Modified FcRIIB selective IgG1 Fc variants and uses thereof

Abstract

In some aspects, mutant or variant Fc domains are provided that can exhibit increased affinity or selectivity for Fc [gamma] RIIB. The variant Fc domain may be a mutant IgG1 Fc domain. In some embodiments, the mutant or variant Fc domain may be present in a therapeutic antibody, such as an agonistic antibody. Additional methods of using and identifying the mutant Fc domains are also provided.

Description

[0001] priority

[0002] 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, both of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] This application contains a sequence listing XML that has been submitted electronically and is hereby incorporated by reference in its entirety. The XML sequence listing was created on June 6, 2023, is named CLFRP0495WO.xml, and is 15,515 bytes in size. Background of the Invention 1. Technical Field

[0006] The present invention relates generally to the field of protein engineering. More particularly, it relates to improved compositions of Fc antibody domains that confer high binding to FcγRIIB and altered effector functions. 2. Background technology

[0007] A wide variety of antibodies have been developed for therapeutic purposes. Currently, the top 25 recombinant therapeutic antibodies on the market have sales of well over $43.5 billion / year. The expected annual growth rate from 2010 to 2015 is 9.2%, and is expected to increase to $62.7 billion / year by 2015 (Elvin et al., 2013). Monoclonal antibodies (mAbs) make up the majority of recombinant proteins currently in the clinic, with 1064 products in company-sponsored clinical trials in the United States or the European Union, of which 164 are in Phase III (Elvin et al., 2013). The mAb market is primarily focused on oncology and inflammatory conditions, and products within these therapeutic areas will continue to be key growth drivers during the forecast period. As a group, genetically engineered mAbs generally have a higher probability of FDA approval success than small molecule drugs. At least 50 biotechnology companies and all major pharmaceutical companies have established active antibody discovery programs. The original method for isolating and producing mAbs was first reported by Milstein and Kohler in 1975 (Kohler and Milstein, 1975). It involves the fusion of mouse lymphocytes with myeloma cells to produce mouse hybridomas. Therapeutic mouse mAbs entered clinical research in the early 1980s; however, problems of insufficient efficacy and rapid clearance due to the production of human anti-mouse antibodies (HAMA) in patients became apparent. These and the time and cost associated with the technology became the driving force for the development of mAb production technology. Polymerase Chain Reaction (PCR) facilitated the cloning of monoclonal antibody genes directly from lymphocytes of immunized animals, as well as the expression of antibody fragment combinatorial libraries in bacteria (Orlandi et al., 1989). Later libraries were generated entirely by in vitro cloning techniques using naive genes with rearranged 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 no longer depends on the immunogenicity of the corresponding antigen. These advantages have promoted the development of antibody fragments against several unique antigens, including small molecule compounds (haptens) (Hoogenboom and Winter, 1992), molecular complexes (Chames et al., 2000), unstable compounds (Kjaer et al., 1998) and cell surface proteins (Desai et al., 1998).

[0008] A method for screening large antibody combinatorial libraries to identify clones that bind to ligands with desired affinity involves expressing and displaying antibody fragments or full-length antibodies on bacterial cells and more specifically E. coli. Cells displaying antibodies or antibody fragments are incubated with fluorescently labeled ligand solutions, and those cells that bind to ligands by virtue of the antibodies displayed on their surfaces are separated by flow cytometry. In particular, Anchored Periplasmic Expression (APEx) is based on anchoring antibody fragments to the periplasmic surface of the E. coli inner membrane, then destroying the outer membrane, incubating with fluorescently labeled targets, and sorting spheroplasts (U.S. Patent No. 7,094,571, Harvey et al., 2004; Harvey et al., 2006).

[0009] Receptors for the Fc domain of antibodies are expressed on different immune cells and are important in both promoting and regulating immune responses to antibody-antigen complexes (called immune complexes). The Fc region of an antibody that has formed an immune complex with a pathogenic target cell binds to different Fc receptors expressed on the surface of leukocytes to induce antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) or complement-mediated reactions, including complement-dependent cytotoxicity (CDC).

[0010] In humans, for IgG class antibodies, there are two major classes of FcγRs: activating receptors, characterized by the presence of a cytoplasmic immunoreceptor tyrosinebased activation motif (ITAM) sequence associated with the receptor, and inhibitory receptors, 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 an activating or pro-inflammatory response, whereas the inhibitory FcγRIIB induces an anti-inflammatory or inhibitory response. Of the activating FcγRs, FcγRIIA and FcγRIIIA have natural allotypes that can affect IgG binding capacity. FcγRIIA H131showed a higher R131 Higher binding affinity to IgG and FcγRIIIA V158 showed a higher F158 Higher binding affinity for IgG. All naturally occurring antibodies and recombinant glycosylated antibodies produced in tissue culture contain an Fc domain that binds to both activating and inhibitory FcγRs (Boruchov et al. 2005; Kalergis et al., 2002).

[0011] As described above, aglycosylated 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 with therapeutic antibodies (e.g., agonistic antibodies), there is a clear need for new Fc domains, such as aglycosylated Fc domains that can selectively bind to FcγRIIB. Summary of the invention

[0012] The present disclosure overcomes the limitations of the prior art by providing Fc domain variants that selectively bind to FcγRIIB without inducing activating FcγRs or triggering pro-inflammatory effector functions such as antibody-mediated phagocytosis or cytotoxicity (ADCP and ADCC, respectively). Compared to previously generated Fc mutants, in some embodiments, the engineered Fc provided herein confers precise selective binding to FcγRIIB, as well as binding to activating receptors (FcγRI, FcγRIIa). H131 , FcγRIIa R131 , FcγRIIIa F158 , FcγRIIIa V158 ) and C1q do not bind or significantly weaken the binding. In addition, the FcγRIIB-specific mutants disclosed herein bind to the neonatal receptor FcRn in a pH-dependent manner comparable to the wild-type Fc domain. The mutant Fc domain may be glycosylated or aglycosylated. The ability of the mutant Fc to selectively bind to FcγRIIB without inducing activating FcγRs or causing antibody-mediated phagocytosis may be particularly advantageous for a variety of treatments, including, for example, therapeutic applications that require minimizing or inhibiting antibody-mediated inflammatory effects caused by binding of activating FcγRs or complement. The disclosed mutant Fc that selectively binds to FcγRIIB comprises SEQ ID NOs: 2 to 5, preferably SEQ ID NO: 5. Also provided are hexameric constructs (e.g., such as Fig.11as shown; SEQ ID NOs: 7 to 8 and 12 to 13, preferably SEQ ID NO: 7 or SEQ ID NO: 8), and related methods using such constructs.

[0013] Some aspects of the present disclosure relate to polypeptides comprising mutant or variant human IgG Fc domains capable of binding to human FcγRIIb, wherein the mutant or variant human IgG Fc domain comprises substitution mutations of valine (E233V) at position 233, leucine (S239L) at position 239, proline (H268P) at position 238, leucine (A327L) at position 327, alanine (L328A) at position 328, and substitution mutations at positions 234 (L234) and 235 (L235); wherein the amino acid position numbering is according to the Kabat system. The mutant or variant human IgG Fc domain may preferably further comprise substitution mutations of glycine (S298G) at position 298 and alanine (T299A) at position 299. The substitution mutation at position 234 may be a proline at position 234 (L234P) or an aspartic acid at position 234 (L234D). In some embodiments, the substitution mutation at position 234 is an aspartic acid at position 234 (L234D). The substitution mutation at position 235 may be a threonine at position 235 (L235T) or a phenylalanine at position 235 (L235F). In some embodiments, the substitution mutation at position 235 is a phenylalanine at position 235 (L235F). In some embodiments, the substitution mutation at position 234 is a proline at position 234 (L234P), and wherein the substitution mutation at position 235 is a threonine at position 235 (L235T). The mutant or variant human IgG Fc domain may further comprise a substitution mutation of an aspartic acid at position 237 (S267D). In some embodiments, the mutant or variant human IgG Fc domain further comprises a substitution mutation of glutamine (I332Q) at position 332 and / or valine (K334V) at position 334. In some embodiments, the mutant or variant human IgG 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 (L234D) at position 234, and wherein the substitution mutation at position 235 is phenylalanine (L235F) at position 235. The mutant or variant human IgG Fc domain may also comprise 1, 2, 3 or all of the following: substitution mutations of arginine (G236R) at position 236, aspartic acid (G237D) at position 237, histidine (A330H) at position 330 and / or isoleucine (E333I) at position 333. The mutant or variant human IgG Fc domain may further comprise a substitution mutation of aspartic acid (S267D) at position 267. In some embodiments, the mutant or variant human IgG Fc domain comprises or consists of Fc 2B18K (SEQ ID NO: 3).The mutant or variant human IgG Fc domain may also include a replacement mutation of glutamine (R292Q) at position 292. The mutant or variant human IgG Fc domain may include Fc 2B18KQ (SEQ ID NO: 4) or consist of it. The mutant or variant human IgG Fc domain may also include a replacement mutation of glutamine (R292Q) at position 292. The mutant or variant human IgG Fc domain may include Fc 2B18KQS (SEQ ID NO: 5) or consist of it. In some embodiments, the mutant or variant human IgG Fc domain is aglycosylated. In some embodiments, the mutant or variant human IgG Fc domain is glycosylated. In some embodiments, the Fc domain does not selectively or detectably bind to 1, 2, 3, 4, or all of the following: human FcγRI, FcγRIIa H131, FcγRIIa F158, and / or 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 selectively or detectably bind to any of human FcγRIIa H131, FcγRIIIa F158, and FcγRIIIa V158. In some embodiments, the Fc domain binds to FcγRIIa as compared to wild-type Fc binding. R131 The binding is at least 30 times or 40 times lower. The Fc domain may also include a substitution mutation at position 299 (e.g., a leucine at amino acid position 299 (T299L)) and / or a substitution mutation at position 298. In some embodiments, the Fc domain includes a serine at position 298 and a threonine at position 299. The polypeptide may also include a non-Fc receptor (non-FcR) binding domain. The non-FcR binding domain can 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 agonist antibody (e.g., an anti-CD40 agonist antibody). The Ig variable domain may include an antibody heavy chain variable domain. The Ig variable domain may be contained in a single domain antibody. The Ig variable domain may include a ScFv. A mutant or variant human IgG Fc domain may be contained in a multimeric oligomer (e.g., a hexameric Fc fusion protein). The hexameric Fc fusion protein may include

[0014]

[0015] As shown in the following examples, these peptides can be used to promote the formation of hexameric Fc fusion proteins. In some preferred embodiments, the hexameric Fc fusion protein comprises PTLYNVSLVMSDTAGTCY (SEQ ID NO: 6). The mutant or variant human IgG Fc domain may comprise a substitution mutation of the Leu residue at position 309, wherein the amino acid position numbering is performed according to the Kabat system. The hexameric Fc fusion protein may be glycosylated or aglycosylated. In some embodiments, the mutant or variant human IgG Fc domain comprises a Gly residue at position 298 and / or an Ala residue at position 299. In some embodiments, the mutant or variant human IgG Fc domain comprises a Ser residue at position 298 and / or a Thr residue at position 299. In some preferred embodiments, the hexameric Fc fusion protein comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 13, and the hexameric Fc fusion protein may even more preferably comprise SEQ ID NO: 7 or SEQ ID NO: 8. The polypeptide or antibody may be chemically conjugated or covalently bound to the toxin. The non-FcR binding region may bind to a cell surface protein. In some embodiments, the non-FcR binding region binds to a soluble protein. The non-FcR binding domain may comprise a single domain antibody, scFv or nanobody. The polypeptide may be aglycosylated or glycosylated. In some embodiments, the Fc domain does not trigger or substantially does not trigger antibody-mediated phagocytosis. In some embodiments, the Fc domain does not trigger or substantially does not trigger antibody-mediated cytotoxicity. In some embodiments, the Fc domain does not cause or substantially does not cause induction of activating FcγRs.

[0016] Another aspect of the present disclosure relates to nucleic acids encoding any polypeptide described above or herein. The nucleic acid can be a DNA segment. In some embodiments, the nucleic acid is an expression vector.

[0017] Another aspect of the present disclosure relates to a host cell comprising the nucleic acid described herein above. The host cell can express the nucleic acid. The host cell can be a eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an insect cell, or a yeast cell.

[0018] Another aspect of the present disclosure relates to a method for preparing an aglycosylated polypeptide, comprising: a) obtaining a host cell as described above or herein; b) incubating the host cell in culture under conditions that promote expression of the aglycosylated polypeptide; and c) purifying the expressed polypeptide from the host cell. The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, or a yeast cell).

[0019] Yet another aspect of the present disclosure relates to a pharmaceutical formulation comprising a polypeptide as described above or herein or a nucleic acid as described above or herein in a pharmaceutically acceptable carrier.

[0020] Another aspect of the present disclosure relates to a method of binding a protein in a mammalian subject, comprising administering an antibody to the subject, wherein the antibody binds to the protein and comprises an Fc domain as described above or herein. In some embodiments, the antibody is capable of specifically binding to human FcγRIIb, and wherein the binding of the antibody to one or more activating Fcγ receptors is reduced compared to a human wild-type IgG Fc domain. The antibody may be aglycosylated or glycosylated. In some embodiments, the antibody does not cause or substantially does not cause antibody-mediated phagocytosis in the subject after administration. In some embodiments, the mammalian subject is a human. In some embodiments, in the subject, the antibody binds to FcγRIIa with an affinity at least about 30 times lower or about 40 times lower than that of a wild-type Fc. R131 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, FcγRIIa 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 therapeutic antibody in an aglycosylated form.

[0021] Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease, comprising administering to the subject an effective amount of a formulation as described above or herein. In some embodiments, the method does not induce antibody-dependent cellular toxicity. The disease can be cancer, infection, or autoimmune disease. In some embodiments, the subject is a human patient.

[0022] In some embodiments, a mutant or variant Fc domain is provided that exhibits, compared to a corresponding wild-type Fc domain: selective binding to FcγRIIB, and (ii) reduced or no detectable binding to all activating human Fcγ receptors (i.e., FcγRI, FcγRIIa H131, FcγRIIa R131, FcγRIIIaF158, or FcγRIIIaV158) and C1q. In some preferred embodiments, the mutant or variant Fc domain is a human mutant or variant Fc domain. The mutant or variant Fc domain may be contained or included in a polypeptide, such as an antibody or fusion protein. In some embodiments, the mutant or variant Fc domain may be contained in a therapeutic antibody, such as an agonist antibody. In some embodiments, there is a composition involving a polypeptide having an aglycosylated Fc domain from a human IgG1 antibody ("antibody Fc domain"). In some embodiments, the Fc domain is a variant of a human IgG1 Fc domain (SEQ ID NO: 1) that is capable of highly selectively binding only to FcγRIIB and not to any effector Fc receptors, namely FcγRI, FcγRIIA and FcγRIIIA. When the Fc domain is expressed in both glycosylated and aglycosylated forms, the Fc domain can highly selectively bind to FcγRIIB and show little or no binding to effector Fc receptors.

[0023] Another aspect of the present invention relates to a pharmaceutically acceptable composition comprising a polypeptide of the present invention and a pharmaceutically acceptable excipient.

[0024] Yet another aspect of the 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 disclosure. In some embodiments, the disease is cancer, infection, bacterial infection, viral infection, or autoimmune disease.

[0025] The antibody Fc domain can be an Fc domain of an IgG antibody or a variant thereof. In addition, the antibody Fc domain can be limited to a human Fc domain. In certain aspects, the Fc domain can be an IgG1 Fc domain, such as an Fc domain of an anti-HER2 antibody, more specifically, an Fc domain of trastuzumab and an Fc domain of an anti-CD20 antibody, more specifically, an Fc domain of rituximab. It is also contemplated that the polypeptide may include a fusion of a modified Fc domain disclosed herein that is fused to a polypeptide that is not derived from an antibody molecule.

[0026] In some cases, the antibody may have a substitution at amino acid position 297 or 299 that impairs N-linked glycosylation when the antibody is expressed in a mammalian cell that recognizes a glycosylation motif in the Fc domain of the antibody. It is contemplated that any mutation in amino acid position 297 or 299 that is known to eliminate glycosylation (e.g., WO2005018572A2) may be used, including, for example, substitution of 299T with a leucine residue.

[0027] In some embodiments, there are multiple amino acid substitutions at one or more positions from the following list: (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 aglycosylated antibody Fc domain described herein may comprise a substitution of alanine at amino acid position 264 (V264A), serine at amino acid position 328 (L328S), cysteine ​​at amino acid position 329 (P329C), tryptophan at amino acid position 330 (A330W), asparagine at amino acid position 332 (I332N), glycine at amino acid position 333 (E333G), valine at amino acid position 336 (I336V), or a combination thereof. The engineered Fc domain may also include one or more substitutions at amino acids 234, 235, 236, 238, and 351; and in some embodiments, the substitution at amino acid 234 is arginine (L234R), the substitution at amino acid 235 is glutamic acid (L235E), the substitution at amino acid 236 is glutamic acid (G236E), the substitution at amino acid 238 is arginine (P238R), and the substitution at amino acid 351 is glutamine (L351Q). In some embodiments, the engineered Fc domain includes an additional amino acid substitution at residue 311, such as lysine (i.e., Q311K) in some preferred embodiments. Combinations of substitution mutations may be present in the mutant or variant Fc domains of the present disclosure. The mutant or variant human IgG Fc domain may comprise 1, 2, 3 or 4 of the following: substitution mutations 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 in combination with 1, 2 or 3 of the following: tryptophan at amino acid position 330 (A330W), asparagine at amino acid position 332 (I332N) and serine at amino acid position 328 (L328S); optionally in combination with threonine at position 299 (T299L). In some embodiments, the variant Fc domain may comprise 1, 2, 3, 4, 5, 6 or all of the following: V264A, L328S, P329C, A330W, I332N, E333G, I336V mutations, optionally in combination with a mutation at position 299, e.g., T299L.In some embodiments, the variant Fc domain may comprise 1, 2, 3, 4, 5 or all of the following: L234R, L235E, G236E, P238R, T299L, and / or L351Q mutations. In some embodiments, the variant Fc domain comprises a Q311K mutation, optionally in combination with a T299L mutation. The engineered antibody Fc domains described herein may also comprise one or more amino acid substitutions disclosed in U.S. Pat. No. 10,526,408.

[0028] Variant Fc domain polypeptides (also referred to as mutants or engineered Fc domains) can be characterized as having a certain percentage of identity compared to an unmodified polypeptide (e.g., a wild-type Fc domain polypeptide, such as a wild-type IgG Fc domain or a human wild-type IgG Fc domain) or any polypeptide sequence disclosed herein. The percentage identity between the unmodified portion of the modified polypeptide (i.e., the sequence of the modified polypeptide excluding any specified substitutions) and the corresponding wild-type polypeptide can be about at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% (or any range derivable therein). For example, for a region of a variant Fc domain excluding a specified substitution mutation (e.g., a substitution mutation at position 299 (e.g., T299L) in addition to any other specified substitution mutations), the variant Fc domain may have, for example, at least 90% (or at least about 95%, etc.) sequence identity compared to a wild-type Fc domain (e.g., a wild-type human Fc domain). In addition to the substitution mutations specified in the mutant Fc domain, the variant Fc domain may include additional mutations compared to the wild-type Fc domain. It is also contemplated that the percentage of identity discussed above may relate to the entirety of a variant Fc domain polypeptide compared to a wild-type Fc domain (e.g., a human IgG Fc domain). For example, a variant Fc domain polypeptide characterized in 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.

[0029] The antibody Fc domain may be an Fc domain of a human IgG antibody or a variant thereof. In certain aspects, the Fc domain may be an IgG1 Fc domain. It is also contemplated that the polypeptide may comprise a fusion of a modified variant Fc domain disclosed herein fused to a polypeptide not derived from an antibody molecule. In some embodiments, the modified Fc domain of the present invention is included in an agonistic antibody, such as an antibody targeting CD40, death receptor 5 (DR5) or TNF receptor (TNFR) molecules.

[0030] In some embodiments, the polypeptide comprising the variant Fc domain described herein may include a linker. In other embodiments, the linker is a conjugated linker. In some embodiments, the polypeptide comprises an Fc domain from an antibody. It may include other regions from an antibody, such as another binding domain. The additional binding domain may not be an FcR binding domain. In some embodiments, the polypeptide may include an antigen binding site or domain from an antibody, such as all or part of the variable region of an antibody. The polypeptide may include an Fc domain from an antibody and another binding domain that is a non-FcR binding domain. In some embodiments, the non-Fc binding region is not an antigen binding site of an antibody, but specifically binds to a cell surface protein or a soluble protein. In some cases, the cell surface protein recognized by the non-Fc binding region is a receptor, such as a receptor expressed on the cell surface.

[0031] Other polypeptides include those having aglycosylated variant Fc domains (e.g., capable of binding to FcγRIIb polypeptides while exhibiting reduced or eliminated binding to activating FcRs) and a second binding domain that is a non-Fc receptor binding domain, wherein the second binding domain is capable of specific binding to a cell surface molecule or a soluble protein. In some embodiments, the second binding domain is an antigen binding domain of an antibody ("Ig variable domain"). In some aspects, the polypeptide may be a full-length antibody. In some cases, the second binding domain is not an antibody antigen binding domain. In some embodiments, the second binding domain is capable of specific binding to a cell surface molecule that is a protein or protein molecule. In some aspects, the second binding domain is capable of specific binding to a soluble protein.

[0032] Some aspects relate to nucleic acids encoding any polypeptide discussed herein. The nucleic acid may be isolated and / or recombinant. It may be an isolated and / or recombinant nucleic acid segment. In some embodiments, the nucleic acid is DNA, and in other embodiments, 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 polypeptide having an Fc binding domain containing one or more replacements that specifically binds to FcγRIIb. The nucleic acid may encode one or more polypeptides herein, which may be glycosylated or may not be glycosylated, depending on the presence or absence of certain mutations and the manner in which the polypeptide is produced.

[0033] In some embodiments, the nucleic acid encodes a polypeptide comprising or consisting of: a variant or mutant Fc domain capable of selectively binding to FcγRIIb as described herein. The nucleic acid can be placed (e.g., transfected or transformed) into a host cell that can express a polypeptide (e.g., a polypeptide without glycosylation). The host cell can be a prokaryotic cell, such as a bacterial cell. Alternatively, the host cell can be a eukaryotic cell, such as a mammalian cell. In some embodiments, the host cell comprises a first expression vector, but it can also comprise a second expression vector. Because some antibodies are composed of a variety of polypeptides, in some embodiments, a host cell comprising an expression vector required for expressing a polypeptide can be utilized. For example, in some embodiments, the host cell comprises a second expression vector, which encodes a polypeptide comprising an immunoglobulin light chain or consisting of it. In some embodiments, the host cell expresses a first expression vector, which encodes a polypeptide comprising an immunoglobulin heavy chain (e.g., comprising a variant or mutant Fc domain that selectively binds to FcγRIIb) or consisting of it. The host cell may comprise, for example, one or two expression vectors to allow the expression of antibodies comprising heavy and light chains.

[0034] In some aspects, a population of host cells is provided, wherein the population comprises a plurality of host cells expressing polypeptides having different Fc domains. It is expected that the identity of the amino acid sequences of any two different Fc domains may differ by less than 20%, 15%, 10%, 5% or less.

[0035] In some aspects, methods of making the polypeptides described herein (e.g., polypeptides having an aglycosylated Fc region that can selectively bind to FcγRIIb) and methods of using these polypeptides are provided. It is contemplated that methods described herein or known to a person of ordinary skill can produce or use any polypeptide described herein.

[0036] In some embodiments, the method for preparing an aglycosylated polypeptide comprises: a) obtaining a host cell capable of expressing an aglycosylated polypeptide comprising an Fc domain capable of selectively binding to FcγRIIb described herein; b) incubating the host cell in culture under conditions that promote expression of the aglycosylated polypeptide; and c) purifying the expressed polypeptide from the host cell. In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. In other embodiments, the host cell is a eukaryotic cell and the polypeptide comprises a substitution mutation at position 299 (e.g., T299L) of a variant or mutant IgG Fc domain. In other embodiments, the method involves collecting the expressed variant polypeptide (e.g., from the supernatant), which can be done before purification.

[0037] In some embodiments, the method involves purifying the polypeptide from the supernatant. This may involve subjecting the polypeptide from the supernatant to filtration, HPLC, anion or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof. In some embodiments, the method involves affinity chromatography using staphylococcal protein A that binds to the IgG Fc region. Other purification methods are well known to those of ordinary skill in the art.

[0038] In some embodiments, a pharmaceutical formulation comprising a polypeptide or nucleic acid of the disclosure in a pharmaceutically acceptable carrier or comprising an excipient is provided.

[0039] In some embodiments, an immune response can be induced in a subject by a method comprising providing or administering (e.g., intravenously, etc.) an antibody to a subject, wherein the antibody is aglycosylated and comprises an Fc domain that selectively binds to FcγRIIb as described herein. In some aspects, the aglycosylated antibody may be able to specifically bind to human FcγRIIb. In some aspects, the aglycosylated antibody may be able to specifically bind to any activating FcγR polypeptide at a level at least 30 or 40 times lower than that of a wild-type human IgG1 antibody. In some embodiments, the antibody may comprise a mutant Fc domain provided herein that does not exhibit specific or detectable binding to an FcγRI polypeptide, does not stimulate antibody-mediated phagocytosis, and / or does not trigger effector functions in a mammalian host. In some aspects, the antibody may be a therapeutic antibody in a glycosylated or aglycosylated form.

[0040] In some aspects, cancer, infection, autoimmune disease or inflammatory disease can be treated by administering a therapeutic polypeptide comprising a variant or mutant Fc domain that selectively binds to FcγRIIb as described herein. Compared to the CDC induced by a polypeptide comprising a wild-type human IgG Fc region, a polypeptide comprising a mutant or variant Fc domain as described herein may exhibit reduced CDC. Compared to a wild-type human IgG antibody, a polypeptide comprising a mutant Fc domain provided herein may exhibit reduced ADCC or ADCP.

[0041] In another embodiment, therapeutic inhibition of a protein target can be achieved by an antibody comprising a variant Fc polypeptide as contemplated herein. In some embodiments involving polypeptides comprising a variant or mutant Fc domain, the polypeptide can exhibit reduced CDC compared to the CDC induced by a polypeptide comprising a wild-type human IgG Fc region, the variant or mutant Fc domain can selectively bind to inhibitory FcγRIIb while exhibiting reduced binding to activating Fc.

[0042] Provided is a method for treating an object suffering from a disease, comprising administering to the object a pharmaceutical preparation of the present disclosure of an effective amount. In some embodiments, the tumor is a solid tumor or a hematological tumor. In some aspects, the object can be a human patient. In some aspects, the pharmaceutical preparation can be administered in the following manner: intratumoral, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intraocular, intranasal, intravitreal, intravaginal, intrarectal, intramuscular, subcutaneous, subconjunctival, intracapsular (intravesicularlly), mucosa, pericardial, umbilical, oral, by inhalation, by injection, by infusion, by continuous infusion, by direct local perfusion bathing target cells, by catheter or by lavage. In some aspects, the method may also include administering to the object at least a second anticancer treatment, such as, for example, surgical treatment, chemotherapy, radiotherapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

[0043] In one embodiment, there is provided a composition comprising a variant Fc domain of an embodiment of the present invention or a nucleic acid encoding a variant Fc domain of an embodiment of the present invention, which is used to treat a disease. Treating a disease may involve combining with a selection protein to achieve a therapeutic effect (e.g., due to the binding of a toxin or the stimulation of a receptor by an agonist antibody, etc.), while producing reduced immune activation or reduced complement-dependent cytotoxicity. In some aspects, the disease may be cancer, an autoimmune disease, an inflammatory disease, or an infectious disease. In another embodiment, there is provided a polypeptide according to an embodiment of the present invention or a nucleic acid encoding a polypeptide according to an embodiment of the present invention for use in the preparation of a drug for treating a disease (e.g., cancer).

[0044] As used herein, "selectively binds to FcγRIIb" or variations thereof refers to the property that a polypeptide (e.g., an Fc domain (e.g., a mutant or variant IgG Fc domain)) has the ability to bind to FcγRIIb, and preferably the polypeptide or Fc domain has the ability to bind to FcγRIIb compared to a wild-type Fc domain (e.g., a wild-type Fc IgG domain). Binding to FcγRIIb may be comparable or reduced (e.g., reduced by 4-fold) compared to wild-type binding, but in all cases, the mutant Fc domain must exhibit detectable selective binding to FcγRIIb. In some embodiments, the Fc domain or polypeptide that selectively binds to FcγRIIb also shows significantly reduced binding compared to the wild type (e.g., a wild-type IgG Fc domain) or no detectable binding to all human activating (proinflammatory) Fcγ receptors. In some embodiments, the mutant Fc domain provided herein binds to FcRn with an affinity similar to or not significantly different from that of the wild-type Fc.

[0045] In some embodiments, the mutant or variant Fc domain provided above or herein is included in an antibody or covalently linked to an antibody fragment (e.g., a heavy chain antibody variable domain, scFv, etc.). Generally, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, single-chain antibodies (comprising a single heavy chain variable region, also referred to as VHH), humanized antibodies, deimmunized antibodies, microantibodies, diantibodies, triantibodies, and antibody fragments, such as Fab', Fab, F(ab')2, single domain antibodies, Fv or single chain Fv (single chain Fv, scFv) antibodies, single domain antibodies, and antibody mimetics, such as anticalins, and any mixture thereof. In some embodiments, single-domain antibodies (single-domainantibody, sdAb) may allow for certain advantages over full-length antibodies, such as smaller size, a greater number of accessible epitopes, and reduced production costs (e.g., Hoey et al., 2019). In some embodiments, the mutant of the variant Fc domain is covalently linked to, or expressed as a fusion protein with, a single chain antibody (scFv) or a single domain antibody.In a related aspect, the cell targeting domain can be an avimer polypeptide.

[0046] As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified components are purposefully formulated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected by standard analytical methods.

[0047] The term "affinity" as used herein refers to the equilibrium constant for the reversible binding of two substances and is expressed as K D The affinity of a binding domain to its target can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM); alternatively, it can be from 100 nM to 1 nM or from 0.1 nM to 10 nM, or any range derivable therein. In addition, when there is an affinity between two substances within the above affinity ranges, the substances are expected to bind specifically.

[0048] As used herein, the term "encoding" or variations thereof with respect to nucleic acids is used to facilitate the understanding of the present invention by those skilled in the art; however, these terms can be used interchangeably with "comprising" or variations thereof, respectively.

[0049] As used herein in the specification, a noun without a quantifier may mean one or more. As used herein in the claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one.

[0050] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, the use of the term "or / or" in the claims is used to mean "and / or / or", but the present disclosure supports a definition referring to only alternatives and "and / or / or". As used herein, "another" may mean at least a second or more.

[0051] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation for the device, the method being employed to determine the value, or the variation that exists between the subjects.

[0052] Through the following detailed description, other purposes, features and advantages of the present invention will become apparent. However, it should be understood that although the detailed description and specific examples indicate some preferred embodiments of the present invention, they are given by way of illustration only, because from this detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0054] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0055] Figure 1 : Results of opsonized SK-BR-3 and FcγR coated bead binding assays with 2b18K Fc and 2b enhanced binding Fc variants. The numbers shown refer to the fold difference in binding affinity compared to wild-type Fc as measured by BLI, and the numbers in the basket (shown in brackets in the figure) are the published values. The table shows the percentage of the positive population and the MFI (median fluorescence intensity) of the results. NA: not determined; NB: no binding.

[0056] Figure 2 : Results of opsonized SK-BR-3 and FcγR coated bead binding assays with 2b18K Fc and silent Fc variants. The percentage of positive population is shown in the figure. Error bars are standard error of the mean of triplicate samples.

[0057] Figure 3 : Raji cell binding assay with antibody coated beads. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test (***P 0.001, ****P 0.0001). Error bars are standard error of the mean of triplicate samples.

[0058] Figure 4 : Antibody-opsonized pHrodo-labeled bead phagocytosis assay with THP-1 cells as effector. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test (***P 0.001). Error bars are standard error of the mean of triplicate samples.

[0059] Figure 5 : SK-BR-3 phagocytosis assay performed with THP-1 cells. Numbers are antibody concentrations in the culture medium (ng / ml). An ET ratio of 20:1 was used. Error bars are standard error of the mean of triplicate samples. non: isotype control. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test (****P 0.0001).

[0060] Figure 6 : SK-BR-3 phagocytosis assay performed with THP-1 cells. Numbers are shown at the bottom of the figure and refer to the concentration of antibody in the culture medium (ng / ml). An ET ratio of 20:1 was used. Error bars are standard error of the mean of triplicate samples. "non" refers to isotype control.

[0061] Figure 7 : Alignment of amino acid sequences.

[0062] Figure 8 : Deposition of C1q on CD20+Raji or Ramos cells measured by FACS. Cells were opsonized with WT rituximab or Fc engineered variants. Fc variants showed low or no C1q deposition on the cell surface. Data for Fc mutants 2b18K (Fc2b-1), 2b18KQ (Fc2b-2), and 2b18KQS (Fc2b-3) are shown.

[0063] Figures 9A to E: In vitro cell-based assays. Data for Fc mutants 2b18K (Fc2b-1), 2b18KQ (Fc2b-2), and 2b18KQS (Fc2b-3) are shown. No complement or activating FcγR-mediated effector function was detected using the Fc variants 2b18K, 2b18KQ, or 2b18KQS. (Figure 9A) CD20+Raji cells or Ramos cells were lysed by complement-dependent cytotoxicity (CDC). Cells were opsonized with various concentrations of rituximab or its Fc-engineered variants and incubated with 10% pooled human serum. (Figure 9B) Antibody-dependent cellular phagocytosis assay (ADCP) using WT or Fc-engineered variants of trastuzumab, with THP-1 cells as effectors and SK-BR-3 cells as targets. (FIG. 9C) Antibody-dependent cellular cytotoxicity assay (ADCC) with peripheral blood mononuclear cells (PBMC). Target cells were Raji cells conditioned with WT rituximab or its Fc engineered variants. (FIG. 9D) ADCP assay using human monocyte-derived M1 macrophages as effector cells and SK-BR-3 cells as targets. Added various concentrations of WT trastuzumab or Fc engineered variants. (FIG. 9E) After CD20+Raji cells were incubated with WT rituximab or Fc engineered variants for 1 hour, phosphorylation of FcγRIIb was analyzed by immunoblotting. In all assays, Fc variants 2b18K, 2b18KQ, and 2b18KQS did not show any complement or activating FcγR-mediated activation, and these variants showed functional binding to FcγRIIb.

[0064] Figures 10A to D: Hexameric Fc expression. (Figure 10A) Schematic diagram of hexameric Fc. (Figure 10B) Reducing SDS-PAGE results of hexameric Fc. (Figure 10C) Non-reducing SDS-PAGE results of hexameric Fc after SEC purification. (Figure 10D) Size exclusion chromatography results of hexameric Fc.

[0065] Fig.11 : Sequence alignment showing point mutations present in different engineered variant Fc regions and hexameric wild-type Fc. Hex-WT (SEQ ID NO: 14) is shown, and mutations in Hex-2bKQS and Hex-2bKQS compared to Hex-WT are shown.

[0066] Fig.12: Binding characteristics of hexameric Fc2b. Results of FcγR coated beads and hexameric Fc binding assays performed with Hex 2b18KQS Fc and glycosylated Hex 2b18KQS-ST Fc are shown. The y-axis shows the mean fluorescence intensity.

[0067] Fig.13 : Raji cell binding assay with antibody coated beads. Error bars are standard error of the mean of triplicate samples. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test (***P 0.001, ****P 0.0001).

[0068] Fig.14 : Blocking FcgR 2b with hexameric Fc enhances phagocytosis of antibody-opsonized SKBR3 cells by THP-1 cells. y axis: percentage of ADCP, error bars are standard error of the mean of triplicate samples. DETAILED DESCRIPTION

[0069] Provided herein are methods and compositions involving polypeptides having engineered antibody Fc domains that exhibit selective binding to FcγRIIB while exhibiting reduced or eliminated effects on activating FcγRs (e.g., reduced or eliminated antibody-mediated phagocytosis, reduced or eliminated effector function). Such a polypeptide may comprise an Fc domain containing one or more substitution mutations compared to a wild-type Fc domain (SEQ ID NO: 1). In addition, some Fc domains may selectively bind to FcγRIIB but not to activating FcγRs (e.g., no detectable binding to FcγRIIaH131, FcγRIIIa, or FcγRIIIb). For example, a polypeptide may comprise an aglycosylated Fc domain that selectively binds to FcγRIIB but not detectably binds to activating FcγRs. In some embodiments, the Fc domain exhibits reduced binding to FcγRIIaR131 (e.g., 40-fold lower binding compared to WT Fc). Reduction or elimination of effector function induced by a mutant Fc may offer significant advantages for treating diseases in which such an immune response (eg, antibody-stimulated phagocytosis) is undesirable.

[0070] I. Antibody Fc domain

[0071] In a variety of different assays, the Fc domain bound by FcγRIIB of IgG has been shown to inhibit the activation of a variety of immune cells (Sidman, CL and Unanue, ER1976; Phillips, NE and Parker, DC1984). FcγRIIB is the only FcγR expressed by B cells, and if it is cross-linked with the B cell receptor (B cell receptor, BCR), the B cell activation threshold is increased, and B cell differentiation and final antibody production are reduced. In other immune cells (including dendritic cells (dendritic cell, DC), macrophages, activated neutrophils, mast cells and basophils), FcγRIIB inhibits functions mediated by activating FcγR, including phagocytosis and proinflammatory cytokine release. When expressed by follicular DC (FDC), FcγRIIB is important for capturing antigen-containing immune complexes, which are considered to be critical for driving germinal center responses (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 disease.

[0072] Importantly, binding to FcγRIIB on effector and stromal cells has been shown to be essential for the agonistic function of TNFRS therapeutic antibodies (agonistic antibodies targeting key TNF receptor (TNFR) molecules). Many TNFRS agonistic antibodies, including anti-CD40 or death receptor 5 (DR5), have been shown to be essential for immune modulation and activation. Signaling to targets such as CD40 by agonistic antibodies has been shown to rely on ligation of the antibody Fc domain through FcγRIIB expressed on neighboring cells in the microenvironment (Nimmerjahn et al. 2005; Wilson et al. 2011).

[0073] The FcγR binding site on IgG1 was determined by the co-crystal structure of the Fc fragment and the extracellular domain of FcγR. The binding site is usually located on the CH2 domain. The Asp265 to Ser267 segment in the lower hinge region of IgG1 (Leu234 to Ser239) and the CH2 domain plays a key role in the interaction with all FcγRs (Christine Gaboriaud et al., 2003 and Jenny M. Woof et al., 2004).

[0074] The CH2 domain has one N-glycosylation site at Apn297, and the N-linked glycosylation at Asn297 bridges the gap between the two CH2 domains. This bridge maintains the correct conformation of the CH2 domain for binding to FcγR. On the other hand, the removal of the glycan at Asn297 significantly improves the conformation of the CH2 domain, allowing the aglycosylated Fc to bind to FcγR with significantly reduced affinity or not at all, thereby significantly reducing ADCC, ADCP, and other biological effects mediated by Fc:FcγR interactions (Borrok et al., 2012).

[0075] In view of the importance of FcγRIIB binding to the biological function of antibodies, a lot of efforts have been made to transform the IgG1 Fc domain so that it binds to the receptor with improved affinity and / or selectivity relative to other Fcγ receptors. These efforts all involve the transformation of glycosylated IgG1 so that it binds to FcγRIIB with higher affinity, because antibodies lacking glycans at position 297 and therefore being aglycosylated do not show any binding to FcγRIIB. Two IgG1 Fc variants with significantly improved binding to FcγRIIB have been reported: the so-called "EF-Fc" variant developed by Xencor and the "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 reportedly has a 430-fold lower KD (equilibrium dissociation constant) for FcγRIIB, while the V-12 variant exhibits a 64-fold higher affinity. However, the Fc domain is selective for FcγRIIB. Specifically, relative to the authentic (wild-type) human IgG1 Fc domain, the EF Fc domain exhibits a 64-fold higher affinity for FcγRI and FcγRIIA. H131 Similar affinity to FcγRIIA R131 The V12-Fc variant reportedly has a significantly enhanced affinity for FcγRIIA relative to the native IgG1 Fc domain. R131 The affinity of H131 The affinity is reduced.

[0076] In certain embodiments, there is a composition comprising a protein molecule that has been modified relative to a natural or wild-type protein. In some embodiments, the protein compound has deleted amino acid residues; in other embodiments, the amino acid residues of the protein compound have been replaced; and in yet other embodiments, both the deletion and replacement of amino acid residues in the protein compound have been performed. In addition, the protein compound may include an amino acid molecule comprising more than one polypeptide entity. "Protein molecule", "protein composition", "protein compound", "protein chain" or "protein material" used herein generally refers to, but is not limited to, a protein of more than about 200 amino acids or a full-length endogenous sequence translated from a gene; a polypeptide of 100 or more amino acids; and / or a peptide of 3 to 100 amino acids. All of the above "protein" terms are used interchangeably herein; however, it is particularly contemplated that the embodiments may be limited to specific types of protein compounds, such as polypeptides. In addition, these terms may also be applied to fusion proteins or protein conjugates. A protein may contain more than one polypeptide. An IgG antibody, for example, has two heavy chain polypeptides and two light chain polypeptides interconnected by disulfide bonds.

[0077] Protein or peptide used herein generally refers to, but is not limited to, proteins greater than about 200 amino acids to the full-length sequence translated from a gene; polypeptides greater 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.

[0078] As used herein, "amino acid residue" refers to any amino acid, amino acid derivative, or amino acid mimetic known to those of ordinary skill in the art. In certain embodiments, the residues of a protein molecule are continuous, without any non-amino acid residues interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In some specific embodiments, the sequence of residues of a protein molecule may be interrupted by one or more non-amino acid moieties.

[0079] "Unique Fc domain" used herein can be defined as a domain that differs from another Fc by as little as one amino acid. Methods for preparing a library of nucleic acids for unique antibody Fc domains or encoding antibodies are well known in the art. For example, in some cases, the Fc domain can be amplified by error-prone PCR. In addition, in some cases, multiple antibody Fc domains may include a randomized section (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of amino acids. In some cases, specific mutations can be engineered into the Fc domain. For example, in some aspects, the residues that are usually glycosylated in the antibody Fc domain can be mutated. In addition, in some aspects, the residues (or adjacent residues) that are usually glycosylated can be used as insertion sites for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0080] The polypeptide may comprise an aglycosylated antibody Fc domain capable of binding to an FcR polypeptide. In some aspects, the aglycosylated Fc domain may be further defined as having a specific affinity for an FcR polypeptide under physiological conditions. For example, under physiological conditions, the Fc domain may have an equilibrium dissociation constant of about 10-6 M to about 10-9 M. Furthermore, in some aspects, the aglycosylated Fc domain may be defined as comprising one or more amino acid substitutions or insertions relative to a wild-type sequence (e.g., a human wild-type sequence).

[0081] Methods for preparing such polypeptides include those discussed in PCT Publication WO 2008 / 137475 (which is incorporated herein by reference). Alternatively, such polypeptides may be prepared directly by genetic engineering techniques, for example by introducing selected amino acid substitutions or insertions into a known Fc background, wherein, as described above, insertion or substitution with an aglycosylated Fc region provides improved FcR binding ability. In some embodiments, the Fc domain is engineered to bind to one or more specific Fc receptors. Additionally, or alternatively, the Fc domain may be engineered so that it does not selectively bind to one or more specific Fc receptors.

[0082] In some embodiments, the aglycosylated Fc domain comprises a specific binding affinity for an FcR, such as human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI, or for C1q. Thus, in some aspects, the aglycosylated Fc domain of the invention is defined as an Fc domain with a specific affinity for FcγRIIB. The binding affinity of an antibody Fc or other binding protein can be determined, for example, by Scatchard analysis of Munson and Pollard (1980). Alternatively, binding affinity can be determined by surface plasmon resonance or any other known method for determining the kinetics and equilibrium constants of protein: protein interactions.

[0083] The amino acid sequence of the Fc domain of an isolated IgG variant having a specific affinity for FcγRIIB exhibiting changes relative to wild-type Fc (SEQ ID NO: 1) is as follows:

[0084] Table 1: Isolated IgG variants with affinity for FcγRIIB (sequence numbering is based on Kabat and is referred to below as There is a mutation)

[0085]

[0086] Specific point mutations of mutant or variant Fc domains are listed in Table 1 above; these mutations indicate the differences between mutant or variant Fc domains and wild-type IgG Fc domains (SEQ ID NO: 1). Some aspects of the present disclosure relate to polypeptides having IgG Fc domains (e.g., aglycosylated IgG Fc domains) or nucleic acids encoding IgG Fc domains, wherein the IgG Fc domains have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the mutant or variant Fc domains of Table 1 or any range derivable therein. In some embodiments, the Fc mutants of Table 1 also include a substitution mutation at T299 (e.g., T299L), for example to allow the production of aglycosylated Fc domains in mammalian cells.

[0087] In some embodiments, the mutant of the variant Fc domain comprises or consists of Fc 2B18K:

[0088]

[0089] Or a polypeptide having a sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein.

[0090] In some embodiments, the mutant of the variant Fc domain comprises or consists of Fc 2B18KQ:

[0091]

[0092] Or a polypeptide having a sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein.

[0093] In some embodiments, the mutant of the variant Fc domain comprises or consists of Fc 2b18KQS:

[0094]

[0095] Or a polypeptide having a sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein.

[0096] In some embodiments, the mutant or variant Fc is contained in a hexameric Fc polypeptide. For example, the mutant or variant Fc (e.g., SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5) may be contained in a polypeptide comprising a human IgM μ-tailpiece (e.g., PTLYNVSLVMSDTAGTCY; SEQ ID NO: 6), which promotes the polymerization of the Fc domain into a hexameric construct. For example, in some preferred embodiments, the mutant of the variant Fc domain (e.g., any one of SEQ ID NO: 3 to 6) is expressed as a fusion construct having a human IgM μ-tailpiece (e.g., SEQ ID NO: 6) at the C-terminus of the mutant of the variant Fc domain or at the C-terminus of the polypeptide.

[0097] It is expected that a variety of polypeptides may be included in the mutant or variant Fc provided herein to produce multimeric oligomers. In some preferred embodiments, the human IgMμ-tail described in (Rowley et al., 2018; Spirig et al., 2018) is included to form a hexamer. Wherein the mutant or variant human IgG Fc domain contains a replacement mutation of the Leu residue at position 309 (according to Kabat numbering) to introduce a disulfide bond between the Fc domains and promote the formation of multimeric oligomers. Multimeric oligomers or hexamers can be glycosylated or aglycosylated. Other polypeptides that can be used to produce multimeric oligomers (e.g., when expressed with a mutant of a variant Fc provided herein) include

[0098]

[0099] It can also be used for hexamer formation, and the two mutations V567I and A572G can contribute to the stability of the hexamer structure. (Yuan, et al., 2022)

[0100] In some embodiments, the hexameric Fc polypeptide comprises or consists of Hex 2b18KQS (aglycosylated Fc):

[0101]

[0102] Or a polypeptide having a sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein.

[0103] In some embodiments, the hexameric Fc polypeptide comprises or consists of Hex 2b18KQS-ST (glycosylated):

[0104]

[0105] Or a polypeptide having a sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or any range derivable therein.

[0106] As used herein, "position" means a position in a protein sequence. Positions may be numbered sequentially, or according to an established format, such as the EU index for antibody numbering.

[0107] For all positions discussed herein, numbering is according to the EU index. The "EU index" or "EU index in Kabat" or "Kabat numbering" or "EU numbering scheme" refers to the EU numbering for antibodies (Edelman et al., 1969; Kabat et al., 1991; both are incorporated herein by reference in their entireties).

[0108] In certain embodiments, the size of at least one Fc polypeptide protein molecule can include, but is not limited to, about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more amino acid residues, and any range derivable therein. The compound may comprise the above number of consecutive amino acids from SEQ ID NO: 1 (human IgG Fc polypeptide) or from the variant Fc domain listed in Table 1, and these may be further identified as having a percent identity or homology with SEQ ID NO: 1 (discussed herein).

[0109] A. Modified Proteins and Peptides

[0110] Some embodiments relate to modified proteins and polypeptides, in particular modified proteins or polypeptides that exhibit at least one functional activity comparable to an unmodified form, but the modified protein or polypeptide has additional advantages relative to the unmodified form, such as inhibiting B cell activation, being easier or cheaper to produce, causing fewer side effects, and / or having better or longer efficacy or bioavailability. Therefore, when the present application relates to the function or activity of a "modified protein" or "modified polypeptide", a person of ordinary skill in the art will understand that this includes, for example, such proteins or polypeptides that 1) perform at least one of the same activities or have at least one of the same specificities as an unmodified protein or polypeptide, but may have different levels of another activity or specificity; and 2) have additional advantages relative to an unmodified protein or polypeptide. The determination of activity can be achieved using assays familiar to those skilled in the art, particularly assays for protein activity, and for comparison purposes, may include, for example, the use of natural and / or recombinant forms of modified or unmodified proteins or polypeptides. It is particularly contemplated that embodiments regarding "modified proteins" may be implemented relative to "modified polypeptides" and vice versa. In addition to the modified proteins and polypeptides discussed herein, embodiments may relate to the domains, polypeptides and proteins described in PCT Publication WO 2008 / 137475, which is specifically incorporated herein by reference.

[0111] Modified proteins may have amino acid deletions and / or replacements; therefore, proteins with deletions, proteins with replacements, and proteins with deletions and replacements are modified proteins. These modified proteins may further comprise inserted or added amino acids, such as proteins with fusion proteins or with joints. This may include inserting a targeting peptide or polypeptide or only a single residue. The end additions referred to as fusion proteins are discussed below.

[0112] A "modified deletion protein" lacks one or more residues of a native protein, but has the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is a protein that has amino acid residues deleted from at least one antigenic region (i.e., a region of a protein determined to be antigenic in a particular organism, such as an organism type to which the modified protein can be administered).

[0113] Replacement or substitution variants generally include an exchange of one amino acid with another amino acid at one or more sites within a protein, and can be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. Replacement may or may not be conservative, i.e., one amino acid is replaced by an amino acid of similar shape and charge. Conservative replacements are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0114] The term "biologically functional equivalent" is well known in the art and is further defined in detail herein. Thus, sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of the native polypeptide are included, as long as the biological activity of the protein is maintained. The modified protein can be biologically functionally equivalent to its native counterpart.

[0115] It is also understood that the amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, but still be substantially as described in one of the sequences disclosed herein, as long as the sequence meets the above criteria, including the maintenance of biological protein activity related to protein expression. The addition of terminal sequences is particularly suitable for nucleic acid sequences, which may, for example, contain various non-coding sequences flanking the 5' or 3' portion of the coding region, or may contain various internal sequences known to exist within genes, i.e., introns.

[0116] The following is a discussion based on changing the amino acids of a protein to produce an equivalent or even improved second-generation molecule. For example, certain amino acids may replace other amino acids in the protein structure, with or without significant loss of interaction binding ability with the structure (e.g., as a binding site for a substrate molecule). Since the interaction ability and properties of a protein define the biological functional activity of the protein, certain amino acid substitutions may be made in the protein sequence and its underlying DNA coding sequence, and proteins with similar properties are nevertheless produced. It is therefore expected that, as discussed below, multiple changes may be made in the DNA sequence of a gene without significant loss of its biological utility or activity. A protein molecule has "homology" or is considered to be "homologous" to a second protein molecule if one of the following "homology criteria" is met: 1) at least 30% of the protein molecule has sequence identity with the second protein molecule at the same position; 2) at the same position as the second protein molecule has some sequence identity, and at different residues relative to the second protein molecule, at least 30% of which are conservative differences described herein; or 3) at least 30% of the protein molecule has sequence identity with the second protein molecule, but there may be gaps of different residues between the same residues. As used herein, the term "homologous" can be applied equally to regions of protein molecules rather than entire molecules. If the term "homology" or "homologous" is qualified by a number, such as "50% homology" or "50% homology", the homology criteria for 1), 2) and 3) are adjusted from "at least 30%" to "at least 50%". Thus, it is contemplated that there may be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homology or sequence identity between two protein molecules or portions of protein molecules.

[0117] Alternatively, the modified polypeptide may be characterized as having a certain percentage of identity with an unmodified polypeptide or with any polypeptide sequence disclosed herein, including a mutant of a variant Fc domain listed in Table 1. The percentage identity between two protein molecules or portions of protein molecules 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). It is contemplated that the percentage of identity discussed above may relate to a specific region of a polypeptide compared to an unmodified region of the polypeptide. For example, a polypeptide may comprise a modified or mutant Fc domain that may be characterized based on the identity of the modified or mutant Fc domain to the amino acid sequence of an unmodified or mutant Fc domain from the same species. A modified or mutant human Fc domain is characterized, for example, as having 90% identity to an unmodified Fc domain, meaning that 90% of the amino acids in the domain are identical to those in an unmodified human Fc domain (SEQ ID NO: 1).

[0118] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic index of amino acids in conferring interactive biological function on proteins is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0119] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the greatest local average hydrophilicity of a protein is determined by the hydrophilicity of its neighboring amino acids, which correlates with the biological properties of the protein. As described in detail in U.S. Pat. No. 4,554,101, the following hydropathic values ​​have been 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 an amino acid can be substituted for another amino acid having a similar hydropathic value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, substitution of amino acids whose hydropathic values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0120] As summarized above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account many of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0121] B. Modified Antibodies and Protein Compounds Having Heterologous Regions

[0122] Once the Fc domain is separated, it is desirable to connect the molecule to at least one substance to form a conjugate to enhance the effectiveness of the molecule. For example, in order to improve the effectiveness of the Fc domain or antibody molecule as a diagnostic or therapeutic agent, it is conventional to connect or covalently bind or compound at least one desired molecule or part. Such a molecule or part can be but is not limited to at least one effector molecule or reporter molecule. The effector molecule comprises a molecule with a desired activity (e.g., cytotoxic activity). Some non-limiting examples of effector molecules connected to antibodies include toxins, antitumor agents, therapeutic enzymes, radiolabeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any part that can be detected using an assay. Some non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent markers, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (e.g., biotin). Another such example is to form a conjugate comprising an antibody connected to a cytotoxic or anticellular agent, and it can be referred to as an "immunotoxin." Techniques for labeling such molecules are known to those skilled in the art and have been described above.

[0123] The labeled proteins (e.g., Fc domains) prepared according to the present disclosure can then also be used, for example, in immunodetection methods for binding, purification, removal, quantification, and / or otherwise generally detecting biological components, such as proteins, polypeptides, or peptides. Some immunodetection methods include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays, and Western blots, among others. The procedures for a variety of useful immunodetection methods have been described in the scientific literature, including, 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.

[0124] Fc domain molecules (including antibodies) can be used, for example, with fresh frozen and / or formalin-fixed paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). Methods for preparing tissue blocks from these pelleted specimens have been successfully used in previous IHC studies of a variety of prognostic factors and / or are well known to those skilled in the art (Abbondanzo et al., 1990).

[0125] Some embodiments relate to Fc polypeptide protein compounds, which may include amino acid sequences from more than one naturally occurring or natural polypeptide or protein. It is expected that some of the embodiments discussed above are applicable to this part, and vice versa. For example, a modified antibody is an antibody comprising a modified Fc domain with an antigen binding domain. In addition, the antibody may have two different antigen binding regions, such as different regions on each of the two heavy chains. Alternatively or additionally, in some embodiments, there are polypeptides comprising multiple heterologous peptides and / or polypeptides ("heterologous" means that they are not derived from the same polypeptide). For example, a protein compound or molecule may include a modified Fc domain with a protein binding region that is not from an antibody. In some embodiments, there are polypeptides comprising a modified Fc domain, the modified Fc domain having a protein binding region that binds to a cell surface receptor. These protein molecules comprising multiple functional domains may be two or more domains chemically conjugated to each other, or they may be fusion proteins of two or more polypeptides encoded by the same nucleic acid molecule. It is expected that a protein or polypeptide may include all or part of two or more heterologous polypeptides.

[0126] Thus, a multi-polypeptide protein compound can be composed of all or a portion of a first polypeptide and all or a portion 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 more polypeptides.

[0127] Amino acids, such as selectively cleavable linkers, synthetic linkers, or other amino acid sequences can be used to separate protein moieties.

[0128] Polypeptides or proteins (including antibodies) having an antigen binding domain or region of an antibody and an aglycosylated Fc domain can be used to target 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, A1 adenosine receptor, A33, ACE, ACE-2, activator protein, activator protein A, activator protein AB, activator protein B, activator protein C, activator protein RIA, activator protein RIAALK-2, activator protein RIB ALK-4, activator protein RIIA, activator protein RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator 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-2BMP-2a, BMP-3 osteogenic protein (BMP-3Osteogenin), BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (carcinoembryonic antigenantigen, CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / ZIP, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CC L9 / 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 perfringensperfringens) toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DA N, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNA enzyme, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin1, EpCAM, EphrinB2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (fibroblast activation protein 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, Gas6, GCP-2, GCSF, G D2, 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 gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MM), HIV gp120, HIVIIIBgp120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (human cytomegalovirus, HCMV), human growth hormone (human growth hormone, HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, i NOS, 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 (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 FactorFactor, KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, 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, Muellerian-inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophic factor 3, 4 or 6, Neurturin, neuronal growth factor (Neuronal growth factor, NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (prostate specific membrane antigen antigen, PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (respiratorysyncytialvirus, RSV)F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific, 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 R1Apo-2, DR4), TNFRSF10B (TRAILR2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID), TNFRSF10D (TRAIL R4DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK RFN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RIICD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFCR), TNFRSF4 (OX40ACT35, TXGP1R), TNFRSF5 (CD40p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1TNFRH1), TNFRSF25 (DR3Apo-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, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis expressionY-related carbohydrate tumor-associated antigen, 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 growth factors. In some embodiments, the polypeptide or protein has an antigen binding domain that is specific for one or more cell surface tumor antigens or B cell antigens. Methods and compositions can be used to target tumor cells or B cells.

[0129] Any antibody with sufficient selectivity, specificity or affinity can be used as the basis for antibody conjugates. Such properties can be evaluated using conventional immunological screening methods known to those skilled in the art. In addition to the classical antigen binding sites, sites in antibody molecules for binding to biologically active molecules also include sites located in the variable domains that can bind to pathogens, B cell superantigens, T cell co-receptor CD4 and HIV-1 envelope

[0130] (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).

[0131] In addition, the variable domain is involved in the self-association of antibodies (Kang et al., 1988) and contains the epitope (idiotope) recognized by anti-antibodies (Kohler et al., 1989).

[0132] Fc domains can bind to FcRs, however, it is expected that the modulation of immune responses can be performed not only by antigen binding domains on polypeptides comprising Fc domains, but also by some other protein binding domains. Therefore, some embodiments may involve Fc domains and heterologous non-antigen binding domains. In certain embodiments, non-antigen binding domains bind to the cell surface. Therefore, these substances need to be chemically conjugated or fused with substances / proteins that can bind to specific target cells. Some embodiments may further include making all or part of the aglycosylated Fc domain adjacent to all or part of any protein listed in Table 2. It is expected that some embodiments include, but are not limited to, the examples provided in Table 2 and the description herein.

[0133] The ligand of the receptor can be used to target the cell of the receptor expressing the ligand on its surface. The ligand also includes, for example, CD95 ligand, TRAIL, TNF (e.g., TNF-α or TNF-β), growth factors (including those discussed above), such as VEGF and cytokines, such as interferons or interleukins, and variants thereof. Some embodiments with multiple domains are also contemplated, such as the VEGF Trap fusion protein comprising the second extracellular domain of VEGF receptor 1 (Flt-1) and the third domain of VEGF receptor 2 (KDR / FIK-1) and IgG Fc region.

[0134] Table 2. Substances / proteins that can bind to specific target cells

[0135]

[0136] C. Antibody Fc Library

[0137] Some examples of techniques that can be used in conjunction with embodiments for producing a variety of antibody Fc domains and / or antibodies comprising such domains can employ techniques similar to those described in U.S. Patent No. 5,824,520 for expressing immunoglobulin heavy chain libraries. Previously used Fc libraries are discussed in PCT Publication WO 2008 / 137475, which is 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).

[0138] II. Antibody Binding Peptide

[0139] A variety of antibody binding domains (e.g., FcR polypeptides) are known in the art and can be used in the methods and compositions of the present invention. For example, in some aspects, FcR can be specific for a particular type or subtype of Ig, such as IgA, IgM, IgE, or IgG (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). Therefore, in some embodiments, the antibody binding domain can be limited to an IgG binding domain. The FcR polypeptide can comprise a eukaryotic, prokaryotic, or synthetic FcR domain. For example, an antibody Fc binding domain can be limited to a mammalian, bacterial, or synthetic binding domain. Some Fc binding domains used in the present invention include, but are not limited to, a binding domain of one of the polypeptides from Table 3. For example, the Fc binding polypeptide may be encoded by the following: FCGR2A, FCGR2B, FCGR2C, FCGR3A, FCGR3B, FCGR1A, Fcgr1, FCGR2, FCGR2, Fcgr2, Fcgr2, FCGR3, FCGR3, Fcgr3, FCGR3, Fcgr3, Fcgr3, FCGRT, mrp4, spa or spg genes. The FcR polypeptide may be an Fc binding region from human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI or C1q. Various Fc receptors that bind to Fc domains are well known in the art, and some examples of receptors are listed in Table 3 below.

[0140] Table 3. Selected FcR polypeptides

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] III. Method for Screening Antibody Fc Domains

[0148] In certain aspects, there are methods for identifying antibody Fc domains that have specific affinity for a target ligand (e.g., an antibody binding polypeptide, such as an Fc receptor). Such methods are described herein and in PCT Publication WO 2008 / 137475, which is specifically incorporated herein by reference in its entirety. In some embodiments, methods for screening using eukaryotic cells (e.g., yeast surface display libraries) can be used.

[0149] The polypeptides screened may comprise a large library of multiple candidate Fc domains, or alternatively, may comprise a particular class of Fc domains (e.g., engineered with point mutations or amino acid insertions) selected for structural attributes believed to make them more likely to bind to a target ligand. In one embodiment, the candidate polypeptide may be a complete antibody, or a fragment or portion thereof comprising an Fc domain.

[0150] To identify a candidate Fc domain capable of binding to a target ligand, the following steps may be performed: providing a population of Gram-negative bacterial cells each expressing a different antibody Fc domain; mixing the bacteria with at least a first labeled or immobilized target ligand (FcR polypeptide) capable of contacting the antibody Fc domain; and identifying at least a first bacterium expressing a molecule capable of binding to the target ligand.

[0151] In some aspects of the above methods, the binding between the antibody Fc domain and the labeled FcR polypeptide will prevent diffusion out of the bacterial cell. In this way, the molecule of the labeled ligand can be retained in the periplasm of the bacteria containing the permeabilized outer membrane. Alternatively, the periplasm can be removed, whereby the Fc domain will cause the retention of the bound candidate molecule because the Fc domain is shown to be associated with the inner membrane. The label can then be used to isolate cells expressing a binding polypeptide capable of binding to the FcR polypeptide, and the gene encoding the Fc domain polypeptide can be isolated. The molecules capable of binding to the target ligand can then be produced in large quantities using in vivo or ex vivo expression methods and subsequently used in any desired application, such as for diagnostic or therapeutic applications. In addition, it should be understood that the identified isolated antibody Fc domain can be used to construct antibody fragments or full-length antibodies comprising an antigen binding domain.

[0152] In other embodiments, the method for screening may include at least two rounds of selection, wherein a subpopulation of bacterial cells obtained in the first round of selection is subjected to at least a second round of selection based on the binding of the candidate antibody Fc domain to the FcR. The subpopulation of bacterial cells obtained in the first round of selection can be grown under permissive conditions (to expand the total number of cells) before the second round of selection. These methods may, for example, include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of selection. In some aspects, the subpopulation of bacterial cells obtained from each round of selection will be grown under permissive conditions before the subsequent round of selection. Cells separated after one or more rounds of such selection may be subjected to additional rounds of mutagenesis. In some cases, the selection will be performed after removing FcR polypeptides that are not bound to the antibody. The stringency of the selection can be changed by adjusting the pH, salt concentration or temperature of the solution containing the bacteria displaying the antibody. For example, bacterial cells can be grown at subphysiological temperatures, for example, at about 25°C.

[0153] Methods for producing bacterial cells are provided, and the bacterial cells may comprise nucleic acid sequences encoding mutant Fc domains provided herein. The bacterial cells produced by the methods provided herein can be used to clone nucleic acid sequences encoding Fc domains with specific affinity for FcR polypeptides. Methods for isolating and amplifying such nucleic acids from cells (e.g., by PCR) are well known in the art and are further described below. The nucleic acid sequences produced by the aforementioned methods comprise some aspects of the present disclosure. The nucleic acids may be expressed in cells to produce Fc domains provided herein or polypeptides comprising Fc domains provided herein. In some embodiments. The antibody Fc domains provided herein are contained in a polypeptide having one or more antibody variable regions (e.g., single domain antibodies, scFv, etc.) with affinity for a specific target ligand.

[0154] B. Periplasmic Expression of Antibody Fc Domains

[0155] In some embodiments, polypeptides comprising antibody Fc domains can be expressed in the periplasmic space of gram-negative bacteria. In addition, in some aspects, antibody Fc domains can be anchored to the periplasmic face of the inner membrane. Methods and compositions for anchoring polypeptides to the inner membrane of gram-negative bacteria have been previously described (U.S. Patent Nos. 7,094,571, 7,419,783, 7,611,866 and U.S. Patent Publication Nos. 2003 / 0219870; Harvey et al., 2004; Harvey et al., 2006). For example, the Fc domain can be directly fused to a transmembrane polypeptide or a membrane-bound polypeptide, or can interact with a transmembrane polypeptide or a membrane-bound polypeptide (e.g., through protein-protein interactions). This technology can be referred to as "anchored periplasmic expression" or "APEx". In some cases, gram-negative bacterial cells can be limited to Escherichia coli (E. coli) cells. Furthermore, in some aspects, Gram-negative bacterial cells can be defined as genetically engineered bacterial cells, such as the Jude-1 strain of E. coli.

[0156] The fusion protein may comprise an N-terminal or C-terminal fusion with an Fc domain and, in some cases, may comprise additional linker amino acids between the membrane anchor polypeptide and the Fc domain. In certain specific cases, the membrane anchor polypeptide may be the first six amino acids encoded by the E. coli NlpA gene, one or more transmembrane α-helices from an E. coli inner membrane protein, a gene III protein of a filamentous bacteriophage 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, such as from

[0157] 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.

[0158] In still other cases, the Gram-negative bacterial population according to the present invention can be defined as comprising at least about 1×10 3 ,1×10 4 ,1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 ,1×10 9 In some specific cases, a Gram-negative bacterial cell population can be produced by a method comprising the following steps: (a) preparing a plurality of nucleic acid sequences encoding a plurality of different antibody Fc domains; and (b) transforming a Gram-negative bacterial population with the nucleic acid, wherein the Gram-negative bacteria comprises a plurality of antibody Fc domains expressed in the periplasm.

[0159] C. Permeabilization of the outer membrane

[0160] Methods for disrupting, permeabilizing or removing the outer membrane of bacteria are well known in the art, for example, see U.S. Pat. No. 7,094,571. For example, the outer membrane of the bacterial cells can be treated with hypertonic conditions, physical stress, lysozyme, EDTA, digestive enzymes, chemicals that disrupt the outer membrane, by infecting the bacteria with bacteriophage, or a combination of the foregoing methods, prior to contacting the bacterial cells with the FcR polypeptides. Thus, in some cases, the outer membrane can be disrupted by lysozyme and EDTA treatment. Furthermore, in certain embodiments, the bacterial outer membrane can be completely removed.

[0161] Some methods can be used to increase the permeability of the outer membrane to one or more labeled ligands. This can allow screening entry of labeled ligands that would otherwise not pass through the outer membrane. However, certain types of molecules, such as hydrophobic antibiotics with an exclusion limit greater than 650 Da, can diffuse through the bacterial outer membrane itself, independent of porins (Farmer et al., 1999). This process can permeabilize the membrane (Jouenne and Junter, 1990). In addition, some long-chain phosphate polymers (100Pi) seem to completely bypass the normal molecular screening activity of the outer membrane (Rao and Torriani, 1988).

[0162] Although conditions have been determined that result in penetration of the ligand into the periplasm without loss of viability or release of the expressed protein from the cell, the present invention can be practiced without maintaining an outer membrane. For Fc domains expressed or anchored in the periplasmic space, it is not necessary to maintain an outer membrane (as a barrier to prevent leakage of the binding protein from the cell) to detect bound labeled ligand. Thus, cells expressing binding proteins anchored to the outer (periplasmic) face of the cytoplasmic membrane can be labeled simply by incubating cells with a solution of labeled ligand with a partially permeabilized membrane or an almost completely removed outer membrane.

[0163] Treatment, such as hyperosmotic shock, can significantly improve labeling. Many substances, including calcium ions (Bukau et al., 1985) and even Tris buffer (Irvin et al., 1981), are known to change the permeability of the outer membrane. In addition, phage infection stimulates the labeling process. Both the filamentous phage inner membrane protein pill and the large multimeric outer membrane protein pIV can change membrane permeability (Boeke et al., 1982), and mutants in pIV are known to improve the entry of maltodextrin, which is usually excluded (Marciano et al., 1999). Combinations of strains, salts, and phages can be used to achieve high permeability (Daugherty et al., 1999). Then, using flow cytometry or other related techniques, cells containing anchored or periplasmic-associated polypeptides bound to the labeled ligand can be easily separated from cells expressing binding proteins that have no affinity for the labeled ligand. However, in some cases, in order to maintain the viability of the cells, it is desirable to use less destructive techniques. EDTA and lysozyme treatments may also be useful in this regard.

[0164] D. Labeled Target Ligand

[0165] As described above, it is generally desirable to provide an FcR polypeptide that has been labeled with one or more detectable substances. This can be done, for example, by linking a ligand to at least one detectable substance to form a conjugate. For example, at least one detectable molecule or moiety is generally linked or covalently bound or complexed. A "label" or "detectable marker" is a compound and / or element that can be detected due to a specific functional property and / or chemical feature, the use of which allows detection and / or further quantification (if desired) of the ligand to which it is attached. Some examples of labels that can be used include, but are not limited to, enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands such as biotin. In some embodiments, streptavidin-biotinylated FcgR tetramers can be used for screening, and FcgRs with Avi-tags can be used for biotinylation.

[0166] In one embodiment of the invention, visually detectable markers are used so that automatic screening of cells can be performed for labeling. Some examples of substances that can be detected by visualization with appropriate instruments are known in the art, and methods for connecting them to desired ligands are also known in the art (see, e.g., U.S. Patent Nos. 5,021,236; 4,938,948 and 4,472,509, each of which is incorporated herein by reference). Such substances may include paramagnetic ions; radioactive isotopes; fluorescent dyes; NMR detectable substances; and substances for X-ray imaging. In particular, fluorescent labels are beneficial because they allow cells expressing desired binding proteins or antibodies to be isolated using flow cytometry.

[0167] Another type of FcR conjugate is one in which the ligand is linked to a second binding molecule and / or to an enzyme (enzyme tag) that will produce a colored product when in contact with a chromogenic substrate. Some examples of such enzymes include urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. In such cases, it is desirable that the selected cells remain viable. Preferred second binding ligands are biotin and / or avidin and streptavidin 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.

[0168] Molecules containing an azide group can be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens and Haley, 1987; Atherton et al., 1985). 2- and 8-azidonucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as ligand binding agents.

[0169] Labeling can be performed by any technique known to those skilled in the art. For example, FcR polypeptides can be labeled by contacting the ligand with the desired label and a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Similarly, a ligand exchange process can be used. Alternatively, a direct labeling technique can be used, for example, by incubating the label, a reducing agent such as SNCl2, a buffer solution such as sodium potassium phthalate solution, and the ligand together. Intermediate functional groups on the ligand can also be used to bind the label to the ligand, for example, in the presence of diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0170] Other methods for linking or conjugating a ligand to its conjugate moiety are also known in the art. Some methods of linking involve the use of organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid or ethylenediaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3α-6α-diphenylglycoluril-3 (U.S. Pat. Nos. 4,472,509 and 4,938,948, each of which is incorporated herein by reference) to link to the ligand. FcR polypeptides can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and detectable imaging moieties are bound to the antibodies using linkers such as methyl p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl) propionate. In still other aspects, the FcR polypeptide can be fused to a reporter protein, such as an enzyme or a fluorescent protein as described above.

[0171] E. Isolation of cells bound to labeled target ligand

[0172] 1. Bead- or column-based immobilization

[0173] Those skilled in the art will appreciate that methods for selecting cells based on the interaction (binding) of cells with FcR are well known in the art. For example, FcR can be fixed on a column or bead (e.g., magnetic bead), and cells (e.g., bacterial cells or eukaryotic cells, such as yeast) bound to FcR can be separated by repeated washing of beads (e.g., magnetic separation) or columns. In addition, the target ligand can be labeled (e.g., with a fluorophore, a radioisotope, or an enzyme). Therefore, in some cases, cells can be selected by detecting the label on the bound FcR. In addition, in some aspects, cells can be selected based on binding or not binding to two or more FcR polypeptides. For example, bacteria that display antibodies that bind to two FcR polypeptides can be selected, wherein each FcR is used to sequentially select bacteria. On the contrary, in some aspects, bacteria that display an antibody Fc domain that binds to an FcR (e.g., an FcR comprising a first label) but does not bind to a second FcR (e.g., comprising a second label) can be selected. The foregoing methods can be used, for example, to identify an antibody Fc domain that binds to a particular FcR but does not bind to a second specific FcR.

[0174] 2. Flow Cytometry

[0175] In one embodiment of the invention, fluorescence activated cell sorting (FACS) screening or other automated flow cytometry techniques can be used to effectively separate bacterial cells containing labeled ligands that bind to the Fc domain. Instruments for performing flow cytometry are known to those skilled in the art and are commercially available to the public. Some examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA); the Epics C from Coulter Epics Division (Hialeah, FL) and the MOFLO from Cytomation (Colorado Springs, CO). TM .

[0176] Flow cytometry techniques generally involve the separation of cells or other particles in a liquid sample. Typically, the purpose of flow cytometry is to analyze one or more characteristics of the separated particles, such as the presence of labeled ligands or other molecules. The basic steps of flow cytometry involve the direction of the fluid sample through the device so that the liquid flows through the sensing area. The particles should pass through the sensor one at a time and be classified based on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.

[0177] Flow cytometry is used not only for cell analysis but also for cell sorting. In U.S. Pat. No. 3,826,364, a device for physically separating particles (e.g., functionally different cell types) is disclosed. In this machine, a laser provides illumination, which is focused on a particle stream through a suitable lens or lens system so that there is a high degree of local scattering from the particles therein. In addition, a high-intensity light source is directed to the particle stream to excite fluorescent particles in the particle stream. Certain particles in the stream can be selectively charged and subsequently separated by deflecting them into a specified container. The classic form of this separation is by fluorescently labeled antibodies, which are used to mark one or more cell types for separation.

[0178] Additional examples of methods for flow cytometry include, but are not limited to, those described in 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.

[0179] A useful aspect of flow cytometry is that multiple rounds of screening can be performed sequentially. Cells can be separated from the initial sorting round and immediately reintroduced into the flow cytometer and screened again to improve the stringency of the screening. Another advantage known to those skilled in the art is that flow cytometry can be used to recycle inactive cells. Because flow cytometry is essentially a particle sorting technique, the ability of cells to grow or reproduce is not necessary. Techniques for recovering nucleic acids from such inactive cells are well known in the art and include, for example, the use of template-dependent amplification techniques, including PCR.

[0180] F. Cloning of Fc domain coding sequences

[0181] After identifying the bacterial cells producing the molecules with the desired specificity, affinity and / or activity, the corresponding coding sequence can be cloned. In this way, conventional methods can be used (e.g., by using oligonucleotide probes that can be specifically bound to the genes encoding antibodies or conjugated proteins) to separate and sequence the DNA encoding the molecules. It will be appreciated by those skilled in the art that nucleic acids can be cloned from living cells or inactive cells. For example, in the case of inactive cells, it is desirable to use the amplification of cloned DNA, for example, using PCR. This can also be carried out using living cells when the cells further grow or do not grow.

[0182] Once isolated, the antibody Fc domain DNA can be placed in an expression vector, which can then be transfected into a host cell such as bacteria. The DNA can also be modified, for example, by adding sequences of human heavy and light chain variable domains, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. In this way, a "chimeric" or "hybrid" binding protein with the desired binding specificity is prepared. For example, the identified antibody Fc domain can be fused to a therapeutic polypeptide or toxin and used to target cells expressing a specific FcR (in vitro or in vivo).

[0183] Chimeric or hybrid Fc domains can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, targeted toxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Some examples of suitable reagents for this purpose include iminothiolate and 4-mercaptobutyrimidic acid methyl ester.

[0184] IV. Nucleic Acid-Based Expression Systems

[0185] In certain embodiments of the invention, nucleic acid-based expression systems can be used to express recombinant proteins.For example, one embodiment of the invention involves transforming Gram-negative bacteria with a coding sequence for an antibody Fc domain or, preferably, a plurality of different Fc domains.

[0186] A. Nucleic Acid Delivery Methods

[0187] Certain aspects of the invention may include delivery of nucleic acids to target cells (e.g., Gram-negative bacteria). For example, bacterial host cells may be transformed with nucleic acids encoding candidate Fc domains that are potentially capable of binding to FcRs. In some specific embodiments of the invention, it may be desirable to target expression to the periplasm of bacteria. Transformation of eukaryotic host cells may similarly be used to express a variety of candidate molecules identified as being capable of binding to a target ligand.

[0188] Suitable methods of nucleic acid delivery for transforming cells are considered to include virtually any method by which nucleic acid (e.g., DNA) can be introduced into cells or even into their organelles. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Pat. No.

[0189] and 5,580,859

[0190] each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, which is incorporated herein by reference), by electroporation (U.S. Pat. No. 5,384,253, which is 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 sonication (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., 1990); al., 1991), by microprojectile bombardment (PCT Publication Nos. WO 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, and each of which is 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 of which is incorporated herein by reference), by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). By using techniques such as these, cells can be stably or transiently transformed.

[0191] B. Carrier

[0192] Vectors can be used in the present invention, for example, for transforming cells with nucleic acid sequences encoding candidate Fc domains. In one embodiment of the invention, the entire heterogeneous "library" of nucleic acid sequences encoding polypeptides can be introduced into a cell population, thereby allowing the entire library to be screened. The term "vector" is used to refer to a vector nucleic acid molecule, wherein a nucleic acid sequence can be inserted to be introduced into a cell where it can be replicated. The nucleic acid sequence can be "exogenous" or "heterologous", which means that it is foreign to the cell into which the vector is introduced, or the sequence is homologous to the sequence in the cell but is in a position in which the sequence is not usually present in the host cell nucleic acid. Vectors include plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors by standard recombinant techniques, which are described in Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference.

[0193] 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 cases, the RNA molecule is then translated into a protein, polypeptide or peptide. An expression vector may contain a variety of "control sequences," which refer to nucleic acid sequences required for transcription and translation of an operably linked coding sequence 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 for other functions.

[0194] 1. Promoters and enhancers

[0195] "Promoter" is a control sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may include genetic elements that can bind to regulatory proteins and molecules (e.g., RNA polymerases and other transcription factors). The phrases "effectively positioned," "effectively connected," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to the nucleic acid sequence to control the transcription initiation and / or expression of the sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence. Those skilled in the art of molecular biology are generally familiar with the use of promoters, enhancers, and cell type combinations for protein expression, e.g., see Sambrook et al. (1989), which is incorporated herein by reference.

[0196] 2. Start signal

[0197] Specific start signals may also be required for the effective translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. One of ordinary skill in the art can easily determine this and provide the necessary signals. It is well known that the start codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and start codons may be natural or synthetic. Expression efficiency may be enhanced by including appropriate transcription enhancer elements.

[0198] 3. Multiple cloning site

[0199] The vector may contain a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any of which can be used in combination with standard recombinant techniques to digest the vector (see Carbonelli et al., 1999, Levenson et al., 1998 and Cocea, 1997, which are incorporated herein by reference). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at a specific position in the nucleic acid molecule. Many of these restriction enzymes are commercially available. Typically, a restriction enzyme that cuts within the MCS is used to linearize or fragment the vector so that an exogenous sequence can be connected to the vector. "Connection" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may be continuous or discontinuous with each other. The techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0200] 4. Termination Signal

[0201] Vectors or constructs prepared according to the present disclosure typically include at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence that participates in the specific termination of an RNA transcript by an RNA polymerase. Therefore, in certain embodiments, a termination signal that terminates the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve a desired message level. Terminators contemplated for use in the present invention include any known transcription terminators known to those of ordinary skill in the art, including but not limited to rho-dependent or rho-independent terminators. In certain embodiments, a termination signal may be a lack of a transcribable or translatable sequence, for example due to sequence truncation.

[0202] 5. Copy starting point

[0203] In order to propagate a vector in a host cell, it may contain one or more origins of replication (commonly referred to as "ori"), which are specific nucleic acid sequences that initiate replication.

[0204] 6. Selectable and screenable markers

[0205] In certain embodiments of the present invention, cells comprising nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by including a marker in an expression vector. Such a marker will confer identifiable changes to the cell, thereby allowing easy identification of cells comprising the expression vector. Typically, a selectable marker is a marker that confers a characteristic that allows selection. A positive selectable marker is a marker in which the presence of the marker allows its selection, while a negative selectable marker is a marker in which the presence of the marker prevents its selection. Some examples of positive selectable markers are drug resistance markers.

[0206] Generally, drug selection markers are included to help clone and identify transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin and histidinol are useful selection markers. In addition to conferring markers that allow the phenotype of transformants to be distinguished based on conditional implementation, other types of markers are also contemplated, including screenable markers, such as GFP, based on colorimetric analysis. Alternatively, screenable enzymes such as chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art also know how to use immune markers that can be used in combination with FACS analysis. It is believed that the marker used is not important as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selectable and screenable markers are well known to those skilled in the art.

[0207] C. Host cells

[0208] In the case of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic cell or eukaryotic cell (e.g., a yeast cell, an insect cell, or a mammalian cell), and includes any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by a vector. Host cells can and have been used as recipients of vectors. Host cells can be "transfected" or "transformed," which refers to the process of transferring or introducing exogenous nucleic acids into host cells. Transformed cells include primary subject cells and their progeny.

[0209] In some specific embodiments of the present invention, host cell is a gram-negative bacterial cell. These bacteriums are suitable for the present invention because they have the periplasmic space between the inner membrane and the outer membrane, particularly the above-mentioned inner membrane between the periplasm and the cytoplasm, also referred to as the cytoplasmic membrane. Therefore, any other cell with such periplasmic space can be used according to the present invention. Some examples of gram-negative bacteria that can be used for the present invention may include but are not limited to Escherichia coli, Pseudomonas aeruginosa, Vibrio choler, Salmonella typhimurium, Shigella flexneri, Haemophilus influenzae, Bordotella pertussi, Erwinia amylovora, Rhizobium sp.

[0210] Those skilled in the art can determine the appropriate host based on the vector backbone and the desired results. For example, plasmids or cosmids 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 many commercially available bacterial hosts, such as Competent cells and Solopack TM Gold cells ( La Jolla). Alternatively, bacterial cells (e.g., E. coli LE392) can be used as host cells for bacteriophage.

[0211] Some examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548), SV40 transformed monkey kidney cells (COS-1; ATCC CRL 1650), murine embryonic cells (NIH-3T3; ATCC CRL 1658), and human embryonic kidney cells (e.g., EXPI293 cells). The foregoing is an exemplary but non-limiting list of many possible host organisms known in the art.

[0212] Mammalian host cells expressing polypeptides are cultured under conditions commonly used to culture parental cell lines. Typically, cells are cultured in standard medium containing physiological salts and nutrients, such as standard RPMI, MEM, IMEM or DMEM, typically supplemented with 5% to 10% serum, such as fetal bovine serum. Culture conditions are also standard, for example, the culture is incubated at 37° C. in fixed or rolling culture until the desired level of protein is obtained.

[0213] Many host cells from various cell types and organisms are available and are known to those skilled in the art. Similarly, viral vectors can be used in conjunction with prokaryotic host cells, particularly prokaryotic host cells that allow vector replication or expression. Some vectors can adopt control sequences that allow them to replicate and / or express in both prokaryotic cells and eukaryotic cells. Those skilled in the art will further understand that hatching all of the above-mentioned host cells to maintain them and allow the conditions for vector replication. It is also understood and known that large-scale production of vectors and production of nucleic acids encoded by vectors and their homologous polypeptides, proteins or peptides are allowed.

[0214] D. Expression system

[0215] There are many expression systems comprising at least a portion or all of the compositions discussed above. Such systems can be used, for example, to produce polypeptide products that are identified as being capable of binding specific ligands according to the present invention. Systems based on prokaryotes can be used in the present disclosure to produce nucleic acid sequences, or their homologous polypeptides, proteins and peptides. Many such systems are commercially available and widely available. Other examples of expression systems include vectors comprising strong prokaryotic promoters such as T7, Tac, Trc, BAD, λpL, tetracycline or Lac promoters, pET expression systems and E. coli expression systems.

[0216] In certain aspects of the present invention, a nucleic acid sequence encoding a polypeptide is disclosed. Depending on the expression system used, the nucleic acid sequence can be selected based on conventional methods. For example, if the polypeptide is derived from a human polypeptide and comprises a plurality of codons that are rarely used in Escherichia coli, it can interfere with expression in Escherichia coli. Therefore, each gene or its variant can be codon optimized for Escherichia coli expression. A variety of vectors can also be used to express the target protein. Some exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, or liposome-based vectors.

[0217] V. Protein Purification

[0218] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, homogenizing and roughly fractionating cells, tissues or organs into polypeptide and non-polypeptide fractions. Unless otherwise indicated, chromatography and electrophoresis techniques may be used to further purify the target protein or polypeptide to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography, reverse phase chromatography, hydroxyapatite chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. Particularly effective methods for purifying peptides are fast-performance liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC). As is generally known in the art, it is believed that the order in which the various purification steps are performed can be changed, or certain steps can be omitted, and still produce a suitable method for preparing a substantially purified protein or peptide.

[0219] Purified protein or peptide is intended to refer to a composition that can be separated from other components, wherein the protein or peptide is purified to any degree relative to its naturally obtainable state. Therefore, a separated or purified protein or peptide still refers to a protein or peptide that is not in an environment where it may exist naturally. Generally, "purified" refers to a protein or peptide composition that has been graded to remove a variety of other components, and the composition substantially retains the biological activity of its expression. In the case of using the term "substantially purified", the indication will refer to such a composition, wherein the protein or peptide forms the main component of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0220] According to the present disclosure, various methods for quantifying the degree of purification of a protein or peptide are known to those skilled in the art. These include, for example, determining the specific activity of an active fraction, or evaluating the amount of polypeptide in a 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 to the specific activity of the initial extract, and thereby calculate the purity assessed by a "purification fold". Of course, the actual units used to express the amount of activity will depend on the specific assay technique selected after purification, and whether the expressed protein or polypeptide exhibits detectable activity.

[0221] There is no general requirement that a protein or peptide will always be provided in its most purified state. In fact, it is expected that substantially less purified products may have utility in certain embodiments. Partial purification may be achieved by combining fewer purification steps or by utilizing different forms of the same general purification scheme. For example, it should be understood that cation exchange column chromatography using HPLC equipment will generally result in greater "fold" purification compared to the same techniques using low pressure chromatography systems. Methods that exhibit a lower relative degree of purification may have advantages in terms of total recovery of a protein product or maintaining the activity of an expressed protein.

[0222] Affinity chromatography is a chromatographic operation that depends on the specific affinity between the substance to be separated and the molecule that can specifically bind to it. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. Then, the column material can specifically adsorb the substance from the solution. Elution is carried out by changing the conditions to conditions where no binding will occur (for example, changing pH, ionic strength, temperature, etc.). The matrix should be a substance that does not adsorb molecules to any significant extent and has a wide range of chemical, physical and thermal stability. The ligand should be coupled in such a way that does not affect its binding characteristics. The ligand should also provide relatively tight binding. The substance should be eluted without destroying the sample or the ligand.

[0223] Size exclusion chromatography (SEC) is a chromatographic method in which molecules in a solution are separated based on their size, or in more technical terms, based on their hydrodynamic volume. It is commonly applied to macromolecules or macromolecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to transfer a sample through a column, the technique is called gel filtration chromatography, as opposed to gel permeation chromatography when an organic solvent is used as the mobile phase. The basic principle of SEC is that particles of different sizes will be eluted (filtered) through the stationary phase at different rates. This results in the separation of particle solutions based on size. Assuming that all particles are loaded simultaneously or nearly simultaneously, particles of the same size should elute together.

[0224] High performance liquid chromatography (or high pressure liquid chromatography, HPLC) is a form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify and quantify compounds. HPLC utilizes a column filled with a chromatographic packing material (stationary phase), a pump to move the mobile phase through the column, and a detector to display the retention time of molecules. The retention time varies depending on the interaction between the stationary phase, the molecule being analyzed, and the solvent used.

[0225] VI. Pharmaceutical Compositions

[0226] In the case of clinical application of a pharmaceutical composition comprising a polypeptide or antibody, it will be generally beneficial to prepare a drug or therapeutic composition suitable for the intended application. Generally, a pharmaceutical composition may include an effective amount of one or more polypeptides or other substances dissolved or dispersed in a pharmaceutically acceptable carrier. In certain embodiments, a pharmaceutical composition may include, for example, at least about 0.1% of a polypeptide or antibody. In other embodiments, a polypeptide or antibody may account for about 2% to about 75% of a unit weight, or, for example, about 25% to about 60%, and any range derivable therefrom. The amount of active compound in each therapeutically useful composition can be prepared in such a way that a suitable dose will be obtained in any given unit dose of the compound. Technicians in the field of preparing such pharmaceutical preparations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore, multiple doses and treatment regimens may be desirable.

[0227] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals (e.g., humans), as appropriate. In light of the present disclosure, one skilled in the art will know the preparation of pharmaceutical compositions comprising antibodies or additional active ingredients, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the formulations should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Bureau of Biological Standards.

[0228] According to certain aspects of the present invention, compositions suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be absorbable and include a liquid, semisolid (i.e., pasty) or solid carrier. Some examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, adhesives, fillers, etc., or combinations thereof. As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, ethanol, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids (e.g., glycine and lysine), carbohydrates (e.g., The invention relates to a pharmaceutical composition comprising a pharmaceutical composition comprising a plurality of components, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.), gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids and nutritional supplements, such materials and combinations thereof, as known to those of ordinary skill in the art. Unless any conventional medium, substance, diluent or carrier is harmful to the recipient or to the therapeutic effect of the composition contained therein, its use in the administrable composition for implementing the method is suitable. The pH and exact concentration of the various components in the pharmaceutical composition are regulated according to known parameters. The composition can be combined with a carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, grinding, etc. For those skilled in the art, such a procedure is conventional.

[0229] Some embodiments of the present disclosure may include different types of carriers, depending on whether it is used in solid, liquid or aerosol form, and whether the route of administration (e.g., injection) needs to be aseptic. Compositions can be formulated into and used as follows: intravenous, intradermal, percutaneous, intrathecal, intraarterial, intraperitoneal, intranasal, intravaginal, intrarectal, intramuscular, subcutaneous, mucosal, oral, surface, local, by suction (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by direct local perfusion bathing target cells, by catheter, by lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the aforementioned methods, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference). Typically, such compositions can be prepared into liquid solutions or suspensions; can also be prepared into a solid form suitable for adding liquid to prepare solutions or suspensions before injection; and, the preparation can also be emulsified.

[0230] The polypeptide can be formulated into the composition in free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of the protein composition, or salts formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides; or organic bases such as isopropylamine, trimethylamine, histidine or procaine.

[0231] In other embodiments, the disclosure may be directed to the use of a pharmaceutical lipid carrier composition comprising a polypeptide, one or more lipids and an aqueous solvent. The term "lipid" used herein is defined as including any of a wide range of substances that are characteristically insoluble in water and can be extracted with an organic solvent. This broad category of compounds is well known to those skilled in the art, and when the term "lipid" is used herein, it is not limited to any specific structure. Some examples include compounds comprising long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by people). 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, glycosphingolipids, glycolipids, sulfolipids, lipids with ether and ester-connected fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds understood by those skilled in the art as lipids other than those specifically described herein are also encompassed by the compositions and methods.

[0232] Those of ordinary skill in the art are familiar with a range of techniques that can be used to disperse the composition in a lipid carrier. For example, the polypeptide or its fusion protein can be dispersed in a solution comprising a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bound to a lipid, contained in a lipid as a suspension, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. Dispersion may or may not result in the formation of liposomes.

[0233] The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the above-mentioned desired response associated with its administration (i.e., appropriate route and treatment regimen). The amount administered, both in terms of the number of treatments and the unit dose, depends on the desired effect. The exact dose of the composition of the present embodiment administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health status and sex, the type of disease to be treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the patient's idiopathic diseases, the route of administration, and the efficacy, stability and toxicity of the specific therapeutic substance. In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 milligram / kg / body weight or more, and any range derivable therein, per administration. In some non-limiting examples of derivable ranges from the numbers listed herein, based on the above numbers, about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. may be administered. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

[0234] It is not intended that the present invention be limited to the special properties of therapeutic preparations. For example, such compositions can be provided in the form of preparations with physiologically tolerable liquids, gels or solid carriers, diluents and excipients. These therapeutic preparations can be applied to mammals such as domestic animals for veterinary use in a manner similar to other therapeutic agents, and for clinical use in people. Usually, the dosage required for therapeutic efficacy will vary according to the specific requirements of the use type and mode of administration and individual objects. The actual dosage of the composition applied to animal patients can be determined by physical and physiological factors, such as body weight, severity of illness, type of disease to be treated, previous or simultaneous therapeutic intervention, patient's idiopathic disease and route of administration. Depending on dosage and route of administration, the number of administrations of preferred dosage and / or effective amount can vary according to the response of the object. In any case, the practitioner responsible for application will determine the concentration of active ingredient in the composition and the appropriate dosage for individual objects.

[0235] VII. Treatment Methods

[0236] Certain aspects of the present disclosure provide polypeptides for treating diseases (e.g., tumors). In particular, the polypeptides may have human polypeptide sequences and thus may prevent allergic reactions in human patients, allowing repeated administration and improving therapeutic efficacy.

[0237] "Treatment" and its variations refer to the administration or application of a therapeutic agent to a subject or the subject's procedure or modality for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment may include administering a pharmaceutically effective amount of an antibody targeting CDC to a cancer cell without triggering cancer cell proliferation.

[0238] "Subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In some specific embodiments, the subject is a human.

[0239] As used throughout this application, the terms "therapeutic benefit" or "therapeutically effective" refer to anything that promotes or enhances the health of a subject with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of disease signs or symptoms. For example, treatment of cancer may involve, for example, reducing tumor size, reducing tumor invasiveness, reducing cancer growth rate, or preventing metastasis. Treatment of cancer may also refer to prolonging the survival of a subject suffering from cancer.

[0240] In some aspects, the disease can be, for example, cancer (e.g., using an agonistic antibody, such as an anti-CD40 agonist antibody), infection, or immune disease. The immune disease can be an autoimmune disease, such as, for example, lupus, rheumatoid arthritis, psoriasis, etc.

[0241] Tumors for which the present treatment methods can be used include any malignant cell type, such as those found in solid tumors or hematological tumors. Some exemplary solid tumors may 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. Some exemplary hematological tumors include myeloid tumors, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Other 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 carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric 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 or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanoma, ovarian cancer, uterine ... The main types of NHL include B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma; small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-dividing cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloid leukemia.

[0242] The cancer may specifically be of the following histological types, although it is not limited thereto: malignant neoplasms; epithelial carcinoma; undifferentiated epithelial carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; solid carcinoma; malignant carcinoid tumors; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin attachments; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceroinous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma 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 thecoma, malignant; malignant granulosa cell tumor; malignant androblastoma; sertoli cell carcinoma; malignant leydig cell tumor, malignant; malignant lipid cell tumor; malignant paraganglioma; malignant paraganglioma of the breast; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus nevus); epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma;Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Wilms tumor; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner tumor (malignant); Malignant phyllodes tumor (malignant); Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant struma thyroid tumor (struma ovarii, malignant; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacorticalosteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma odontosarcoma); malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease disease); Hodgkin's paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemias; lymphoid leukemias; plasma cell leukemias; erythroleukemias; lymphosarcoma cell leukemias; myeloid leukemias; basophilic leukemias; eosinophilic leukemias; monocytic leukemias; mast cell leukemias; megakaryoblastic leukemias; myeloid sarcomas; and hairy cell leukemias.

[0243] The polypeptides can be used as anti-tumor agents in various ways herein to trigger complement activation in tumor tissue or to trigger complement activation when needed. In a specific embodiment, the present invention contemplates a method of using a polypeptide as an anti-tumor agent, thus comprising contacting a tumor cell population with a therapeutically effective amount of the polypeptide for a time sufficient to inhibit tumor cell growth.

[0244] In one embodiment, in vivo contact is achieved by administering to the patient a therapeutically effective amount of a physiologically tolerated composition comprising a polypeptide of the invention by intravenous, intraperitoneal or intratumoral injection. The polypeptide can be administered parenterally by injection or gradual infusion over time. The polypeptide can be administered intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavitary, transdermally, dermally, can be delivered by a peristaltic device, or can be injected directly into a tissue containing tumor cells.

[0245] Therapeutic compositions comprising polypeptides are conventionally administered intravenously, for example by injection, such as unit doses. The term "unit dose" when used in reference to therapeutic compositions refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required diluent (i.e., carrier or vehicle).

[0246] The composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount administered depends on the subject to be treated, the subject's systemic ability to utilize the active ingredient, and the desired degree of therapeutic action. The exact amount of active ingredient to be administered depends on the practitioner's judgment and is specific to each individual. However, suitable dosage ranges suitable for systemic administration are disclosed herein and depend on the route of administration. Suitable regimens for initial administration and booster administration are also contemplated, and are characterized by initial administration followed by repeated administration at intervals of one or more hours by subsequent injections or other administrations. Exemplary multiple administrations are described herein, and it is particularly preferred to maintain sustained high serum and tissue levels of the polypeptide. Alternatively, continuous intravenous infusion sufficient to maintain the concentration in the blood within the range prescribed for in vivo treatment is contemplated.

[0247] It is expected that the polypeptide of the present invention can be administered systemically or locally to treat a disease, such as inhibiting tumor cell growth or killing cancer cells in cancer patients with locally advanced or metastatic cancer. It can be administered intravenously, intrathecally and / or intraperitoneally. It can be administered alone or in combination with an antiproliferative drug. In one embodiment, it is administered before surgery or other procedures to reduce the cancer load of the patient. Alternatively, it can be administered after surgery to ensure that any remaining cancer (e.g., cancer that was not eliminated by surgery) cannot survive.

[0248] The therapeutically effective amount of a polypeptide is a predetermined amount calculated to achieve the desired effect, i.e., triggering CDC in tumor tissue, and thereby mediating the ablation of the pro-inflammatory response of the tumor. Therefore, the dosage range of the polypeptide of the present invention is large enough to produce the desired treatment (e.g., cancer cell division is reduced, or cancer cell death is improved, or other clinical benefits). The dosage should preferably not be too large to cause significant adverse side effects, such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, nervous system effects, etc. Generally, the dosage will vary with the age, condition, sex, and degree of disease in the patient, and can be determined by those skilled in the art. In the case of any complication, the dosage can be adjusted by an individual physician. In some embodiments, the dosage can be from about 0.1 mg / kg to about 10 mg / kg.

[0249] VIII. Combination Therapy

[0250] In certain embodiments, the compositions and methods of the present invention embodiments involve administering a polypeptide or antibody in combination with a second or additional treatment. Such treatment can be used to treat any disease that is responsive to CDC. For example, the disease can be cancer.

[0251] The methods and compositions comprising combined treatments enhance the therapeutic or protective effects and / or improve the therapeutic effects of another anti-cancer or anti-hyperproliferative treatment. Therapeutic and preventive methods and compositions can be provided in a combined amount that effectively achieves the desired effect (e.g., killing cancer cells and / or inhibiting cell hyperproliferation). The process may involve the administration of a polypeptide or antibody and a second treatment. The second treatment may or may not have a direct cytotoxic effect. For example, the second treatment may be an agent that upregulates the immune system without a direct cytotoxic effect. Tissues, tumors, or cells may be exposed to one or more compositions or pharmacological preparations comprising one or more agents (e.g., polypeptides or anticancer agents), or by exposing tissues, tumors, and / or cells to two or more different compositions or preparations, wherein one composition provides 1) polypeptides or antibodies, 2) anticancer agents, or 3) both polypeptides or antibodies and anticancer agents. In addition, it is expected that such combined treatments can be used in combination with chemotherapy, radiotherapy, surgical treatment, or immunotherapy.

[0252] When applied to cells, the terms "contacted" and "exposed" are used herein to describe the process of delivering a therapeutic polypeptide or antibody and a chemotherapeutic or radiotherapeutic agent to or in direct juxtaposition with a target cell. To achieve cell killing, for example, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0253] Polypeptide or antibody can be used before, during, after or in multiple combinations relative to anticancer treatment.The interval between administration can be from simultaneously to several minutes to several days to several weeks.In the embodiment that polypeptide or antibody and anticancer agent are separately provided to patient, usually will ensure that meaningful time period between each delivery time does not expire, so that two compounds can still play a favorable combination effect to patient.In such case, it is expected that polypeptide and anticancer treatment can be provided to patient within about 12 to 24 or 72 hours each other, more particularly within about 6 to 12 hours each other.In some cases, it can be expected to significantly extend the time period of treatment, wherein between each administration, several days (2,3,4,5,6 or 7 days) to several weeks (1,2,3,4,5,6,7 or 8 weeks) are spaced.

[0254] In certain embodiments, the course of treatment will last for 1 to 90 days or longer (this range includes the number of days between intervals). It is expected that one agent can be given on any day from the 1st to the 90th day (this such range includes the number of days between intervals) or any combination thereof, and another agent can be given on any day from the 1st to the 90th day (this such range includes the number of days between intervals) or any combination thereof. Within a day (within 24 hours), one or more agent administrations can be given to the patient. In addition, after the course of treatment, it is expected that there is a time period in which anticancer treatment is not administered. This time period can last for 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or longer (this such range includes the number of days between intervals), depending on the patient's condition, such as its prognosis, strength, health, etc. It is expected that the treatment cycle can be repeated as needed.

[0255] A variety of combinations can be used. For the following examples, the polypeptide or antibody is "A" and the anti-cancer treatment is "B":

[0256] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0257] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0258] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0259] Any polypeptide or treatment of the present invention embodiment is administered to the patient and will follow the general scheme of administering such compound in the case of considering the toxicity (if any) of the medicament.Therefore, in some embodiments, there is a step of monitoring the toxicity attributable to the combination therapy.In some embodiments related to treating cancer in the subject, the second treatment can be, for example, chemotherapy, radiotherapy, immunotherapy (for example, checkpoint inhibitors, such as anti-PD1 antibodies or anti-PDL1 antibodies), gene therapy, anti-inflammatory drugs, antibiotics or surgery.

[0260] A. Chemotherapy

[0261] According to some embodiments of the present invention, a wide variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to represent a compound or composition used in the treatment of cancer. These agents or drugs are classified by their intracellular activity patterns (e.g., whether and at which stage they affect the cell cycle). Alternatively, agents can be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0262] Some examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins such as phenoxyethanol; n) (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard mustard; nitrosoureas, for example carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, for example enediyne antibiotics (for example calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 11); dynemicins, including dynemicin A; bisphosphonates, for example clodronate; esperamicin;and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, actinomycin dactinomycin, daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin antimetatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine;Pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents, such as mitotane and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate 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; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; m itolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes complexe, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinums; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide (1,2-dimethoate); poside; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids or derivatives thereof. ;

[0263] B. Radiation therapy

[0264] Other factors that cause DNA damage and have been widely used include commonly known gamma rays, X-rays and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damage factors, such as microwaves, proton beam irradiation (USPs 5,760,395 and 4,870,287) and UV irradiation are also considered. Most likely, all of these factors produce extensive damage to DNA, DNA precursors, DNA replication and repair, and the assembly and maintenance of chromosomes. The dosage range of X-rays is from a daily dose of 50 to 200 roentgens for a prolonged period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake of neoplastic cells.

[0265] C. Immunotherapy

[0266] The skilled artisan will appreciate that immunotherapy may be combined or used in conjunction with the methods of some embodiments. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and inhibit immune cells. Blinatumomab It is such an example. Checkpoint inhibitors such as ipilimumab are another such example. Immune effectors can be, for example, antibodies that are specific to some markers on the surface of tumor cells. A single antibody can serve as an effector of treatment, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.), and are used only as targeting agents. Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0267] In one aspect of immunotherapy, tumor cells must carry some markers suitable for targeting (i.e., not present on most other cells). There are many tumor markers, and in the case of embodiments of the present invention, any of these tumor markers may be suitable for targeting. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligands.

[0268] Some examples of immunotherapies currently under investigation or in use are immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998); and immunotherapy. al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 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. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

[0269] D. Surgery

[0270] About 60% of people with cancer will undergo some type of surgery, which includes preventive, diagnostic or staged, curative and palliative surgery. Curative surgery includes resection, in which all or a portion of the cancerous tissue is physically removed, excised and / or destroyed, and can be used in conjunction with other treatments such as the treatment of some embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy and / or alternative treatment. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, treatment by surgery also includes laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohs' surgery).

[0271] After removing part or all of the cancer cells, tissues or tumors, a cavity can be formed in vivo. Treatment can be accomplished by perfusion, direct injection or local application of other anticancer treatments to the region. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also have different dosages.

[0272] E. Other medicines

[0273] It is expected that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cytostatics and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Improving intercellular signaling by increasing the number of GAP connections can increase the anti-hyperproliferative effect on neighboring hyperproliferative cell groups. In other embodiments, cytostatics or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Consider cell adhesion inhibitors to improve the efficacy of the embodiments of the present invention. Some examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is also considered that other agents (e.g., antibody c225) that increase the sensitivity of hyperproliferative cells to apoptosis can be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy.

[0274] IX.Medicine Box

[0275] Certain aspects of the invention may provide kits, such as therapeutic kits. For example, the kit may include one or more pharmaceutical compositions described herein and optionally instructions for use thereof. The kit may also include one or more devices for completing the administration of such compositions. For example, the subject kit may include a pharmaceutical composition and a catheter for completing the intravenous injection of the composition directly into a cancerous tumor. In other embodiments, the subject kit may include an ampoule of a pre-filled polypeptide, optionally formulated as a drug or lyophilized, for use with a delivery device.

[0276] The kit may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed of a variety of materials (e.g., glass or plastic). The container may hold a composition comprising a polypeptide effective for, for example, the above-mentioned treatment or non-therapeutic applications. The label on the container may indicate that the composition is used for a specific treatment or non-therapeutic application, and may also indicate directions for in vivo or in vitro use, such as those described above. The kit of the present invention will typically include the above-mentioned container and one or more other containers, which contain materials desired from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0277] X. Examples

[0278] The following examples are included to illustrate some preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, it will be appreciated by those skilled in the art that, according to this disclosure, many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention, and the same or similar results may still be obtained.

[0279] Example 1

[0280] Library construction strategy for isolating IgG1 Fc domains that bind to FcγRIIB

[0281] The Fc receptor binding residues in the aglycosylated V8.2Fc were mutagenized, and additional random mutations were introduced in the Fc by error-prone PCR. The FcγRIIb binding residues in the Fc (ELLGG (233 to 237, EU numbering), SH (267, 268), NST (297 to 299), and ALPAPIE (327 to 333)) were used as site-saturation mutagenesis residues for the FcγRIIb selective Fc library. The library was displayed on the yeast surface to screen for FcγRIIb selective variants. The N-terminal Aga2-linked Fc mutants were expressed and displayed on the cell surface of yeast cells, which were linked to Aga1 via two disulfide bonds. The population showing binding affinity for FcγRIIb was sorted with other streptavidin-coated tetramer-FcγRs used as competitors. During the screening, in the presence of high concentrations of FcγRIIa R131 -tetramer as competitor, the first and second libraries were sorted for binding to fluorescently labeled FcγRIIb-tetramer. In order to sort only the FcγRIIb selective population, a two-step sorting method was used for the third and fourth library sorting. First, the library was compared with FcγRIIa R131 (the most competitive FcγR) and the negative population was sorted to remove FcγRIIa R131 FcγRIIa was isolated from this step. R131 Non-binders. Next, these sorted cells were incubated with FcγRIIb tetramers and sorted for the 2b positive population. After sorting the fourth library, 91 individual clones were selected from the final library and 8 variants were expressed in HEK293 cells for affinity measurements.

[0282] Example 2

[0283] Binding characteristics of Fc2b variants

[0284] Table 4 shows a list of modified Fc variants used in other experiments described below. V8.2 is an Fc variant produced using the method described in Example 1. EF, V11 and V12 are published Fc variants that show enhanced binding affinity to FcγRIIb (Teige et.al., 2019) V11 and V12: (Mimoto, et al., 2013), and these variants were evaluated for comparison purposes. In the 2b18K variant, the G237D mutation overlaps with V12 and V11. E233V is the same site as V12 but a different mutation, but not with V11. H268, A330 are the same sites as V12 and V11 but different mutations. L328 is the same site as EF but a different mutation. The substitution mutations in Table 4 are shown using Kabat numbering.

[0285] Table 4 Mutations of Fc2b variants

[0286]

[0287] In order to verify the binding affinity to Fcγ receptors, biolayer interferometry (BLI) was measured on the Octet RED96 system (ForteBio Inc., California, USA). High-precision streptavidin (SAX) biosensor (Sartorius Inc., 18-5117) was used and measured under shaking at 1,000 rpm at 25 ° C. For KD measurement, biotinylated FcgR was fixed on the biosensor until enriched to 0.5 or 1.0nm displacement. The monoclonal antibody was associated for 2 minutes and dissociated for 2 minutes. A 1: 1 binding model with a drift baseline was used in the BIAevaluation software to calculate KD. The results are shown in Table 5.

[0288] Table 5 Binding properties of engineered antibody variants measured by BLI

[0289]

[0290] a: previously published data, NA: not determined, NB: no binding

[0291] By BLI, the FcγRIIb selective Fc variant candidates (2b18K, 2b18KQ, and 2b18KQS) showed binding affinity to FcγRIIb and no or very low binding affinity to activating receptors (Table 5). In contrast, the previously reported 2b enhanced binders (i.e., EF, V11, and V12) showed binding affinity to activating receptors, especially to FcγRIIa. R131 Binding affinity (FcγRIIa R131 It is 95% identical to the extracellular domain of FcγRIIb and differs only in three residues located at the binding interface).

[0292] In order to more accurately measure the binding affinity of 2b18KQS Fc variants, surface plasmon resonance (SPR) assays were performed using 2b18KQS variants. The results are shown in Table 6 below. The method described previously in Lee et al., 2017 was used.

[0293] Table 6 Affinity of 2b18KQS Fc variants for Fc receptors measured by SPR (by steady-state analysis)

[0294]

[0295] The 2b18KQS Fc variant did not show any detectable binding affinity to activating receptors and even to the high affinity Fc receptor FcγRI (Table X3). The variant showed reduced binding to FcγRIIb (4-fold lower than WT Fc) and no significant binding to FcγRIIa. R131 There was almost no detectable binding (40-fold lower than WT Fc). The binding affinity of 2b18KQS Fc to FcRn was similar to WT Fc, which may be related to the circulating half-life.

[0296] Due to differences in avidity, the binding characteristics of Fc variants in BLI may not always accurately reflect the binding properties of immune complexes. To measure the binding profile under high-avidity conditions, opsonized SK-BR-3 and FcγR-coated bead binding assays were performed with the 2b18K variant. Biotinylated Fcγ receptors were coated on beads to mimic the geometry of Fc receptors on effector cells, and binding was measured by flow cytometry. Results are presented in Figure 1 Shown in.

[0297] exist Figure 1 In the first column, the results for cancer cells opsonized with WT antibodies are shown, and the following columns show data for V8.2, 2b18K, V12, and EF Fc variants. The numbers shown refer to the fold difference in binding affinity compared to wild-type Fc as measured by BLI, and the basket ( Figure 1 The numbers in parentheses (in ) are published values ​​from other laboratories. WT showed strong binding to all Fcγ receptors. V8.2 showed strong 2aR and 2b binding, even though it showed a 3-fold lower binding affinity than WT in BLI. 2b18K showed weak binding to 2aR and strong binding to 2b. V12 and EF showed strong binding to 2a and 2b as well as 3aV.

[0298] Binding assays of opsonized SK-BR-3 and FcγR-coated beads were performed with 2b18KQ and 2b18KQS Fc variants ( Figure 2 ). The LALAPG Fc variant was included as a negative control (it is a well-known scientific Fc and completely abolishes Fc-mediated effector function). The 2b18K variant showed no binding to FcγRIIa H131 , FcγRIIIa and FcγRIIIb, but maintained binding to FcγRIIb. In the 2b18K variant, 2b18KQS was shown to bind to FcγRIIa. R131 The binding affinity is the lowest.

[0299] To examine actual cell binding, IC binding assays were performed using B Burkitt lymphoma Raji cells (ATCC CCL-86). Raji cells express FcγRIIb only in FcγRs on the surface. Therefore, FcγRIIb binding was measured using B cells. Antibody-coated beads were used as ICs, and binding was measured by flow cytometry. The results are in Figure 3 To generate immune complexes, 1 μm fluorescent polystyrene beads were coated with antibodies. These ICs were incubated with Raji cells at 4°C for 1 hour. The binding of ICs to Raji cells was detected by flow cytometry.

[0300] The 2b18K variant showed similar binding to WT Fc and significantly better binding than the LALAPG Fc variant ( Figure 3 These results are consistent with and further support those obtained using binding assays with opsonized SK-BR-3 and FcγRIIb-coated beads.

[0301] Example 3

[0302] Thermal stability of Fc2b variants

[0303] Measure the melting temperature (T m ) to evaluate the thermodynamic stability of the engineered Fc variants without glycosylation. T was measured by differential scanning fluorimetry (DSF). m Compared with WT Fc, the T of 2b18K variant m showed lower values ​​(Table 7). These results are consistent with the view that aglycosylation can induce loss of thermal stability. The method described in Lee et al., 2019 was used.

[0304] Table 7 Tm measured by differential scanning fluorimetry

[0305] Variants Her WT Her 2b18K Her 2b18KQ Her 2618KQS <![CDATA[T m (℃)]]> 68.70±0.17 57.44±0.40 53.66±0.14 56.28±0.32

[0306] Example 4

[0307] Validation of phagocytic effector function in effector cells

[0308] The lack of binding affinity for activating receptors in ICs attenuates phagocytic effector function. To measure the phagocytic rate of the 2b18K variant Fc IC, a phagocytic assay using pHrodo red beads coated with antibodies was performed using the THP-1 (FcγRIIa H / H homozygous) cell line. pHrodo is a pH-sensitive dye that increases the signal at low pH. At normal pH, little or no signal is detected. The pHrodo fluorescence signal increases when the phagosome contains the target cell and becomes acidic as detected on the flow cytometer. Using this dye, actual phagocytosis can be detected and only surface-bound signals can be removed. THP-1 cells (50k / well) were mixed with antibody-coated beads (50-fold excess) in RPMI-1640 (Gibco, 11875135) in a TC-treated round-bottom plate (Corning, 07-200-95) and incubated for 4 hours at 37C 5% CO2. Cells were washed once with DPBS and immediately analyzed in a BD LSRII flow cytometer equipped with a high throughput sampler. The extent of phagocytosis was expressed as the percentage of bead+ / pHrodo+ cells multiplied by the Dragon Green geometric MFI of the double positive population.

[0309] THP-1 cells expressed FcγRIIaH131, to which 2b18K, 2b18KQ, and 2b18KQS did not detectably bind (Table 5). 2b18K, 2b18KQ, and 2b18KQS variants were used, and LALAPG Fc-coated beads were used as negative controls. No FcyR-mediated phagocytosis was detected with 2b18K, 2b18KQ, and 2b18KQS ICs, which is consistent with the binding assay results. Figure 4 Shown in.

[0310] SK-BR-3 ADCP assay was performed with THP-1 cells, revealing phagocytosis mediated by null antibodies against 2b18K and V8.2 ( Figure 5 ). However, high phagocytic rates were detected in V12 and EF variants with higher binding affinity for FcγRIIb. These results indicate that higher binding affinity for FcγRIIb compared to activating FcγRs does not guarantee low phagocytic activity (Table 5). The phagocytic rate of the V12 variant was significantly higher than that of WT and EF, despite its lower affinity for FcγRIIaH131. These results indicate that the absence of activating receptors is indispensable for the abrogation of immune cell activation.

[0311] The 2b18K, 2b18KQ, and 2b18KQS variants were also tested using the SK-BR-3 ADCP assay with THP-1 cells. ICs that bind only to FcγRIIb do not induce activating FcγRs and trigger effector functions. Figure 6Shown in.

[0312] Additional experiments were performed to measure immune complex binding activity. Deposition of C1q on CD20+ Raji or Ramos cells was measured by FACS. Cells were opsonized with WT rituximab or Fc engineered variants. All 2b18K, 2b18KQ, and 2b18KQS variants showed low or no C1q deposition on the cell surface. Results Figure 8 Shown in.

[0313] Additional experiments using cell-based in vitro assays were performed using 2b18K, 2b18KQ, and 2b18KQS variants. No complement or activating FcγR-mediated effector function was detected using the Fc2b variants. As shown in FIG9A , complement-dependent cytotoxicity (CDC) against CD20 was measured. + Lysis of Raji cells or Ramos cells. The cells were opsonized with various concentrations of rituximab or its Fc-engineered variants and incubated with 10% pooled human serum. Antibody-dependent cellular phagocytosis (ADCP) assays were performed using WT or Fc-engineered variants of trastuzumab, THP-1 cells as effectors, and SK-BR-3 cells as targets ( FIG. 9B ). Antibody-dependent cellular cytotoxicity (ADCC) assays were measured using peripheral blood mononuclear cells (PBMCs) ( FIG. 9C ). The target cells were Raji cells opsonized with WT rituximab or its Fc-engineered variants. ADCP assays were performed using human monocyte-derived M1 macrophages as effectors and SK-BR-3 cells as targets ( FIG. 9D ). Various concentrations of WT trastuzumab or Fc-engineered variants were added. After CD20 + After incubation of Raji cells with WT rituximab or Fc engineered variants for 1 hour, phosphorylation of FcγRIIb was analyzed by immunoblotting (Figure 9E). In all assays, Fc2b variants 2b18K, 2b18KQ and 2b18KQS did not show any complement or activating FcγR-mediated activation, and these variants showed functional binding to FcγRIIb.

[0314] Example 5

[0315] Hexameric engineered Fc variants that bind to FcγRIIB

[0316] Hexameric mutant Fc regions were generated as follows. The 18-aa residues of the human IgMμ-tail (PTLYNVSLVMSDTAGTCY; SEQ ID NO: 6) were fused to the C-terminus of the human IgG1 Fc fragment (Rowley et al., 2018; Spirig et al., 2018). Additional mutations were added at the 309th (EU numbering) Leu residue of the mutant IgG Fc to introduce a disulfide bond between Fc. A modified hexameric construct (hexameric Fc2b) was generated, and the results are shown in Figures 10A to D.

[0317] The following sequences were generated and observed to form hexamers. Hex 2b18KQS (aglycosylated Fc):

[0318]

[0319] Hex 2b18KQS-ST (glycosylated form of 2b18KQS):

[0320]

[0321] Hex2b218K (aglycosylated hexameric form of the hexameric construct of Fc variant 2b18K):

[0322] Hex2b218K-ST (glycosylated hexameric form of the hexameric construct of Fc variant 2b18K):

[0323]

[0324] exist Fig.11 A sequence alignment showing the point mutations present in the hexameric wild-type Fc and different engineered variant Fc regions is shown in .

[0325] The platelet activation assay will be tested by the following method. Platelets express FcγRIIa on their surface, and when they come into contact with immune complexes, FcγRIIa is responsible for activating platelets. In order to test whether there is any platelet activation mediated by FcγRIIa in the presence of hexamer 2b selective Fc (Hex2b18KQS or Hex 2b18KQS-ST), platelet activation can be assessed by co-incubation with hexamer 2b selective Fc. It is expected that in the presence of hexamer 2b selective Fc, FcγRIIa will mediate little or no activation of platelets.

[0326] Complement dependent cytotoxicity (CDC) assays were performed as described in Lee et al. (2017). Hexameric IgG1 has better binding affinity for C1q. However, hexameric Fc-mediated CDC has not been shown previously. Hexameric 2b-selective Fc (Hex 2b18KQS or Hex2b18KQS-ST) was observed to have reduced C1q binding affinity, and it is expected that hexameric 2b-selective Fc will have no CDC activity. A CDC assay will be performed to demonstrate the absence of hexameric 2b-selective Fc-mediated CDC.

[0327] B cell proliferation and differentiation assays were performed using hexameric 2b-selective Fc (Hex 2b18KQS and Hex 2b18KQS-ST). To verify the effect of FcγRIIb engagement alone on B cell proliferation and differentiation, hexameric Fc will be treated during B cell proliferation and differentiation and analyzed for the effect. B cell proliferation and differentiation assays can be performed as described in Khoenkhoen et al. (2020).

[0328] Primary monocyte and macrophage-mediated ADCP assays were performed using hexamer 2b-selective Fc (Hex 2b18KQS and Hex 2b18KQS-ST). A large body of evidence suggests that blocking FcγRIIb can improve the therapeutic efficacy of therapeutic antibodies. The tumor microenvironment (TME) expresses elevated levels of FcγRIIb, at least in part due to the hypoxic conditions of the TME. To mimic the TME, monocytes and macrophages were incubated under physiological and pharmacological hypoxic conditions, and overexpression of FcγRIIb was determined (Hussain et al., 2022). Phagocytic function was significantly restored by blocking FcγRIIb. Hexamer 2b selective Fc (Hex 2b18KQS and Hex2b18KQS-ST) was tested using primary monocyte and macrophage mediated ADCP assay to measure the blocking effect of FcγRIIb on monocytes and macrophages incubated under hypoxic conditions. Primary monocyte and macrophage mediated ADCP assay was performed as described in Kang et al. (2019).

[0329] Fig.12 The binding characteristics of the hexameric Fc2b constructs are shown in FIG. The results of the FcγR coated beads and hexameric Fc binding assays using Hex2b18KQS Fc and glycosylated Hex 2b18KQS-ST Fc are shown. The y-axis shows Fig.12 The average fluorescence intensity in .

[0330] The binding of hexameric Fc to Fcγ receptors was confirmed by bead binding assay. Hexameric Fc has six valencies, which provides sufficient avidity to bind to cells expressing Fcγ receptors or multimers of receptors. By using Fcγ receptor-coated beads, the binding characteristics of the hexamer can be measured ( Fig.12 ). Hex2b18KQS showed selective binding to FcγR2b and some binding to FcγR2aR. Glycosylated hexameric 2b18KQS-ST Fc was observed to show increased selectivity for FcγR2b, but decreased binding compared to Hex 2b18KQS. The LALAPG Fc variant was expressed as a fusion protein with human IgMμ-tail (SEQ ID NO:6) and included as a negative control (because this Fc variant is a well-known scientific Fc and completely eliminates Fc-mediated effector function).

[0331] Fc variant 2b18K is hexamerized to construct Hex 2b18K. Glycosylated variants of Hex 2b18K are also produced and referred to as Hex 2b18K-ST, and include G298S and A299T substitution mutations in 2b18K Fc variants. Hex LALAPG-2 is a LALAPG (L234A, L235A, P329G) variant hexameric Fc. LALAPG Fc variants are silent variants that do not bind to any Fcγ receptors, and therefore the hexameric variants are used as negative controls in experiments. Hex LALAPG-2 is produced as a hexameric Fc based on LALAPG Fc variants, but V567I and A572G point mutations are also included in the IgMμ-tail to stabilize the structure and increase expression.

[0332] Hexameric Fc was tested for binding to Fcγ receptors using a Raji cell binding assay with antibody-coated beads and the results were Fig.13 The error bars are the standard error of the mean of triplicate samples. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test (***P < 0.001, ****P < 0.0001). Binding to FcγR2b was further analyzed using B cell lines. Cellular binding of hexameric Fc was tested using Raji cells that express only FcγR2b in the Fc receptor ( Fig.13 ). Concentration-dependent binding of Hex2b18KQS Fc and glycosylated Hex 2b18KQS-ST Fc was observed.

[0333] It was observed that hexameric Fc blocking Fcγ receptor 2b increased phagocytic activity in monocytes and the results were Fig.14To examine the effect of FcγR2b blockade on monocyte phagocytosis, THP-1 cell antibody-dependent cellular phagocytosis (ADCP) assay was performed with opsonized SK-BR-3 cells. Hex WT showed negligible ADCP rates because it also blocks activating Fc receptors. Blockade of FcγR2b by Hex 2b18KQS and Hex 2b18KQS-ST showed increased ADCP rates compared to Her WT alone.

[0334] ***

[0335] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present invention have been described according to some preferred embodiments, it is obvious to those skilled in the art that the methods and steps or the order of steps of the methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be obvious that the medicaments described herein can be replaced by medicaments related to both certain chemical and physiological aspects, and the same or similar results will be obtained at the same time. 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 defined by the appended claims.

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Claims

1. A polypeptide comprising a mutant or variant human IgG Fc domain capable of binding to human FcγRIIb, wherein the mutant or variant human IgG Fc domain comprises substitution mutations of 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 position numbering is based on the Kabat system.

2. The polypeptide of claim 1, wherein the mutant or variant human IgG Fc domain further comprises substitution mutations of glycine at position 298 (S298G) and alanine at position 299 (T299A).

3. The polypeptide of any one of claims 1 to 2, wherein the substitution mutation at position 234 is proline at position 234 (L234P) or aspartic acid at position 234 (L234D).

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

5. The polypeptide of any one of claims 1 to 4, wherein the substitution mutation at position 235 is threonine at position 235 (L235T) or phenylalanine at position 235 (L235F).

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

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

8. The polypeptide of claim 7, wherein the mutant or variant human IgG Fc domain further comprises a substitution mutation of aspartic acid at position 237 (S267D).

9. The polypeptide of claim 8, wherein the mutant or variant human IgG Fc domain further comprises a substitution mutation of glutamine at position 332 (I332Q) and / or valine at position 334 (K334V).

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

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

12. The polypeptide of claim 11, wherein the mutant or variant human IgG Fc domain further comprises 1, 2, 3 or all of the following: substitution mutations of 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 of claim 12, wherein the mutant or variant human IgG Fc domain further comprises a substitution mutation of aspartic acid at position 267 (S267D).

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

15. The polypeptide of claim 13, wherein the mutant or variant human IgG Fc domain further comprises a substitution mutation of glutamine at position 292 (R292Q).

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

17. The polypeptide of claim 12, wherein the mutant or variant human IgG Fc domain further comprises a substitution mutation of glutamine at position 292 (R292Q).

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

19. The polypeptide of any one of claims 1 to 18, wherein the mutant or variant human IgG Fc domain is aglycosylated.

20. The polypeptide of any one of claims 1 to 18, wherein the mutant or variant human IgG Fc domain is glycosylated.

21. The polypeptide of any one of claims 1 to 20, wherein the Fc domain does not selectively or detectably bind to 1, 2, 3, 4 or all of the following: human FcγRI, FcγRIIa H131, FcγRIIIa F158, FcγRIIIa V158, and / or C1q polypeptide.

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

23. The polypeptide of 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 polypeptide of any one of claims 1 to 23, wherein the Fc domain binds to FcγRIIa compared to wild-type Fc. R131 The binding of β-lactamase is at least 30- or 40-fold lower.

25. The polypeptide of any one of claims 3 to 24, wherein the Fc domain further comprises a substitution mutation at position 298 and / or a substitution mutation at position 299.

26. The polypeptide of claim 25, wherein the Fc domain comprises a leucine at amino acid position 299 (T299L).

27. The polypeptide of any one of claims 3 to 9, 11 to 13, 15, 17 or 19 to 24, wherein the Fc domain comprises a serine at position 298 and a threonine at position 299.

28. The polypeptide of any one of claims 1 to 27, further comprising a non-Fc receptor (non-FcR) binding domain.

29. The polypeptide of claim 28, wherein the non-FcR binding domain is an Ig variable domain, an antibody variable domain, or an antibody heavy chain variable domain.

30. The polypeptide of claim 29, wherein the polypeptide is a full-length antibody.

31. The polypeptide of claim 30, wherein the antibody is an agonistic antibody.

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

33. The polypeptide of claim 29, wherein the Ig variable domain comprises an antibody heavy chain variable domain.

34. The polypeptide of claim 33, wherein the Ig variable domain is comprised in a single domain antibody.

35. The polypeptide of claim 29, wherein the Ig variable domain comprises a ScFv.

36. The polypeptide of any one of claims 1 to 35, wherein the mutant or variant human IgG Fc domain is contained in a multimeric oligomer.

37. The polypeptide of claim 36, wherein the multimeric oligomer is further defined as a hexameric Fc fusion protein.

38. The polypeptide of claim 37, wherein the hexameric Fc fusion protein comprises PTLYNVSLVMSDTAGTCY (SEQ ID NO:6), PTLYNVSLIMSDTGGTCY (SEQ ID NO:9), PTLYNVSLIMSDTAGTCY (SEQ ID NO:10), or PTLYNVSLVMSDTGGTCY (SEQ ID NO:11).

39. The polypeptide of claim 38, wherein the hexameric Fc fusion protein comprises PTLYNVSLVMSDTAGTCY (SEQ ID NO: 6).

40. The polypeptide of any one of claims 37 to 39, wherein the mutant or variant human IgG Fc domain comprises a substitution mutation of a Leu residue at position 309, wherein the amino acid position numbering is according to the Kabat system.

41. The polypeptide of any one of claims 37 to 40, wherein the hexameric Fc fusion protein is glycosylated.

42. The polypeptide of any one of claims 37 to 40, wherein the hexameric Fc fusion protein is aglycosylated.

43. The polypeptide of any one of claims 37 to 42, wherein the mutant or variant human IgG Fc domain comprises a Gly residue at position 298 and / or an Ala residue at position 299.

44. The polypeptide of claims 37 to 43, wherein the mutant or variant human IgG Fc domain comprises a Ser residue at position 298 and / or a Thr residue at position 299. E 45. The polypeptide of E 37, wherein the hexameric Fc fusion protein comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, or SEQ ID NO:

13.

46. ​​The polypeptide of claim 45, wherein the hexameric Fc fusion protein comprises SEQ ID NO:7 or SEQ ID NO:

8.

47. The polypeptide of any one of claims 1 to 46, wherein the polypeptide or antibody is chemically conjugated or covalently bound to a toxin.

48. The polypeptide of claim 28, wherein the non-FcR binding region binds a cell surface protein.

49. The polypeptide of claim 28, wherein the non-FcR binding region binds a soluble protein.

50. The polypeptide of claim 28, wherein the non-FcR binding domain comprises a single domain antibody, a scFv, or a nanobody.

51. The polypeptide of any one of claims 1 to 50, wherein the polypeptide is aglycosylated.

52. The polypeptide of any one of claims 1 to 50, wherein the polypeptide is glycosylated.

53. The polypeptide of any one of claims 1 to 52, wherein the Fc domain does not trigger or does not substantially trigger antibody-mediated phagocytosis.

54. The polypeptide of any one of claims 1 to 53, wherein the Fc domain does not trigger or does not substantially trigger antibody-mediated cytotoxicity.

55. The polypeptide of any one of claims 1 to 54, wherein the Fc domain does not induce or does not substantially induce induction of activating FcγRs.

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

57. The nucleic acid of claim 56, wherein the nucleic acid is a DNA segment.

58. The nucleic acid of claim 56, wherein the nucleic acid is an expression vector.

59. A host cell comprising the nucleic acid of any one of claims 56 to 58.

60. The host cell of claim 59, wherein the host cell expresses the nucleic acid.

61. The host cell of any one of claims 59 to 60, wherein the host cell is a eukaryotic cell.

62. The host cell of any one of claims 59 to 60, wherein the host cell is a mammalian cell, an insect cell, or a yeast cell.

63. A method for preparing aglycosylated polypeptide, comprising: a) obtaining a host cell according to any one of claims 60 to 62; b) incubating the host cell in culture under conditions that promote expression of the aglycosylated polypeptide; and c) purifying the expressed polypeptide from the host cell.

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

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

66. A pharmaceutical formulation comprising a polypeptide as claimed in any one of claims 1 to 55 or a nucleic acid as claimed in any one of claims 56 to 58 in a pharmaceutically acceptable carrier.

67. A method of binding a protein in a mammalian subject comprising administering to the subject an antibody, wherein the antibody binds to the protein and comprises an Fc domain according to any one of claims 1 to 55.

68. The method of claim 67, wherein the antibody is capable of specifically binding to human FcγRIIb, and wherein the antibody has reduced binding to one or more activating Fcγ receptors compared to a human wild-type IgG Fc domain.

69. The method of claim 68, wherein the antibody is aglycosylated.

70. The method of claim 68, wherein the antibody is glycosylated.

71. The method of any one of claims 67-70, wherein following administration, the antibody does not cause or does not substantially cause antibody-mediated phagocytosis in the subject.

72. The method of any one of claims 67 to 71, wherein the mammalian subject is a human.

73. The method of any one of claims 67 to 72, wherein in the subject, the antibody binds FcγRIIa with at least about 30-fold lower or about 40-fold lower affinity than wild-type Fc R131 Receptor. E 74. The method of any one of E 67-73, wherein the antibody does not selectively or detectably bind one or more activating human Fc gamma receptor polypeptides in the subject. E 75. The method of any one of E 73-74, wherein the activating human Fcγ receptor polypeptide is FcγRI, FcγRIIa H131, FcγRIIIa F158, and / or FcγRIIIa V158.

76. The method of any one of claims 67 to 75, wherein the antibody does not specifically or detectably bind one or more activating human CIq.

77. The method of claim 67, wherein the antibody is a therapeutic antibody in an aglycosylated form.

78. A method of treating a subject suffering from a disease, comprising administering to the subject an effective amount of the formulation of claim 66.

79. The method of claim 71, wherein the method does not induce antibody-dependent cellular cytotoxicity.

80. The method of claim 78, wherein the disease is cancer, infection, or autoimmune disease.

81. The method of claim 78, wherein the subject is a human patient.

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