FcγRIIb was measured using B cells. Antibody-coated beads were used as ICs, and the binding was measured by flow cytometry. The results are shown in Figure 3. To prepare immune complexes, 1-μm fluorescent polystyrene beads were coated with an antibody. These ICs were incubated with Raji cells at 4 °C for 1 hour. The binding between ICs and Raji cells was detected by flow cytometry.

[0211] The 2b18K variant showed a binding similar to that of WT Fc and showed significantly better binding compared to the Fc variant of LALAPG (Figure 3). These results were consistent with and further supported the results obtained using the binding assay between opsonized SK-BR-3 and beads coated with FcγRIIb.

[0212] Example 3 Thermal stability of the Fc2b variant To evaluate the thermodynamic stability of the engineered Fc variants that are not glycosylated, the melting temperature (T m ) was measured. T m was measured by differential scanning fluorimetry (DSF). The T m of the 2b18K variant showed a lower value compared to WT Fc (Table 7). These results are consistent with the view that the loss of thermal stability can be induced when not glycosylated. The method used was as described below: Lee et al., 2019.

[0213] (Table 7) Measurement of Tm by differential scanning fluorimetry TIFF2025519556000021.tif16128

[0214] Example 4 Verification of the effector function of phagocytic activity in effector cells In ICs, when there is no binding affinity for the activating receptor, the phagocytic effector function decreases. To measure the phagocytosis rate of the IC of the 2b18K variant Fc, a phagocytosis assay using antibody-coated pHrodo Red beads was performed using the THP-1 (FcγRIIa H / H homozygous) cell line. pHrodo is a pH-sensitive dye whose signal increases at low pH. At neutral pH, little or no signal is detected. When the phagosome encapsulates the target cell and becomes acidic, which is detected on the flow cytometer, the fluorescence signal of pHrodo increases. Using this dye, it was possible to detect actual phagocytosis and remove signals that were simply bound to the surface. THP-1 cells (50,000 per well) were mixed with RPMI-1640 (Gibco, 11875135) containing antibody-coated beads (50-fold excess) in TC-treated round-bottom plates (Corning, 07-200-95) and incubated at 37 °C and 5% CO2 for 4 hours. The cells were washed once with DPBS and immediately analyzed in a BD LSRII cytometer equipped with a high-throughput sampler. The degree of phagocytosis is expressed as the percentage of bead+ / pHrodo+ cells multiplied by the geometric MFI of Dragon Green of the double-positive population.

[0215] THP-1 cells express FcγRIIaH131, and 2b18K, 2b18KQ, and 2b18KQS did not bind detectably to it (Table 5). The variants 2b18K, 2b18KQ, and 2b18KQS were used, and beads coated with the Fc of LALAPG were used as a negative control. When the ICs of 2b18K, 2b18KQ, and 2b18KQS were used, FcγR-mediated phagocytosis was not detected, which is consistent with the results of the binding assay. The results are shown in Figure 4.

[0216] The ADCP assay of SK-BR-3 using THP-1 cells was performed, and it was revealed that antibody-mediated cell phagocytosis was zero when 2b18K was used and when V8.2 was used (Figure 5). However, high phagocytosis rates were detected in the V12 variant and the EF variant, which have high binding affinity for FcγRIIb. These results demonstrate that even if the binding affinity for FcγRIIb is higher than that for activating FcγRs (Table 5), that does not guarantee low phagocytosis activity. The V12 variant had a significantly higher phagocytosis rate than WT and EF, despite having a lower affinity for FcγRIIaH131. These results indicate that inactivation of activating receptors is essential to abrogate the activation of immune cells.

[0217] The variants 2b18K, 2b18KQ, and 2b18KQS were also tested using the ADCP assay of SK-BR-3 with THP-1 cells. ICs that bind only to FcγRIIb do not induce activating FcγRs and do not cause effector functions. The results are shown in Figure 6.

[0218] Additional experiments were performed to measure the binding activity of the immune complex. Deposition of C1q in Raji or Ramos cells that are CD20+ was measured by FACS. The cells were opsonized with the WT or Fc-engineered variants of rituximab. All of the variants 2b18K, 2b18KQ, and 2b18KQS showed a decrease in C1q deposition on the cell surface or no deposition. The results are shown in Figure 8.

[0219] Additional experiments were performed using an in vitro cell-based assay with the variants 2b18K, 2b18KQ, and 2b18KQS. When using the Fc2b variant, neither complement-mediated effector function nor activating FcγR-mediated effector function was detected. As shown in Figure 9A, lysis of Raji or Ramos cells that are CD20 + by complement-dependent cytotoxicity (CDC) was measured. The cells were opsonized with various concentrations of rituximab or its Fc-engineered variants and incubated with 10% human pooled serum. The antibody-dependent cell phagocytosis (ADCP) assay was performed using THP-1 cells as effectors and SK-BR-3 cells as targets with the WT or Fc-engineered variants of trastuzumab (Figure 9B). Measurement of the antibody-dependent cell cytotoxicity (ADCC) assay using peripheral blood mononuclear cells (PBMC) was performed (Figure 9C). The target cells were Raji cells opsonized with the WT or Fc-engineered variants of rituximab. The ADCP assay was performed using human monocyte-derived M1 macrophages as effectors and SK-BR-3 cells as targets (Figure 9D). The WT or Fc-engineered variants of trastuzumab were added at various concentrations. CD20 +Phosphorylation of FcγRIIb after incubation of Raji cells for 1 hour with either the WT or Fc-engineered variants of rituximab was analyzed by immunoblotting (Figure 9E). In all assays, the Fc2b variants 2b18K, 2b18KQ, and 2b18KQS, which are hexameric, showed no complement-mediated activation or activation mediated by activating FcγRs, and these variants showed functional binding to FcγRIIb.

[0220] Example 5 Engineered hexameric Fc variants that bind to FcγRIIB The hexameric mutant Fc region was generated as follows. The 18 amino acid residue μ-tailpiece of human IgM (PTLYNVSLVMSDTAGTCY; SEQ ID NO:6) was fused to the C-terminus of the Fc fragment of human IgG1 (Rowley et al., 2018; Spirig et al. 2018). To introduce a disulfide bond between Fc and Fc, an additional mutation was added to the Leu residue at position 309 (EU numbering) in the mutant Fc of IgG. Engineered hexameric constructs (hexameric Fc2b) were generated, and the results are shown in Figures 10A - 10D.

[0221] The following sequences were generated and observed to form hexamers. Hex 2b18KQS (non-glycosylated Fc): TIFF2025519556000022.tif31145; Hex 2b18KQS-ST (glycosylated version of 2b18KQS): TIFF2025519556000023.tif30144; Hex2b218K (non-glycosylated hexameric version of the hexameric construct of Fc variant 2b18K): TIFF2025519556000024.tif30145; and Hex2b218K-ST (glycosylated hexameric version of the Fc variant 2b18K hexameric construct): TIFF2025519556000025.tif30146。

[0222] The sequence alignment showing the hexameric wild-type Fc and the point mutations present in the various engineered variant Fc regions is shown in Figure 11.

[0223] The platelet activation assay can be tested by the following method. Platelets express FcγRIIa on their surface, which plays a role in activating platelets when in contact with immune complexes. In the presence of the 2b-selective hexameric Fc (Hex 2b18KQS or Hex 2b18KQS-ST), it is possible to evaluate platelet activation by incubating with the 2b-selective hexameric Fc to test whether there is any activation of platelets mediated by FcγRIIa. In the presence of the 2b-selective hexameric Fc, it is expected that platelet activation mediated by FcγRIIa is either minimal or not mediated at all.

[0224] The complement-dependent cytotoxicity (CDC) assay is performed as described below: Lee et al. (2017). The hexamer of IgG1 has a better binding affinity for C1q. However, CDC mediated by the hexameric Fc has not been shown in the past. The 2b-selective hexameric Fc (Hex 2b18KQS or Hex 2b18KQS-ST) has been observed to have a reduced binding affinity for C1q, and it is expected that the 2b-selective hexameric Fc does not have CDC activity. A CDC assay is performed to demonstrate the absence of CDC mediated by the 2b-selective hexameric Fc.

[0225] Assays for B cell proliferation and differentiation are performed using the 2b-selective hexameric Fc (Hex 2b18KQS and Hex 2b18KQS-ST). To verify the effect of engaging only FcγRIIb on B cell proliferation and differentiation, treatment with hexameric Fc is performed during the process of B cell proliferation and differentiation, and the effect is analyzed. Assays for B cell proliferation and differentiation can be performed as described below: Khoenkhoen et al. (2020).

[0226] Assays for ADCP mediated by primary monocytes and macrophages are performed using the 2b-selective hexameric Fc (Hex 2b18KQS and Hex 2b18KQS-ST). Substantial evidence has shown that blocking FcγRIIb can increase the therapeutic effect of therapeutic antibodies. The tumor microenvironment (TME) expresses increased levels of FcγRIIb, at least in part, due to the hypoxic conditions in the TME. To mimic the TME, monocytes and macrophages are incubated under physiological and pharmacological hypoxic conditions, and high expression of FcγRIIb is confirmed (Hussain et al., 2022). By blocking FcγRIIb, the phagocytic function was significantly restored. Using the assay for ADCP mediated by primary monocytes and macrophages, the 2b-selective hexameric Fc (Hex 2b18KQS and Hex 2b18KQS-ST) was tested to measure the effect of blocking FcγRIIb on monocytes and macrophages incubated under hypoxic conditions. Assays for ADCP mediated by primary monocytes and macrophages are performed as described below: Kang et al. (2019).

[0227] The binding properties of the hexameric Fc2b construct are shown in Figure 12. The results of the binding assay between beads coated with FcγR and the hexameric Fc are shown using Hex 2b18KQS Fc, and glycosylated Hex 2b18KQS-ST Fc. In Figure 12, the y-axis indicates the mean fluorescence intensity.

[0228] The binding of the hexameric Fc to Fcγ receptors was confirmed by a binding assay with beads. The hexameric Fc has a hexavalency, which provided sufficient avidity to bind to cells expressing Fcγ receptors or to multimers of the receptor. By using beads coated with Fcγ receptors, it was possible to measure the binding properties of the hexamer (Figure 12). Hex 2b18KQS showed selective binding to FcγR2b and some binding to FcγR2aR. The glycosylated hexameric 2b18KQS-ST Fc showed increased selectivity for FcγR2b, but a decrease in binding was observed compared to Hex 2b18KQS. The Fc variant of LALAPG was expressed as a fusion protein with the μ-tailpiece of human IgM (SEQ ID NO:6) and included as a negative control (this Fc variant is the well-known science Fc and has completely lost the effector functions mediated by Fc).

[0229] The Fc variant 2b18K was hexamerized to construct Hex 2b18K. Glycosylated variants of Hex 2b18K were also prepared, which are designated as Hex 2b18K-ST and contain substitution mutations of G298S and A299T in the Fc variant 2b18K. Hex LALAPG-2 is a hexameric Fc of the LALAPG (L234A, L235A, P329G) variant. The Fc variant of LALAPG is a silent variant that does not bind to any Fcγ receptor, and thus this hexameric variant was used as a negative control in the experiment. Hex LALAPG-2 was prepared as a hexameric Fc based on the Fc variant of LALAPG and also contains point mutations of V567I and A572G in the μ-tailpiece of IgM to stabilize the structure and increase the expression yield.

[0230] The binding of hexameric Fc to Fcγ receptors was tested using a binding assay of Raji cells with beads coated with antibodies, and the results are shown in Figure 13. Error bars are the standard error of the mean of triplicate samples. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test (***P < 0.001, ****P < 0.0001). The binding to FcγR2b was further analyzed using B cell lines. The binding of hexameric Fc to cells was tested using Raji cells that express only FcγR2b among Fc receptors (Figure 13). Concentration-dependent binding was observed for Hex 2b18KQS Fc and glycosylated Hex 2b18KQS-ST Fc.

[0231] Blocking Fcγ receptor 2b with hexameric Fc was observed to increase the phagocytic activity of monocytes, and the results are shown in Figure 14. To examine the effect of FcγR2b blockade on monocyte phagocytosis, an antibody-dependent cell phagocytosis (ADCP) assay using THP-1 cells was performed with opsonized SK-BR-3 cells. Since Hex WT also blocks activating Fc receptors, it showed only a slight ADCP rate. Blockade of FcγR2b by Hex 2b18KQS and the same blockade by Hex 2b18KQS-ST showed increased ADCP rates compared to Her WT alone.

[0232] All of the methods disclosed and claimed herein can be practiced and executed without undue experimentation from the perspective of the present disclosure. Although the compositions and methods of the present invention have been described in connection with certain preferred embodiments, it will be apparent to those skilled in the art that changes can be applied to the methods described herein, to the steps of such methods, or to the order of the steps of such methods without departing from the concept, spirit, and scope of the present invention. More specifically, it is obvious that both any chemically related agent and any physiologically related agent can be used in place of the agents described herein, and in so doing, the same or similar results can be achieved. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0233] References The following references are specifically incorporated herein by reference to the extent that they provide details of exemplary procedures or other details that supplement the details shown herein. TIFF2025519556000026.tif177128TIFF2025519556000027.tif21734TIFF2025519556000028.tif21750TIFF2025519556000029.tif215146TIFF2025519556000030.tif217135TIFF2025519556000031.tif217146TIFF2025519556000032.tif217146TIFF2025519556000033.tif217121TIFF2025519556000034.tif217146TIFF2025519556000035.tif217146TIFF2025519556000036.tif217146TIFF2025519556000037.tif30128

Claims

1. A polypeptide comprising a human IgG variant Fc domain or variant Fc domain capable of binding to human FcγRIIb, The human IgG variant Fc domain or variant Fc domain includes substitution mutations where the domain is valine at position 233 (E233V), leucine at position 239 (S239L), proline at position 268 (H268P), leucine at position 327 (A327L), and alanine at position 328 (L328A), as well as substitution mutations at positions 234 (L234) and 235 (L235). The amino acid position numbering follows the Kabat system. The polypeptide.

2. The polypeptide according to claim 1, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is glycine at position 298 (S298G) and alanine at position 299 (T299A).

3. The polypeptide according to claim 1, wherein the substitution mutation at position 234 is proline at position 234 (L234P) or aspartic acid at position 234 (L234D).

4. The polypeptide according to claim 3, wherein the substitution mutation at position 234 is aspartic acid (L234D) at position 234.

5. The polypeptide according to claim 1, wherein the substitution mutation at position 235 is threonine at position 235 (L235T) or phenylalanine at position 235 (L235F).

6. The polypeptide according to claim 5, wherein the substitution mutation at position 235 is phenylalanine (L235F) at position 235.

7. The polypeptide according to claim 1, wherein the substitution mutation at position 234 is proline at position 234 (L234P), and the substitution mutation at position 235 is threonine at position 235 (L235T).

8. The polypeptide according to claim 7, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is aspartic acid (S267D) at position 267.

9. The polypeptide according to claim 8, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is glutamine (I332Q) at position 332 and / or valine (K334V) at position 334.

10. The polypeptide according to claim 9, wherein the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc V8.2 (SEQ ID NO:2).

11. The polypeptide according to claim 1, wherein the substitution mutation at position 234 is aspartic acid at position 234 (L234D), and the substitution mutation at position 235 is phenylalanine at position 235 (L235F).

12. The polypeptide according to claim 11, further comprising one, two, three, or all of the substitutional mutations in the human IgG variant Fc domain or variant Fc domain, which are arginine at position 236 (G236R), aspartic acid at position 237 (G237D), histidine at position 330 (A330H), and / or isoleucine at position 333 (E333I).

13. The polypeptide according to claim 12, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is aspartic acid (S267D) at position 267.

14. The polypeptide according to claim 13, wherein the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc 2B18K (SEQ ID NO:3).

15. The polypeptide according to claim 13, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is glutamine (R292Q) at position 292.

16. The polypeptide according to claim 15, wherein the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc 2B18KQ (SEQ ID NO:4).

17. The polypeptide according to claim 12, further comprising a substitution mutation in which the human IgG variant Fc domain or variant Fc domain is glutamine (R292Q) at position 292.

18. The polypeptide according to claim 17, wherein the human IgG variant Fc domain or variant Fc domain comprises or consists of Fc 2B18KQS (SEQ ID NO: 5).

19. The polypeptide according to claim 1, wherein the human IgG variant Fc domain or variant Fc domain is not glycosylated.

20. The polypeptide according to claim 1, wherein the human IgG variant Fc domain or variant Fc domain is glycosylated.

21. The polypeptide according to claim 1, wherein the Fc domain does not selectively or detectably bind to one, two, three, four, or all of the human FcγRI, FcγRIIa H131, FcγRIIIa F158, FcγRIIIa V158, and / or C1q polypeptides.

22. The polypeptide according to claim 21, wherein the Fc domain does not selectively or detectably bind to human FcγRI.

23. The polypeptide according to claim 22, wherein the Fc domain does not selectively or detectably bind to any of human FcγRIIa H131, FcγRIIIa F158, and FcγRIIIa V158.

24. The Fc domain has a binding affinity of at most 1 / 30 or 1 / 40 compared to wild-type Fc, FcγRIIa R131 The polypeptide according to claim 1, having binding ability to a target.

25. The polypeptide according to claim 3, wherein the Fc domain further comprises a substitution mutation at position 298 and / or a substitution mutation at position 299.

26. The polypeptide according to claim 25, wherein the Fc domain contains leucine (T299L) at amino acid position 299.

27. The polypeptide according to claim 3, wherein the Fc domain contains serine at position 298 and threonine at position 299.

28. The polypeptide according to claim 1, further comprising a domain that binds to a receptor other than the Fc receptor (FcR).

29. The polypeptide according to claim 28, wherein the domain that binds to a domain other than FcR is an Ig variable domain, an antibody variable domain, or an antibody heavy chain variable domain.

30. The polypeptide according to claim 29, which is a full-length antibody.

31. The polypeptide according to claim 30, which is an agonist antibody.

32. The polypeptide according to claim 31, wherein the agonist antibody is an anti-CD40 agonist antibody.

33. The polypeptide according to claim 29, wherein the Ig variable domain includes an antibody heavy chain variable domain.

34. The polypeptide according to claim 33, wherein the Ig variable domain is contained in a single-domain antibody.

35. The polypeptide according to claim 29, wherein the Ig variable domain constitutes ScFv.

36. The polypeptide according to claim 1, wherein the human IgG variant Fc domain or variant Fc domain is contained in a multimeric oligomer.

37. The polypeptide according to claim 36, wherein the multimeric oligomer is further defined as a hexamer-type Fc fusion protein.

38. The aforementioned hexamer-type Fc fusion protein The polypeptide according to claim 37, comprising:

39. The aforementioned hexamer-type Fc fusion protein The polypeptide according to claim 38, comprising:

40. The polypeptide according to claim 37, wherein the human IgG variant Fc domain or variant Fc domain comprises a substitution mutation in which the Leu residue is at position 309, and the amino acid position numbering follows the Kabat system.

41. The polypeptide according to claim 37, wherein the hexamer-type Fc fusion protein is glycosylated.

42. The polypeptide according to claim 37, wherein the hexamer-type Fc fusion protein is not glycosylated.

43. The polypeptide according to claim 37, wherein the human IgG variant Fc domain or variant Fc domain comprises a Gly residue at position 298 and / or an Ala residue at position 299.

44. The polypeptide according to claim 37, wherein the human IgG mutant Fc domain or variant Fc domain comprises a Ser residue at position 298 and / or a Thr residue at position 299.

45. The polypeptide according to claim 37, wherein the hexamer-type Fc fusion protein comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, or SEQ ID NO:

13.

46. The polypeptide according to claim 45, wherein the hexamer-type Fc fusion protein comprises SEQ ID NO:7 or SEQ ID NO:

8.

47. The polypeptide according to claim 1, wherein the polypeptide is chemically conjugated with or covalently bonded to a toxin.

48. The polypeptide according to claim 28, wherein the region that binds to a protein other than FcR binds to a cell surface protein.

49. The polypeptide according to claim 28, wherein the region that binds to a protein other than FcR binds to a soluble protein.

50. The polypeptide according to claim 28, wherein the domain that binds to a domain other than FcR constitutes a single-domain antibody, scFv, or nanobody.

51. The polypeptide according to claim 1, which is not glycosylated.

52. The polypeptide according to claim 1, which is glycosylated.

53. The polypeptide according to claim 1, wherein the Fc domain does not cause or essentially does not cause antibody-mediated phagocytosis.

54. The polypeptide according to claim 1, wherein the Fc domain does not cause or essentially does not cause antibody-mediated cytotoxicity.

55. The polypeptide according to claim 1, wherein the Fc domain does not induce, or essentially does not induce, activated FcγR.

56. A nucleic acid encoding any of the polypeptides described in claims 1 to 55.

57. The nucleic acid according to claim 56, which is a DNA segment.

58. The nucleic acid according to claim 56, which is an expression vector.

59. A host cell comprising the nucleic acid described in claim 56.

60. A host cell according to claim 59 that expresses the nucleic acid.

61. The host cell according to claim 59, which is a eukaryotic cell.

62. The host cell according to claim 59, which is a mammalian cell, an insect cell, or a yeast cell.

63. A method for preparing an unglycosylated polypeptide, including the following steps: a) The step of obtaining the host cell described in claim 60; b) Incubating the host cells under conditions that promote the expression of unglycosylated polypeptides during culture; and c) A step of purifying the expressed polypeptide from the host cell.

64. The method according to claim 63, wherein the host cell is a eukaryotic cell.

65. The method according to claim 64, wherein the host cell is a mammalian cell, an insect cell, or a yeast cell.

66. A pharmaceutical preparation comprising a polypeptide according to any one of claims 1 to 55 in a pharmaceutically acceptable carrier.

67. A pharmaceutical preparation comprising the nucleic acid described in claim 56 in a pharmaceutically acceptable carrier.

68. A pharmaceutical product comprising an antibody for use in a method of binding to a protein in a mammalian subject, A pharmaceutical product comprising the step of administering the antibody to a subject, wherein the antibody is bound to the protein, and the antibody comprises the polypeptide described in any one of claims 1 to 55.

69. The pharmaceutical product according to claim 68, wherein the antibody can specifically bind to human FcγRIIb, and the antibody has reduced binding affinity to one or more activated Fcγ receptors compared to the human wild-type IgG Fc domain.

70. The pharmaceutical product according to claim 69, wherein the antibody is not glycosylated.

71. The pharmaceutical product according to claim 69, wherein the antibody is glycosylated.

72. The pharmaceutical product according to claim 68, wherein, after the administration step, the antibody does not, or essentially does not, cause antibody-mediated phagocytosis in the subject.

73. The pharmaceutical product according to claim 68, wherein the target mammal is a human.

74. The aforementioned antibody has an affinity of at most about 1 / 30 or about 1 / 40 compared to wild-type Fc, and in the aforementioned subject, FcγRIIa R131 The pharmaceutical agent according to claim 68, which binds to a receptor.

75. The pharmaceutical product according to claim 68, wherein the antibody does not selectively or detectably bind to one or more activated human Fcγ receptor polypeptides in the subject.

76. The pharmaceutical product according to claim 74, wherein the activated human Fcγ receptor polypeptide is FcγRI, FcγRIIa H131, FcγRIIIa F158, and / or FcγRIIIa V158.

77. The pharmaceutical product according to claim 68, wherein the antibody does not specifically or detectably bind to one or more types of activated human C1q.

78. The pharmaceutical product according to claim 68, wherein the antibody is an unglycosylated version of a therapeutic antibody.

79. A pharmaceutical preparation according to claim 66 for use in a method of treating a subject having a disease.

80. The pharmaceutical product according to claim 72, which does not induce antibody-dependent cell-mediated cytotoxicity.

81. The pharmaceutical preparation according to claim 79, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

82. The pharmaceutical preparation according to claim 79, wherein the subject is a human patient.

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