Compositions and Uses Thereof

Abstract

Molecules, particularly polypeptides, including immunoglobulins (e.g., antibodies) containing mutations that reduce maternal-to-fetal transfer, as well as nucleic acids encoding such polypeptides, methods of making and using the molecules, including therapeutic and diagnostic compositions, formulations, kits, and methods of treatment.

Description

[Technical Field]

[0001] The present disclosure relates to molecules, particularly polypeptides, including but not limited to immunoglobulins (e.g., antibodies), that comprise mutant IgG Fc domains containing mutations that reduce maternal-to-fetal transfer. The disclosure also includes nucleic acids encoding such polypeptides, expression vectors, host cells, and methods of making and using them, including therapeutic and diagnostic compositions, formulations, and kits. [Background technology]

[0002] The development of new therapeutic agents is extremely challenging because drugs administered to the mother may be dangerous to the fetus. Very few animal models mirror the physiology and pathology of human pregnancy, and therapeutic effects in these models do not adequately reflect those in humans. Maternal metabolism can change during different stages of pregnancy and in pathological conditions, meaning that fetal exposure is diverse and difficult to define. Fetal susceptibility also changes as development and growth progress. Furthermore, tools for monitoring fetal safety during pregnancy are very limited, making it difficult to easily detect or characterize drug-related adverse effects. These limitations and safety concerns combine to leave few drugs approved for use during pregnancy and few in development, severely limiting the prospects for new treatments.

[0003] Therefore, there is an unmet need for a new class of therapeutic agents that can address medical conditions in pregnant women (e.g., pre-existing autoimmune disorders or pregnancy-related hypertension or eclampsia / pre-eclampsia) and are safe for the fetus.

[0004] This important problem is solved by the present invention by providing polypeptides, particularly antibodies, that are not transported across the placenta, thereby enabling the development of new drugs that can take advantage of the broad therapeutic applicability and specificity of antibodies without fetal safety concerns.

[0005] No attempt has been made to intentionally reduce the placental transfer ability of a polypeptide to solve the above problems. Placental transfer has been studied extensively (as discussed below), but the attempt has always been to promote or increase placental transfer rather than inhibit it.

[0006] The placenta functions to exchange all vital nutrients between the early embryo and the developing fetus and to remove waste products resulting from fetal metabolism. It selectively permits the exchange of dissolved gases, small molecules, and nutrients, but provides a nearly complete barrier to larger molecules and proteins, thereby preventing the mixing of maternal and fetal blood components.

[0007] In women, maternal antibodies of the IgG subclass cross the placenta in increasing amounts beginning around the 20th week of pregnancy. This allows for the transfer of maternal antibodies, providing a certain level of immunity to the fetus until its own immune system develops. The efficiency of antibody transfer depends on the IgG subclass, with IgG-1 being the most efficient. Administered therapeutic antibodies can be measured at higher concentrations (1.5–4-fold) in the fetal circulation compared to the mother, making this an active process (Pham-Huy, A., et al., From mother to baby: antenatal exposure to monoclonal antibody biologics. Expert Review of Clinical Immunology. Taylor and Francis Ltd; 2019, 15: 221–9).

[0008] The transfer of antibodies from mother to fetus is not necessarily beneficial. The presence of autoantibodies against Ro (SS-A) and La (SS-B) in pregnant women, regardless of the presence or absence of a full-blown autoimmune disease, significantly increases the risk of neonatal lupus syndrome. Similarly, prospective studies of anticardiolipin autoantibodies in pregnant women have shown that women with high levels of these autoantibodies have a significantly higher incidence of fetal death during the second trimester (Elkon K., et al., Nature and functions of autoantibodies. Nat. Clin. Pract. Rheumatol. 2008, 9:491-8).

[0009] The majority of therapeutic antibodies in clinical use are of the IgG class, and placental transfer has been widely reported (Pham-Huy, KA, et al., The use and impact of monoclonal antibody biologics during pregnancy. CMAJ 2021, 193: 1129-1136). For example, azulimumab is detected in newborns at levels up to 1.5 times higher than in the maternal circulation, and the drug may persist in the neonatal circulation for 3–5 months. Placental transfer of therapeutic antibodies is a real medical concern due to known or lack of information regarding fetal risks. An example of a known risk is bevacizumab, which inhibits angiogenesis and causes fetal abnormalities in animals. According to the drug label, it is not recommended for use during pregnancy. Assessing the risk of serious adverse effects on the fetus after treatment in pregnant women, despite their low incidence, is extremely difficult. Currently, only products with long-term use can be evaluated for fetal safety using large registry databases. The risk of adverse effects from fetal exposure limits the use of therapeutic antibodies in pregnant women, meaning that treatment for ongoing maternal conditions is usually discontinued towards the end of the first trimester, after which maternal antibody transfer becomes significant, unless the maternal benefits outweigh the fetal risks. Therefore, therapeutic antibodies currently in development can only be administered to pregnant women who are willing to accept the fetal risks, and the antibody treatments available to pregnant women are quite limited.

[0010] Native IgG antibodies are tetrameric proteins containing two identical heavy chains and two identical light chains, with the variable regions of each heavy / light chain pair juxtaposed to form two identical antigen-binding sites. The carboxy-terminal portions of the heavy chains, beyond the so-called "hinge region," are not paired with the light chains and are connected to each other by disulfide bridges, comprising the so-called Fc region. The Fc region contains binding sites that interact with a range of different Fcγ and FcRn receptors, as described below. The translocation of IgG across the maternal syncytial chorion and the fetal placental endothelial cell layer is a complex process that is not fully understood, and the role of different Fc receptors is under discussion.

[0011] The FcRn receptor is known to play a major role in the placental transfer of IgG, which was demonstrated, although not for the purpose of providing the polypeptides according to the present invention, by introducing the following single mutation into the Fc region of an antibody to inhibit binding to the FcRn receptor: Disruption of Fc-FcRn binding by substituting alanine for histidine at amino acid 435 has been shown to reduce maternal-fetal transfer by as much as 90% in both pregnant mice and ex vivo human placental transfer (Firan, M., et al, The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of gamma-globulin in humans. Int. Immunol. 2001, 13: 993-1002). - Single FcRn mutations I253A, H310A, or H435A reduce maternal-to-fetal transfer by 80-90% in pregnant mice, but their effect on maternal-to-fetal transfer when used in combination was not tested (see U.S. Patent No. 6,277,375). A modified version of bevacizumab with the H435A Fc mutation was tested in a rat model and found to block maternal-fetal transfer by over 90% [Thorn, M., et al, Embryo-fetal transfer of bevacizumab (Avastin) in the rat over the course of gestation and the impact of neonatal Fc receptor (FcRn) binding. Birth Defects Res B Dev Reprod. Toxicol. 2012, 95: 363-75].

[0012] Although FcRn plays a major role in IgG transfer, no studies have demonstrated that inhibition of maternal-to-fetal transfer is reduced by more than 90% following alterations in FcRn binding. This suggests that other receptors must be involved. Furthermore, FcRn is not present on fetal endothelium, which would be expected for an FcRn-only transport process. However, selective maternal-to-fetal transfer of IgG dependent on glycosylation or antigen specificity has been reported, which cannot be explained by FcRn binding selectivity. Accordingly, FcγR receptors IIa and IIIb have been reported to play a role in maternal-to-fetal antibody transfer (Jennewein, MF, et al., Fc Glycan-Mediated Regulation of Placental Antibody Transfer. Cell. 2019, 178: 202-215). Based on the current state of research, it cannot be excluded that other FcγRs, in addition to IIa and IIIb, play a role in maternal-to-fetal transfer of IgG antibodies. Summary of the Invention [Problem to be solved by the invention]

[0013] The above cited prior art has not demonstrated greater than 90% inhibition of maternal-to-fetal transfer, and such a level of inhibition is insufficient to provide safe treatment for pregnant women. [Means for solving the problem]

[0014] The development of any therapeutic agent for the treatment of pregnant women, or women who may become pregnant, faces significant medical issues due to potential exposure and safety implications for the fetus. This important issue is resolved in the present invention by designing antibodies that do not cross the placenta, allowing the development of new drugs that can take advantage of the broad therapeutic applicability, specificity, and known manufacturing techniques of antibodies without concerns about fetal safety. Such "maternal-only" antibodies enable the creation of an entirely new class of therapeutic agents that address unmet medical needs in pregnant women, such as hypertension-related conditions and eclampsia / preeclampsia, as well as other pregnancy disorders.

[0015] The antibodies described herein, and other IgG Fc-containing molecules, may also be developed for conditions unrelated to pregnancy, in which case they may be used by women of childbearing potential and pregnant women.

[0016] It is therefore understood that the present invention is not limited to any particular disease or treatment, but rather provides a platform for drug design and drugs by which pregnant women can be safely treated for a variety of different diseases with no risk of harm to the fetus or with significantly reduced risk to the fetus.

[0017] In a preferred aspect, the invention relates to the treatment or prevention of a disorder in a pregnant woman using a therapeutic agent, wherein the therapeutic agent has a variant Fc domain, e.g., a variant polypeptide, e.g., a variant antibody, disclosed herein, and the therapeutic agent is suitable for treating or preventing the disorder, e.g., suitable for treating a pregnancy disorder, such as treating pre-eclampsia or hypertension.

[0018] The present disclosure is directed to recombinant polypeptides comprising a variant Fc domain having amino acid substitutions that result in a reduction in maternal-to-fetal transfer of greater than 90%, preferably greater than 95%, preferably greater than 96%, greater than 97%, or greater than 98%.

[0019] Polypeptides of the present disclosure comprise human variant IgG Fc domains (which may be wild-type domains) that comprise amino acid substitutions relative to the human parent Fc domain, numbered according to the Kabat EU index numbering system, wherein: a) (i) positions 234 and 235 are each substituted with alanine, or (ii) positions 234 and 235 are each substituted with alanine, and position 331 is substituted with serine; or (iii) position 234 is substituted with phenylalanine, position 235 is substituted with glutamic acid, and position 331 is substituted with serine, or (iv) position 328 is substituted with arginine and an arginine is inserted after position 236; and b) (i) position 253 is substituted with alanine, or (ii) position 435 is substituted with alanine, or (iii) positions 235, 310, and 435 are substituted with alanine; or (iv) Position 310 is substituted with alanine and position 435 is substituted with glutamine.

[0020] In addition to its role in maternal-fetal transfer of antibodies, the FcRn receptor is also important for maintaining the long half-life of IgG in the circulation. Blood proteins are endocytosed and catabolized by endothelial cells. However, IgG binds to the FcRn receptor in the low pH of endosomes, recycles to the endothelium, and is released in this neutral pH environment. Inhibition of FcRn binding prevents this rescue mechanism from operating, shortening the half-life of IgG from 5 days to 2 days, as demonstrated, for example, in mice (see U.S. Patent No. 6,277,375). The typical half-life of native-sequence antibodies in humans is approximately 10–20 days, and inhibition of recycling shortens it by 2–10-fold.

[0021] A short half-life is advantageous for treating pregnant women with "maternal-only" antibodies. The relatively short duration of pregnancy means that prolonged treatment is not necessary, and therapeutic antibodies can be given via short-term infusions when higher doses are beneficial. Taking sVEGFR-1 (vascular endothelial growth factor receptor-1) during pregnancy as an example, one of the primary modalities of therapeutic antibodies is to bind to soluble target proteins and remove them from the circulation. A short half-life means that such targets are rapidly removed from the maternal circulation upon binding, allowing for the antibody to be tapered if concentrations are easily measured, such as sVEGFR-1. Infusion of short-half-life antibodies also offers safety advantages, as treatment can be interrupted if a significant safety issue arises and the antibody is rapidly cleared. A further advantage of a short half-life for the mother is that therapeutic antibodies do not persist after delivery, when treatment may no longer be beneficial or even harmful to the mother. Preferably, the polypeptides of the invention, e.g., therapeutic antibodies, have a half-life that is at least 2-, 3-, 5-, 10- or more times reduced compared to the same molecule having a "parent" Fc domain.

[0022] Antibodies transferred from mother to fetus can persist for weeks, for example during treatment of a chronic maternal disease, indicating that the FcRn recycling mechanism that sustains IgG in the circulation is active in the newborn. Conversely, ablation of FcRn binding means that even low levels of "maternal-only" antibodies transferred across the placenta are rapidly cleared in the newborn, further reducing the potential risk.

[0023] Disabling FcγRs is also a desirable feature for treatments directed against targets that are soluble proteins but have membrane equivalents, in that immune effector-dependent activities such as antibody-directed cellular cytotoxicity, which can mediate undesirable off-target side effects, are greatly reduced or eliminated.

[0024] The present disclosure also provides an isolated nucleic acid comprising a sequence encoding a polypeptide of the present disclosure. Also provided are compositions, expression vectors, and host cells comprising a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure. The host cell may comprise an isolated nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, a composition comprising a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, or an expression vector comprising a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure.

[0025] The present disclosure also provides a method of making a polypeptide of the present disclosure, comprising: (a) culturing a host cell containing a nucleic acid comprising a sequence encoding the polypeptide of the present disclosure; and (b) isolating the polypeptide. The present disclosure also provides a composition comprising a polypeptide of the present disclosure and a carrier.

[0026] The present disclosure also provides a conjugate comprising a polypeptide of the present disclosure and a therapeutic moiety.

[0027] The present disclosure also provides a method of treating a mammal, preferably a human, preferably a female, more preferably a pregnant female, comprising administering to a patient in need of treatment an effective amount of (a) a polypeptide of the present disclosure, (b) an isolated nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, (c) a composition, expression vector, or host cell comprising a nucleic acid comprising a sequence encoding a polypeptide of the present disclosure, (d) a composition comprising a polypeptide of the present disclosure and a carrier, or (e) a conjugate comprising a polypeptide of the present disclosure and a therapeutic moiety.

[0028] The present disclosure also provides a method for reducing binding to at least one FcγR receptor and FcRn in a parent polypeptide comprising an Fc domain, the method comprising the steps of: a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) a substitution at position 310 with alanine and at position 435 with glutamine.

[0029] Other preferred features of the present invention include: An isolated polypeptide for use in the medical care of pregnant women, comprising a human mutant IgG Fc domain having a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR, and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-fetal transfer by at least 95% relative to the parent sequence.

[0030] An isolated polypeptide for use in treating or preventing a pregnancy disorder in a pregnant woman, comprising a human mutant IgG Fc domain having a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR, and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-fetal transfer by at least 95% relative to the parent sequence.

[0031] An isolated polypeptide for use as disclosed herein, or a method of treatment or a nucleic acid or a cell or a vector for use as disclosed herein, wherein the use is in the treatment or prevention of a pregnancy disorder, for example (i) the disorder or disease is a hypertension-related condition or eclampsia / pre-eclampsia; or (ii) the disorder or disease is pre-eclampsia / eclampsia and the polypeptide is an anti-VEGFR-1 antibody that inhibits binding of VEGF and / or PGF to sVEGFR-1; or (iii) An isolated polypeptide for use, or a method of treatment or a nucleic acid or a cell or a vector for use, wherein the disorder or disease is pre-eclampsia / eclampsia and the polypeptide comprises or consists of the polypeptide of SEQ ID NO: 13, provided together with the antibody light chain of SEQ ID NO: 12, in the form of an antibody, optionally in the form of a pharmaceutically acceptable composition in combination with a pharmaceutically acceptable excipient.

[0032] An antibody comprising the polypeptide of SEQ ID NO: 13 together with the antibody light chain of SEQ ID NO: 12, optionally formulated as a pharmaceutically acceptable composition together with an excipient or carrier. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a gel electrophoresis of purified antibodies WBP70323_1 (MOm301) and WBP70323_2 (MOm303). [Figure 2] Figure 2 shows the fold change in total fluorescent signal in pregnant mice and fetuses compared to the PBS control. Panel A: Pregnant mouse before dissection of the fetus. Panel B: Fetus [Figure 3] Figure 3 shows the plasma concentrations of BB301 and BB303 in pregnant mice and fetuses 24 hours after administration. Plasma concentrations in the PBS control were negative and are not shown. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description The present disclosure is directed to recombinant polypeptides comprising mutant Fc domains with amino acid substitutions that reduce maternal-to-fetal transfer. The present disclosure relates to polypeptides, more particularly immunoglobulins, comprising an IgG Fc domain (e.g., a human IgG Fc domain) or fragments thereof (preferably, an Fc or hinge-Fc domain) that contain one or more amino acid modifications relative to a parent IgG, which may be a wild-type IgG sequence, such modifications greatly reducing both binding to FcγRs and binding to FcRn.

[0035] In some aspects, the present disclosure relates particularly to modifications of human IgG or humanized IgG and other biologically active molecules containing the FcRn-binding portion of the human IgG Fc domain, particularly for use in therapy, prophylaxis, and diagnosis. In some aspects, the polypeptide comprises an IgG Fc domain, or a fragment thereof (preferably the Fc or hinge-Fc domain), containing a modification that inhibits binding to FcγR and binding to FcRn.

[0036] definition It should be noted that the term "a" or "an" refers to one or more of that entity; for example, "a polypeptide sequence" is understood to refer to one or more polypeptide sequences. Therefore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. Furthermore, when used herein, "and / or" is construed as specifically disclosing each of the two specified features or components, regardless of the presence or absence of the other feature or component. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the technical field to which this disclosure pertains.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with the general dictionary of many terms used in this disclosure.

[0038] Units, prefixes, and symbols are expressed in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino to carboxy direction. The headings provided herein are not intended to limit the various aspects that can be had by reference to the entire specification. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0039] Where embodiments are described herein using the word "comprising," it is understood that analogous embodiments described using the phrases "consisting of" and / or "consisting essentially of" are also provided.

[0040] Amino acids may be referred to herein by either their name, their commonly known three-letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are also referred to by their commonly accepted one-letter codes.

[0041] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not imply a specific length. As used herein, the term "protein" is intended to encompass molecules composed of one or more polypeptides, which may optionally be associated by bonds other than amide bonds. Alternatively, a protein may be a single polypeptide chain. In this latter example, a single polypeptide chain may optionally contain two or more polypeptide subunits fused together to form the protein. The terms "polypeptide" and "protein" also refer to products of post-expression modifications, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide or protein may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0042] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form that does not exist in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0043] Reference herein to an isolated polypeptide comprising a human variant IgG Fc domain may be a reference to a single polypeptide, or more preferably refers to a combination of two separate polypeptides that together form an Fc domain, for example via one or more disulfide bonds, which may also be described herein as a protein comprising a human variant IgG Fc domain.

[0044] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0045] Also included in the present disclosure are fragments, variants, or derivatives of polypeptides, and any combination thereof. The term "fragment" when referring to the polypeptides and proteins of the present disclosure includes any polypeptide or protein that retains at least some of the properties of the reference polypeptide or protein. Polypeptide fragments include proteolytic fragments and deletion fragments.

[0046] The term "variant," as used herein, refers to a polypeptide sequence that differs from a parent polypeptide sequence by at least one amino acid modification. The parent polypeptide may be a naturally occurring polypeptide, including known allotypes, i.e., a "wild-type" ("wt") polypeptide, or may be a modified version of a wild-type polypeptide that does not already contain all of the Fc domain amino acid substitutions disclosed herein, in which any one of the substitutions disclosed herein may be introduced, resulting in an altered Fc domain sequence.

[0047] Examples of modifications to the native wild-type IgG sequence that have been incorporated into currently approved antibodies include, but are not limited to, the following: IgG1(N297A), IgG1(N297G), IgG1(L234F / L235E / P331S), IgG1(L234A / L235A), IgG1(L235V / F243L / R292P / Y300L / P396L), IgG1(L234F / L235E / P331S), IgG1(L234A / L235A / P329G), IgG1(S354C / T366W), IgG 1(Y349C / T366S / L368A / Y407V), IgG1(M252Y / S254T / T256E), IgG1 / 2(S239D / I332E), IgG2(C131S / R133K / C219S), IgG2(H268Q / R355Q / Q419E / N4 34A), IgG2 / 4(M428L / N434S), IgG4(S228P), IgG4(S228P / F234A / L235A), IgG4(S228P / L235E), IgG4(S241P / F234A / L235A), IgG4(F405L / R409K). Thus, an IgG sequence that is a naturally occurring wild-type allotype having any of these modifications, or a combination of modifications, is a "parent" polypeptide or Fc domain as defined herein, and the parent amino acid sequence can be modified to include the Fc domain amino acid substitutions disclosed herein. Known human allotype sequences are listed in several databases, for example, ImMunoGeneTics (http: / / www.imgt.org).

[0048] The term variant polypeptide can refer to the polypeptide itself, a composition comprising the polypeptide, or the amino acid sequence encoding it. Preferably, a variant polypeptide (e.g., a polypeptide comprising a variant IgG Fc domain) has at least one amino acid modification compared to the parent polypeptide, e.g., about 1 to about 10 amino acid modifications, preferably about 1 to about 6 amino acid modifications, compared to the parent polypeptide. Variant polypeptide sequences herein generally have at least about 90% sequence identity with the parent polypeptide sequence, and most commonly, at least about 95% sequence identity, e.g., when considered across the entire antibody chain region.

[0049] Variants of the polypeptides or proteins of the present disclosure include fragments as described above, as well as polypeptides or proteins having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants may be generated using mutagenesis techniques known in the art. Variant polypeptides may contain conservative or non-conservative amino acid substitutions, deletions, or additions.

[0050] The term "derivative" as applied to a polypeptide or protein refers to a polypeptide or protein that has been altered so as to exhibit additional characteristics not found in the native polypeptide or protein. An example of a "derivative" of a variant Fc domain is a fusion or conjugate with a second polypeptide or a chelating chemical structure capable of binding to another molecule (e.g., a polymer, a chromophore, or a fluorophore) or an atom (e.g., a radioisotope).

[0051] The terms "polynucleotide" or "nucleotide," as used herein, are intended to encompass a single nucleic acid and multiple nucleic acids, and refer to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide contains conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)).

[0052] The term "nucleic acid" refers to one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment; for example, a recombinant polynucleotide encoding a polypeptide comprising a variant Fc domain contained in a vector would be considered isolated for purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. Additionally, polynucleotides or nucleic acids may include regulatory elements such as promoters, enhancers, ribosomal binding sites, or transcription termination signals.

[0053] As used herein, the term "host cell" refers to a cell or population of cells that harbors or is capable of harboring a recombinant nucleic acid. Host cells can be prokaryotic cells (e.g., E. coli), or alternatively, host cells can be eukaryotic cells, such as fungal cells (e.g., yeast cells, e.g., Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3, PERC6).

[0054] The present disclosure also encompasses polypeptides comprising variant IgG Fc domains containing one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is substituted with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, strings of amino acids can be conservatively substituted with structurally similar strings that differ in the order and / or composition of side chain family members.

[0055] The term "percent sequence identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are neither nucleotides nor amino acids. Similarly, gaps present in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, not nucleotides or amino acids from the reference sequence.

[0056] The percentage of sequence identity is calculated by determining the number of positions in both sequences where identical amino acid residues or nucleic acid bases occur to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the resulting value by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of percent sequence identity between two sequences can be performed using software readily available online or for download. Suitable software programs are available from various sources for aligning protein and nucleic acid sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to perform comparisons between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) (www.ebi.ac.uk / Tools / psa).

[0057] Different regions in a single polynucleotide or polypeptide target sequence that aligns with polynucleotide or polypeptide reference sequence can have different sequence identity percentages.Please note that sequence identity percentage values ​​are rounded to the nearest tenth.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.It should also be noted that length values ​​are always integers.

[0058] Those skilled in the art will understand that the generation of sequence alignment for calculating percent sequence identity is not limited to binary sequence-sequence comparison driven only by primary sequence data.Sequence alignment can be derived from multiple sequence alignment.One suitable program for generating multiple sequence alignment is ClustalW2, available from www.clustal.org.Another suitable program is MUSCLE, available from www.drive5.com / muscle / .ClustalW2 and MUSCLE are also available, for example, from EBI.

[0059] It will also be understood that sequence alignments can be created by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to create multiple sequence alignments is T-Coffee, available at www.tcoffee.org or, for example, from EBI. It will also be understood that the final alignment used to calculate percent sequence identity can be inspected automatically or manually.

[0060] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies may belong to one of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional structures. Antibodies may be naked or conjugated to other molecules such as toxins, radioisotopes, etc. The terms "antibody" or "immunoglobulin," used interchangeably herein, include whole antibodies and any antigen-binding fragments or single chains thereof.

[0061] The term "IgG," as used herein, refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4. In mice, this class includes IgG1, IgG2a, IgG2b, and IgG3.

[0062] The term "antigen-binding fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. It is known in the art that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0063] The term "monoclonal antibody" refers to a homogeneous antibody population that is responsible for highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to antibodies produced by any method, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0064] The term "human antibody" refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technique known in the art. This definition of a human antibody includes intact antibodies, full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide. The term "humanized antibody" refers to an antibody derived from a non-human (e.g., murine) immunoglobulin that has been engineered to contain minimal non-human (e.g., murine) sequence.

[0065] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) and have the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences of antibodies derived from another species (usually human) to avoid eliciting an immune response from that species.

[0066] IgG immunoglobulins naturally contain a pair of heavy chain polypeptides, each containing approximately 450 amino acids in IgG1, and a pair of light chains, each containing approximately 110 amino acids in IgG1. These heavy chains are linked amino-terminally to each other in a cartoon "Y" shape. The arms of the Y contain "complementarity-determining regions," which are the binding sites for the antibody's molecular target formed between the "so-called" variable regions of each pair of heavy and light chains. These "variable" regions of the protein can have thousands of different sequences depending on the structure of the binding site and the nature of the target, and typically determine the antibody's binding characteristics. The carboxy-terminal regions of the two heavy chains join to form the stem of the Y. Treating a whole antibody with the enzyme "papain" cleaves it at the Y junction, releasing two target-binding arms known as Fab fragments and one stem fragment known as the Fc (fragment crystallizable) region because it is easily crystallizable. Thus, the amino-terminal regions of the light and heavy chains contain variable regions that determine the "complementarity regions," designated VL and VH, respectively. The light chain also contains a so-called "constant" region, CL, while the heavy chain contains three "constant" regions known in human IgG nomenclature as CH1, CH2, and CH3. "Constant" in this context is to distinguish them from the highly variable complementarity-determining region (CDR) sequences, which are subject to natural variation due to population genetic polymorphism and allotypes. In intact antibodies, the constant region CL of the light chain and the first constant region of the heavy chain, below the arms of the "Y" and "hinge region" of the antibody, are usually located between CH1 and CH2.

[0067] Treating an intact antibody with the enzyme papain cleaves both heavy chains between the CH1 and CH2 constant domains, and the resulting Fc fragment contains a dimer of two heavy chains with a portion of the hinge region sequence and the complete constant domains CH2 and CH3. The sequence of a human Fc variant is presented herein as the entire heavy chain constant region, including the CH1, hinge region, and CH2 and CH3 regions, and typically begins with the sequence alanine-serine-threonine-lysine-glycine. However, it will be apparent to those skilled in the art that "Fc domain" typically refers to a dimer comprising the CH2 and CH3 regions from two individual heavy chains, and for clarity and consistency, the entire sequence of the constant heavy chain region is provided across antibody types, e.g., IgG1 to IgG4.

[0068] The term "Kabat EU Index numbering system" refers to Kabat et al., Sequences of Immunological Interest, 5 th This refers to the numbering system for human IgG1 EU antibodies as described in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Accordingly, the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CHI, Hinge, CH2, and CH3). For example, "L234" and "EU L234" both refer to the amino acid leucine at position 234 according to the Kabat EU index numbering system.

[0069] The terms "Fc domain" and "IgG Fc domain," as used herein, refer to the Fc region of an immunoglobulin, e.g., an IgG molecule, which comprises the C-terminal halves of the two heavy chains of the IgG molecule linked by disulfide bonds. It lacks antigen-binding activity but contains carbohydrate moieties and binding sites for complement and Fc receptors (including the FcRn receptor). For example, the Fc region contains the entire second constant domain CH2 (residues 231-340 of human IgG1 according to the Kabat EU index numbering system) and the third constant domain CH3 (residues 341-447), in addition to a portion of the hinge region. Fc can refer to this region alone or in the context of an antibody, antibody fragment, or Fc fusion protein.

[0070] Although the CH1-3 region of a native antibody sequence is a "constant" region, subtle variations in sequence exist between individuals and populations. These variations, known as allotypes and polymorphisms, often occur as single amino acid changes and are observed at several positions in the Fc domain, both between and within classes of IgG1, 2, 3, and 4. For example, natural IgG1 allotypes (variants) include the G1m3, G1m17,1, and G1m17,1,2 allotypes, as well as the G1m(f), G1m(z,a), and G1m(z,a,x) allotypes, which differ by one to ten or more amino acid changes in the heavy chain constant region (DeTaeye, SW, et al., FcγR Binding and ADCC Activity of Human IgG Allotypes, Frontiers in Immunology 2020, 11: 1-16). Several databases provide examples of known polymorphisms, such as the international ImMunoGeneTics information system (http: / / www.imgt.org). Thus, "wild-type IgG Fc domain" or "wt IgG Fc domain" refers to any naturally occurring IgG Fc region, as well as all polymorphisms, allotypes, and alleles.

[0071] The sequences of the heavy chain constant regions of human IgG1, IgG2, IgG3, and IgG4 can be found in several sequence databases, such as the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN), respectively. The sequences of the heavy chain constant regions of these specific alleles of IgG1 to IgG4 are designated starting at position 119 according to the Kabat EU index numbering system: IgG1 (SEQ ID NO: 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3), and IgG4 (SEQ ID NO: 4).

[0072] IgG light chains can take the form of either the so-called Kappa or Lamba sequences. The light chains presented in this application include either form, and also polymorphisms in the constant light chain region.

[0073] The terms "mutant IgG Fc domain" and "IgG Fc variant domain," as used herein, refer to an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions, or modifications introduced anywhere within the Fc domain. In certain embodiments, the variant IgG Fc domain comprises one or more amino acid substitutions that result in a reduced or decreased binding affinity to FcγR and FcRn compared to a parent Fc domain (which may be a wild-type domain) that does not comprise the one or more amino acid substitutions.

[0074] The term "Fc fusion" as used herein refers to a protein in which one or more polypeptides or small molecules are operably linked to an Fc domain or its variant or derivative.Fc fusion combines the Fc region of immunoglobulin with a fusion partner, and generally can be any protein or small molecule.The role of the non-Fc part of Fc fusion, i.e., the fusion partner, can be to mediate binding with target, and therefore can be functionally similar to the variable region of antibody.

[0075] The term "parent" polypeptide, as used herein, refers to a polypeptide (e.g., a parent Fc domain, or a polypeptide comprising an Fc domain, such as an antibody or Fc fusion) that is subsequently modified to generate a variant (e.g., a variant Fc domain, or a variant polypeptide comprising an Fc domain, such as a variant antibody or variant Fc fusion). A parent polypeptide can be a naturally occurring polypeptide (e.g., a wild-type Fc domain), or a variant or engineered version of a naturally occurring polypeptide. The term parent polypeptide can refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it. Thus, a "parent Fc domain," as used herein, refers to an Fc domain that has been modified to generate a variant, and a "parent antibody," as used herein, refers to an antibody that has been modified to generate a variant antibody that comprises an IgG variant Fc domain.

[0076] An "Fc variant" comprises an Fc domain and may exist alone or in the context of an antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptide. Fc variant may refer to the Fc polypeptide itself, a composition comprising the Fc variant polypeptide, or the amino acid sequence encoding it. The variant IgG Fc domains described herein are defined according to the amino acid modifications that comprise them. For all amino acid positions discussed herein, numbering always follows the Kabat EU index numbering system. Thus, for example, L234A is an Fc variant in which leucine (L) at EU position 234 is substituted with alanine (A) relative to the parent Fc domain. Similarly, for example, L234A / L235A / L328R defines a variant Fc variant with substitutions at EU positions 234 (L to A), 235 (L to A), and 328 (L to R) relative to the parent Fc domain.

[0077] The term " Fc gamma receptor " or " FcγR " as used herein refers to any member of the protein family that binds to IgG antibody Fc region and is encoded by FcγR gene.In humans, this family includes but is not limited to FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb and FcγRIc; FcγRIIa (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and any unidentified human FcγR or FcγR isoform or allotype. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRIIb (CD32), FcγRIII (CD16), and FcγRIV (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0078] The terms "FcRn" or "FcRn receptor," as used herein, refer to an Fc receptor (the "n" indicates the neonate based on the function first identified) known to be involved in the transfer of maternal IgG to the fetus via the human or primate placenta or yolk sac (rabbit, rat, and mouse), and from colostrum to the neonate via the small intestine. FcRn is also known to be involved in maintaining constant serum IgG levels by binding IgG molecules and recycling them into the serum. The binding of FcRn to IgG molecules is pH-dependent, with optimal binding at pH 6.0 and weak binding above pH 7.0. While IgG binding to FcγR receptors can trigger effector functions (e.g., ADCC), pH-dependent binding to FcRn can extend the half-life of IgG antibodies in serum. Effector function may be undesirable for molecules with extended serum half-lives or molecules that target soluble versions of cell-presented proteins or receptors.

[0079] The term "effector function," as used herein, refers to a biochemical event resulting from the interaction of an Fc domain with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. "Effector cells," as used herein, refer to cells of the immune system that express one or more Fc receptors and mediate one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γδ T cells, and can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys.

[0080] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide containing an Fc domain, such as an antibody, binds to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., primarily NK cells, neutrophils, and macrophages), and these cytotoxic effector cells specifically bind to antigen-bearing "target cells" and then kill the target cells with cytotoxins. (Hogarth et al., Nature review Drug Discovery 2012, 11: 313) In addition to antibodies and fragments thereof, it is contemplated that other polypeptides containing an Fc domain capable of specifically binding to antigen-bearing target cells, such as Fc fusion proteins and Fc conjugate proteins, can effect cell-mediated cytotoxicity.

[0081] Briefly, cell-mediated cytotoxicity resulting from the activity of a polypeptide containing an Fc domain is also referred to herein as ADCC activity. The ability of any particular polypeptide of the present disclosure to mediate target cell lysis by ADCC can be assayed. To evaluate ADCC activity, a polypeptide of interest (e.g., an antibody) is added to target cells in combination with immune effector cells, resulting in target cell lysis. Cytolysis is generally detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0082] The term "ADCP" as used herein refers to antibody-directed cellular phagocytosis, i.e., clearance of viruses and virus-infected cells, and provides a mechanism for stimulating downstream adaptive immune responses by promoting antigen presentation or by stimulating the secretion of inflammatory mediators. The term "CDC" as used herein refers to complement-dependent cytotoxicity, i.e., the biochemical event of target cell destruction mediated by the complement system.

[0083] The term "half-life" or "in vivo half-life," as used herein, refers to the biological half-life of a particular type of polypeptide of the present disclosure in the circulation of a given animal, and is expressed as the time required for half of the amount administered to the animal to be eliminated from the circulation and / or other tissues of the animal.

[0084] The term "subject," as used herein, refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. The terms "subject" and "patient" are used interchangeably in reference to a human subject.

[0085] The term "pharmaceutical composition," as used herein, refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0086] An "effective amount" of a polypeptide, e.g., an antibody, disclosed herein is an amount sufficient to accomplish a specifically stated purpose. An "effective amount" can be determined empirically and routinely in relation to the stated purpose. The term "therapeutically effective amount," as used herein, refers to an amount of a polypeptide, e.g., an antibody, or other drug effective to "treat" a disease or disorder in a subject or mammal.

[0087] The term "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to a polypeptide, such as an antibody, to produce a "labeled" polypeptide. The label may be itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0088] The terms "treating," or "treatment," or "to treat," or "alleviating," or "to alleviate," and the like, refer to both (1) therapeutic measures that cure, delay, lessen the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and / or delay the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder, those prone to having the disorder, and those in whom the disorder is to be prevented.

[0089] The treatment or prevention of pregnancy disorders in pregnant women described herein refers to treatment to prevent disorders in pregnant women and is not intended to cover the treatment of pregnancy itself.

[0090] The term "vector" refers to a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors conjugated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0091] Mutant IgG Fc domain In some embodiments, mutant IgG Fc domains are provided that have mutations that reduce maternal-to-fetal transfer, preferably by more than 90%, more preferably by more than 95%, more preferably by more than 98%, compared to the same polypeptide comprising the parent Fc domain (which may be the wild-type domain). These mutant IgG Fc domains can be incorporated into therapeutic antibodies to ensure fetal safety during treatment of pregnant women.

[0092] Although FcRn plays a major role in IgG transfer, it is not present on fetal endothelium, suggesting that this is an FcRn-only transport process. Furthermore, selective transfer of IgG from mother to fetus has been reported depending on glycosylation or antigen specificity, and does not reflect selectivity of binding to FcRn. Therefore, the inventors hypothesized that other receptors are likely to be involved (Jennewein, MF, et al, Fc Glycan-Mediated Regulation of Placental Antibody Transfer. Cell. 2019, 27: 202-215).

[0093] Therefore, the present inventors investigated disruption of Fc-FcγR binding. FcγRIIb, which can bind monomeric IgG like FcRn, is the only Fc receptor localized on the fetal placental endothelium and has been reported to be responsible for IgG transfer to the fetal circulation in the absence of FcRn (Ishikawa, T., et al., FcγaRIIb participates in maternal IgG trafficking of human placental endothelial cells. Int. J. Mol. Med. 2015, 35: 1273-89). FcγRIIIa has been detected in maternal syncytial chorion and is involved in the selective transfer of digalactosylated IgG, and possibly IgG-3, which is poorly FcRn-binding, through the placenta (Jennewein, MF, et al., Fc Glycan-Mediated Regulation of Placental Antibody Transfer. Cell. 2019, 178: 202-215).

[0094] The I253A, H310A, and H435A mutations disrupt binding of FcRn to the Fc region and have been shown to individually inhibit maternal-to-fetal transfer by up to 90%. The triple I253A, H301A, and H435A mutation in an erythropoietin-Fc fusion protein inhibited transfer through the intestine and lungs of neonatal mice by up to 50%. This is the first report using all three mutations in the same Fc construct (Spiekermann, GM, et al., J Exp Med. 2002, 196:303-10). The I253A, H301A, and H435A triple mutation has not been tested in the prior art for inhibiting maternal-to-fetal transfer of modified IgG antibodies.

[0095] The FcγR binding site is located in the CH2 domain of the Fc region, and binding can be blocked or greatly reduced by a combination of known mutations.

[0096] It is known that introduction of the mutations L234A and L235A into the Fc region of IgG significantly reduces binding by the FcγR receptors tested (Hezareh, M., et al, J Virol. 2001, 75:12161-8).

[0097] Other modifications known to significantly reduce binding to FcγR include the insertion of an arginine after G236 and the change of L328 to arginine (Chu, SY, et al, Molecular Immunology 2008, 45: 3926-33) or the changes L234F / L235E / P331S (Oganesyan, V., et al Acta Crystallogr D Biol Crystallogr. 2008, 64: 700-4).

[0098] Thus, in some aspects, a polypeptide is provided comprising a human variant IgG Fc domain comprising amino acid substitutions relative to a human wild-type Fc domain numbered according to the Kabat EU index numbering system, wherein a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) Position 310 is substituted with alanine and position 435 is substituted with glutamine.

[0099] In some aspects, a polypeptide is provided comprising a human variant IgG-1 Fc domain comprising an amino acid sequence that is at least 80%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO: 5 and comprising amino acid substitutions numbered according to the Kabat EU index numbering system, wherein: a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) Position 310 is substituted with alanine and position 435 is substituted with glutamine.

[0100] The resulting variant Ig Fc domain polypeptides have reduced binding to at least one Fcγ receptor (FcγR) and FcRn compared to the same polypeptide comprising the parent Fc domain (which may be a wild-type domain).

[0101] An Ig Fc domain may have the same modification in both polypeptides, or may have a modification in only one of the two polypeptides comprising the Fc domain, or may have different substitutions in each chain. Preferably, the modification is identical in both polypeptides of the Fc domain.

[0102] In one embodiment, the resulting variant Ig Fc domain polypeptide exhibits at least a 2-fold, at least a 5-fold, or at least a 10-fold increase in the concentration of antibody required to give 50% binding or activation in a binding or cell activation assay, and / or a 2-fold, 5-fold, or 10-fold or greater decrease in the Km binding constant for at least one Fcγ receptor and FcRn by SPR or equivalent methods, when compared to the same polypeptide comprising the parent Fc domain (which may be the wild-type domain).

[0103] In one embodiment, the at least one FcγR is selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb, preferably FcγRIIb or FcγRIIIa.

[0104] In this way, the resulting polypeptide exhibits reduced maternal-to-fetal transfer when compared to the same polypeptide comprising the parent Fc domain (which may be the wild-type domain), preferably greater than 90%, more preferably greater than 95%, even more preferably greater than 98% reduced maternal-to-fetal transfer when compared to the same polypeptide comprising the parent Fc domain (which may be the wild-type domain), but also exhibits a shorter half-life, which is also a safety advantage since treatment can be discontinued and the antibody can be quickly cleared if a significant safety issue arises.

[0105] In one aspect, the polypeptides, antibodies, and compositions of the present invention can be administered in therapeutically effective doses more frequently than polypeptides and antibodies lacking the mutations disclosed herein, for example, once daily, once every 2, 3, 4, 5, 6 days, or once a week.

[0106] In one embodiment, the human mutant IgG Fc domain contains alanines (A) at positions 234 and 235 to significantly reduce FcγR binding, and alanines (A) at positions 253, 310, and 435 to significantly reduce FcRn binding. This mutant IgG Fc domain and set of amino acid substitutions is hereinafter referred to as "L234A / L235A+I253A / H301A / H435A."

[0107] In another embodiment, there is provided a polypeptide comprising a human mutant IgG Fc domain comprising alanine (A) at positions 234 and 235, serine (S) at position 331, and alanine (A) at positions 253, 310, and 435. Hereinafter, this mutant IgG Fc domain and set of amino acid substitutions is referred to as "L234A / L235A / P331S+I253A / H301A / H435A."

[0108] In another embodiment, a polypeptide is provided comprising a human mutant IgG Fc domain, comprising a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, a serine (S) at position 331, and an alanine (A) at positions 253, 310, and 435. Hereinafter, this mutant IgG Fc domain and set of amino acid substitutions is referred to as "L234F / L235E / P331S+I253A / H301A / H435A."

[0109] In another aspect, a polypeptide is provided comprising a human mutant IgG Fc domain comprising an arginine (R) inserted after position 236 and an arginine (R) at position 328, and alanines (A) at positions 253, 310, and 435. This mutant IgG Fc domain and set of amino acid substitutions is hereinafter referred to as "^236R / L328R+I253A / H301A / H435A."

[0110] In another embodiment, a polypeptide is provided comprising a human mutant IgG Fc domain and having one of the following FcγR binding-reducing mutation sets: L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S, or ^236R / L328R, each containing an alanine (A) at position 253. Hereinafter, this mutant IgG Fc domain and set of amino acid substitutions will be referred to as the combination of the FcγR mutation set and "I253A," e.g., "L234A / L235A / P331S+I253A."

[0111] In another aspect, a polypeptide is provided comprising a human variant IgG Fc domain and having any of the following FcγR binding-reducing mutation sets: L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S, or ^236R / L328R, which contain an alanine (A) at position 435. Hereinafter, this variant IgG Fc domain and set of amino acid substitutions will be referred to as the combination of the FcγR mutation set and "H435A," e.g., "L234A / L235A / P331S+H435A."

[0112] In another embodiment, a polypeptide is provided comprising a human mutant IgG Fc domain and having any of the FcγR binding-reducing mutation sets L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S, or ^236R / L328R, comprising an alanine (A) at position 310 and a glutamine (Q) at position 435. Hereinafter, this mutant IgG Fc domain and amino acid substitutions will be referred to as the combination of the FcγR mutation set and "H301A / H435Q," e.g., "L234A / L235A / P331S+H301A / H435Q."

[0113] Thus, provided are sets of polypeptides comprising human mutant IgG Fc domains having any combination of one of the four amino acid substitution sets L234A / L235A or L234A / L235A / P331S or L234F / L235E / P331S or ^236R / L328R with any one of the four amino acid substitutions or substitution sets I253A / H301A / H435A or I253A or H435A or H301A / H435Q. In some embodiments, the parent polypeptide of the mutant IgG Fc domain already contains one or more amino acids corresponding to the above-described substitutions; for example, the parent Fc polypeptide may contain a phenylalanine (F) at position 234, as found in IgG4. In such embodiments, no modification of the amino acid(s) already containing one or more of the disclosed substitutions is necessary.

[0114] In some embodiments, the mutant IgG Fc domain is human. In some other embodiments, the mutant IgG Fc domain is non-human. The non-human IgG Fc domain can be derived from, for example, a rodent (e.g., a rat or mouse), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Preferably, the IgG Fc domain is selected from the group consisting of a human immunoglobulin G class 1 (IgG1) Fc domain, a human immunoglobulin G class 2 (IgG2) Fc domain, a human immunoglobulin G class 3 (IgG3) Fc domain, and a human immunoglobulin G class 4 (IgG4) Fc domain, preferably a human immunoglobulin G class 1 (IgG1) Fc domain. When the mutant IgG Fc domain is a mouse IgG Fc domain, the domain can be, for example, a subclass IgG1, IgG2a, IgG2b, or IgG3 domain.

[0115] In some embodiments, polypeptides are provided comprising a mutant IgG Fc domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. SEQ ID NO: 5 is a naturally occurring "wild-type" sequence corresponding to the human IgG1 allotype G1m3. SEQ ID NOs: 6 to 9 include variants of SEQ ID NO: 5 that contain a mutation or set of mutations that significantly reduce FcγR and / or FcRn binding, as follows: SEQ ID NO: 6: L234A / L235A / P331S and I253A / H301A / H435A; SEQ ID NO: 7: L234A / L235A / P331S and H301A / H435Q; SEQ ID NO: 8: L234F / L235E / P331S and I253A / H301A / H435A; and SEQ ID NO: 9: L234F / L235E / P331S and H301A / H435Q.

[0116] In some aspects, there is suitably provided a polypeptide comprising a variant IgG Fc domain comprising an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:9 when considered over the length of SEQ ID NO:5 to SEQ ID NO:9, respectively.

[0117] In some other embodiments, polypeptides are provided that comprise a variant IgG Fc domain consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 9. Based on the teachings provided herein, one of skill in the art will understand that the variant IgG Fc domains provided in SEQ ID NO: 5 to SEQ ID NO: 9 represent one particular allelic variant. Thus, in some embodiments, polypeptides are provided that comprise different allelic variants of the variant IgG Fc domains provided in SEQ ID NO: 5 to SEQ ID NO: 9. Sites of known allelic variation are available, for example, in the following literature and various databases: (Jefferies, R., et al, Human immunoglobulin allotypes: Possible implications for immunogenicity, mAbs 2009, 1: 332-338 and the IMGT database: http: / / www.imgt.org).

[0118] Fc receptor binding Polypeptides comprising the mutant IgG Fc domains provided herein (e.g., antibodies or fragments thereof comprising the mutant IgG Fc domains) have reduced or abolished binding to at least one FcγR receptor (e.g., FcγR IIb, FcγR IIIa) and the FcRn receptor.

[0119] Those skilled in the art will appreciate that polypeptides comprising mutant IgG Fc domains may have altered FcγR and FcRn binding properties (compared to unmodified molecules), including, but not limited to, binding specificity, dissociation and association rates (koff and kon, respectively), equilibrium dissociation constant (KD, defined as the ratio of koff divided by kon), binding affinity and / or avidity.

[0120] The affinity and binding properties of polypeptides comprising a variant IgG Fc domain for a receptor or ligand can be determined by a variety of in vitro assay methods (biochemical or immunological) known in the art for determining Fc-FcγR and Fc-FcRn interactions, i.e., specific binding of an Fc region to an FcγR. Such methods include equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., surface plasmon resonance, such as BIACORE® analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration).

[0121] These and other methods may utilize labels on one or more of the components being tested and / or may employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).

[0122] In one aspect, a polypeptide is provided comprising a mutant IgG Fc domain, which exhibits reduced binding affinity to at least one Fcγ receptor, including but not limited to, FcγRI (including isoforms FcγRIa, FcγRIb, and FcγRIc); FcγRII (including isoforms FcγRIIa, FcγRIIb, and FcγRIIc); and FcγRIII (including isoforms FcγRIIIa and FcγRIIIb), and to the FcRn receptor, compared to a parent polypeptide comprising a wild-type or modified wt Fc domain. In another embodiment, binding of a polypeptide comprising a variant IgG Fc domain to one or more of the above-mentioned Fcγ receptors and FcRn is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold less than a parent polypeptide comprising a wild-type or modified wt Fc domain, or is reduced to undetectable levels.

[0123] In another embodiment, binding of the polypeptide comprising the mutant IgG Fc domain to one or more of the above-mentioned Fcγ receptors and FcRn is completely abolished.

[0124] In one aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit reduced affinity for FcγRI compared to a parent polypeptide comprising a wild-type or modified wt Fc domain. In another aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit affinity for the FcγRI receptor that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower, or reduced to undetectable levels, than a parent polypeptide comprising a wild-type or modified wt Fc domain.

[0125] In another embodiment, a polypeptide is provided comprising a mutant IgG Fc domain that exhibits affinity for the FcγRI receptor that is at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% lower than a parent polypeptide comprising a wild-type or modified wt Fc domain. In some embodiments, the FcγRI is the isoform FcγRIa. In other embodiments, the FcγRI is the isoform FcγRIb. In yet other embodiments, the FcγRI is the isoform FcγRIc.

[0126] In one aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit reduced affinity for FcγRII compared to a parent polypeptide comprising a wild-type or modified wt Fc domain. In another aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit affinity for the FcγRII receptor that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold less than a parent polypeptide comprising a wild-type or modified wt Fc domain.

[0127] In another embodiment, a polypeptide comprising a mutant IgG Fc domain is provided that exhibits affinity for the FcγRII receptor that is at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% lower than a parent polypeptide comprising a wild-type or modified wt Fc domain. In some embodiments, the FcγRII is the isoform FcγRIIa. In another embodiment, the FcγRIIa isoform is the allotype H131. In yet another embodiment, the FcγRIIa isoform is the allotype R131. In other embodiments, the FcγRII is the isoform FcγRIIb. In some embodiments, the FcγRIIb isoform is FcγRIIb-1. In other embodiments, the FcγRIIb isoform is FcγRIIb-2. In yet another embodiment, the FcγRII is the isoform FcγRIIc.

[0128] In one aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit reduced affinity for FcγRIII compared to a parent polypeptide comprising a wild-type or modified wt Fc domain. In another aspect, polypeptides comprising a variant IgG Fc domain are provided that exhibit affinity for FcγRIII that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 6-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than a parent polypeptide comprising a wild-type or modified wt Fc domain.

[0129] In another aspect, polypeptides are provided comprising a variant IgG Fc domain that exhibits affinity for the FcγRIII receptor that is at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% lower than a parent polypeptide comprising a wild-type or modified wt Fc domain. In some aspects, the FcγRIII is the isoform FcγRIIIa. In other aspects, the FcγRIIIa is the allotype 158V (F158V allelic variant). In other aspects, the FcγRIII is the isoform FcγRIIIb. In other aspects, the FcγRIIIb is the allotype NA1. In other aspects, the FcγRIIIb is the allotype NA2.

[0130] In one embodiment, polypeptides comprising a mutant IgG Fc domain are provided that exhibit reduced affinity for FcRn compared to a parent polypeptide comprising a wild-type or modified wt Fc domain. In another embodiment, polypeptides comprising a mutant IgG Fc domain are provided that exhibit affinity for the FcRn receptor that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold less than a parent polypeptide comprising a wild-type or modified wt Fc domain. In another embodiment, polypeptides comprising a mutant IgG Fc domain are provided that exhibit affinity for the FcRn receptor that is at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% less than a parent polypeptide comprising a wild-type or modified wt Fc domain.

[0131] In one aspect, polypeptides comprising a mutant IgG Fc domain are provided that exhibit affinity for FcγR receptors of about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certain aspects, polypeptides comprising a mutant IgG Fc domain are provided that exhibit affinity for FcγR receptors of greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM. In another aspect, a polypeptide comprising a variant IgG Fc domain is provided that exhibits an affinity for an FcγR receptor that is less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 2.5 μM, less than 1 μM, or less than 100 nM, or less than 10 nM.

[0132] In one embodiment, a polypeptide comprising a mutant IgG Fc domain is provided that exhibits an affinity for the FcRn receptor of about 100 nM to about 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In a specific embodiment, a polypeptide comprising a mutant IgG Fc domain is provided that exhibits an affinity for the FcRn receptor of greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM. In another embodiment, a polypeptide comprising a variant IgG Fc domain is provided that exhibits an affinity for the FcRn receptor of less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 2.5 μM, less than 1 μM, or less than 100 nM, or less than 10 nM.

[0133] In a particular aspect, there is provided a polypeptide comprising L234A / L235A or L234A / L235A / P331S or L234F / L235E / P331S or ^236R / L328R substitutions in an IgG Fc domain that exhibits reduced affinity for FcγR compared to a parent polypeptide comprising a wild-type or modified wt IgG Fc domain, and further comprising I253A / H301A / H435A or I253A or H435A or H301A / H435Q substitutions in the IgG Fc domain that exhibits reduced affinity for FcRn.

[0134] In a particular embodiment, there is provided a polypeptide comprising L234A / L235A or L234A / L235A / P331S or L234F / L235E / P331S or ^236R / L328R substitutions in an IgG Fc domain that exhibit completely abolished binding to FcγR compared to a parent polypeptide comprising a wild-type or modified wt IgG Fc domain, and further comprising I253A / H301A / H435A or I253A or H435A or H301A / H435Q substitutions in the IgG Fc domain that exhibit reduced affinity for FcRn.

[0135] In a particular embodiment, there is provided a polypeptide comprising L234A / L235A or L234A / L235A / P331S or L234F / L235E / P331S or ^236R / L328R substitutions in an IgG Fc domain that exhibits reduced affinity for FcγR compared to a parent polypeptide comprising a wild-type or modified wt IgG Fc domain, and further comprising I253A / H301A / H435A or I253A or H435A or H301A / H435Q substitutions in the IgG Fc domain that exhibit completely abolished binding to FcRn.

[0136] In a particular embodiment, there is provided a polypeptide comprising L234A / L235A or L234A / L235A / P331S or L234F / L235E / P331S or ^236R / L328R substitutions in an IgG Fc domain that exhibits completely abolished binding to FcγR compared to a parent polypeptide comprising a wild-type or modified wt IgG Fc domain, and further comprising I253A / H301A / H435A or I253A or H435A or H301A / H435Q substitutions in the IgG Fc domain that exhibits completely abolished binding to FcRn.

[0137] In one aspect, the invention provides a molecule such as an antibody or fragment thereof comprising, from N-terminal to C-terminal direction, a first polypeptide and a second polypeptide, respectively, comprising at least a portion of an immunoglobulin hinge region, comprising one or more cysteine ​​residues, an immunoglobulin CH2-domain and an immunoglobulin CH3-domain, wherein the first polypeptide and the second polypeptide respectively comprise a mutation from group A below and a mutation from group B below: A (i) a substitution of alanine at positions 234 and 235, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and B (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) relating to a molecule in which position 310 is substituted with alanine and position 435 is substituted with glutamine.

[0138] Here, the first and second polypeptides may have the same or different mutations (preferably the same), where the mutations are defined relative to the wild-type or modified wt sequence as defined herein.

[0139] All features of the present disclosure apply to the above aspects.

[0140] method In some embodiments, a method for inhibiting the ability of a parent polypeptide comprising an Fc domain to be transferred from mother to fetus comprises the steps of: a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) Position 310 is substituted with alanine and position 435 is substituted with glutamine.

[0141] Antibodies and fragments thereof In some embodiments, the polypeptide comprising the mutant IgG Fc domain comprises an antigen-binding domain. In certain embodiments, the antigen-binding domain can be an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. The antigen-binding domain can be a full-length antibody, such as a human antibody, a humanized antibody, or a chimeric antibody, or a fragment thereof.

[0142] The term "antibody variant" refers to a polypeptide containing a variant IgG Fc domain provided herein, wherein the polypeptide is an antibody. Antibody variants include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, anti-idiotypic (anti-Id) antibodies, and fragments containing the Fc domain of any of the above. In some embodiments, antibody variants include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen-binding site, which fragments may be fused or conjugated to another immunoglobulin domain containing a variant IgG Fc domain provided herein. In one embodiment, the antibody variant is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

[0143] The antibody variants and fragments thereof comprising the variant IgG Fc domains provided herein can be derived from any animal, including birds and mammals (e.g., humans, rodents such as mice or rats, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In certain embodiments, antibody variants are provided that are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and also include antibodies isolated from human immunoglobulin libraries, synthetic immunoglobulin libraries in microorganisms, or mice or other birds or mammals that express antibodies from human genes.

[0144] Antibody variants may be monospecific, bispecific, trispecific, or of greater specificity (multispecific antibodies). Multispecific antibody variants may specifically bind to different epitopes of a desired target molecule, or may specifically bind to both the target molecule as well as a heterologous epitope, such as a heterologous polypeptide or solid support material.

[0145] Specific therapeutic targets Virtually any molecule can be targeted by a binding molecule, such as an antibody, fusion protein, or conjugate comprising a mutant IgG Fc domain according to the present invention. Furthermore, virtually any molecule can be incorporated into a fusion protein or conjugate comprising the mutant IgG Fc domain provided herein. However, as explained above, the mutant IgG Fc domain of the present invention is particularly useful for treating pregnant women.

[0146] Pregnancy carries the risk of serious complications, morbidity, and even death. The Centers for Disease Control and Prevention (CDC) lists the following serious morbidity conditions: acute myocardial infarction, aneurysm, acute renal failure, adult respiratory distress syndrome, amniotic fluid embolism, cardiac arrest / ventricular fibrillation, cardiac rhythm conversion, disseminated intravascular coagulation, eclampsia, heart failure / cardiac arrest during surgery or procedures, postpartum cerebrovascular accident, pulmonary edema / acute heart failure, severe anesthesia complications, sepsis, and shock (https: / / www.cdc.gov / reproductivehealth / maternalinfanthealth / severematernalmorbidity.html). While treatments for these conditions are generally available in the general population, they are likely not suitable or unproven for use in pregnant women due to maternal-fetal transfer risks and potential adverse effects on the fetus. Many pregnancy morbidity conditions can be safely and effectively treated using antibodies, fusion proteins, or conjugates comprising a variant IgG Fc domain according to the present invention.

[0147] Hypertension is a common condition during pregnancy and is the underlying cause of many pregnancy-related morbidities. According to the U.S. Centers for Disease Control, approximately 10% of hospitalized mothers in 2004 had hypertension. Hypertension during pregnancy can lead to lifelong circulatory problems, seizures, and stroke in the mother and serious problems for the baby through preterm birth and low birth weight. Hypertension, particularly preeclampsia / eclampsia, is a leading cause of morbidity and mortality for mothers and babies worldwide, and new treatments are clearly needed (Ives, CW, et al., J. Am Coll Cardiol. 2020, 76: 1690-1702).

[0148] High blood pressure, pre-eclampsia / eclampsia, and other pregnancy disorders seen in pregnant women are well known and are described in common general knowledge textbooks, such as: Sankaran S. Creasy and Resnik's Maternal-Fetal Medicine: Principles and Practice Sixth edition. Obstet Med. 2012 Jun;5(2):88-9. Doi: 10.1258 / om.2011.11E005. Epub 2012 Jun 19. PMCID: PMC4989620. Magee, LA, von Dadelszen, P., Stones, W., & Mathai, M. (2016). The FIGO Textbook of Pregnancy Hypertension: an evidence-based guide to monitoring, prevention and management. Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin, Number 222. Obstetrics & Gynecology 135(6):p e237-e260, June 2020. | DOI: 10.1097 / AOG.0000000000003891 Saito, Shigeru 2018. Preeclampsia Basic, Genomic, and Clinical: Basic, Genomic, and Clinical DOI - 10.1007 / 978-981-10-5891-2

[0149] Thus, the present invention relates to any of the therapeutic agents described herein, e.g., the polypeptides and antibodies having an Fc modification described and claimed herein, for the treatment of any one of the following in pregnant women: bacterial and parasitic infections, maternal and fetal viral infections, sexually transmitted diseases, maternal-fetal infections, cardiac diseases, coagulation disorders, thromboembolic diseases, anemia, malignancies, renal diseases, respiratory diseases, diabetes, thyroid diseases and other endocrine disorders, digestive diseases, diseases of the liver, biliary system, pancreas, and rheumatic diseases.

[0150] These disorders seen in pregnant women are described in Creasy and Resnik, supra, the list of disorders and treatments of which is specifically incorporated herein by reference.

[0151] Hypertension complicates 5-10% of pregnancies, and hypertensive disorders of pregnancy can be classified as chronic, gestational, or preeclampsia. Preeclampsia is particularly serious and is among the top five causes of maternal and perinatal mortality. In 2005, it was estimated that preeclampsia claimed the lives of more than 70,000 women and more than 500,000 fetuses and newborns annually worldwide (Sabai, B., et al, Lancet 2005, 365: 785-99).

[0152] Many compounds are available for the treatment of hypertension in the general population, and in 2011, the FDA listed 69 approved drugs from 15 different drug classes (https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / hypertension-indication-drug-labeling-cardiovascular-outcome-claims). However, none of these have been specifically tested and approved for use during pregnancy, and most, if not all, are small molecule or small peptide drugs, which are likely to cross the placenta and have unknown effects on the fetus.

[0153] Although conventional medical treatments for controlling hypertension are sometimes used to treat pregnant women, all of these pose risks to the mother or fetus, and their efficacy is limited, particularly in the treatment of preeclampsia, with very limited data making risk-benefit assessment difficult. Thus, there is a clear unmet need for therapies that treat hypertension and preeclampsia without harming or risking harm to the fetus. This problem is solved by providing antibodies, fusion proteins, or conjugates comprising the human variant IgG Fc domain of the present invention. The conjugates can be developed to target and affect soluble proteins, receptors, or small molecules that reduce, regulate, eliminate, or otherwise beneficially interfere with the mechanisms of hypertension and preeclampsia.

[0154] Currently available drugs for treating hypertension include diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-blockers, alpha2 receptor agonists, alpha- and beta-blocker combinations, central nervous system agonists, peripheral adrenergic inhibitors, and vasodilators. New drug classes, including inhibitors of vasopeptidases, aldosterone synthase, and soluble epoxide hydrolase, agonists of natriuretic peptide A and vasoactive intestinal peptide receptor 2, and novel mineralocorticoid receptor antagonists, are in phase II / III clinical development, while inhibitors of aminopeptidase A, dopamine β-hydroxylase, and intestinal Na+ / H+ exchanger 3, and agonists of components of the angiotensin-converting enzyme 2 / angiotensin(1-7) / Mas receptor axis are in earlier stages of development. Many of the underlying mechanisms of action of these classes of drugs may be amenable to the development of antibodies, fusion proteins or conjugates comprising mutant IgG Fc domains according to the invention, and therefore safe for treatment of pregnant women.

[0155] Targeting VEGFR-1 to treat preeclampsia Preeclampsia is a pregnancy disorder characterized by new-onset hypertension, often occurring after the 20th week of gestation and often near term. Several disease mechanisms have been proposed for preeclampsia, including chronic utero-ovarian ischemia, immune maladaptation, very-low-density lipoprotein toxicity, increased chorioblast apoptosis or necrosis, and an enhanced maternal inflammatory response to chorioblast shedding. It is now believed that an imbalance of angiogenic factors is important in the pathogenesis of preeclampsia, and that several other potential mechanisms may combine to induce the clinical spectrum of preeclampsia. For example, clinical and experimental evidence suggests that uteroplacental ischemia increases circulating levels of antiangiogenic factors, including sVEGFR-1, which promotes angiogenic imbalance.

[0156] The developing placenta requires a complex network of new blood vessels to manage communication between the mother and fetus and meet the increasing demands of the growing fetus. Preeclampsia is thought to result from an imbalance in this process, resulting in an inadequate supply of nutrients and oxygen to the fetus and an imbalance in pro- and anti-angiogenic factors. The sequelae of this imbalance can lead to global destruction of the maternal endothelium and trigger an inappropriate immune response. Thus, there are many potential targets for antibodies that can treat the mother without affecting the fetus. The anti-angiogenic factor soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) is thought to be most important in the pathogenesis of preeclampsia and therefore represents a prime target for treatment using antibodies comprising a mutant IgG Fc domain according to the present invention.

[0157] Experimental and epidemiological studies support the pathological role of sVEGFR-1 in the imbalance between circulating angiogenic and antiangiogenic factors in the pathogenesis of the maternal syndrome of preeclampsia. sVEGFR-1 is a soluble circulating protein that functions to regulate the activity of VEGF and placenta-derived growth factor (PGF) by sequestering and preventing binding to functional membrane-bound cell surface receptors (Rana, S., et al., Am. J. Obstet. Gynecol. 2022, 226: S1019-S1034). Increased levels of sVEGFR-1 and decreased levels of PGF correlate with the development of preeclampsia. Removal of sVEGFR-1 by apheresis has been reported to prolong pregnancy in women with preeclampsia, demonstrating its potential as a therapeutic modality. Ideally, a drug that reduces sVEGFR-1 levels while simultaneously releasing VEGF and PGF, partially restoring normal levels, would be desirable. Normal IgG antibodies that bind to sVEGFR-1 have been proposed for the treatment of hypertensive conditions in pregnant women. However, they can cross the placenta and potentially disrupt fetal development by altering the availability of VEGF and PGF. In fact, Avastin, an antibody that binds to and blocks the effects of VEGF, carries a warning against its use in pregnant women due to safety concerns regarding fetal exposure. Similarly, antibodies with unmodified Fc regions that target sVEGFR-1 are not suitable for use in pregnant women due to fetal safety concerns. On the other hand, modified placenta-impermeable antibodies against sVEGFR-1, ideally those that inhibit ligand binding and release bound VEGF or PGF, are suitable for the treatment of pregnancy conditions, particularly eclampsia and preeclampsia. The therapeutic effect is to bind to sVEGFR-1, displacing bound VEGF and PGF, followed by endosomal degradation of the antibody-sVEGFR-1 complex. Additionally, plasma levels of sVEGFR-1 can be measured by many readily available clinical assays, and with the added benefit of inhibiting FcRn binding to the Fc domain, rapid clearance allows antibodies to be titrated accordingly to individual patient sVEGFR-1 levels.

[0158] Excessive levels of the anti-angiogenic factor sVEGFR-1, produced in the placenta and released into the maternal circulation, induce maternal endothelial dysfunction and lead to the symptoms of preeclampsia. sVEGFR-1 is a soluble splice variant of the membrane-bound receptor VEGFR-1 and binds to the pro-angiogenic proteins VEGF and PGF; thus, sVEGFR-1 acts as a ligand trap, antagonizing ligand-mediated angiogenic signaling via its cell surface receptor. In rodents, overexpression of sVEGFR-1 leads to the symptoms of preeclampsia, and in humans, high maternal sVEGFR-1 levels are associated with more severe forms of the disease. High plasma levels of sVEGFR-1 and / or sVEGFR-1 relative to PGF indicate that inhibition of the angiogenic process is a strong predictor of the severity and adverse clinical outcomes of preeclampsia. Drugs that inhibit angiogenic signaling, such as the VEGF-blocking antibody bevacizumab (Avastin; Genentech), the VEGF trap (aflibercept; Regeneron), and small-molecule inhibitors of VEGF receptors, are associated with major side effects of preeclampsia-like symptoms in nonpregnant women, including hypertension, proteinuria, and renal glomerular changes. Together, these findings indicate that high circulating levels of sVEGFR-1 and low circulating levels of proangiogenic factors (VEGF and PGF) create an antiangiogenic state that contributes to the clinical manifestations of preeclampsia.

[0159] Thus, in one embodiment, a polypeptide comprising a human variant IgG Fc domain according to the present invention is an anti-VEGFR-1 antibody that inhibits the binding of VEGF and / or PGF to sVEGFR-1. In this embodiment, the anti-sVEGFR-1 antibody binds with high affinity to the VEGF and / or PGF binding site of sVEGFR-1, thereby preventing sVEGFR-1 from binding to VEGF and / or PGF and releasing previously bound ligands. The antibody thus prevents VEGF and / or PGF from binding to the sVEGFR-1 receptor and displaces any bound VEGF and / or PGF, thereby eliminating the negative effects of circulating levels of sVEGFR-1.

[0160] IMC-18F (disclosed in U.S. Patent No. 7,972,596, the disclosure of which is specifically incorporated by reference) is an IgG1 antibody that has high binding affinity to sVEGFR-1 and can displace bound VEGF and PDG, and can be modified according to the present invention. The sequences of the heavy and light chains are set forth as SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0161] In another embodiment, the anti-VEGFR-1 antibody comprises an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence of a heavy chain comprising SEQ ID NO: 10 of the variable heavy chain sequence joined to a human mutant IgG Fc domain sequence according to the invention, e.g., in combination with SEQ ID NO: 8 to give the complete heavy chain SEQ ID NO: 13 and light chain SEQ ID NO: 12, constituting an IgG-1 that binds to sVEGFR-1 and exhibits very low or undetectable levels of maternal-to-fetal transfer.

[0162] In another embodiment, the anti-VEGFR-1 antibody comprises a variable heavy chain sequence SEQ ID NO: 10 joined to a human variant IgG Fc domain sequence selected from SEQ ID NO: 6, 7, or 9, and a light chain of SEQ ID NO: 12.

[0163] In another embodiment, the anti-VEGFR-1 antibody comprises a modified IgG-1 sequence (N297A) SEQ ID NO: 14, or an IgG-4 sequence SEQ ID NO: 15, or an IgG-4 sequence in which serine 228 has been replaced with proline SEQ ID NO: 16, or an IgG-4 sequence in which serine 228 has been replaced with proline and leucine 253 has been replaced with glutamic acid SEQ ID NO: 17, or an IgG-2 sequence SEQ ID NO: 18, or a hybrid IgG-2 / IgG-4 sequence SEQ ID NO: 19 and a variable heavy chain sequence SEQ ID NO: 10 joined to a human variant IgG Fc region comprising a light chain of SEQ ID NO: 13.

[0164] In another embodiment, a polypeptide comprising a human variant IgG Fc domain according to the invention is an anti-VEGFR-1 antibody comprising a variant Fc domain from SEQ ID NO: 5 in the heavy chain comprising any one of four mutation sets L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S or ^236R / L328R designed to reduce binding to FcγR, in combination with SEQ ID NO: 10 in the heavy chain variable region and a light chain of SEQ ID NO: 12, in combination with any one of four mutations or mutation sets I253A / H301A / H435A or I253A or H435A or H301A / H435Q designed to reduce binding to FcRn.

[0165] For completeness, a polypeptide comprising a human variant IgG Fc domain according to the invention may comprise any of the four mutation sets L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S designed to reduce binding to FcγR in combination with one of the four mutation sets, i.e., four mutations or mutation sets I253A / H301A / H435A or I253A or H435A or H301A / H435Q designed to reduce binding to FcRn. The present invention therefore relates to polypeptides (which may be in the form of heavy chains or antibodies) comprising any of these 16 individualized mutation sets.

[0166] In another embodiment, a polypeptide comprising a human variant IgG Fc domain according to the invention is an anti-VEGFR-1 antibody comprising a heavy chain variable region of SEQ ID NO: 10 and a light chain of SEQ ID NO: 12, together with a variant Fc domain from any of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, with a heavy chain sequence comprising any one of four mutation sets L234A / L235A, L234A / L235A / P331S, L234F / L235E / P331S or ^236R / L328R designed to reduce binding to FcγR (or equivalent changes based on mutations in the IgG4 sequence), in combination with any one of four mutations or mutation sets I253A / H301A / H435A or I253A or H435A or H301A / H435Q designed to reduce binding to FcRn (or equivalent changes based on mutations in the IgG4 sequence). The sequence positions and changes that correspond to substitutions between IgG1 and IgG4 sequences are readily determined by one of skill in the art.

[0167] In another embodiment, the antibody variable heavy chain SEQ ID NO: 10 is optimized for reduced immunogenic potential, stability and manufacturability by one or more of the following substitutions: alanine-2 substituted with valine, valine-4 substituted with leucine, serine-14 substituted with proline, tryptophan-52 substituted with serine, tyrosine or phenylalanine, aspartic acid-53 substituted with alanine, threonine or glutamic acid, or glycine-54 changed to alanine or serine, and by selected sequences in combination with any of the human Fc variant regions of the invention, and is optimized relative to the light chain SEQ ID NO: 12 to include optimized antibodies with improved therapeutic and manufacturability properties and very low or undetectable levels of maternal-to-fetal transfer.

[0168] Other drug treatments The present invention is particularly applicable to antibodies or IgG Fc-containing molecules that are known to bind to VEGFR1 and have therapeutic effect, such as any anti-VEGFR-1 antibody or fragment thereof that inhibits the binding of VEGF and / or PGF to sVEGFR-1, or antibodies or fragments thereof disclosed and claimed in WO2017175054 (referring to, for example, antibodies or synthetic or recombinant fragments thereof that can recognize and bind to an epitope comprised in the sequence from aa.149 to aa.161 of VEGFR-1 as defined in WO2017175054, such as D16F7, which is incorporated by reference), and to the use of such antibodies for the prevention or treatment of diseases, such as pregnancy disorders in pregnant women.

[0169] The present invention is also applicable to antibodies or IgG Fc-containing polypeptides known to be useful or potentially useful in treating disorders in pregnant women, such as eculizumab (Soliris) for pre-eclampsia.

[0170] In another aspect, the approaches and uses described herein also apply to the treatment of pregnant companion animals such as dogs or cats or horses, or pregnant livestock such as cattle or sheep or goats, etc. Thus, it is understood that such animals may be treated, and the invention is not limited to pregnant human women.

[0171] Methods for producing antibodies containing mutant IgG Fc domains Antibody variants or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, chemical synthesis or recombinant expression techniques.

[0172] Monoclonal antibody variants can be prepared using a wide variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or a combination thereof. For example, monoclonal antibody variants can be generated using hybridoma technology, including those known in the art. Methods for producing and screening specific antibodies using hybridoma technology are routine and known in the art.

[0173] Antibody variants can be generated by numerous methods well known to those skilled in the art. Non-limiting examples include isolating an antibody coding region (e.g., from a hybridoma) and introducing one or more Fc domain amino acid substitutions into the isolated antibody coding region. Alternatively, the variable region can be subcloned into a vector encoding the mutant IgG Fc domain provided herein.

[0174] Antibody variant fragments that recognize specific epitopes can be generated by any technique known to those skilled in the art. For some applications, including in vivo use of antibody variants in humans and in vitro detection assays, it may be advantageous to use human or chimeric antibody variants. For therapeutic treatment of human subjects, fully human antibodies are particularly desirable. Human antibodies or fragments thereof comprising the variant IgG Fc domains provided herein can be generated by various methods known in the art.

[0175] Chimeric antibody variants or fragments thereof comprising the variant IgG Fc domains provided herein can also be produced by various methods known in the art. In certain cases, humanized antibody variants or fragments thereof can comprise the variant IgG Fc domains provided herein. Humanized antibody variants can be produced using various techniques known in the art, including, but not limited to, CDR grafting, veneering, or resurfacing.

[0176] Human antibody variants can also be generated using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional separately or simultaneously by introducing human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. These chimeric mice are then bred to generate homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen or an immunogenic fragment thereof.

[0177] Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Therefore, such technology can be used to produce therapeutically useful antibodies.

[0178] Polynucleotides Polynucleotides encoding polypeptides comprising mutant IgG Fc domains are provided, as are polynucleotides that hybridize to polynucleotides encoding polypeptides comprising mutant IgG Fc domains under high stringency, medium stringency, or low stringency hybridization conditions.

[0179] In some embodiments, a polynucleotide sequence encoding a polypeptide comprising a variant IgG Fc domain can be generated from a parent polynucleotide sequence obtained from a suitable source. Once the polynucleotide sequence is obtained, it can be manipulated using methods known in the art for manipulating nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are incorporated herein by reference in their entireties), to generate a polypeptide comprising a variant IgG Fc domain with a different amino acid sequence, e.g., to generate amino acid substitutions, deletions, and / or insertions.

[0180] In other embodiments, polynucleotide sequences encoding polypeptides comprising variant IgG Fc domains can be assembled from chemically synthesized oligonucleotides (e.g., Kutmejer et al. BioTechniques 1994, 17:242), which briefly involves the synthesis of overlapping oligonucleotides containing portions of the coding sequence, annealing and ligation of those oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0181] Conjugates and Derivatives In some embodiments, the variant IgG Fc domains provided herein may be conjugated or fused to one or more moieties including, but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules.

[0182] In some embodiments, polypeptides comprising mutant IgG Fc domains include derivatives modified, for example, by the covalent attachment of any type of molecule to the polypeptide or by chemical or enzymatic modification. For example, derivatives include polypeptides modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, derivatives can contain one or more non-classical amino acids.

[0183] Conjugates are provided that include a polypeptide comprising a mutant IgG Fc domain chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids). Conjugation need not necessarily be direct, but can occur via a linker. Such linker molecules are generally known in the art and are described in Denardo et al. Clin Cancer Res 1998, 4:2483; Peterson et al. Bioconjug. Chem. 1999, 10:553; Zimmerman et al. Nucl. Med. Biol. 1999, 26:943; Garnett, Adv. Drug Deliv. Rev. 2002, 53:171.

[0184] Compositions comprising a heterologous protein, peptide or polypeptide conjugated to a polypeptide comprising a variant IgG Fc domain are also provided.

[0185] In some embodiments, the polypeptide comprising the mutant IgG Fc domain is conjugated to a diagnostic or detection agent. Such conjugates may be useful for monitoring the onset or progression of inflammatory disorders or determining prognosis as part of a clinical testing procedure, such as determining the effectiveness of a particular treatment. Such diagnosis and detection can be achieved by binding the polypeptide comprising the mutant IgG Fc domain to a detectable substance.

[0186] In some embodiments, polypeptides comprising mutant IgG Fc domains are conjugated to therapeutic agents. Polypeptides comprising mutant IgG Fc domains can be conjugated to therapeutic moieties such as cytotoxins, therapeutic agents, radioactive metal ions, etc. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Therapeutic agents or drug moieties are not limited to classical chemical therapeutic agents. For example, drug moieties can be proteins or polypeptides with desired biological activity. Such proteins can include, for example, toxins, cytokines, or growth factors. Furthermore, polypeptides comprising mutant IgG Fc domains can be conjugated to therapeutic moieties such as macrocyclic chelators useful for conjugating radioactive materials or radioactive metal ions. Radioactive metals can emit destructive radiation, such as alpha particles for therapy or penetrating gamma rays for diagnosis. (https: / / world-nuclear.org / information-library / non-power-nuclear-applications / radioisotopes-research / radioisotopes-in-medicine.aspx).

[0187] Antibodies, i.e., antibody variants, comprising the mutant IgG Fc domains described herein can be conjugated to a therapeutic moiety. Techniques for conjugating therapeutic moieties to antibodies are well known, and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2 nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);”Analysis, See "Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. Immunol. Rev. 62:119-58 (1982). Alternatively, the antibody variant can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in US Pat. No. 4,676,980.

[0188] In some embodiments, a polypeptide comprising a mutant IgG Fc domain comprises one or more engineered glycoforms, i.e., carbohydrate compositions covalently attached to the polypeptide. Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, reducing effector function. Engineered glycoforms can be produced by any method known to those skilled in the art, such as by using engineered or mutant expression strains, by coexpression with one or more enzymes, e.g., DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a polypeptide comprising a mutant IgG Fc domain in various organisms or cell lines derived from various organisms, or by modifying carbohydrates after expression of a polypeptide comprising a mutant IgG Fc domain. Methods for producing engineered glycoforms are known in the art.

[0189] Fusion proteins An Fc fusion protein is a combination of an immunoglobulin Fc domain or a fragment thereof with a fusion partner, which can generally be any protein, polypeptide, peptide, or small molecule. The role of the non-Fc portion of an Fc fusion protein, i.e., the fusion partner, is often, but not necessarily, to mediate target binding, and thus is functionally similar to the variable region of an antibody. Thus, provided are fusion proteins, i.e., polypeptides comprising a mutant IgG Fc domain and a fusion partner that specifically binds to a molecule (e.g., a cell surface receptor, a chemokine, etc.).

[0190] In some embodiments, the fusion protein may comprise a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, TandaB, or antibody mimetic fused to a polypeptide comprising a mutant IgG Fc domain. In some embodiments, the fusion protein may comprise a linker region connecting the peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, TandaB, or antibody mimetic to the polypeptide comprising a mutant IgG Fc domain. The use of naturally occurring and artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature.

[0191] In some embodiments, the fusion protein may combine a mutant IgG Fc domain with a fusion partner, which may generally be a protein including, but not limited to, a ligand, an enzyme, a ligand portion of a receptor, an adhesion protein, or other protein or domain.

[0192] In another embodiment, the fusion protein comprises a biologically active molecule fused to a mutant IgG Fc domain described herein. Biologically active molecules that can be fused to the mutant IgG Fc domain described herein include, but are not limited to, peptides, polypeptides, proteins, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules. In one embodiment, the biologically active molecule is a polypeptide comprising at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive amino acid residues, and is heterologous to the amino acid sequence of the mutant IgG Fc domain described herein.

[0193] Fusion proteins comprising the mutant IgG Fc domains described herein can be fused to a marker sequence, such as, but not limited to, a peptide, to facilitate purification. In some embodiments, the marker amino acid sequence is a His6 tag, a "flag" tag, a hemagglutinin "HA" tag, or one of many other commercially available tags.

[0194] A variety of linkers can be used to covalently link a polypeptide comprising a variant IgG Fc domain to a fusion partner to produce a fusion protein. Alternatively, polypeptides, proteins and fusion proteins can be produced by standard recombinant DNA techniques or by protein synthetic techniques, for example, using a peptide synthesizer.

[0195] Expression of recombinant polypeptides Recombinant expression of a polypeptide comprising a variant IgG Fc domain, derivative, analog or fragment thereof, e.g., an antibody variant or fusion protein comprising a variant IgG Fc domain described herein, can be achieved by construction of an expression vector containing a polynucleotide encoding the polypeptide. Once a polynucleotide encoding a polypeptide (e.g., an antibody variant or fusion protein) comprising a variant IgG Fc domain has been obtained, a vector for producing the polypeptide can be generated by recombinant DNA technology using techniques well known in the art.

[0196] Thus, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding polypeptides (e.g., antibody mutants or fusion proteins) comprising mutant IgG Fc domains are described herein. Expression vectors containing coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, replicable vectors are provided that contain a nucleotide sequence encoding a polypeptide comprising a mutant IgG Fc domain, operably linked to a promoter.

[0197] The expression vector is transfected into host cells by conventional methods, and the transfected cells are cultured by conventional methods to produce a polypeptide comprising a mutant IgG Fc domain. Thus, a host cell is provided comprising a polynucleotide encoding a polypeptide comprising a mutant IgG Fc domain, operably linked to a heterologous promoter.

[0198] A variety of host-expression vector systems can be utilized to express polypeptides comprising variant IgG Fc domains. Such host-expression systems represent not only the vehicle in which a coding sequence of interest may be produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing a polypeptide comprising a variant IgG Fc domain in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing sequence(s) encoding a polypeptide comprising a mutant IgG Fc domain; yeast (e.g., Saccharomyces picia) transformed with a recombinant yeast expression vector containing sequence(s) encoding a polypeptide comprising a mutant IgG Fc domain; insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing sequence(s) encoding a polypeptide comprising a mutant IgG Fc domain; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing sequence(s) encoding a polypeptide comprising a mutant IgG Fc domain; or mammalian cell lines (e.g., COS, CHO, BHK, 293, NS0, 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell or a mammalian virus.

[0199] A host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, PER.C6, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NS0, CRL7O3O, and HsS78Bst cells.

[0200] For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express polypeptides comprising variant IgG Fc domains can be engineered using methods known in the art.

[0201] Once a polypeptide (e.g., an antibody variant or fusion protein) comprising a variant IgG Fc domain is produced by recombinant expression, it can be purified by any method known in the art for purifying proteins, for example, chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for specific antigens following Protein A, and sizing column chromatography), centrifugation, differential lysis, or any other standard technique for purifying proteins.

[0202] Characterization and functional assays Polypeptides comprising the mutant IgG Fc domains described herein can be characterized in a variety of ways. In particular, polypeptides comprising the mutant IgG Fc domains can be assayed for the ability to specifically bind to a ligand, e.g., FcγRIIb, FcγRIIIa(158V), or FcRn. Such assays can be performed in solution (see, e.g., Houghten, Bio / Techniques 13:412-421 (1992)), on beads (see, e.g., Lam, Nature 354:82-84 (1991)), on chips (see, e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (see, e.g., U.S. Pat. No. 5,223,409), on plasmids (see, e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)), or on phage (see, e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991). Molecules identified to specifically bind to a ligand, e.g., FcγRIIb, FcγRIIIa, FcRn, can be assayed for affinity for the ligand.

[0203] Polypeptides comprising mutant IgG Fc domains can be assayed for specific binding to molecules such as antigens (e.g., cross-reactivity with cancer antigens and other antigens) or ligands (e.g., FcγRs) by any method known in the art. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, agglutination assays, complement fixation assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routinely performed and are well known in the art. See, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York.

[0204] The binding affinity of a polypeptide comprising a mutant IgG Fc domain to an antigen or a ligand, such as an FcγR, and the dissociation rate (off-rate) of the interaction can be determined by competitive binding assays. The kinetic parameters of a polypeptide comprising a mutant IgG Fc domain can also be determined using any surface plasmon resonance (SPR)-based assay known in the art (e.g., BIAcore or ProteOn kinetic analysis). See, for example, Mullet et al. Methods 22: 77-91 (2000); Dong et al. Rev. Mol. Biotech. 82: 303-23 ​​(2002); Fivash et al. Curr. Opin. Biotechnol. 9: 97-101 (1998); Rich et al. Curr. Opin.Biotechnol. 11: 54-61 (2000). Additionally, any of the SPR instruments and SPR-based methods for measuring protein-protein interactions described in U.S. Patent Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; and 6,268,125 are contemplated in the methods of the present disclosure.

[0205] Fluorescence-activated cell sorting (FACS) can be used to characterize binding of polypeptides comprising a variant IgG Fc domain to molecules expressed on cell surfaces (e.g., FcγRs) using any of the techniques known to those of skill in the art.

[0206] Polypeptides comprising variant IgG Fc domains can be assayed for their ability to mediate FcγR-mediated effector cell functions, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), C1q binding, and complement-dependent cell-mediated cytotoxicity (CDC). Any cell-based or cell-free assay known to those skilled in the art can be used to determine effector cell functional activity (see, for example, Perussia et al. Methods Mol. Biol. 121: 179-92 (2000); Baggiolini et al. Experientia 44: 841-8 (1998); Lehmann et al. J. Immunol. Methods 243: 229-42 (2000); Brown, Methods Cell Biol. 45: 147-64 (1994); Munn et al. J. Exp. Med. 172: 231-237 (1990); Abdul-Majid et al. Scand. J. Immunol. 55:70-81 (2002); Ding et al. Immunity 8:403-411 (1998)). In particular, polypeptides comprising the variant IgG Fc domain can be assayed for FcγR-mediated ADCC activity on effector cells, such as natural killer cells, using any of the standard methods known to those of skill in the art (see, e.g., Perussia et al. Methods Mol. Biol. 121: 179-92 (2000)).

[0207] Pharmaceutical Compositions and Methods of Administration In another aspect, there is provided a pharmaceutical composition comprising a polypeptide comprising a variant IgG Fc domain as described herein, or a conjugate as described herein, or a vector as described herein, or a combination thereof, formulated together with a carrier and a pharmaceutically acceptable excipient.

[0208] In another aspect, compositions are provided that include a polypeptide comprising a mutant IgG Fc domain described herein, a nucleic acid encoding a polypeptide comprising a mutant IgG Fc domain described herein, or a combination thereof, formulated with a carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, fusion proteins, or conjugates. In some aspects, such compositions are physiologically acceptable, and such compositions are suitable for therapeutic, prophylactic, or diagnostic administration to a subject.

[0209] In another embodiment, a composition comprising a polypeptide (e.g., an antibody variant, a fusion protein, or a conjugate) comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain, may include one or more pharmaceutically acceptable salts.

[0210] Examples of suitable aqueous and non-aqueous carriers that can be used in the contemplated compositions include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0211] In another embodiment, compositions comprising a polypeptide (e.g., an antibody variant, fusion protein, or conjugate) comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain, may also contain agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the composition. Furthermore, the absorption of injectable pharmaceuticals can be prolonged by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0212] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in pharmaceutical compositions is contemplated.A supplementary active compound can also be incorporated into the composition.In some embodiments, acceptable carriers include excipients that are approved for administration to humans and animals or are considered safe, i.e., GRAS substances (generally regarded as safe).GRAS substances are listed by the Food and Drug Administration in 21 CFR 182 and 21 CFR 184 of the Code of Federal Regulations (CFR), and are incorporated herein by reference.

[0213] The actual dosage level of the active ingredient in a pharmaceutical composition comprising a polypeptide (e.g., an antibody variant, fusion protein, or conjugate) comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain, can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and is not toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition or ester, salt, or amide thereof employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0214] A therapeutically effective dose of a polypeptide comprising a mutant IgG Fc domain, a nucleic acid encoding a polypeptide comprising a mutant IgG Fc domain, or a pharmaceutical composition thereof reduces the severity of disease symptoms, increases the frequency and duration of symptom-free periods, or prevents disability or insufficiency due to disease. A therapeutically effective dose may also prevent or delay the onset of disease. Therefore, any clinical or biochemical monitoring assay can be used to determine whether a particular treatment is a therapeutically effective dose. One skilled in the art would be able to determine such an amount based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or administration route selected.

[0215] Compositions comprising a polypeptide (e.g., an antibody variant, fusion protein, or conjugate) comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain, can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Selected routes of administration for compositions comprising a polypeptide comprising a variant IgG Fc domain, nucleic acids encoding a polypeptide comprising a variant IgG Fc domain, and pharmaceutical compositions thereof, include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as administration by injection or infusion. Parenteral administration can refer to modes of administration other than enteral and topical administration, and is typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, compositions comprising polypeptides comprising variant IgG Fc domains, nucleic acids encoding polypeptides comprising variant IgG Fc domains, and pharmaceutical compositions thereof may be administered via a parenteral route, e.g., a topical, epidermal, or mucosal administration route, e.g., nasal, oral, intravaginal, rectal, sublingual, or topical administration route.

[0216] Treatment method Polypeptides comprising variant IgG Fc domains (e.g., antibody variants, fusion proteins, or conjugates), or nucleic acids encoding polypeptides comprising variant IgG Fc domains, can be administered to animals, particularly mammals, particularly humans, preferably females, more preferably pregnant women, to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection.

[0217] Polypeptides comprising variant IgG Fc domains, or nucleic acids encoding polypeptides comprising variant IgG Fc domains, may be particularly useful in the treatment or prevention of pregnancy-related diseases or disorders.

[0218] Polypeptides comprising mutant IgG Fc domains, or nucleic acids encoding polypeptides comprising mutant IgG Fc domains, and compositions thereof, may be particularly useful in treating or preventing hypertension-related conditions or pre-eclampsia / eclampsia.

[0219] Polypeptides comprising mutant IgG Fc domains, or nucleic acids encoding polypeptides comprising mutant IgG Fc domains, may be provided in pharmaceutically acceptable compositions as known in the art or described herein. As described in more detail below, polypeptides comprising mutant IgG Fc domains, or nucleic acids encoding polypeptides comprising mutant IgG Fc domains, may be used in methods of treating or preventing hypertension-related conditions or eclampsia / pre-eclampsia.

[0220] Polypeptides comprising a variant IgG Fc domain, or nucleic acids encoding polypeptides comprising a variant IgG Fc domain, and compositions thereof, may also be advantageously utilized in combination with other therapeutic agents known in the art for the treatment or prevention of hypertension-related conditions or eclampsia / pre-eclampsia. Polypeptides comprising a variant IgG Fc domain, or nucleic acids encoding polypeptides comprising a variant IgG Fc domain, and compositions thereof may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection, such as, for example, an anti-cancer agent, an anti-inflammatory agent, or an anti-viral agent.

[0221] In some embodiments, methods are provided for preventing, treating, or ameliorating one or more symptoms associated with hypertension-related conditions or pre-eclampsia / eclampsia and related conditions by administering a polypeptide comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain.

[0222] The present disclosure also encompasses methods for treating or preventing a hypertension-related condition or eclampsia / pre-eclampsia in a subject comprising administering a therapeutically or prophylactically effective amount of a polypeptide comprising a variant IgG Fc domain.

[0223] In another aspect, there is provided a polypeptide comprising a variant IgG Fc domain, or a nucleic acid encoding a polypeptide comprising a variant IgG Fc domain, or conjugates and compositions thereof, for use in therapy of a mammal, preferably a human, more preferably a female, even more preferably a pregnant woman, preferably for the treatment or prevention of a hypertension-related condition or pre-eclampsia / eclampsia.

[0224] kit Also provided are pharmaceutical packs or kits comprising one or more containers filled with one or more of the pharmaceutical compositions disclosed herein. Such containers may optionally be accompanied by a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, the notice reflecting approval by the government agency of the manufacture, use, or sale for human administration. The present disclosure provides kits that can be used in the above-described treatment and administration methods. In one aspect, the kit comprises a polypeptide (e.g., an antibody variant, fusion protein, or conjugate) comprising a variant IgG Fc domain, preferably in purified form, in one or more containers. [Example]

[0225] Materials and General Methods A summary of the nucleotide sequences encoding human immunoglobulin light and heavy chains is provided in: Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5 thed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of antibody chains are numbered according to the EU numbering system established for "EU antibodies" and designated accordingly [Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)]. Specifically, the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CH1, Hinge, CH2, and CH3).

[0226] In accordance with the present invention, conventional molecular biology, microbiology, protein expression and purification, antibody, and recombinant DNA techniques may be employed that are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Sambrook, J., et al. (2001) Molecular Cloning: A Laboratory Manual. 3 rded. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York;Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ;Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ;Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ;Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc. : Hoboken, NJ;Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ;およびEnna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.;Nucleic Acid Hybridization, Hames & Higgins eds.(1985);Transcription And Translation, Hames & Higgins, eds.(1984);Animal Cell Culture Freshney, ed. (1986);Immobilized Cells And Enzymes, IRL Press (1986);Perbal, A Practical Guide To Molecular Cloning (1984);およびHarlow and Lane.See Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press: 1988). Antibodies are usually expressed and purified from mammalian cells, typically HEK293 and CHO, although other expression systems such as yeast, plant cells, and E. coli can also be used (Frenzel, A., et al.; Expression of Recombinant Antibodies; Frontiers in Immunology: 2013, 4, Article 271: 1-20).

[0227] Gene constructs and plasmids Gene sequences for expression of antibody chains and other desired sequences can be codon-optimized, synthesized, and sequence-verified by contractors such as Geneart Services provided by Thermofisher Scientific (https: / / www.thermofisher.com / ch / en / home / life-science / cloning / gene-synthesis.html). The desired sequences for DNA synthesis are provided electronically, codon-optimized for the appropriate expression system, then synthesized and checked by DNA sequencing.

[0228] The confirmed sequence is cloned into an appropriate plasmid, such as Thermofisher Scientific's pcDNA3.4-TOPO, which is engineered to support transient expression of the target protein in mammalian cell culture. pcDNA3.4-TOPO incorporates features and control sequences that allow for: PCR amplification and insertion of the target gene sequence; E. coli amplification and selection of the correct plasmid; plasmid purification and linearization; and mammalian cell transfection and high-level production and secretion of the target protein. In the specific example of the pcDNA3.4-TOPO plasmid, the required features are: a WPRE (woodchuck posttranscriptional regulatory element) downstream of the cloning site to enhance transcript expression; a full-length human cytomegalovirus (CMV) immediate-early promoter / enhancer for high-level gene expression in mammalian cells, such as HEK 293 or CHO; a TOPO cloning site for rapid and efficient cloning of Taq-amplified PCR products; a herpes simplex virus thymidine kinase polyadenylation signal for proper termination and processing of recombinant transcripts; a neomycin resistance gene for selection of stable cell lines with the antibiotic geneticin; a pUC origin for high-copy replication and maintenance of the plasmid in E. coli; and an ampicillin (bla) resistance gene for selection in E. coli. Other plasmids with similar design features are available and publicly available.

[0229] In the examples below, the antibody having native sequence heavy chains (SEQ ID NO: 11) and light chains (SEQ ID NO: 12) is designated MOm301 and is also known as WBP70323_1 and BB301. The antibody having substitutions I253A+H310A+H435A / L234F+L235E+P331S in the heavy chain (SEQ ID NO: 13) and light chain (SEQ ID NO: 12) is designated MOm303 and is also known as WBP70323_2 and BB303.

[0230] Data from the following examples are presented in the figures below:

[0231] Figure 1. Gel electrophoresis of purified antibodies WBP70323_1 (MOm301) and WBP70323_2 (MOm303).

[0232] Figure 2. Fold change in total fluorescent signal in pregnant mice and fetuses

[0233] Figure 3 Plasma concentrations of BB301 and BB303 in pregnant mice and fetuses 24 hours after administration.

[0234] Example 1. Production of MOm301 and MOm303 by transient expression in CHO cells. Codon-optimized DNA sequences were synthesized and cloned into expression vectors designed to express approximately equimolar amounts of each heavy and light chain in transient expression in CHO K1 cells. Pilot expression was performed to confirm the functionality of the expression vector. The vectors were then cloned and purified in sufficient quantities for transient expression in a 2-liter culture. CHO-K1 host cells were thawed and prepared for transfection by culturing them in BM001H medium (WuXi Bio internal catalog number) containing 4 mM glutamine (JT Baker, 2078-06) and 1% HT Supplement (Gibco, 11067-030). Separate cultures were transfected with an equal mass mixture of light and heavy chain vectors WBP70323_1 (vectors PWX4.1-HC-70323_1 and PWX4.1-LC-70323_1) and WBP70323_2 (vectors PWX4.1-HC-70323_2 and PWX4.1-LC-70323_2). Cells were expanded in BM022H medium (WuXi Bio internal catalog number) containing 6 mM glutamine (ITW Reagents A1420,1000) and 1% HT Supplement (Gibco, 11067-030), and the same medium was used for transfection and antibody production. The feed media used to support production were FM020a (Hyclone-SH31026.01) and FM020b (Hyclone-SH31027.01). For WBP70323_1 and WBP70323_2, transient transfections were performed in individual 5 L shake flasks by mixing CHO-K1 host cells with polyethyleneimine (PEI, BIOHUB) and plasmid DNA. 96 hours prior to transfection, 1.8–2.0 × 10 cells were cultured in BM024H medium. 6Host cells were seeded at 100 cells / mL. For cell density, cells were counted using a Vi-CELL counter and diluted in prewarmed BM022H before transfection. The diluted host cells were incubated in a Kühner shaker (36.5°C, 6% CO2, 150 rpm, 50 mm diameter) before use. 1.25 mg of vector DNA, 1.25 mg of the appropriate heavy and light chain vectors for each antibody, and 12 mg of PEI were added to the diluted host cells. The transfected cultures were incubated with shaking for 2 hours before adding feed medium. The incubation was continued for 4 days, at which point the antibodies were harvested. On the day of harvest, the cell cultures were centrifuged at 10,000 x g for 40 minutes and clarified by sterile filtration through a 0.22 μm filter. WBP70323_1 was captured from the supernatant and purified by protein A chromatography using MabSelect SuRe (Cytiva, 17543803). WBP70323_2 is Capto (商標) L (Cytiva, 17547802) was used for capture and purification.

[0235] Chromatography was performed using an AKTA Pure M150 system. MabSelect SuRe was packed in a column with a diameter of 5.0 cm, a bed height of 7.3 cm, and a column volume of 140 ml. (商標)L was packed into a column with a diameter of 5.0 cm, a bed height of 7.7 cm, and a column volume of 150 ml. All runs were performed in bind-elute mode, with bound protein eluted at low pH. Elution fractions of each molecule were tested for concentration and purity. Affinity-purified WBP70323_1 and separately pooled fractions of WBP70323_2 were further purified by SEC chromatography using Superdex 200 (Cytiva, 17104302). The Superdex 200 resin was packed into a column with a diameter of 5.0 cm and a bed height of 87.6 cm, for a packed volume of 1720 ml. Fractions containing individual purified antibodies were concentrated to approximately 20 mg / mL in 20 mM histidine acetate, 150 mM NaCl, pH 5.5 buffer using 30 kDa Amicon Ultra-15 mL Centrifugal Filter Units (Millipore UFC903096) and clarified by sterile filtration through a 0.22 μm filter. Antibodies were assayed for protein content and endotoxin and analyzed by SEC, gel electrophoresis, and mass spectrometry. SDS-PAGE profiles under non-reducing and reducing conditions are shown in Figure 1. The mass of the non-reduced antibody matched the known mass of an intact IgG antibody, and the masses of the individual heavy and light chains after separation under reducing conditions also matched.

[0236] Example 2. Binding properties to VEGFR-1, FcγRIIb, FcγRIIIa and FcRn. Surface plasmon residence (SPR) is commonly used to measure the binding affinity of antibodies to ligands and Fc receptors. SPR measures the change in refractive index caused by mass differences due to protein binding or unbinding from a gold-coated sensor. Biocore (商標) The 8k (Cytivia) is widely used to measure antibody binding interactions, and the real-time increase or decrease in mass, reported as resonance units (RU), is used to measure the binding rate constant (k a ) and dissociation rate constant (k d) and determine the equilibrium constant, KD. Biocore is used to measure antibody binding to its ligand, VEGFR-1, and its Fc receptor. (商標) Biocore equipment (商標) Multi-cycle kinetic analysis was performed using Insight software running.

[0237] To assess the binding of BB301 (MOm301, WBP70323_1) and BB303 (MOm303, WBP70323_2) to their target ligands, VEGFR-1, Fcγ receptors IIb and IIIa, and FcRn, binding measurements were performed at two different pH values. In parallel, BB301 was stained with the Alexa Fluor dye. (商標) A third sample, modified with 647 and identified as BB301-AF647, was analyzed. This analysis was performed to determine whether the dye modifications described in Example 3 affected binding to any of the target ligands VEGFR-1, Fcγ receptor IIb or IIIa, or the FcRn receptor compared to the unmodified BB301 antibody.

[0238] Binding to VEGFR-1. Activator solution was prepared by mixing 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS) immediately prior to injecting and activating a CM5 sensor chip at a flow rate of 10 μL / min for 420 seconds. VEGFR-1 (H.pro.1 His: Sinobiologics, 10136-H08H1) at a concentration of 0.8 μg / mL in 10 mM NaAc (pH 5.0) was injected into flow cell 2 (Fc2) at a flow rate of 10 μL / min for 60 seconds. The reference channel, Fc1, was blocked and not exposed. The chip was deactivated with 1 M ethanolamine-HCl at a flow rate of 10 μL / min for 420 seconds. Six concentrations (0.625, 1.25, 2.5, 5, 10, and 20 nM) of analyte BB301, BB303, or BB301-AF647 in running buffer (1x HBS-EP+; 0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA, and 0.5% v / v Surfactant P20; Cytiva BR100669) were injected at a flow rate of 30 μL / min for a 240-s association phase followed by a 3600-s dissociation phase. After each dissociation phase, the chip was regenerated with an injection of 10 mM glycine (pH 1.5) buffer.

[0239] The sensorgrams for the reference and buffer channels were subtracted from the test sensorgrams, and the experimental data were fitted to a 1:1 binding model. A molecular weight of 146,508 Da was used to calculate the molar concentrations of analytes BB301 and BB301-AF647. A molecular weight of 146,239 Da was used to calculate the molar concentration of analyte BB303.

[0240] [Table 1]

[0241] The dissociation constants of BB301 and BB303 are 1.64 × 10 -11 M and 1.52 x 10 -11The dissociation constant of BB301-AF647 was 1.93 x 10. -11 M, and there is no difference between BB301 and this method within the range of variation. (商標) It is confirmed that modification at 647 does not affect VEGFR-1 binding.

[0242] Binding to Fcγ receptor IIb. The CM5 sensor chip was activated with activator solution at a flow rate of 10 μL / min for 420 seconds. Anti-His tag antibody (THE (商標) , Genescript, A00186-100) at a concentration of 30 μg / mL in 10 mM NaAc pH 4.5 was injected for 400 seconds at a flow rate of 30 μL / min. The chip was deactivated with 1 M ethanolamine-HCl (Cytiva) at a flow rate of 10 μL / min for 420 seconds. His-tagged FcγRIIb (AcrobioSystems, CDB-H5228) at a concentration of 0.7 μg / mL in running buffer (1× HBS-EP+) was injected over Fc2 at a flow rate of 10 μL / min for 30 seconds. Eight concentrations of analytes BB301, BB303, or BB301-AF647 (160, 320, 640, 1280, 2560, 5120, 10240, and 20480 nM) in running buffer were injected at a flow rate of 30 μL / min for a 60-s association phase followed by a 90-s dissociation phase. Glycine (10 mM, pH 1.5) regeneration buffer was injected into the flow cell after every dissociation phase.

[0243] The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. The experimental data were fitted by a steady-state affinity model. A molecular weight of 146,508 Da was used to calculate the molar concentrations of the analytes BB301 and BB301-AF647. A molecular weight of 146,239 Da was used to calculate the molar concentration of the analyte BB303.

[0244] [Table 2]

[0245] The dissociation constants of BB301 and BB303 are 4.1 × 10 -6 M and 1.17 x 10 -5 The results demonstrate that the amino acid substitutions introduced into the Fc region of BB303 reduced the dissociation constant, a measure of affinity, by a factor of three compared to the unmodified antibody. The dissociation constant of BB301-AF647 was 4.28 x 10 -6 M, which is very similar to BB301 within the variability of the method. (商標) It is confirmed that modification at 647 does not affect Fcγ receptor IIb binding.

[0246] Binding to FcγRIIIa(V176) The CM5 sensor chip was activated for 420 seconds with activator solution at a flow rate of 10 μL / min. 30 μg / mL of THE in 10 mM NaAc (pH 4.5) was added. (商標) Anti-His tag antibody was injected for 400 seconds at a flow rate of 30 μL / min. The chip was deactivated with 1 M ethanolamine-HCl (Cytiva) for 420 seconds at a flow rate of 10 μL / min. His-tagged FcγRIIIa (AcrobioSystems, CD8-H52H4) 0.5 μg / mL in running buffer (1× HBS-EP+) was injected over Fc2 at a flow rate of 10 μL / min for 30 seconds. BB301 and BB301-AF647 were injected at concentrations of 5, 10, 20, 40, 80, 160, 320, and 640 nM in running buffer, and BB303 was injected at concentrations of 80, 160, 320, 640, 1280, 2560, 5120, and 10240 nM at a flow rate of 30 μL / min for a 300-s association period followed by a 300-s dissociation period. Glycine (10 mM, pH 1.5) regeneration buffer was injected into the flow cell after each injection.

[0247] [Table 3]

[0248] The dissociation constants of BB301 and BB303 are 9.74 × 10 -8 M and 3.9 x 10 -6 The results demonstrate that the amino acid substitutions introduced into the Fc region of BB303 reduced the dissociation constant, a measure of affinity, by 40-fold compared to the unmodified antibody. The dissociation constant of BB301-AF647 is 9.86 x 10 -8 M, which is very similar to BB301 within the variability of the method. (商標) It is confirmed that modification at 647 does not affect Fcγ receptor IIIa binding.

[0249] Binding to human FcRn at pH 6.0 and pH 7.4 In preparation, human FcRn (AcrobioSystems, FCM-H5286) was buffer-exchanged into either pH 6.0 phosphate-buffered saline with Tween (PBST pH 6.0, 50 mM NaHPO / NaHPO, 150 mM NaCl, 0.05% Tween-20, pH 6.0) or pH 7.4 PBST (50 mM NaHPO / NaHPO, 150 mM NaCl, 0.05% Tween-20, pH 7.4) running buffer using a desalting column (Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL; Thermofisher, Pierce-89882). Concentrations were determined using a NanoDrop 2000 spectrophotometer.

[0250] The sensor chip activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. A CM5 sensor chip was activated with the mixture for 420 seconds at a flow rate of 10 μL / min. In separate experiments using dedicated chips, BB301, BB303, or BB301-AF647 at a concentration of 10 μg / mL in 10 mM NaAc (pH 5.5) was injected over Fc2 at a flow rate of 10 μL / min for 60 seconds. The chip was deactivated with 1 M ethanolamine-HCl at a flow rate of 10 μL / min for 420 seconds. In separate experiments, the binding properties of BB301, BB303, or BB301-AF647 were analyzed at both pH 6.0 and pH 7.4. Eight concentrations of human FcRn analyte (46.9, 93.7, 187.5, 375, 750, 1500, 3000, and 6000 nM) in running buffer (PBST, pH 6.0 or 7.4) were injected over Fc1 and Fc2 at a flow rate of 30 μL / min for a 60-second association phase, followed by a 90-second dissociation period for each antibody at each pH. Regeneration buffer, PBS (50 mM NaHPO / NaHPO, 150 mM NaCl, pH 7.4), was injected over the flow cell for each dissociation phase.

[0251] The sensorgrams of the reference and buffer channels were subtracted from the test sensorgrams. The experimental data were fitted by a steady-state affinity model. The molar concentration of human FcRn was calculated using a molecular weight of 45 kDa.

[0252] [Table 4]

[0253] The dissociation constant of BB301 at pH 6.0 is 2.06 × 10 -6 Although the FcRn binding of BB301 and BB303 was not detected at pH 7.4, the binding of BB301 and BB303 was not detected at pH 7.4. This result demonstrates that the amino acid substitutions introduced into the Fc region of BB303 significantly reduced binding to the FcRn receptor.

[0254] The dissociation constant of BB301-AF647 at pH 6.0 is 1.66 × 10 -6 M, which is very similar to BB301 within the variability of the method. BB301-AF647 binding is also not detected at pH 7.4.

[0255] Example 3. Fluorescent labeling of antibodies. A BB301 stock solution containing 16 mg of protein was diluted, distributed among three Amicon Ultra-0.5 ml units, concentrated by centrifugation, recovered by centrifugation in an inverted unit, pooled, and brought to a final volume of 450 μL with 1× borate buffer. After three additional centrifugations, the original buffer was replaced, and the final concentrate was diluted to 8 mL with 1× borate buffer. For labeling, 40 μL of Alexa Fluor 1000 (Alexa Fluor 1000) was added. (商標) 647 NHS Ester was added, mixed, and incubated at room temperature for 2 hours. Free dye was removed by dialysis, and the solution was diluted and the antibody and dye concentrations were measured using a Nanodrop spectrophotometer (Thermofisher). -1 M -1 The protein concentration of AF647-BB301 measured at 280 nm using an extinction coefficient of 239,000 cm was 2.04 mg / mL (13.9 μM). -1 M -1 The concentration of AF647 measured at 650 nm using an extinction coefficient of 0.05 was 32.2 μM. The final yield of AF647-BB301 was 14.0 mg in a volume of 6.9 mL, with a dye-to-protein ratio of 2.3 to 1.

[0256] BB303 was modified by the same method employed to accommodate the initial antibody concentration. -1 M -1 The protein concentration after modification, dialysis, and dilution of AF647-BB303 was 2.15 mg / mL (14.7 μM) measured at 280 nm using an extinction coefficient of 239,000 cm -1 M -1The concentration of AF647 measured at 650 nm using an extinction coefficient of 1.0 μM was 33.1 μM. The final yield of AF647-BB303 was 14.6 mg in a volume of 6.8 mL, with a dye-to-protein ratio of 2.3 to 1 mol / mol.

[0257] Store the labeled antibody in the dark at 2-8°C and is stable under these conditions for at least one month.

[0258] In Example 2, SPR analysis of AF647-BB301 demonstrates that the dye-modified antibody has binding properties for VEGFR-1, FcγIIb, FcγIIIa, and FcRn similar to those of the unmodified BB301 antibody. The labeled antibodies AF647-BB301 and AF647-BB303 have the same degree of modification in terms of moles of dye per mole of antibody, meaning that AF647-BB303 is expected to have the same binding properties as unmodified BB303.

[0259] Example 4 Maternal-to-fetal transfer using fluorescently labeled antibodies. The inhibition of maternal-to-fetal transfer by the introduction of mutations that significantly reduce or eliminate receptor-binding affinity was demonstrated by comparing the maternal-to-fetal transfer of labeled MOm301 (AF647-BB301), which has no receptor-binding modifications, with the maternal-to-fetal transfer of labeled MOm303 (AF647-BB303), which contains the modifications I253A+H310A+H435A / L234F+L235E+P331S.

[0260] Male and female BALB / c mice were housed under specific, non-pregnancy-free conditions with appropriate attention to their welfare and in compliance with all local regulations. Males were mated at 18 weeks and females at 16 weeks. Pregnant females were identified the morning after mating by the presence of a viscous vaginal plug. These females were designated as gestational day 0.5 (D0.5), and treatment occurred on D16.5. Pregnant mice were divided into three groups based on body weight: Group 1 received phosphate-buffered saline (PBS) alone, Group 2 received AF647-BB301 at 30 mg / kg, and Group 3 received AF647-BB303 at 30 mg / kg. 24 hours after treatment, mice were anesthetized with isoflurane and then IVIS-administered.(登録商標) Fluorescence imaging was performed using a Spectrum (Revvity, Inc.). After imaging, pregnant mice were sacrificed under anesthesia, and fetuses were dissected from the placenta and surrounding tissues before imaging. For each mouse, the fluorescence efficiencies of three fetuses were averaged to control for fetal variability. The fold change in fluorescence was calculated relative to the PBS control. The laser excitation filter was set at 640 nm, and the emission filter was set at 680 nm.

[0261] The mean fold change in fluorescence of pregnant females and fetuses was measured relative to the PBS control (Figure 2). Females administered AF647-BB301 showed a 70.4-fold increase in fluorescence, while females administered AF647-BB303 showed a 21.2-fold increase. The lower fold increase for AF647-BB303 is consistent with increased clearance due to impaired binding to FcRn, as confirmed by the pharmacokinetic analysis in Example XXX. Fetuses from mice administered AF647-BB301 showed a 104.3-fold increase in fluorescence, while fetuses from mice administered AF647-BB303 had only a 1.74-fold increase, not statistically different from the PBS control.

[0262] In conclusion, comparison of fluorescently labeled BB301 (MOm301) and BB303 (MOm303) indicates that the amino acid substitution I253A+H310A+H435A / L234F+L235E+P331S resulted in a reduction in maternal-to-fetal transfer of at least 98.3% [100 minus (1.74 divided by 104.3 multiplied by 100)].

[0263] Example 5 Pharmacokinetics of MOm301 and MOm303 in BALB / c mice BALB / c female mice were housed under specific-pathogen-free conditions with appropriate attention to their welfare and in compliance with all local regulations. Eight to ten-week-old female mice were randomly divided into four groups of five mice each. Groups 1 and 2 received 10 mg / kg BB301 (MOm301), while groups 3 and 4 received 10 mg / kg BB303 (MOm303). Blood samples were collected by ophthalmic vein puncture at the indicated time points. Mice were fully anesthetized with isoflurane before collection: whole blood was collected into 1.5 mL disposable anticoagulant tubes and centrifuged at 8000 rpm for 5 minutes at 4°C. Plasma supernatants were collected for analysis by ELISA. Sampling times were as follows: Group 1: 1 hour, 24 hours, 72 hours, 168 hours; Group 2: 6 hours, 48 ​​hours, 120 hours, 168 hours; Group 3: 1 hour, 6 hours, 24 hours, 72 hours; Group 4: 3 hours, 8 hours, 48 ​​hours, 96 hours.

[0264] Antibody concentrations in plasma were measured by ELISA. Each sample was tested in duplicate against a standard curve. Briefly, goat anti-human IgG-F(ab')2 antibody (Bethyl Laboratories, A80-249A) was coated at 1 μg / mL onto a 96-well ELISA plate (100 μL per well) for 2.5 hours at 37°C. The plate was then washed once with PBST (300 μL per well) and blocked with 2% BSA (bovine serum albumin, 200 μL per well) for 2 hours at ambient temperature. After washing three times with PBST (300 μL per well), serially diluted standards or plasma samples in 2% BSA were added to the plate (100 μL per well) and incubated for 1 hour at ambient temperature. After washing three times with PBST (300 μL per well), goat anti-human Ig Fab-HRP antibody (Southern Biotech, 2085-05) was added to the plate at 0.09 μg / mL (100 μL per well) and incubated at ambient temperature for 1 hour. After washing three times with PBST (300 μL per well), tetramethylbenzidine substrate was added to the plate and allowed to develop for 8 minutes (100 μL per well). The reaction was then stopped by adding 2 M HCl (100 μL per well). Absorbance was measured at 450 nm and 540 nm using a microplate spectrophotometer (SpectraMax® M5e). A standard curve was generated based on the standard samples using SoftMax Pro software.

[0265] [Table 5]

[0266] In conclusion, BB301 (MOm301) showed a half-life of 380 hours in female BALB / c mice, whereas BB303 (MOm303) had a reduced half-life of 17 hours due to the amino acid substitutions I253A+H310A+H435A / L234F+L235E+P331S.

[0267] Example 6 Maternal-to-fetal transfer measured by enzyme-linked immunosorbent assay (ELISA) The inhibition of maternal-to-fetal transfer by the introduction of mutations that significantly reduce or eliminate receptor-binding affinity was demonstrated by comparing the maternal-to-fetal transfer of BB301 (MOm301), which has no receptor-binding modifications, with that of BB303 (MOm303), which contains the modifications I253A+H310A+H435A / L234F+L235E+P331S.

[0268] Male and female BALB / c mice were housed under specific, non-pregnancy-free conditions with appropriate welfare considerations and in compliance with all local regulations. Males were mated at 18 weeks and females at 16 weeks. Pregnant females were identified the morning after mating by the presence of a viscous vaginal plug. These females were designated as day 0.5 of pregnancy (D0.5), and treatment occurred on D16.5. Pregnant mice were divided into three groups of four mice based on body weight. Group 1 received 70 mg / kg BB301, Group 2 received 70 mg / kg BB303, and Group 3 received phosphate-buffered saline (PBS) as a control. 24 hours after BB301 and BB303 treatment, samples from pregnant females and fetuses were analyzed.

[0269] Pregnant mice were fully anesthetized with isoflurane before blood collection from the ophthalmic venous plexus. Blood was collected from decapitated fetuses and then pooled. Whole blood was collected into 1.5 mL disposable anticoagulant tubes and centrifuged at 8000 rpm at 4°C for 5 minutes. Plasma supernatant was collected and analyzed by ELISA according to the method described in Example 5.

[0270] Figure 3 shows the plasma levels of BB301 and BB303 in pregnant mice and fetuses. After 24 hours, the plasma levels of BB301 in pregnant females were 271 (SD 58.7) and in fetuses were 127 (SD 6.73); for BB303, the plasma levels in pregnant females were 97.0 (SD 2.10) and in fetuses were 0.27 (SD 0.02).

[0271] In conclusion, based on quantitative ELISA analysis, comparison of BB301 (MOm301) and BB303 (MOm303) shows that the amino acid substitutions I253A+H310A+H435A / L234F+L235E+P331S resulted in a reduction in maternal-to-fetal transfer of at least 99.8% [100 minus (0.27 divided by 127 multiplied by 100)].

[0272] [Table 6]

[0273] [Table 7-1] [Table 7-2] [Table 7-3]

[0274] Description 1. A human variant IgG Fc domain, comprising a mutation or combination of mutations relative to the parent polypeptide sequence that reduces binding of the Fc domain to any FcγR, and a mutation or combination of mutations relative to the parent polypeptide sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-to-fetal transfer, preferably by at least 95% or more, relative to the parent sequence.

[0275] 2. An isolated polypeptide comprising a human variant IgG Fc domain according to statement 1, wherein the Fc domain has a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-to-fetal transfer, preferably by at least 95% or more relative to the parent sequence.

[0276] 3. The isolated polypeptide of statement 2, comprising a human variant IgG Fc domain comprising amino acid substitutions numbered according to the Kabat EU index numbering system relative to a human wild-type Fc domain, a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) a substitution at position 310 with alanine and at position 435 with glutamine; An isolated polypeptide, wherein the polypeptide has reduced binding to at least one Fc gamma receptor (FcγR) and FcRn when compared to an unsubstituted polypeptide comprising the parent Fc domain.

[0277] 4. A polypeptide according to any one of statements 2 or 3, or a variant IgG Fc domain according to statement 1, wherein the polypeptide forms part of an antibody or fragment thereof, and when compared to the same polypeptide comprising the Fc domain, exhibits such as at least a 10-fold increase in the concentration of antibody or antibody fragment required to give 50% binding or activation in a parent binding or cell activation assay, respectively, and / or exhibits a 10-fold or greater reduction in the Km binding constant for at least one Fcγ receptor and FcRn, e.g. by SPR or equivalent methods.

[0278] 5. A polypeptide according to any one of statements 2, 3 and 4, or a variant IgG Fc domain according to statement 1 or 4, wherein at least one FcγR is selected from FcγRi, FcγRIIa, FcγRIIb, FcγIIIa and FcγRIIIb, preferably FcγIIb and FcγIIIa.

[0279] 6. A polypeptide or variant IgG Fc domain according to any preceding statement, wherein the IgG Fc domain is selected from the group consisting of a human immunoglobulin G class 1 (IgG1) Fc domain, a human immunoglobulin G class 2 (IgG2) Fc domain, a human immunoglobulin G class 3 (IgG3) Fc domain, and a human immunoglobulin G class 4 (IgG4) Fc domain, preferably a human immunoglobulin G class 1 (IgG1) Fc domain.

[0280] 7. The parent polypeptide sequence is the wild-type sequence appropriate for the IgG subclass of the Fc domain or one of the following mutations: IgG1(N297A), IgG1(N297G), IgG1(L234F / L235E / P331S), IgG1(L234A / L235A), IgG1(L235V / F243L / R292P / Y300L / P396L), IgG1(L234F / L235E / P331S), IgG1(L234A / L235A / P329G), IgG1(S354C / T366W), IgG1(Y349C / T366S / L368A / Y407V), IgG1(M2 IgG1 / 2(S239D / I332E), IgG2(C131S / R133K / C219S), IgG2(H268Q / R355Q / Q419E / N434A), IgG2 / 4(M428L / N434S), IgG4(S228P), IgG4(S228P / F234A / L235A), IgG4(S228P / L235E), IgG4(S241P / F234A / L235A), IgG4(F405L / R409K), or a modified wild-type sequence comprising any of the following: IgG1 / 2(S239D / I332E), IgG2(C131S / R133K / C219S), IgG2(H268Q / R355Q / Q419E / N434A), IgG4 / 4(M428L / N434S), IgG4(S228P), IgG4(S228P / F234A / L235A), IgG4(S241P / F234A / L235A), IgG4(F405L / R409K).

[0281] 8. A polypeptide or variant IgG Fc domain according to any preceding statement, comprising an amino acid sequence which is at least 80%, preferably at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 9.

[0282] 9. A polypeptide or variant IgG Fc domain according to any preceding statement, wherein the polypeptide further comprises an antigen-binding domain, for example the antigen-binding domain of a monoclonal antibody or an antigen-binding fragment thereof.

[0283] 10. The polypeptide of any of statements 2 to 8, comprising an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence of the heavy chain of SEQ ID NO: 13, such as the polypeptide of SEQ ID NO: 13, optionally together with the antibody light chain of SEQ ID NO: 12.

[0284] 11. An antibody comprising a heavy chain having a combination of SEQ ID NO: 10 as the variable heavy chain sequence and SEQ ID NO: 5 as the heavy chain constant region, and a light chain having the sequence of SEQ ID NO: 12, said antibody further comprising a substitution as described in statement 3.

[0285] 12. A conjugate comprising a variant IgG Fc domain, polypeptide or antibody according to any preceding statement and a therapeutic moiety.

[0286] 13. A nucleic acid comprising a nucleotide sequence encoding a mutant IgG Fc domain or polypeptide according to any of statements 1 to 9, an antibody according to statement 10, or a conjugate according to statement 11.

[0287] 14. A vector comprising the nucleic acid of statement 13.

[0288] 15. A host cell comprising a nucleic acid according to statement 13 or 14.

[0289] 16. A pharmaceutical composition comprising a mutant IgG Fc domain according to statement 1, a polypeptide according to any one of statements 2-10, an antibody according to statement 11, a conjugate according to statement 12, a nucleic acid according to statement 13, or a vector according to statement 14, and a pharmaceutically acceptable excipient.

[0290] 17. A variant IgG Fc domain according to statement 1, a polypeptide according to any one of statements 2-9, an antibody according to statement 11, a conjugate according to statement 12, a nucleic acid according to statement 13, or a vector according to statement 14, or a pharmaceutical composition according to statement 16, for use in treating a mammal, preferably a human, more preferably a female, such as a female of reproductive age 15-45 years of age, even more preferably a pregnant woman.

[0291] 18. A method for reducing binding to at least one Fcγ receptor and FcRn in a parent polypeptide comprising an Fc domain, comprising: a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) substitutions at positions 234 and 235 with alanine, and at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) substitution of arginine at position 328 and insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) a substitution at position 310 with alanine and at position 435 with glutamine; The polypeptide containing the substitution may be suitably expressed in the form of an antibody or fragment thereof, The method may be combined with a pharmaceutically acceptable carrier or excipient.

[0292] 19. A method for treating a mammal, preferably a human, more preferably a female, for example a female of reproductive age between 15 and 45 years of age, even more preferably a pregnant female, comprising administering to a mammal an effective amount of (a) a mutant IgG Fc domain or polypeptide according to any one of claims 1 to 10; (b) the antibody of statement 11; (c) a conjugate according to statement 12; (d) a nucleic acid according to statement 13; (e) a vector according to statement 14; (f) the cell of statement 15; or (g) the pharmaceutical composition of statement 16 The method comprises administering any one or more of the following:

Claims

1. An isolated polypeptide for use in the medical care of pregnant women, comprising a human variant IgG Fc domain having a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR, and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-fetal transfer by at least 95% relative to the parent sequence.

2. 1. An isolated polypeptide for use in treating or preventing a pregnancy disorder in a pregnant woman, comprising a human variant IgG Fc domain having a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR, and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, thereby inhibiting maternal-fetal transfer by at least 95% relative to the parent sequence.

3. 1. A method of treating a pregnant woman, comprising administering an effective amount of an isolated polypeptide, wherein the polypeptide comprises a human variant IgG Fc domain having a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to any FcγR and a mutation or combination of mutations relative to the parent sequence that reduces binding of the Fc domain to FcRn, whereby maternal-fetal transfer is inhibited by at least 95% or more relative to the parent sequence.

4. the polypeptide comprises a human variant IgG Fc domain comprising amino acid substitutions relative to a human wild-type Fc domain numbered according to the Kabat EU index numbering system; a) (i) a substitution at positions 234 and 235 with alanine, respectively; or (ii) a substitution at positions 234 and 235 with alanine, and a substitution at position 331 with serine; or (iii) a substitution at position 234 with phenylalanine, a substitution at position 235 with glutamic acid, and a substitution at position 331 with serine; or (iv) a substitution of arginine at position 328 and an insertion of arginine after position 236; and b) (i) a substitution at position 253 with alanine, or (ii) a substitution at position 435 with alanine, or (iii) substitutions at positions 235, 310, and 435 with alanine; or (iv) a substitution at position 310 with alanine and at position 435 with glutamine; 10. An isolated polypeptide for use in medical care of a pregnant woman as claimed in claim 1, or for use in treating a pregnant woman as claimed in claim 2, or a method of treating a pregnant woman as claimed in claim 3, wherein the polypeptide has reduced binding to at least one Fc gamma receptor (FcγR) and FcRn when compared to an unsubstituted polypeptide comprising the parent Fc domain.

5. 5. An isolated polypeptide for use in the medical care of pregnant women as claimed in claim 1 or 4, or for use in the treatment of pregnant women as claimed in claim 2 or 4, or a method of treating pregnant women as claimed in claim 3 or 4, wherein the polypeptide forms part of an antibody or fragment thereof and the polypeptide exhibits an increase in the concentration of antibody or antibody fragment required to give 50% binding or activation in a binding or cell activation assay, respectively, and / or exhibits a 10-fold or greater reduction in the Km binding constant for at least one Fcγ receptor and FcRn, e.g. by SPR or equivalent methods, when compared to the same polypeptide comprising the parent Fc domain.

6. 6. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 or 5, or for use in treating a pregnant woman according to any one of claims 2 or 4 or 5, or a method of treating a pregnant woman according to any one of claims 3 or 4 or 5, wherein at least one FcγR is selected from FcγRi, FcγRIIa, FcγRIIb, FcγIIIa, and FcγRIIIb, preferably FcγIIb and FcγIIIa.

7. 7. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 6, or for use in treating a pregnant woman according to any one of claims 2 or 4 to 6, or a method of treating a pregnant woman according to any one of claims 3 to 6, wherein the IgG Fc domain is selected from the group consisting of a human immunoglobulin G class 1 (IgG1) Fc domain, a human immunoglobulin G class 2 (IgG2) Fc domain, a human immunoglobulin G class 3 (IgG3) Fc domain, and a human immunoglobulin G class 4 (IgG4) Fc domain, preferably a human immunoglobulin G class 1 (IgG1) Fc domain.

8. The parent polypeptide sequence is the wild-type sequence appropriate for the IgG subclass of the Fc domain or has the following mutations: IgG1(N297A), IgG1(N297G), IgG1(L234F / L235E / P331S), IgG1(L234A / L235A), IgG1(L235V / F243L / R292P / Y300L / P396L). , IgG1 (L234F / L235E / P331S), IgG1 (L234A / L235A / P329G), IgG1 (S354C / T366W), IgG1 (Y34 9C / T366S / L368A / Y407V), IgG1 (M252Y / S254T / T256E), IgG1 / 2 (S239D / I332E), IgG2 (C131S 8. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 7, or for use in treatment of a pregnant woman according to any one of claims 2 or 4 to 7, or a method of treating a pregnant woman according to any one of claims 3 to 7, wherein the isolated polypeptide is a modified wild-type sequence comprising any of IgG2(H268Q / R355Q / Q419E / N434A), IgG2 / 4(M428L / N434S), IgG4(S228P), IgG4(S228P / F234A / L235A), IgG4(S228P / L235E), IgG4(S241P / F234A / L235A), IgG4(F405L / R409K).

9. 9. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 8, or for use in treating a pregnant woman according to claims 2 or any one of claims 4 to 8, or a method of treating a pregnant woman according to any one of claims 3 to 8, wherein the polypeptide comprises an amino acid sequence which is at least 80%, preferably at least 90%, identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO:

9.

10. 10. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 9, or for use in treating a pregnant woman according to any one of claims 2 or 4 to 9, or a method of treating a pregnant woman according to any one of claims 3 to 9, wherein the polypeptide further comprises an antigen-binding domain, such as an antigen-binding domain of a monoclonal antibody or an antigen-binding fragment thereof.

11. 11. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 10, or for use in treating a pregnant woman according to any one of claims 2 or 4 to 10, or a method of treating a pregnant woman according to any one of claims 3 to 10, wherein the polypeptide comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95% identical to the amino acid sequence of the heavy chain of SEQ ID NO: 13, for example the polypeptide of SEQ ID NO: 13, optionally together with an antibody light chain of SEQ ID NO:

12.

12. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 11, or for use in treating a pregnant woman according to any one of claims 2 or 4 to 11, or a method of treating a pregnant woman according to any one of claims 3 to 11, wherein the polypeptide is an antibody comprising a heavy chain having a combination of SEQ ID NO: 10 as a variable heavy chain sequence, the sequence of SEQ ID NO: 5 as a heavy chain constant region, and a light chain having the sequence of SEQ ID NO: 12, and further comprising any of the substitutions according to claim 4.

13. 13. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 12, or for use in treating a pregnant woman according to claims 2 or any one of claims 4 to 12, or a method of treating a pregnant woman according to any one of claims 3 to 12, wherein the polypeptide is in the form of a conjugate comprising a variant IgG Fc domain, polypeptide or antibody according to any preceding claim and a therapeutic moiety.

14. A nucleic acid comprising a nucleotide sequence encoding a polypeptide as disclosed in any one of claims 1 to 13 for use in treating pregnant women, for example in the treatment or prevention of pregnancy disorders.

15. 16. A vector comprising the nucleic acid of claim 15 for use in treating a pregnant woman, for example in treating or preventing a pregnancy disorder.

16. 16. A host cell comprising a nucleic acid according to claim 14 or 15 for use in the treatment of pregnant women, for example in the treatment or prevention of pregnancy disorders.

17. 17. An isolated polypeptide for use in medical care of a pregnant woman according to any one of claims 1 or 4 to 12, or for use in treating a pregnant woman according to any one of claims 2 or 4 to 12, or a method of treating a pregnant woman according to any one of claims 3 to 12, or a nucleic acid for use according to claim 14, a vector for use according to claim 15, or a cell for use according to claim 16, wherein the polypeptide is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

18. The disorder or disease may be a hypertension-related condition or pre-eclampsia / eclampsia; or The disorder or disease may be pre-eclampsia / eclampsia and the polypeptide is an anti-VEGFR-1 antibody that inhibits binding of VEGF and / or PGF to sVEGFR-1; or The disorder or disease may be pre-eclampsia / eclampsia, and the polypeptide comprises or consists of the polypeptide of SEQ ID NO: 13, provided in the form of an antibody, optionally in the form of a pharmaceutically acceptable composition in combination with a pharmaceutically acceptable excipient, together with the antibody light chain of SEQ ID NO:

12.

20. An isolated polypeptide for use, a method of treatment, a nucleic acid for use, a vector for use or a cell for use according to any one of claims 1 to 17 in the treatment or prevention of pregnancy disorders.

19. An antibody comprising the polypeptide of SEQ ID NO: 13 together with the antibody light chain of SEQ ID NO: 12, which may be formulated as a pharmaceutically acceptable composition together with an excipient or carrier.

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