Engineered antibody constant region polypeptides and related methods

Abstract

Provided are engineered human IgG Fc polypeptides incorporating amino acids substitutions conferring beneficial properties, such as altered in vivo half-life and / or binding affinity for human FcRn, as compared to the polypeptides without the one or more amino acid substitutions. Also provided are various molecules and molecular complexes, such as biologic therapeutics incorporating such engineered polypeptides, methods of generating such polypeptides, nucleic acids and nucleic acids constructs encoding the engineered polypeptides, therapeutic methods using molecules and molecular complexes, such as biologic therapeutics incorporating the engineered polypeptides, as well as the related systems and kits.

Description

Attorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355ENGINEERED ANTIBODY CONSTANT REGION POLYPEPTIDES AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U. S. Provisional Application No. 63 / 736,783 and 63 / 844,557, filed December 20, 2024, and July 15, 2025, respectively, which are incorporated by reference in their entirety for all purposes.SEQUENCE LISTING

[0002] This application is accompanied by a sequence listing entitled, created >, which is approximately in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted herewith via Patent Center and is m compliance with 37 C. F. R. § 1.824(a)(2)-(6) and (b).BACKGROUND

[0003] Biologic therapeutics, including therapeutic antibodies, that incorporate Fc domain of antibodies are an important and growing class of drugs that have revolutionized the treatment of a broad set of diseases--- ranging from infectious diseases to cancer and autoimmunity. However, across most applications, the clinical viability of biologic therapeutics is undermined by their relatively short half-life in vivo, requiring frequent administration to maintain efficacious levels in serum. Thus, there remains a critical unmet need for a broadly applicable solution that can extend the half-life of biologic therapeutics.BRIEF SUMMARY

[0004] The terms “invention,” “the invention,” “this invention” and “the present invention,” as used in the present disclosure, are intended to refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent1US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355claims below. Covered embodiments of the invention are defined by the claims, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary' is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures and each claim. Some of the exemplary embodiments of the present invention are discussed below.

[0005] Included among exemplary embodiments of the present invention are polypeptides comprising a human Fc polypeptide ammo acid sequence comprising one or more amino acid substitutions L251V, L251Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, wherein the one or more amino acid substitutions are numbered with respect to the human Fc polypeptide amino acid sequence. In some exemplary embodiments, the one or more amino acid substitutions comprise one or more of V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, and may further comprise at least one of (a) amino acid substitutions M252Y, S254T, and T256E, or (b) amino acid substitutions (i) M428L and (ii) N434S, N434A, or N434G (e.g., M428L and N434S, M428L and N434A, or M428L and N434G).

[0006] In some exemplary embodiments, the human Fc polypeptide amino acid sequence is a human IgGFc amino acid sequence. In some exemplary embodiments, the human Fc polypeptide amino acid sequence has at least 90% sequence similarity to SEQ ID NO: 1. In some exemplary’ embodiments, the polypeptide has increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has decreased binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has increased binding2US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355affinity for human FcRn at pH from about 5.5 to about 6,5, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary7embodiments, the polypeptide has substantially unaltered binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has a substantially unaltered dissociation rate for human FcRn at pH from about 7.0 to about 7.5, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has an increased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide ammo acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has a decreased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has increased binding affinity for human FcRn at pH about 5.5 to about 6.5 and a substantially unaltered dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide has a substantially unaltered binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, a polypeptide has altered binding to one or more human FcyRs, as compared to the polypeptide without the one or more amino acid substitutions according to the present disclosure.

[0007] In some exemplary embodiments, an antibody or antibody fragment comprising a polypeptide according to the embodiments of the present invention has one or more altered effector functions (such as, but not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as compared to a corresponding antibody or antibody fragment comprising a polypeptide without the one or more amino acid substitutions according to the present disclosure.3US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0008] Antibodies or antibody fragments comprising polypeptides according to the embodiments of the present invention and having one or more altered effector functions are included among the embodiments of the present invention. In some exemplary embodiments, an antibody or antibody fragment comprising a polypeptide according to the embodiments of the present invention has improved expression, as compared to a corresponding antibody or antibody fragment comprising a polypeptide without the one or more amino acid substitutions according to the present disclosure. Antibodies or antibody fragments comprising polypeptides according to the embodiments of the present invention and having improved expression are included among the embodiments of the present invention.

[0009] In some exemplary embodiments, a polypeptide according to the embodiments of the present invention is linked to at least one other polypeptide and / or to at least one non-peptide moiety. In some exemplary embodiments, the at least one non-peptide moiety is one or more of a radioactive isotope, a molecule comprising a radioactive label, fluorophore, a drug molecule, a toxin, or a nucleic acid.

[0010] Included among exemplary embodiments of the present invention are molecules or molecular complexes comprising at least one of the polypeptides according to exemplary embodiments of the present invention. In some exemplary embodiments, a molecule or a molecular complex is an antibody or the antibody fragment. In some exemplary embodiments, a molecule or a molecular complex is linked to at least one other polypeptide and / or to at least one non-peptide moiety. In some exemplary embodiments, the at least one non-peptide moiety is one or more of a radioactive isotope, a molecule comprising a radioactive label, fluorophore, a drug molecule, a toxin, or a nucleic acid.

[0011] Included among exemplary embodiments of the present invention are recombinant nucleic acid molecules encoding the polypeptides according to exemplary embodiments of the present invention. In some exemplary’ embodiments, a recombinant nucleic acid molecule is a synthetic sequence designed for expression in a host cell. Also included among exemplary' embodiments of the present invention are DNA constructs comprising at least one recombinant nucleic acid according to exemplary embodiments of the present invention, the at least one recombinant nucleic acid operably linked to a promoter. Also included among exemplary4US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355embodiments of the present invention are vectors comprising at least one recombinant nucleic acid molecule according to exemplary embodiments of the present invention and / or at least one DNA construct according to exemplary embodiments of the present invention. Also included among exemplary embodiments of the present inventi on are host cells comprising one or more of: at least one recombinant nucleic acid molecule according to exemplary embodiments of the present invention; at least one DNA construct according to exemplary embodiments of the present invention; or at least one vector according to exemplary embodiments of the present invention. In some exemplary embodiments, a host cell is a bacterial cell. In some exemplary embodiments, a host cell is a eukaryotic cell.

[0012] Included among exemplary embodiments of the present invention are compositions comprising one or more of: at least one polypeptide according to exemplary embodiments of the present invention; at least one molecule or molecular complex according to exemplary- embodiments of the present invention; at least one recombinant nucleic acid molecule according to exemplary embodiments of the present invention; at least one DNA construct according to exemplary embodiments of the present invention; or at least one vector according to exemplary embodiments of the present invention. In some exemplary embodiments, a composition comprises a pharmaceutically acceptable carrier.

[0013] Included among exemplary embodiments of the present invention are methods of treating a subject with a disease or a condition, comprising administering to the subject a composition cording to exemplary embodiments of the present invention, in an amount effective to alleviate the disease or the condition. In some exemplary embodiments, a disease or a condition is one or more of an autoimmune disease or condition, a genetic disease or condition, a cancer, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a gastrointestinal disease or condition, an endocrine disease or condition, a neurological disease or condition, an ophthalmological disease or condition, a musculoskeletal disease or condition, or an infection.

[0014] Included among exemplary- embodiments of the present invention are methods of modifying a polypeptide comprising a human Fc polypeptide amino acid sequence, the method comprising introducing into the human Fc polypeptide amino acid sequence one or more amino5US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355acid substitutions L251V, L251 Y, M252D, S254T, V266L, H268K, H268S, 1N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, wherein the one or more amino acid substitutions are numbered with respect to the human Fc polypeptide amino acid sequence, thereby producing a modified human Fc polypeptide ammo acid sequence, wherein a polypeptide comprising the modified human Fc polypeptide amino acid sequence has an altered in vivo half-life and / or binding affinity for human FcRn, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the one or more amino acid substitutions comprise one or more of L251V, L251 Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421 V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, and may further comprise at least one of (a) amino acid substitutions M252Y, S254T, and T256E, or (b) amino acid substitutions (i) M428L and (li) N434S, N434A, or N434G (e.g., M428L and N434S, M428L and N434A, or M428L and N434G). In some exemplary embodiments, the altered in vivo half-life is increased half-life. In some exemplary embodiments, the altered in vivo half-life is reduced half-life. In some exemplary embodiments, the binding affinity for human FcRn is increased binding affinity'. In some exemplary embodiments, the binding affinity for human FcRn is decreased binding affinity. In some exemplary embodiments, a polypeptide comprising the modified human Fc polypeptide amino acid sequence has increased binding affinity' for human FcRn at pH from about 5.5 to about 6.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, a polypeptide comprising the modified human Fc polypeptide amino acid sequence has decreased binding affinity for human FcRn at pH from about 5.5 to about 6,5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, a polypeptide comprising the modified human Fc polypeptide amino acid sequence has substantially unaltered binding affinity for human FcRn at pH from about 5.5 to about 6,5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid 6US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355substitutions according to the present disclosure. In some exemplary embodiments, the dissociation rate for human FcRn is an increased dissociation rate. In some exemplary embodiments, the dissociation rate for human FcRn is a decreased dissociation rate. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide amino acid sequence has an increased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide ammo acid sequence has a decreased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide ammo acid sequence has a substantially unaltered dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide ammo acid sequence without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide amino acid sequence has increased binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide amino acid sequence has increased binding affinity' for human FcRn at pH about 5.5 to about 6.5 and a substantially unaltered dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide ammo acid sequence has a substantially unaltered binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions. In some exemplary embodiments, the polypeptide comprising the modified human Fc polypeptide ammo acid sequence has altered binding to FcyRs, as compared to the polypeptide comprising human Fc polypeptide ammo acid sequence without the one or more amino acid substitutions according to the present disclosure. In some 7US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355exemplary embodiments, the human Fc polypeptide ammo acid sequence has at least 90% sequence similarity to SEQ ID NO: 1.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a schematic illustration of structures of FcRn (gray) and IgGFc (black) polypeptides. The residues predicted by inverse folding (IF) to alter interaction with FcRn are highlighted as spheres.

[0016] FIG. 2 is a dot plot illustrating the binding of Fc variant antibodies with single mutations predicted by IF to FcRn at pH 6.0. Each dot represents a single variant, and the y-axis shows the fold change in enzyme immunoassay binding (ECso), as compared to wild-type antibody. A higher value on the y-axis indicates stronger binding to FcRn.

[0017] FIG. 3 is a dot plot illustrating the binding of Fc variant antibodies with single mutations predicted by IF to FcRn at pH 6.0. Each dot represents a single variant, and the y-axis shows the fold change in affinity (KD), as compared to wild-type antibody. A higher value on the y-axis indicates stronger binding to FcRn.

[0018] FIG. 4 is a dot plot illustrating the binding of Fc variant antibodies with single mutations predicted by IF to FcRn at pH 7.4. Each dot represents a single variant, and the y-axis shows the fold change in enzyme immunoassay (ELISA) maximum response, as compared to wild-type antibody. A higher value on the y-axis indicates increased retention at pH 7.4.

[0019] FIG. 5 is a bar graph illustrating the binding to FcRn at pH 6,0 of Fc variant antibodies with single mutations, predicted by IF, in the WT or YTE background. Each bar represents a single variant, and the y-axis shows the fold change in BIT signal relative to the corresponding background Fc antibody. A higher value on the y-axis indicates stronger binding to FcRn,

[0020] FIGS. 6A-6B are bar graphs illustrating relative changes in FcRn binding of IgG Fc variants combined with YTE, normalized to YTE background binding, in the context of an anti-RSV IgGl antibody (FIG 6A) or an anti-Pf-CSP antibody (FIG, 6B) as determined by BIT Each bar represents a single variant, and the y-axis shows the fold change in BIT signal relative to the corresponding background YTE Fc antibody. Higher values at pH 6.0 (black bars) indicate8US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355stronger FcRn binding, while higher values at pH 7.4 (grey bars) indicate slower dissociation. For KD at pH 6, a higher fold-change reflects stronger binding. For koff at pH 7.4, a smaller fold-change reflects faster dissociation.

[0021] FIGS. 7A-7B show bar graphs of FcRn binding for IgG Fc variants with LS, LA, or LG substitutions. Binding was measured by BLI and normalized to the LS background in either an anti-RSV IgGl antibody (FIG. 7A) or an anti-Pf-CSP antibody (FIG. 7B). The y-axis indicates fold change relative to the LS Fc antibody. At pH 6.0 (black bars), a higher fold change means stronger FcRn binding. At pH 7.4 (grey bars), a lower fold change in koff means faster dissociation (more efficient release from FcRn).

[0022] FIG. 8 A shows a bar graph of FcRn binding at pH 6.0 (KD) and dissociation at pH 7.4 (koff) for Fc variant antibodies with double and triple mutations, predicted by IF, in the YTE background of an anti-proprotein convertase subtilisin / kexin type 9 (anti-PCSK9) IgG2 antibody. Binding was measured by BLI and normalized to the corresponding WT Fc antibody. The y-axis indicates fold change relative to the WT Fc antibody. At pH 6.0 (black bars), a higher fold change means stronger FcRn binding. At pH 7.4 (grey bars), a lower fold change in koff means faster dissociation (more efficient release from FcRn).

[0023] FIG. 8B shows a bar graph of FcRn binding at pH 6.0 (KD) and dissociation at pH 7.4 (koff) for Fc variant antibodies with double and triple mutations, predicted by IF, in the YTE background of an anti-programmed cell death protein 1 (anti-PD-1) IgGl antibody. Binding was measured by BLI and normalized to the corresponding WT Fc antibody. The y-axis indicates fold change relative to the corresponding background Fc antibody. At pH 6.0, a higher fold change means stronger FcRn binding. At pH 7.4, a lower fold change in koff means faster dissociation (more efficient release from FcRn).

[0024] FIG. 9 shows a bar graph of FcRn binding at pH 6.0 (KD) and dissociation at pH 7.4 (koff) for Fc variant antibodies various single mutations in the LS background of an anti-respiratory syncytial virus (anti-RSV) antibody having an IgGl subclass. Binding was measured by BLI and normalized to the corresponding LS Fc antibody. The y-axis indicates fold change relative to the corresponding LS Fc antibody. At pH 6.0, a higher fold change means stronger9US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355FcRn binding. At pH 7.4, a lower fold change in koff means faster dissociation (more efficient release from FcRn),

[0025] FIG. 10 shows a bar graph of FcRn binding at pH 6,0 (KD) and dissociation at pH 7,4 (koff) for Fc variant antibodies various single mutations in the LG background of an anti-respiratory syncytial virus (anti-RSV) antibody having an IgGl subclass. Binding was measured by BLI and normalized to the corresponding LS Fc antibody. The y-axis indicates fold change relative to the corresponding LS Fc antibody. At pH 6.0, a higher fold change means stronger FcRn binding. At pH 7.4, a lower fold change in koff means faster dissociation (more efficient release from FcRn).DETAILED DESCRIPTIONOverview

[0026] Among the various classes of immunoglobulins, immunoglobulins G (IgGs) are most often developed for antibody therapies. The pharmacokinetics of the resulting therapeutic antibodies are predominantly mediated by their interaction with the neonatal Fc receptor (FcRn), which provides the antibodies in vivo with protection from catabolism. IgG antibodies evade endolysosomal degradation by binding to FcRn in a pH-dependent manner. Binding to FcRn occurs at a site on IgG located between the CH2 and CH3 domains of the fragment crystallizable (Fc) region1. The antibodies are then recycled back to the plasma membrane and re-enter the circulation. The IgG recycling process involves several pH-dependent steps. After the internalization via pinocytosis, IgG antibodies bind to FcRn within endosomes at an acidic pH of about 5.5-6.0, which prevents the trafficking to lysosomes for degradation. Upon exocytosis, as the pH shifts to a more neutral physiological level of about 7.4 in the extracellular environment, the antibodies dissociate from FcRn and re-enter circulation. The ability of IgGFc region to bind FcRn at acidic pH while avoiding the interaction at physiological pH is essential for this recycling process. Thus, simply engineering Fc region to have high affinity for FcRn at acidic pH is not sufficient for extending in vivo half-life of the therapeutic antibodies and other biologic therapeutics incorporating IgGFc domain. Some Fc region variants with improved affinity for FcRn at both pH 6.0 and physiological pH failed to exhibit increased half-life2’7 To increase the10US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355half-life of the engineered therapeutic antibodies and other biologic therapeutics incorporating IgG Fc domain in vivo, it is important to maintain their pH-dependent binding to FcRn.

[0027] Beyond the interaction with FcRn to facilitate recycling, IgG Fc region is also responsible for mediating a separate set of immunological functions known as effector functions, such as, but not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC)4,5. Previous efforts to increase the affinity of Fc for FcRn have included mutations that ultimately compromised the interaction of Fc with other Fc receptors as well as the effector functions. Thus, for the antibody¬ based therapeutics and other biologic therapeutics incorporating IgGFc domain, it not only important to identify novel Fc region mutations that can enhance in vivo half-life of such molecules. It is also important to identify the mutations that allow for simultaneous fine-tuning of both effector functions of interest and pharmacokinetic properties. Furthermore, it crucial to identify minimal set of desirable substitutions in Fc region, so as to limit the risks of introducing immunogenicity and polyspecificity.

[0028] Previously, antibody design has harnessed the natural IgG recycling pathway via the neonatal Fc receptor (FcRn) to extend therapeutic antibody half-lives by introducing targeted mutations into the Fc region that increase the half-life of antibodies have been identified through a combination of rational design and high-throughput protein engineering techniques7 1!. These rational design approaches primarily targeted ammo acid residues that were in direct contact with FcRn or were located within the protein-protein interface between Fc domain and FcRn. Among the most extensively studied and clinically validated variants are the YTE, LS, and LA mutations7'8'27’28, which have been utilized in therapeutic antibodies approved for clinical use. However, another Fc mutation, the REW mutation, also led to undesirable effects, because it decreased the binding to Protein G11, which is used for antibody purification.

[0029] The “YTE” mutations are comprised by three amino acid substitutions m the Fc region (M252Y / S254T / T256E) and are among the first and most well-known sets of mutations for halflife extension discovered via phage display. The YTE (M252Y / S254T / T256E) mutation was included in nirsevimab (MEDI8897), a therapeutic antibody against respiratory syncytial virus (RSV)'2, and in Evusheld, a combination of antibodies tixagevimab and cilgavimab used against11US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355SARS-CoV-213. The YTE variant was reported to enhance the binding affinity to FcRn by approximately 10-fold at an acidic pH of 6.04. This improved, pH-specific binding translated to more efficient recycling of the antibody. In preclinical studies with cynomolgus monkeys, antibodies with the YTE mutations showed a nearly four-fold increase in serum half-life compared to their wild-type counterparts4. In human clinical trials, the YTE mutations have been shown to extend the half-life of antibodies by two- to four-fold25. However, a notable drawback of the YTE mutations is decreased FcyRIIIA binding4, a reduction in antibody-dependent cell- mediated cytotoxicity (ADCC)4, and a decrease in the thermal stability of the CH2 domain24.

[0030] In contrast, the “LS” pair of mutations M428L / N434S confer enhanced antibody half-life in preclinical studies in cynomolgus macaques and human FcRn transgenic mice demonstrated by about three- to four-fold, but without appreciably impacting ADCC effector functions8. This breakthrough has been translated into several clinically approved therapies. The LS mutation was included on Ultomiris (ravulizumab-cwvz), an antibody therapeutic against paroxysmal nocturnal hemoglobinuria (PNH)14, and in Sotrovimab, used against SARS-CoV-215.Ravulizumab, a C5 complement inhibitor, exhibits a markedly extended serum half-life. The LS mutations have also been used in monoclonal antibodies developed to combat SARS-CoV-2, such as tixagevimab, cilgavimab, and sotrovimab. Beyond extending serum half-life, these mutations have also been shown to increase antibody concentrations in mucosal tissues, enhancing protection against pathogens like HIV in animal models. While the “LS” mutations are well-documented, another notable substitution is N434A, which identified through alanine scanning, which also improves FcRn binding when paired with M428L and is known by “LA” and was incorporated in admtrevimab, a therapy for SARS-CoV-2 infection20, and crovalimab (Piasky™), a long lasting C5 neutralizing for PNH27.

[0031] The inventors discovered, and the current disclosure describes new amino acid modifications in IgGFc region, including surprising beneficial mutations in the sites that are not strictly within the FcRn-Fc interface and do not make direct contacts ’ith the FcRn. Based on their discoveries, the inventors conceived, and the present disclosure provides, engineered human IgGFc polypeptides incorporating amino acids substitutions conferring beneficial properties, such as altered in vivo half-life and / or binding affinity for human FcRn, as compared to the polypeptides without the one or more amino acid substitutions. The inventors also conceived12US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355various molecules and molecular complexes, such as biologic therapeutics incorporating such engineered polypeptides, methods of generating such polypeptides, nucleic acids and nucleic acids constructs encoding the engineered polypeptides, therapeutic methods using molecules and molecular complexes, such as biologic therapeutics incorporating the engineered polypeptides, as well as the related systems and kits.Terms and concepts

[0032] A number of terms and concepts are di cussed below. They are intended to facilitate the understanding of various embodiments of the invention in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present invention, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present invention, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present invention and interpreted in the context of the present document and / or the accompanying figures.A. General

[0033] Unless otherwise dictated by context, singular terms include pluralities, and plural terms include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry are those well-known and commonly used. Known methods and techniques are generally performed according to conventional methods well-known and as described in various general and more specific references, unless otherwise indicated. The nomenclatures used in connection with the laboratory procedures and techniques described in the present disclosure are those well-known and commonly used.

[0034] As used herein, the terms “a”, “an”, and “the” can refer to one or more unless specifically noted otherwise.13US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0035] The use of the term “or” is used to mean “and / or,” unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” can mean at least a second or more.

[0036] The terms “about” and “approximately” as used herein shall generally mean an acceptabl e degree of error for the quantity measured given the nature or preci si on of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Alternatively, in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0037] The terms “individual,” “subject,” “person,” and “patient” can be used interchangeably in the present disclosure to refer to a non-human animal or a human. Examples of subjects include, but are not limited to: humans and other primates, including non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs; birds, including domestic, wild and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms individual, subject, and patient, by themselves, do not denote a particular age, sex, race, or clinical status. Thus, subjects of any age, whether male or female, are intended to be covered by the present disclosure. Likewise, the methods of the present invention can be applied to any human race, including, for example, Caucasian (white), African- American (black), Native American, Native Hawaiian, Hispanic, Latino, Asian, and European.14US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0038] The terms “administering” or “administration,” when using in the context of the present disclosure (and the related terms and expression), encompass act of physically delivering a substance as it exists outside the body (for example, an immunogenic composition described in the present disclosure) into a subject. Administration can be by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery and / or by any other known methods of physical delivery. Administration encompasses direct administration, such as administration to a subject by a medical professional or self-administration, or indirect administration, which may be the act of prescribing a composition described m the present disclosure. In the above context, administration may refer to administration of pharmaceuticals, including, but not limited to, chemotherapeutic or immunotherapeutic pharmaceutical to a patient, such as an NSCLC patient.B. Polypeptides and sequences

[0039] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to ammo acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. The terms encompass ammo acid chains of any length, including full-length proteins, wherein the ammo acid residues are linked by covalent peptide bonds.

[0040] An “isolated” or “purified” polypeptide or protein, or biologically active portion a polypeptide or a protein, is substantially or essentially free from components that normally accompany or interact with the polypeptide or protein as found in its naturally occurring environment. Thus, an isolated or purified polypeptide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (total protein) of contaminating protein. When the protein of the invention or its biologically active portion is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (by concentration) of chemical precursors or non-protein-of-interest chemicals.15US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0041] The term “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Ammo acids include naturally occurring a-amino acids and their stereoisomers, as well as unnatural (non -naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).

[0042] Naturally occurring amino acids are those encoded by the genetic code, as well as those ammo acids that are later modified, e.g., hydroxyproline, y-carboxyglutainate, and O-phosphoserine. Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and their combinations. Stereoisomers of a naturally occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonme (D-Thr), D-valme (D-Val), D-tryptophan (D-I'rp), D-tyrosine (D-Tyr), and their combinations.

[0043] Unnatural (non-naturally occurring) ammo acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic ammo acids, A-substituted glycines, and A-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to by either the16US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355commonly known three letter symbols or by the one-letter symbols recommended by the TUPAC -TUB Biochemical Nomenclature Commission.

[0044] The expression “conservatively modified variant” and related expression may apply to ammo acid sequences, as well to nucleic acid sequences encoding amino acid sequence.Substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar ammo acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain ammo acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M).

[0045] The terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” “homology” and the related terms and expressions used in the context of describing nucleic acid or ammo acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are17US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman “Identification of common molecular subsequences.” J Mol Biol. 147(1): 195-7 (1981)) by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch “A general method applicable to the search for similarities in the ammo acid sequence of two proteins.” J Mol Biol. 48(3):443-53 (1970)), by the search for similarity method of Pearson and Lipman (Pearson and Lipman “Improved tools for biological sequence comparison.” Proc. Natl. Acad. Sci. USA 85(8):2444-8 (1988)), by computerized implementations of these algorithms (for example, BLAST,), or by manual alignment and visual inspection.

[0046] Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. “Basic local alignment search tool.” J. Mol. Biol. 215:403-410 (1990), and Altschul etal., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402 (1997), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching18US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity' X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, “Ammo acid substitution matrices from protein blocks.” Proc. Nail. Acad. Sei. USA 89:10915-10919 (1989)). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc. Nat'l. Acad. Sei. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10’5, and most preferably less than about 10‘20.

[0047] A “domain” of a protein or a polypeptide refers to a region of the protein or polypeptide defined by structural and / or functional properties. Exemplary' functional properties include enzymatic activity and / or the ability to bind to or be bound by another protein or non-protein entity. For example, an Fc domain of human IgGs is capable of specifically binding to FcRn and / or FcyRs.

[0048] A “binding site” of a protein or a polypeptide refers to a location of a protein or a polypeptide molecule at which another molecule (which can be referred to as a “target,” a “ligand,” or a “binding partner”) can bind with specificity. In proteins and polypeptides, binding sites are formed in tertiary structure, with some residues actually participating in binding the ligand and other residues acting as a framework to provide correct conformation and orientation. For example, an antigen-binding site of an antibody is capable of specifically binding an epitope.19US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355which is a target of the antibody’s antigen-binding site. For example, an Fc domain of human IgGs includes binding sites for FcRn and FcyRs.

[0049] The terms “fusion protein,” “fusion polypeptide,” and the related terms relate to polypeptide molecules, including artificial or engineered polypeptide molecules, that include two or more ammo acid sequences previously found in separate polypeptide molecule, that, are joined or linked in a fusion protein ammo acid sequence to form a single polypeptide. For example, a fusion protein can be an engineered recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein. In this context, proteins are considered unrelated, if their ammo acid sequences are not normally found joined together via a peptide bond m their natural environment, for example, inside a cell. For example, the present disclosure describes fusion proteins that include an amino acid sequence of a coronavirus receptor polypeptide and an amino acid sequence of an antibody, which are unrelated proteins. The amino acid sequences of a fusion protein are encoded by corresponding nucleic acid sequences that are joined “in frame,” so that they are transcribed and translated to produce a single polypeptide. The ammo acid sequences of a fusion protein can be contiguous or separated by one or more spacer, linker, or hinge sequences. Fusion proteins can include additional amino acid sequences, such as, for example, signal sequences, tag sequences, and / or linker sequences. For example, engineered Fc polypeptides according to the present disclosure may be incorporated into fusion proteins, such as engineered antibodies, comprising an engineered Fc polypeptide sequence and an antigen-binding site sequence.C. Antibodies

[0050] The term “antibody” and the related terms refer to an immunoglobulin or its fragment that binds to a particular spatial and polar organization of another molecule. Immunoglobulins include various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgG4, IgM, etc. An antibody can be monoclonal or recombinant, and can be prepared by laboratory' techniques, such as by preparing continuous hybrid cell lines and collecting the secreted protein, or by cloning and expressing nucleotide sequences or their mutagenized versions coding at least for the amino acid sequences required for binding. Antibodies as referenced herein may have sequences derived from non-human antibodies, human sequence, chimeric sequences, and wholly synthetic sequences. The term “antibody” encompasses natural,20US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355artificially modified, and artificially generated antibody forms, such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies and their fragments. The term “antibody” also includes composite forms including but not limited to fusion proteins containing an immunoglobulin moiety. “Antibody” also refers to non-quatemary antibody structures (such as camelids and camelid derivatives) and antigen¬ binding fragments of antibodies, minibodies, bispecific antibodies, nanobodies (also referred to as VHH fragments), and diabodies. Antibodies and antigen-binding portions thereof include domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains. / Antibody fragments may include Fab, Fv, F(ab’)2, Fab’, scFv, dsFv, ds-scFv, Fd, dAb, Fc, and the like. A natural antibody digested by papain yields three fragments: two Fab fragments and one Fc fragment. The Fc fragment is dimeric and contains two CH2 and two CH3 heavy chain domains. CH3 domains interact to form a homodimer. Fc domains in antibodies may also be optimized to alter antibody characteristics of interest (e.g., bioavailabilty, serum half-life). In addition, aggregates, polymers and conjugates of immunoglobulins or their fragments can be used where appropriate. Additional details of antibodies useful in the context of this disclosure are provided below.

[0051] As used herein, the term antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. A natural immunoglobulin G (IgG) antibody molecule is a tetramer that contains two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (ATI) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also21US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355including a “D” region of about 10 more amino acids. See generally. Fundamental Immunology, Paul, W., ed,, 3rd ed. Raven Press, NY (1993), SH. 9 (incorporated by reference in its entirety' for all purposes). Antibody sequences and structural information is widely available. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and Ig / \-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. As used herein, the term antibody also encompasses an antibody fragment, for example, an antigen-binding fragment. Antigen- binding fragments comprise at least one antigen- binding domain, which, in turn, comprises at least one antigenbinding site. One example of an antigen- binding domain is an antigen-binding domain formed by a VH-VL dimer. Antibodies and antigen-binding fragments can be described by the antigen to which they specifically bind.

[0052] The terms “antigen- binding portion” and “antigen-binding fragment” are used interchangeably in the present disclosure and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. The fragments are not necessarily generated from contiguous antibody sequences, but can be engineered proteins containing antibody sequences needed to form an antigen-binding site. Examples of antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CHI domains), F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv) fragment (a fusion protein of the VH and VL regions), a disulfide-linked Fv (dsFv), complementarity determining regions (CDRs), VL (light chain variable region), ATI (heavy chain variable region), nanobodies, and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen.

[0053] Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”),22US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Each VI I and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C- terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. SeeKabat et al. Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991). CDR sequences on the heavy chain (VH) may be designated as CDRH1, 2, 3, while CDR sequences on the light chain ( VL) may be designated as CDRL1, 2, 3.

[0054] The term “variable region” refers to a domain in an antibody heavy chain or light chain that gives an antibody its specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”10055] The term “complementarity determining region” or “CDR” refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDRS, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0056] As noted, the part of a variable region not contained in the CDRs is called the framework. The “framework regions” of different light or heavy chains are relatively conserved23US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355within a species. The framework region of an antibody, that, is the combined framework regions of the constituent light, and heavy chains, serves to position and align the CDRs in three-dimensional space. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DN / X sequences for human heavy and light chain variable region genes can be found in the “VBASE2” germline variable gene sequence database for human and mouse sequences. The amino acid sequences of the CDRs and framework regions can be determined using various well- known methods, some of which are described elsewhere in the present disclosure.

[0057] A term “monoclonal antibody” refers to antibodies produced by a single clone of cells or a single cell line and consisting of or consisting essentially of antibody molecules that are identical in their primary amino acid sequence. In some embodiments, a monoclonal antibody preparation comprises a population of antibodies that are identical and bind to the same epitope of an antigen, except for mutations that arise during monoclonal antibody production. Unless otherwise specified or clear from context, the term “monoclonal antibody” includes synthetic antibodies and antigen- binding fragments thereof.

[0058] “Fc region,” “Fc domain,” or “Fc sequence” and the related terms and expressions can all refer the “tail” region of an antibody sequence. Fc region contains at least the amino acid sequences of the CH2 and CH3 domains of an antibody (and, m some antibody isotypes, can also contain CH4 domain sequence).10059] “Fc fragment” contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. An Fc domain introduced into a fusion or other type of engineered protein may promote dimerization. In some instances, an Fc domain introduced into a fusion or other type of engineered protein may improve its stability in vitro or upon administration in vivo. For examples, engineered Fc polypeptides according to the present disclosure may be incorporated into therapeutic agents to increase their in vivo half-life.

[0060] A “Fab fragment” is comprised of one light chain, and the CHI and variable regions of one heavy chain and can specifically recognize a target epitope, such as an epitope of a spike24US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355protein. A Fab domain introduced into a fusion protein according to the present disclosure results in binding of the fusi on protein to the target. Such a fusion protein may be referred to as “Fab fusion protein,” “Fab-based fusion protein,” and by other related expressions.

[0061] A “single-chain variable fragment” or “scFv fragment” is a fusion protein comprising the variable regions of a heavy chain and a light chain from an antibody. The heavy chain and light chain portions may be connected by a linker peptide. An scFv fragment may retain the binding specificity of the antibody from which it is derived. A scF v domain introduced into a fusion protein according to the present disclosure results in binding of the fusion protein to the target. Such a fusion protein may be referred to as “an scFv fusion protein,” “scFv-based fusion protein,” and by other related expressions.Polypeptides and related methods

[0062] Embodiments of the present invention include polypeptides, which may be referred to as “engineered,” that incorporate an ammo acid sequence derived from human IgGFc domain ammo acid sequence having one or more amino acid substitutions listed in Tables 4-10, in which the substitutions are numbered with respect to human IgG Fc polypeptide amino acid sequence shown below as SEQ ID NO: 1, following Eu-IMGT positions29. SEQ ID NO: I is a partial amino acid sequence of the Fc domain of human IgGl, residues Al 18 to K447, which correspond to the constant region of the Fc domain. In other words, the first ammo acid residue as shownSEQ ID NO: 1 is numbered 118, and the last one is numbered 447.SEQ ID NO:1 - Human IgGl constant region(118)ASTKGPSVTTLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQ S SGLYSLS S WTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSWLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVI. T\T. HQDWL> NGKEYKCKVSNKALPAPIEKTISKAK GQPREPQWTITPSRDELTKNQVSLTCIATCGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSI< LTVDKSRWQQGNVFSCSVK4HEALHNHYTQKSLSLSPGK(447)25US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0063] The amino acid substitutions discovered by the inventors, identified in reference to SEQ ID NO:1, include L251V, L251Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, and L441C. It is to be understood that engineered polypeptides according to the present disclosure may include one of the above substitutions, or more than one (such as one or more, two or more, three or more, four or more, five or more), etc. of the above substitutions, in reference to SEQ ID NO: 1. It is also to be understood that engineered polypeptides according to the present disclosure may include other mutations, such as substitutions, deletions or additions, in reference to SEQ ID NO: 1. In some embodiments, engineered polypeptides according to the present disclosure include one or more of amino acid substitutions V266L, II268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, and at least one of (a) amino acid substitutions M252Y, S254T, and T256E, or (b) amino acid substitutions (i) M428L and (ii) N434S, N434A, or N434G (e.g., M428L and N434S, M428L and N434A, or M428L and N434G). In some embodiments, engineered polypeptides according to the present disclosure include one or more of amino acid substitutions V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, and (a) amino acid substitutions M252Y, S254T, and T256E, and (b) amino acid substitutions (i) M428L and (ii) N434S, N434A, or N434G (e.g., M428L and N434S, M428L and N434A, or M428L and N434G).

[0064] In some embodiments, a polypeptide according to the present disclosure comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, an amino acid sequence of a polypeptide according to the present disclosure is derived from the Fc domain of human IgGI. It has been shown that Fc mutations leading to better recycling or in vivo half-life of IgGI may be successfully transferred to IgG2, IgG3, and IgG416. The sequences of the Fc domain constant region of IgGI, IgG2, IgG3, and IgG4, each26US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355corresponding to residues A 118 to K447 of the IgGl Fc domain, are set forth in SEQ ID NOs: 1-4, respectively. In some embodiments, the ammo acid sequence of a polypeptide according to the present disclosure is derived from Fc domain amino acid sequence a of human IgG2, IgG2, IgG3, or IgG4, in which case the location of the substitutions discovered by the inventors may be determined by aligning amino acid sequence of an Fc domain of one or more of IgG2 (SEQ ID NO:2), IgG3 (SEQ ID NO:3), IgG4 (SEQ ID NO:4) with SEQ ID NO:1, and then, through the analysis of the alignment, determining location of one or more of the substitutions discovered by the inventors in the Fc amino acid sequence of one or more of IgG2, IgG2, IgG3, or IgG4. For example, of the specific Fc mutations identified herein, the ammo acid residue position of each in IgG2, IgG2, IgG3, or IgG4 are set forth in the table below.

[0065] Thus, in some embodiments, a polypeptide according to the present disclosure comprises or consists of an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the ammo acid sequence of SEQ ID NOs: 1-4. Thus, some embodiments of the present invention are methods of modifying a polypeptide incorporating a human Fc polypeptide amino acid sequence of SEQ ID NOs: 1-4, or an amino acid sequence that that is at least 80% identical, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, to the amino acid sequence of SEQ ID NOs: 1-4. In some embodiments, an ammo acid sequence of a polypeptide according to the present disclosure is derived from the Fc domain of human IgG2. In some embodiments, an ammo acid sequence of a polypeptide according to the present disclosure is derived from the Fc domain of human IgG3. In some embodiments, an amino acid sequence of a polypeptide according to the present disclosure is derived from the Fc domain of human IgG4.SEQ ID NO:2 - Human IgG2 constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVPSSNFGTQTYTCNVDIIKPSNTKVDKTVERKCCVECPPCPAPPVAG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVIIQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP27US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355PSREEM TEN QV SLTCLVKGFYPSDIAVEWESNGQPENN YK TTPPMLDSDGSFFL YSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:3 - Human IgG3 constant region ASTKGPSVFPLAPCSRSTSGGTAALGCIAZK. DYFPEPVTVSWNSGALTSGVHTFPAVI., Q SSGLYSLSSW7TVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPE PKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKP KDTIAlISim>EVT(: VVVDVSHEDPEVQFKWYVD(3VEVHNAKTKPREEQYNSTFRVV SVLTVLIIQDWLNGKEYKCKVSNKALPAPIEK'nSKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMIJDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLS VCYSATVTFFKVKW1FSSVVDLKQTIIPDYRNMIGQGASEQ ID NO:4 - Human IgG4 constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ S SGLYSLS S WTVPS S SLGTKTYTCNYTJHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PS\TLFPPKPKDTENflSRTPEVTCVWDVSQEDPEVQFNWYYT)GVEVHNAKTKPREE QFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSD1 AVEWESNGQPENNYKTTPPVLDSDGSFFEYSRL TXTJKSRWQEGNYTSCSWIHEALHNHYTQKSLSLSLGK

[0066] / Xny molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure are included among the embodiments of the present invention. In some embodiments, a polypeptide according to the present disclosure may be linked to at least one other polypeptide and / or to at least one non-peptide moiety. In some embodiments, a polypeptide according to the present disclosure contain additional polypeptide domain or domains. In some embodiments, a polypeptide according to the present disclosure may be included in a fusion protein along with one or more other polypeptide domains. Some examples of such fusion proteins may include at least one antibody domain capable of specifically binding an epitope of interest. In some embodiments, fusion proteins including one or more polypeptides according to the present disclosure may contain additional domain or28US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355domains. Non-limiting examples of such additional domain are multimerization domains, such as, but not limited to, dimerization, trimerization, or tetramerization domains. In some embodiments, fusion proteins include linkers, which can be polypeptide linkers, between different domains. In some embodiments, a polypeptide according to the present disclosure may be included as a dimerization domain in a fusion protein in which the fusion protein may comprise one or more other polypeptide domains. Fusion proteins may also include various sequences useful in their purification, expression, etc. Some examples of such additional amino acid sequences are signal sequences, purification tags, etc.

[0067] In some embodiments, a polypeptide according to the present disclosure may be incorporated in a biological therapeutic, such as, but not limited to, a therapeutic antibody or a therapeutic antibody fragment. Such therapeutic antibodies or therapeutic antibody fragments are included among the embodiments of the present invention. Therapeutic antibodies or therapeutic antibody fragments according to the embodiments of the present invention may be used to treat various diseases or conditions (or their combinations), such as, but not limited to, autoimmune diseases or conditions, genetic diseases or conditions, cancers, respiratory diseases or conditions, cardiovascular diseases or conditions, renal diseases or conditions, gastrointestinal diseases or conditions, endocrine diseases or conditions, neurological diseases or conditions, ophthalmological s or conditions, musculoskeletal diseases or conditions, or infections. Some non-limiting examples of therapeutic antibodies and / or therapeutic antibody fragments (which can be collectively referred as “antibody therapeutics”) that can be modified to include engineered polypeptides according to the embodiments of the present invention are as follows: Humira (adalimumab) (target: TNF-a; indications: rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, plaque psoriasis); Keytruda (pembrolizumab) (target: PD-1; indications: various cancers, including melanoma, lung cancer, head and neck cancer); Stelara (ustekinumab) (target: IL-12 / IL-23; indications: plaque psoriasis, psoriatic arthritis, Crohn’s disease); Dupixent (dupilumab) (target: IL-4 receptor; indications: atopic dermatitis, asthma, chronic rhinosinusitis with nasal polyposis); opdivo (nivolumab) (target: PD-1; indications: various cancers, including melanoma, lung cancer, renal cell carcinoma); Darzalex (daratumumab) (target: CD38; indications: multiple myeloma); Ocrevus (ocrelizumab) (target: CD20; indications: multiple sclerosis); Entyvio (vedolizumab) (target:29US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355a407 integrin; indications: ulcerative colitis, Crohn’s disease); Skyrizi (risankizumab) (target: IL-23; indications: plaque psoriasis); Cosentyx (secukinumab) (target: IL-17A; indications: plaque psoriasis, psoriatic arthritis, ankylosing spondylitis); Perjeta (pertuzumab) (target: HER2; indications: HER2-positive breast cancer); Hemlibra (emicizumab) (target: Factors IXa and X; indications: hemophilia / X); Tecentnq (atezolizumab) (target: PD-LI; indications: various cancers, including bladder cancer, lung cancer); Soliris (eculizumab) (target: Complement protein C5; indications: paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome); Prolia (denosumab) (target: RANKL; indications: osteoporosis, bone loss); A vastin (bevacizumab) (target: VEGF-A; indications: various cancers including colorectal, lung, and ovarian cancer); Actemra / RoActemra (tocilizumab) (target: IL-6 receptor; indications: rheumatoid arthritis, giant cell arteritis, cytokine release syndrome); Xevudy (sotrovimab) (target: SARS-CoV-2 spike protein; indications: COVID-19); Imfinzi (durvalumab) (target: PD-LI; indications: barious cancers including lung cancer, bladder cancer); Tremfya (guselkumab) (target: IL-23; indications: plaque psoriasis, psoriatic arthritis).

[0068] In some embodiments, a polypeptide according to the present disclosure may be incorporated in any biologic therapeutic that includes an IgGFc domain. Such biologic therapeutics are included among the embodiments of the present invention. Biologic therapeutics according to the embodiments of the present invention may be used to treat various diseases or conditions (or their combinations), such as, but not limited to, autoimmune diseases or conditions, genetic diseases or conditions, cancers, respiratory diseases or conditions, cardiovascular diseases or conditions, renal diseases or conditions, gastrointestinal diseases or conditions, endocrine diseases or conditions, neurological diseases or conditions, ophthalmological s or conditions, musculoskeletal diseases or conditions, or infections. Some non-limiting examples of such biological therapeutics that can be modified to include polypeptides according to the embodiments of the present invention are as follows: Belatacept (target: CD80 / CD86); indications: prevention of kidney transplant rejection); Aflibercept (target: VEGF-A, VEGF-B, P1GF); indications: wet age-related macular degeneration, metastatic colorectal cancer); Rilonacept (target: IL-la, IL-1 P); indications: cryopyrin-associated periodic syndromes, recurrent pericarditis); Romiplostim (target: Thrombopoietin receptor (c-Mpl)); indications: chronic immune thrombocytopenia); Abatacept (target: CD80 / CD86); indications:30US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis); Alefacept (target: CD2); indications: moderate-to- severe chronic plaque psoriasis); Etanercept (target: TNF-a); indications: rheumatoid arthritis juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis); Trebananib (target: Angiopoietm-1 and -2); indications: ovarian cancer (investigational); Sotatercept (target: Activin receptor type IIA); indications: betathalassemia, myelodysplastic syndromes); Luspatercept (target: Activin receptor type IIB); indications: beta-thalassemia, myelodysplastic syndromes).

[0069] In some embodiments, a polypeptide according to the present disclosure, or a molecule or molecular complex incorporating one or more polypeptides according to the present disclosure (such as, but not limited to, an antibody therapeutic or a biological therapeutic) may be linked to one more non-one non-peptide moieties. Some non-limitmg examples of such moieties are one or more of a radioactive isotope, a molecule comprising a radioactive label, fluorophore, a drug molecule, a toxin, or a nucleic acid. In some instances, a polypeptide according to the present disclosure, an antibody therapeutic according to the present disclosure, or a biological therapeutic according to the present disclosure can be labeled, conjugated, or fused with a therapeutic agent or diagnostic agent (such as an imaging agent). The linkage can be covalent or nonco valent (e.g., ionic). Such conjugates may be useful for targeted delivery of therapeutic agents, such as, but not limited to cytotoxic or cytostatic agents. For example, such conjugates may be used for the delivery of drugs to kill or inhibit tumor cells m the treatment of cancer by allowing targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated. Therapeutic agents include but are not limited to toxins, including but not limited to plant and bacterial toxins, small molecules, peptides, polypeptides and proteins.

[0070] Also included among the embodiments of the present invention are methods of modifying a polypeptide incorporating a human Fc polypeptide amino acid sequence (such as, but not limited to SEQ ID NO: 1, or an amino acid sequence that that is at least 80% identical, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, to the amino acid sequence ofSEQ ID NO: 1), Embodiments of such methods involve introducing into a human Fc polypeptide31US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355amino acid sequence one or more of the following amino acid substitutions: L251 V, L251 Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302E, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, and L441 C. In some embodiments, the methods involve introducing into a human Fc polypeptide ammo acid sequence amino acid substitutions M252Y, S254T, and T256E, and one or more of the following amino acid substitutions: V266L, H268K, H268S, N276S, A287L, A287M, V302L. S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421 V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441 C. As discussed above, the substitutions are numbered with respect to human IgG Fc polypeptide ammo acid sequence shown in the present disclosure as SEQ ID NO: 1, which is a partial amino acid sequence of the Fc domain of human IgGl, residues Al 18 to K447, corresponding to the constant region of the Fc domain. The sequences of the Fc domain constant region of lgG2, IgG3, and IgG4 correspond to residues Al 18 to K447 as set forth in SEQ ID NO:2-4, and corresponding amino acid positions for the mutations disclosed herein with respect to IgGl Fc domain may be determined through analysis of alignment with the sequences of IgG2, IgG3, and IgG4 as set forth in SEQ ID NOs:2-4. In some embodiments, one or more of the above substitutions are introduced into an amino acid sequence of Fc domain of IgG2, IgG3, or IgG4. Thus, some embodiments of the present invention are methods of modifying a polypeptide incorporating a human Fc polypeptide amino acid sequence of SEQ ID NOs: 1-4, or an amino acid sequence that that is at least 80% identical, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, to the amino acid sequence of SEQ ID NOs: 1-4. In some embodiments, one or more of the above substitutions are introduced into human Fc polypeptide amino acid sequence that has at least 80% (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence similarity (identity) to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. It is to be understood that the above methods may include introducing other mutations, such as substitutions, deletions or additions, into a human Fc polypeptide ammo acid sequence. Techniques for introducing substitution or other mutations are known and can include site specific mutagenesis of nucleic acids encoding a polypeptide according to the present disclosure.32US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0071] The above methods result in a modified or engineered polypeptide that includes a modified human Fc polypeptide amino acid sequence. In some embodiments, the modified or engineered polypeptide has an altered (increased or decreased) in vivo half-life and / or binding affinity (increased or decreased binding affinity) for human FcRn, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure.

[0072] In some embodiments, the modified or engineered polypeptide has decreased binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has substantially unaltered binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, as compared to a polypeptide without the one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has an increased dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has a decreased dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has a substantially unaltered dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has an increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, and a faster dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from33US2008 32326720 1Attorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355about 7.3 to about 7,5, from about 7,35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has an increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, and substantially unaltered dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure. In some embodiments, the modified or engineered polypeptide has a substantially unaltered binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, and a faster dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure.

[0073] In vivo half-life, which can also be referred to as biological half-life, elimination half-life, and / or pharmacological half-life, can be denoted by an abbreviation ti / 2, and is the time for concentration of a substance to decrease from its maximum concentration (Cmax) to half of Cmax in blood plasma. In some embodiments, a molecule or a molecular complex that incorporates a modified or engineered polypeptide has an altered (increased or decreased) in vivo half-life and / or binding affinity (increased or decreased binding affinity) for human FcRn, as compared to a molecule or a molecular complex, respectively, that incorporates the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some embodiments, a molecule or a molecular complex that incorporates a modified or engineered polypeptide according to the present disclosure has an increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0, and substantially unaltered dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4, as compared to a molecule or a molecular complex, respectively, that incorporates the polypeptide without the one or more amino acid substitutions according to the present disclosure. In some embodiments, a molecule or a molecular complex that incorporates a modified or engineered polypeptide according to the present disclosure has substantially unaltered binding affinity for human FcRn34US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6,1, or at about 6.0, and a faster dissociation rate from human FcRn at pH from about 7.0 to about 7.5, from about 7,3 to about 7.5, from about 7.35 to about 7.45, or at about 7,4, as compared to a molecule or a molecular complex, respectively, that incorporates the polypeptide without the one or more amino acid substitutions according to the present disclosure.

[0074] In some embodiments, a molecule or a molecular complex that incorporates a modified or engineered polypeptide according to the present disclosure has altered human FcyRs, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0075] Binding affinity of a modified or engineered polypeptide according to the present disclosure for human FcRn can be expressed as the equilibrium dissociation constant (Kd or KD). In some embodiments, the modified or engineered polypeptide has an increased KD for human FcRn, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the modified or engineered polypeptide has a KD for human FcRn that is increased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a KD for human FcRn that is increased at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6.1, or at about 6.0 by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a decreased KD for human FcRn, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the modified or35US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355engineered polypeptide has a KD for human FcRn that is decreased at pH from about 5.5 to about 6.5, from about 5.8 to about 6.2, from about 5.9 to about 6,1, or at about 6.0 by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0076] Retention of a modified or engineered polypeptide according to the present disclosure at human FcRn can be expressed as the dissociation rate constant (koff). In some embodiments, the modified or engineered polypeptide has an increased koff for human FcRn, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a Koff for human FcRn that is increased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a koff for human FcRn that is increased at pH from about 7.0 to about 7.5, from about 7.3 to about 7.5, from about 7.35 to about 7.45, or at about 7.4 by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a decreased koff for human FcRn, as compared to the polypeptide without one or more amino acid substitutions according to the present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the modified or engineered polypeptide has a koff for human FcRn that is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, as compared to the polypeptide without one or more amino acid substitutions according to the36US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355present disclosure, such as the corresponding wild-type polypeptide comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NON.

[0077] In some embodiments, the modifications of Fc amino acid sequence according to the present disclosure may lead to modifications of one or more other properties (in addition or instead of modifying in vivo half-life) of a molecule or molecular complex incorporating a modified or engineered polypeptide, as compared to a molecule or a molecular complex, respectively, that incorporates the polypeptide without the one or more amino acid substitutions according to the present disclosure. Accordingly, methods of modifying such properties are contemplated and included among the embodiments of the present invention, along with methods of modifying half-life of molecules or molecular complexes incorporating the polypeptides according to the present disclosure. For example, antibody therapeutics incorporating modified polypeptides may have altered (increased or decreased) effector functions17, such as, but not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or opsonization phagocytosis killing (OPK). Molecules or molecular complexes incorporating modified or engineered polypeptides according to the present disclosure with altered (increased or decreased) effector functions, such as, but not limited to, ADCC, ADCP, CDC, or OPK, are included among the embodiments of the present invention. Methods of modifying a polypeptide comprising a human Fc polypeptide amino acid sequence to alter one or more effector functions of an antibody incorporating the modified human polypeptide are also included among the embodiments of the present invention. In one more example, molecules or molecular complexes incorporating modified or engineered polypeptides according to the present disclosure may have altered (increased or decreased) placental transfer. Increasing placental transfer may be desirable in various applications, such as administering a therapeutic molecule to a subject during pregnancy to take advance of placental transfer to the fetus. In one more example, molecules incorporating modified or engineered polypeptides according to the present disclosure may have increased transcytosis in human epithelia, which may be desirable for drug delivery’ (such as transmucosal or oral delivery)16, 18, 19In one more example, molecules or molecular complexes incorporating modified or engineered polypeptides according to the present disclosure may have enhanced delivery7across blood-brain barrier20. Creating the molecules or molecular complexes with37US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355various altered properties, such as, but. not limited to those discussed above, may involve modifying Fc human polypeptide sequences included in the molecules. Molecules or molecular complexes with various altered properties may also be created by incorporating previously created sequences of modified or engineered polypeptides according to the present, disclosure into various biologically active molecules.

[0078] In some embodiments, the modifications of Fc ammo acid sequence according to the present disclosure may lead to improved expression of a recombinant molecule or molecular complex incorporating a modified or engineered polypeptide, as compared to a molecule or a molecular complex, respectively, that incorporates the polypeptide without the one or more amino acid substitutions according to the present disclosure. Accordingly, methods of modifying improving expression of such molecules or molecular complexes are contemplated and included among the embodiments of the present invention. Molecules and molecular complexes incorporating modified or engineered polypeptides according to the present disclosure with improved expression are included among the embodiments of the present invention.Improvements in expression of molecules or molecular complexes incorporating modified or engineered polypeptides according to the present disclosure improve manufacturing costs, and may reduce the cost molecules or molecular complexes used as therapeutics.Nucleic acids, vectors, host ceils, and related methods

[0079] Molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure can be recombinantly produced and purified or isolated from a host cell or population of host cells. For example, a recombinant nucleic acid including a sequence or sequences encoding one or more polypeptides according to the present disclosure can be introduced into a host cell under conditions that allow protein expression. In some embodiments, the recombinant nucleic acid is codon-optimized for expression. After expression in the host cell, a recombinant protein can be isolated or purified using purification methods known in the art. Recombinant nucleic acids that include sequences encoding any of the polypeptides according to the present disclosure are provided and included among the embodiments of the present invention. In some embodiments, a nucleic acid sequence according to the present disclosure encodes one or more polypeptides comprising an amino acid sequence that that is at least 80% identical (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,38US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-35591%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to the amino acid sequence of SEQ ID NO:1. In some embodiments a nucleic acid sequence according to the present disclosure encodes one or more engineered polypeptides according to the present disclosure containing one or more substitutions according to the present disclosure. Such engineered polypeptides are described elsewhere in the present disclosure. In some embodiments, a nucleic acid sequence according to the present disclosure encodes an engineered polypeptide comprising one or more of the following amino acid substitutions: L251 V, L251Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, and L441C. In some embodiments, a nucleic acid sequence according to the present disclosure encodes an engineered polypeptide comprising amino acid substitutions M252Y, S254T, and T256E, and one or more of the following amino acid substitutions: V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C. Nucleic acids encoding any molecules or molecular complexes incorporating one or more engineered polypeptides according to the present disclosure are included among the embodiments of the present invention.

[0080] Also included among the embodiments of the present invention are DNA constructs comprising a promoter operably linked to a recombinant nucleic acid according to the present disclosure. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A promoter is a region or a sequence located upstream and / or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter can be a eukaryotic or a prokaryotic promoter. In some embodiments the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter.

[0081] A recombinant nucleic acids according to the present disclosure can be included in expression cassettes for expression in a host cell or an organism of interest. The cassette includes 5' and 3' regulatory sequences operably linked to a recombinant nucleic acid provided herein that allows for expression of the modified polypeptide. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism. Where additional39US2008 32326720 1Attorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355genes or elements are included, the components are operably linked. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette will include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide disclosed herein, and a transcriptional and translational termination region (i.e., termination region) functional in the cell or organism of interest. The promoters are capable of directing or driving expression of a coding sequence in a host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0082] Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See Sambrook et al. Molecular Cloning:A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2012) (“Sambrook”); Davis et al., eds. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1980), and the references cited therein.10083] The expression cassette can also include a selectable marker gene for the selection of transformed cells. Marker genes include genes conferring antibiotic resistance, such as those conferring hygromycm resistance, ampicillin resistance, gentamicin resistance, neomycin resistance, to name a few. Additional selectable markers are known, and any can be used.10084] In preparing the expression cassette, various DM A fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis,40US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0085] Also included among the embodiments of the present invention are vectors comprising a nucleic acid or expression cassette according to the present disclosure, A vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted nucleic acid. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers that can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region that may serve to facilitate the expression of the inserted gene or hybrid gene. The vector can be a plasmid.

[0086] There are numerous E. coli expression vectors that can be used for the expression of a nucleic acid according to the present disclosure. Other microbial hosts suitable for such use include bacilli, such as Bacillus suhtilis, and other Enter obacteriaceae, such as Salmonella, Senatia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.Additionally, yeast expression can be used. In one example, a nucleic acid encoding a polypeptide according to the present disclosure can be expressed by a yeast cell. More specifically, the nucleic acid can be expressed by Pichia pastoris or S. cerevisiae.

[0087] Mammalian cells also permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. V ectors useful for the expression of active proteins in mammalian cells are known in the art and can contain genes conferring hygromycin resistance, geneticin or G418 resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. A41US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355number of suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include CHO cells, HEK293 cells, HeLa cells, COS-7 cells, myeloma cell lines, Jurkat cells, derivatives of any of the above (e.g., Expi-HEK cells, Expi-CHO cells), etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.

[0088] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids m mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes, such as E. coll gpt. The selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection. A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus, CMV, polyoma virus, or SV40 virus.

[0089] The expression vectors according to the present disclosure can also include nucleic acids according to the present disclosure under the control of an inducible promoter such as the tetracycline inducible promoter or a glucocorticoid inducible promoter. The nucleic acids according to the present disclosure can also be under the control of a tissue-specific promoter to promote expression of the nucleic acid m specific cells, tissues, or organs. Any regulatable promoter, such as a metallothionein promoter, a heat-shock promoter, and other regulatable promoters, of which many examples are well known in the art are also contemplated.Furthermore, a Cre-loxP inducible system can also be used, as well as a Flp recombinase inducible promoter system.

[0090] The DNA constructs and vectors according to the present disclosure may include the nucleic acids described above in a variety of configurations to allow expression and purification of polypeptides, or molecules or molecular complexes incorporating one or more polypeptides42US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355according to the present disclosure. Hosts cells comprising nucleic acids, DNA constructs, or vectors according to the present disclosure are also included among the embodiments of the present invention. The host cell can be an in vitro, ex vivo, or in vivo host cell. Host cells include yeast, bacteria, insect, plant, and mammalian cells. Populations of any of the host cells described above are also included among the embodiments of the present invention. Cell cultures including one or more host cells according to the present disclosure are included among the embodiments of the present invention.

[0091] A host cell can be a prokaryotic cell, including, for example, a bacterial cell.Alternatively, the cell can be a eukaryotic cell, for example, a mammalian cell. In some embodiments, the cell can be an HEK293T cell, a Chinese hamster ovary (CHO) cell, a COS-7 cell, a HeLA cell, an avian cell, a myeloma cell, a Pichia cell, an insect cell, or a plant cell. A number of other suitable host cell lines have been developed and include myeloma cell lines, fibroblast cell lines, and a variety of tumor cell lines such as melanoma cell lines. The vectors containing the nucleic acid segments of interest can be transferred or introduced into the host cell by well-known methods, which vary depending on the type of cellular host. Insect cells also permit the expression of the polypeptides. Recombinant proteins produced in insect cells such as Sf9 with baculovirus vectors undergo post-translational modifications similar to those of wild¬ type mammalian proteins.

[0092] Polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains may be produced by recombinant expression in a human or non-human cell. In some instances, the cell is a synthetic antibody-producing cell, such as non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells that express heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy chains or light chains, but not both heavy and light chains. Polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure may be heterologously expressed, in vitro or in vivo, in cells other than human B cells, such as non-human cells and human cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.43US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0093] Polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains can be produced from the cells by culturing a host cell containing the nucleic acid encoding a suitable amino acid sequence or sequences, under conditions and for an amount of time sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell. Methods for the culture and production of many cells are available in the art. See e.g., Sambrook, Freshney Culture of Animal Cells, a Manual of Basic Technique, 3rdEd., Wiley-Liss, New York (1994) and the references cited therein; Doyle and Griffiths Mammalian Cell Culture: Essential Techniques John Wiley and Sons, NY (1997); and Humason Animal Tissue Techniques, 4thEd. W. H. Freeman and Company (1979).

[0094] The nucleic acids, DNA constructs, and expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. In the context of the present disclosure, the phrase “introducing” in in reference to introducing a nucleic acid into a cell refers to the translocation of the nucleic acid sequence from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPO4 precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection. Various methods of translocation are contemplated, including but not limited to, electroporation, nanoparticle delivery', viral delivery, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, DEAE dextran, lipofectamine, calcium phosphate or any method now known or identified in the future for introduction of nucleic acids into prokaryotic or eukaryotic cellular hosts. A targeted nuclease system (e.g., an RNA-guided nuclease (CRISPR-Cas9), a transcription activator- like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a megaTAL (MT) can also be used to introduce a nucleic acid, for example, a nucleic acid encoding a recombinant protein described herein, into a host cell,44US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0095] In some embodiments, polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains can be expressed in, and purified from, transgenic animals (e.g,, transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk.

[0096] Following expression, polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains can be isolated or purified in a variety of ways, depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase IIPLC chromatography. For example, a fusion protein or modified protein comprising an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes Protein Purification, 3rdedition, Springer- Verlag, New York City, New York (1994). The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof is necessary.Other methods of generating polypeptides

[0097] In vitro methods are also suitable for preparing polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains. Some exemplary methods may be used to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxy carbonyl) or Boc (tert- butyloxy carbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). Polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the 45US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. See Grant Synthetic Peptides: A User Guide. W. H. Freeman and Co., N. Y. (1992); Bodansky M and Trost B,, Ed. Principles of Peptide Synthesis. Springer Verlag Inc,, NY (1993). Alternatively, the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to a lager polypeptide via similar peptide condensation reactions.

[0098] For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains. Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction. The first step is the chemoselective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change m the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.

[0099] In some embodiments, production of polypeptides involves chemical linkage of unprotected peptide segments where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond. This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et al., Techniques in Protein Chemistry TV. Academic Press, New York, pp. 257-267 (1992)). Various chemical linkers may be used to link two amino acid residues. For example, two amino acid residues may be cross linked via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Hetero bifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which46US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355link two ammo groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an ammo group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccininnde ester), an ammo group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidimum group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate). In some embodiments, a neutralizing polypeptide or an antibody described herein, one of which is attached to a linker having a chemical functional group at the free / unattached end is produced and joined to another neutralizing polypeptide domain or antibody comprising a complementary reactive chemical functional group (e.g., at an end or internally). In some embodiments, two domains of the modified protein having a full or partial linker sequence with a chemical functional group at the end are produced and chemically linked in vitro via the free / unattached ends of the full or partial linker. Non-peptide linkers may be used to generate polypeptides according to the present disclosure, molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, as well as their domains. In some embodiments, non-peptide linkers comprise functional groups on at least one terminus to allow attachment to a polypeptide domain. For example, PEG linkers can be designed with N-hydroxy-succinimide (NHS) esters at one end or both ends that react specifically and efficiently with lysine and N-terminal amino groups to form amide bonds. In another example, linkers can also be designed with sulfhydrylreactive crosslinkers at one end or both ends that react with reduced sulfhydryls to form stable thioether bonds.Compositions and formulations

[0100] Compositions comprising one or more polypeptides according to the present disclosure, as well as molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, are also included among the embodiments of the present invention. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions may be referred to as “pharmaceutical compositions,” “therapeutic compositions,” “formulations,” “pharmaceutical formulations,” “therapeutic formulations,” and by other non-limiting terms and expressions.

[0101] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation 47US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355materials are for subcutaneous and / or intravenous admini tration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrm); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, tliimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride,. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the fusion protein or modified protein, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. The information regarding pharmaceutical formulation is available. See, for example, Remington - The Science and Practice of Pharmacy, 23d Edition, Adeboye Adejare, ed,, Academic Press (2021) (“Remington”),48US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0102] In certain embodiments, the primary vehicle or carrier m a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buff ered saline. In certain embodiments, a composition comprising a fusion protein or modified protein disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Remington) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising a fusion protein or modified protein disclosed herein can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some instances, the cryo-preservative may be sucrose or trehalose. In some instances, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0103] In certain embodiments, a pharmaceutical composition can include an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds, beads, or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. International Patent Publication No. WO1993015722 describes the controlled release of porous polymeric microparticles for the delivery' of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, including, for example, chemically synthesized polymers, starch-based polymers, andpoly hydroxy alkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate,49US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355ethylene vinyl acetate. In certain embodiments, implantable drug delivery' devices can be used to deliver a pharmaceutical composition.

[0104] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. For example, a pharmaceutical composition can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. For example, pulmonary administration is further described in International Patent PublicationNo. WO 1994020069, which describes pulmonary delivery of chemically modified proteins.

[0105] In certain embodiments, a pharmaceutical composition can be administered orally. A composition comprising one or more polypeptides according to the present disclosure, as well as molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure that are administered in this fashion can be formulated with or without carriers customarily used in compounding solid dosage forms, such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized, and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of a fusion protein or modified protein. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

[0106] Pharmaceutical compositions to be used for in vivo administration typically are sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, a pharmaceutical composition can be stored in lyophilized form or in a solution. In certain embodiments, a pharmaceutical composition can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. In certain embodiments, once a pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such50US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration,

[0107] In certain embodiments, the components of a pharmaceutical composition are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5-6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 62-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.Methods of use

[0108] Methods of using molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, or corresponding pharmaceutical compositions, are included among the embodiments of the present invention. In some embodiments, provided are methods of treating a subject a disease or a condition, comprising administering to the subject a therapeutically effective amount of one or more of: a polypeptide of according to the present disclosure; a molecule or a molecular complex according to the present disclosure, such as an antibody or an antibody fragment or a biologic therapeutic; a recombinant nucleic acid according to the present disclosure; a DNA construct according to the present disclosure; or a vector according to the present disclosure. It is understood that each of the foregoing can be incorporated into a suitable pharmaceutical composition. Such methods may be referred to as “methods of treatment,” “treatment methods,” or “therapeutic methods.”

[0109] As used in the present disclosure, “treating” or “treatment” of any disease or condition encompasses preventing or ameliorating a disease or disorder, or one or more of its symptoms, in a subject. The term “ameliorating” refers to any therapeutically beneficial result in the treatment51US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355of a disease or a condition, lessening in the severity or progression of the disease or a condition, or curing thereof. Treating or treatment also encompass prophylactic treatments that reduce the incidence of a disease or a condition in a subject, reduce the incidence, or reduce severity of a symptom or a disease of a condition in a subject. Treating or treatment includes ameliorating at least one physical parameter or symptom of a disease or a condition. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. Thus, a therapeutically effective amount may be an amount effective to achieve various treatment goals. For example, it may be an amount effective, to alleviate the disease or the condition, increase the likelihood of remission of the disease or the condition, reduce the likelihood of worsening or relapse of a disease or a condition, or to stimulate immunity of the subject.

[0110] A therapeutically effective amount depends, for example, upon therapeutic context and objectives. Appropriate dosage levels for treatment vary depending, in part, upon the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of a subject. A clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the fusion protein or modified protein in the formulation used. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0111] Treatment methods according to the present disclosure are not limited by any particular disease or the condition, since the polypeptides according to the present disclosure may be beneficially incorporated in various molecules or molecular complexes, such as, but not limited to, antibody or biologic therapeutics. Some non-limiting examples of diseases or conditions that may be treated according to the treatment methods according to the present disclosure are as follows: an autoimmune disease or condition, a genetic disease or condition, a cancer, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or52US2008 32326720 1Attorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355condition, a gastrointestinal disease or condition, an endocrine disease or condition, a neurological disease or condition, an ophthalmological disease or condition, a musculoskeletal disease or condition, or an infection.

[0112] In the treatment methods according to the present disclosure administration to the subject can be accomplished using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) injection, intramuscular injection (IM), intradermal injection (ID), subcutaneous, transdermal, intracavity, oral, intracranial injection, or intrathecal injection (IT). The injection can be in a bolus or a continuous infusion. Administration can be achieved by¬ oral administration, topical administration, local administration, injection, by means of an implant.Systems and kits

[0113] Included among the embodiments of the present invention are systems and kits containing one or more of: a polypeptide a according to the present disclosure; a molecule or a molecular complex incorporating one or more polypeptides according to the present disclosure, such as an antibody or an antibody fragment or a biologic therapeutic, a recombinant nucleic acid according to the present disclosure, a DNA construct according to the present disclosure, or a vector according to the present disclosure. It is understood that, in the systems and kits, each of the foregoing can be incorporated into a suitable pharmaceutical composition. Some embodiments of the systems and kits may contain two or more polypeptides according to the present disclosure. Some embodiments of the systems and kits may contain two or more molecules or molecular complexes incorporating one or more polypeptides according to the present disclosure, two or more DNA construct according to the present disclosure, and / or two or more vectors according to the present disclosure.

[0114] Systems and kits according to the present disclosure may include one or more components or ingredients in addition to those discussed above. For example, a system or a kit may include a storage container, such as a bag or a vial. Such a container may have a sterile access port, for example, a bag or vial having a stopper pierceable by a hypodermic injection needle. In another example, a system or a kit may include a diluent. In such a kit, a diluent may53US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355also be a pharmaceutically acceptable carrier or excipient, as described elsewhere in the present disclosure. Examples of diluents that may be included in such systems or kits are saline, buffered saline, water, or sucrose. In another example, a system or a kit. may include an administration device for administering the composition. A device for administering the composition may be a syringe for injection or oral administration (for example, the kit may be a pre-filled syringe), a microneedle device, such as a microneedle patch, an inhaler, or a nebulizer. In some embodiments, a system or a kit may contain multiple vials, syringes or microneedle patches. In some embodiments, a system or a kit may contain single and / or multi-chambered pre-filled syringes. Systems and kits according to the present disclosure may include a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used the methods according to the present disclosure. In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0115] Materials, compositions, components, and ingredients described in the present disclosure can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every' combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety' of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps54US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.EXAMPLESExample 1: MethodsA. Inverse Folding Predictions

[0116] To identify amino acid substitutions within the Fc region of IgGthat improve the affinity to FcRn, a language model-based method was employed.6The method is described, for example, in International Patent Publication No. WO 2023 / 196658. Using an inverse folding (IF) framework, the model was used to predict amino acid substitutions that, have increased sequence likelihood over the target protein chain. A reference structure from RCSB Protein Data Bank (RCSB PDB) PDB 4N0U of the ternary complex between Neonatal Fc receptor, serum albumin and Fc was used. The reference structure pre-processing involved the removal of atom coordinates from the serum albumin chain to focus attention of the protein design to the FcRn-Fc interaction. The corresponding structure was input into the inverse folding model, and the top 31 scoring mutations were used for experimental testing.B. Antigen cloning, expression and purification

[0117] A DNA sequence encoding the extracellular domain (Met. 1-Met 119, NP_004039.1) of human Beta-2-microglobulin (B2M) was cloned into a pADD2 vector using In-Fusion FID Cloning Kit Master Mix (Clontech). A DNA sequence encoding the extracellular domain (Met 1-Ser 297, NP__004098.1 ) of human Fc grammar receptor and transporter (FcGRT) was cloned into a pADD2 vector with AviTag and 6xHis tag at the C-terminus. The FcRn was produced in Expi293F cells (Thermo Fisher Scientific) with BirA enzyme for spontaneous biotinylation by co-transfection of B2M and FcGRT at a 1:1 ratio using FectoPRO (Polyplus). The cells were incubated with under 8% CO2 at 37°C with shaking (120 rpm). The cells were harvested 4-6 days post-transfection by spinning at 4,000 x g for 10 min and filtered through a 0.22-pm filter. The FcRn proteins with His-tag were purified using HisPur Ni-NTA resin (Thermo Fisher Scientific). Cell supernatants were diluted with a 1 / 3 volume of wash buffer containing 20 mM imidazole in Phosphate-buffered saline (PBS), and Ni-NTA resin was added to the supernatants. The supernatants were incubated overnight at 4°C with stirring. Supernatants containing the resin 55US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355were added to chromatography columns for gravity flow. The resin was washed with the wash buffer, followed by elution with 250 mM imidazole in PBS. The eluted fractions were concentrated using centrifugal concentrators, followed by size-exclusion chromatography on AKTA FPLC system using GE Superdex 200 increase 10 / 300 GL column (GE Healthcare).C. Antibody cloning, expression and purification

[0118] Genes encoding the light chain (LC) and heavy chain (HC) of VRC01 and HC variants were synthesized by Integrated DNA technologies (IDT). The genes were cloned into the CMV7R plasmid backbone. Antibodies with Fc variants were produced in Expi293F cells by a co-transfection of HC and LC at a 1:1 ratio using Fectopro (PolyPlus). The cells were harvested as above, and the antibodies were purified using a 5 mL MabSelect Sure PRISM column (Cytiva) on AKTA pure chromatography system.D. Quantification of protein expression

[0119] The antibodies incorporating Fc variants according to the present disclosure are expressed and purified as described above. Their expression levels are quantified by measuring absorbance at 280 nm using NanoDrop spectrophotometer (Fisher Scientific). Corresponding wild-type (WT) antibodies are used as controls.E. Enzyme immunoassay

[0120] Nunc 96- well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with 200 ng of streptavidin in phosphate-buffered saline (PBS). Plate wells were washed once with distilled water and blocked with 150 pL of ChonBlock blocking buffer (Chondrex) for 1 hour at 37°C. Biotinylated FcRn (1 pg / mL) in ELISA buffer (1% BSA / PBS) was added to the wells and incubated for 2 hours at 37°C. The plates were washed three times with 0.05% (v / v) Tween 20 in PBS (PBST), followed by incubation with 50 pL of antibody Fc variants (0.00064- 10 pg / mL) in ELISA buffer for 1 hour at 37°C. The plates were then washed with PBST and incubated with anti-kappa light chain-horseradish peroxidase (Southern Biotech) diluted 1:2,000 in ELISA buffer. After the final wash with PBST six times, 3,3',5,5'-tetramethylbenzidine (TMB)-ELISA substrate solution (Thermo Fisher Scientific) was added, followed by 2 M sulfuric acid stop solution. Absorbance was measured at 450 nm using BioTek Synergy FIT56US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355Microplate Reader, For binding studies at pH 6.0, the pH of the buffers was adjusted accordingly after the addition of FcRn and before the addition of TMB. The EC50 value of the WT was divided by the values from the Fc variants to get the fold change at pH 6.0, where a higher value indicates stronger binding to FcRn. For binding studies at pH 7.4, the Fc variants were initially incubated at pH 6.0 and the pH of the buffers in the subsequent steps were adjusted to pH 7.4. The maximum response values of the Fc variants at 10 pg / mL were divided by the values from the antibody with WT Fc to get the fold change at pH 7.4. A higher value indicated enhanced retention at pH 7.4.F. Biolayer interferometry (BLI)

[0121] Biotinylated FcRn (50 nM) was loaded onto streptavidin biosensors (Sartorius) to a load threshold of 0.8 nm on Octet RED96 system (Pall ForteBio). The biosensors were first equilibrated with octet buffer (0.5% wt / vol bovine serum albumin (BSA) in PBS containing 0.05% vol / vol Tween 20), then dipped into the solutions of Fc variant antibodies (20 nM) in octet buffer (pH 6.0) for association (200 seconds) followed by dissociation (200 seconds) into the octet buffer (pH 6.0 or pH 7.4). A biosensor on which biotinylated antigen was loaded but did not associate with any antibodies was used for baseline subtraction. The data were processed on ForteBio Data Analysis software to calculate equilibrium dissociation constant [KD], association rate constant [kon], and dissociation rate constant [koff], The KD values of the corresponding control (WT or mutant) were divided by those of the Fc variants to calculate the change at pH 6.0. A higher value indicated stronger binding to FcRn. Similarly, the koff values of the corresponding control were divided by those of the Fc variants to calculate the change in koff at pH 7.4. A higher value indicated slower dissociation at pH 7.4 (i.e. greater binding retention). For both the KD and koff, fold-change is computed by dividing the wild-type value by the variant value. For KD at pH 6, a smaller value is desirable, such that that a “better binder” will have a lower KD and, thus, smaller denominator, and will result in a higher fold-change (desirable). For koff, a larger value is desirable, such that a “better releaser” will have a higher koff at pH 7.4 and, thus, larger denominator and will result in a smaller fold-change (desirable). These parameters are summarized in the table below. Higher fold-change values at pH 6.0 indicate increased FcRn binding, while higher fold-change values at pH 7.4 indicate decreased release (i.e. greater binding retention).57US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0122] For the expanded screen of Fc variants combined in LS background as described in Example 7, biotinylated and His-tagged FcRn (50 nM) was loaded onto Ni-NTA biosensors (Sartorius) to a load threshold of 0.8 nm on Octet RED96 system (Pall ForteBio), The remainder of the assay and analysis was carried out as described above.Direction of Value Fold-Change Parameter Behavior Value(wt / variant) Increased binding Smaller Larger (desirable)KD pH 6 DecreasedLarger SmallerbindingIncreased release Larger Smaller (desireable) koff pH 7.4Decreased release Smaller LargerG. Antibody Backbone Sequences

[0123] The antibody backbone sequences used to produce the variant antibodies described in the Examples of this application are shown in Table 1 below.Table 1. Antibody sequences.Anti-RSV Antibody (Nirsevimab / Beyfortus™)V ariable Heavy Chain QVQLVQSGAEVKKPGSSVNWSCQASGGLLEDY1INWVRQA PGQGPEWMGGIIPVLGTVHYGPKFQGRVTITADESTDTAYM ELSSLRSEDTAMYYCATETALWSETYI., PHYFDNWGQGTI> VTVSS (SEQ ID NO:5)Variable Light Chain DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPG KAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVA TYYCQQYDNLPLTFGGGTKVEIKR (SEQ ID NO:6)WT IgGl Heavy Chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW Constant Region NSGALTSGVHTFPAVI. QSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNT1< VDKI< VEPKSCDI< THTCPPCPAPELLGGPSVF LFPPKPKDTLMlSRTPEVTCVVVDVSklEDPEVKFNWYVDClV EVHNAKTKPREEQYNSTYRVVS\TT\T. HQDWL> NGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:6)58US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355YTE IgGl Heavy Cham ASTKGPSVFPLAl’SSKSTSGGTAALGCLVKDYFPEPVTVSW Constant Region NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSN TKVDKKVEPKSCDK THTCPPCPAPELLGGPS VF LFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLT\T)KSRWQQGNVTSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 7)LS IgGl Heavy Cham ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW Constant Region NS GALTS G VHTFP A VLQ S S GL YS LS S WT VPS S SLGTQT YIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLWLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVLIIEALHSHYTQKSLSLSPGK (SEQ ID NO: 8)LA IgGl Heavy Chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW Constant Region NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVUNAKTKPREEQTOSTYRWSVTTVLHQDWLNGKEYKC KVSNKAI. PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SETCL. VK(}FYPSDL'-\VE\VESN(}QPENNYKTTPPVEr)SD(}SFF LYSKLTVDKSRWQQGNATSCSVTHEALHAHYTQKSLSLSP GK (SEQIDNO:9)LG IgGl Heavy Chain ASIXGPSVFPLAI’SSKSTSGGTAALGCLVKDYFPEPVTVSW Constant Region NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNIKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKI’KDTLNHSRITEVTCVVV'DVSHEDPEVKFNWYVDGV EVHNAKTKl’REEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKT1TPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVLHEALHGHYTQKSLSLSP GK (SEQ ID NO: 10)Anti-Pf-CSP Antibody ( L9)Variable Heavy Cham QVKLVESGGGVVQPGRSLRLSCEASGF1FSTYGMHWVRQA PGKGLEWVAVIWFDGSNIY YADS VKGRFTISRDN SKN TVFM QMDSLRAEDTAVYYCHRNFYDGSGPFDYWGQGTLVIVSS(SEQ ID NO: 11)59US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355Variable Light Chain DIQMTQSPSTLSASVGDRVTITCRASQFISRWLAWYQQKPG KAPKLLIYKASSLESGVPSRFSGSGSETHFTLTISSLQPDDVA TYYCQEY TS YGRTFGQGTK VEIK (SEQ ID NO: 12)WT IgGl Heavy Chain (SEQ ID NO: 1)Constant RegionYTE IgGl Heavy Chain (SEQ ID NO: 7)Constant RegionLS IgGl Heavy Chain (SEQ ID NO: 8)Constant RegionLA IgGl Heavy Chain (SEQ ID NO: 9)Constant RegionLG IgGl Heavy Chain (SEQ ID NO: 10)Constant RegionAnti-PCSK9 Antibody ( Evolocumab / Repatha®)V ariable Heavy Chain EVQLVQSGAEVKKPGASVKVSCKASGYTLTSYGISWVRQA PGQGLEWMGWVSFYNGNTNYAQKLQGRGTMTTDPSTSTA YMELRSLRSDDTAVYYCARGYGMDVWGQGTTVTVSS(SEQ ID NO: 13)Variable Light Chain ESALTQPASVSGSPGQSITISCTGTSSDVGGYNSVSWYQQHP GKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAED EADYYCNSYTSTSMVFGGGTKLTVLGQP (SEQ ID NO: 14) WT IgG2 Heavy Cham (SEQ ID NO:2)Constant RegionYTE IgG2 Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN Constant Region SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLYITREPEVTCWVDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFN STFRV VS VLTVVHQD WLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 15)Anti-PDl Antibody (Pembrolizumab / Keytruda®)Variable Heavy Cham QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQ APGQGLEW1GGINPSNCJGTNFNEKFKNRVTLTTDSSTTTAY MELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 16)60US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355Variable Light Chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQ KPGQAPRLLIYLASYLESGVPARFSGSGSGTOFTLTISSLEPE DF A VY YCQHSRDLPL TFGGGTKVEIKR TV (SEQ ID NO: 17) WT IgG4 Heavy Chain (SEQ ID NO: 3)Constant RegionYTE IgG4 Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN Constant Region SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNS T YRVVS VLTVLHQDWLN GKE YKCKV SNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 18)Example 2: Predicted Fc variants show increased binding to FcRn at acidic pH, with a minimal change at physiological pH

[0124] The inverse folding model discussed in Example one was used to predict mutations in the Fc region of IgG to enhance its binding to FcRn at acidic pH. The locations of the predicted mutations are schematically illustrated in FIG. 1. Antibodies with the predicted mutations were generated. In a preliminary experiment, the binding of these antibodies to FcRn (KD) at pH 6.0 and retention at FcRn at pH 7,4 were determined. Many variants showed increased binding to FcRn at pH 6.0, as measured by ELISA (FIG. 2) and BIT (FIG. 3), while showing minimal changes in retention at FcRn at pH 7,4, as measured by ELISA (FIG. 4). All data are shown relative to WT. Higher value at pH 6.0 indicates stronger FcRn binding, while higher value at pH 7.4 indicates enhanced retention.

[0125] Fc mutations reported previously to alter antibody half-life were located at the site or near the interface to FcRn (see Table 2). Surprisingly, many of the mutations according to the present disclosure, which were discovered to have increased binding to FcRn at pH 6.0 with a minimal change in retention at pH 7.4, were distant from the binding interface to FcRn (FIG. I and Table 3). Many variants showed increased binding to FcRn at pH 6.0, as measured by ELISA (binding) and by BLI, while showing minimal changes in relative maximum response measured by ELISA at pH 7.4 to assess retention. All data are shown relative to WT. Higher 61US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355values at pH 6.0 indicated stronger FcRn binding, while higher values at pH 7.4 indicated enhanced retention.

[0126] As our focus was on the binding at pH ~6 and the kinetics of dissociation at pH ~7.4, we subsequently measured the KD and koff using BLI only, rather than ELISA,Table 2. Distance of wild-type Fc amino acids to FcRn (A) in previously identified variants.Distance of wild-type residue toMutation VariantFcRn (A)M252Y 3.17 YTES254T 2.99 YTET256E 8.17 YTEM428L 6.73 LSH433K 3.69 KFN434S 1.93 LSN434F 1.93 KFTable 3. Relative change in binding of IgG Fc variants to FcRn compared to wild-type protein, as determined by enzyme immunoassay and BLI.Fold Fold Distance Relative maximumchange change of WT Variant IgG Fv response (binding)(binding) (KD) at Residue to at pH 7.4at pH 6,0 pH 6.0 FcRn (A) L251V IgGl Anti-RSV 0.96 1.00 1.01 3.57 L251Y IgGl Anti-RSV 0.35 0.40 0.79 3.57 M252D IgGl Anti-RSV 0.00 0.00 0.33 3 17 S254T IgGl Anti-RSV 0.63 0.13 0.63 2.99 V266L IgGl Anti-RSV 0.87 1.51 1.47 30.87 H268K IgGl Anti-RSV 0.58 0.62 0.51 33.87 H268S IgGl Anti-RSV 0.81 0.00 1 96 33 87 N276S IgGl / Xnti-RSV 0.73 1.41 1.06 16.95 A287L IgGl Anti-RSV 1.68 1.28 1.67 14.97 A287M IgGl Anti-RSV 2.94 1.03 085 ] 4 97 V302L IgGl / Xnti-RSV 2.23 1.01 0.69 24, 80 S304A IgGl Anti-RSV 1.57 1.96 1.58 17.88T307N IgGl Anti-RSV 0.36 0.88 1.02 7.9662US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355L309G IgGl Anti-RSV 0.06 0.00 0.42 3 04 K320T IgGl Anti-RSV 0.02 0.69 0.44 16.61 K320S IgGl Anti-RSV 4.46 1.16 082 1661 N325S IgGl Anti-RSV 2.13 1.96 1.65 25.84 Q342P IgGl Anti-RSV 0.98 1.67 0.70 16.66 Y349T IgGl Anti-RSV 1.23 1.13 1.14 17.66 R416T IgGl Anti-RSV 2.48 1.51 1.12 21.68 R416V IgGl Anti-RSV 2.87 1.57 1.04 21.68 N421E IgGl Anti-RSV 3.34 0.92 0.71 19.52 N421V IgGl Anti-RSV 9.28 1.75 0.72 19.52 S426V IgGl Anti-RSV 2.56 1.93 0.88 10.41 S426R IgGl Anti-RSV 2.66 1.18 1.27 10.41 E430R IgGl Anti-RSV 2.25 2.83 0.90 7.63 L432S IgGl Anti-RSV 1.97 1.64 079 5.42 L432C IgGl Anti-RSV 3.70 1.89 0.77 5.42 H433K IgGl Anti-RSV 4.95 3.29 0.61 3.69 N434G IgGl Anti-RSV 15.72 2.33 1 21 1.93L441C IgGl Anti-RSV 4.94 1.39 1.01 17.78Example 3: Predicted Fc variants in the YTE background show different and greater impact on binding to FcRn at acidic pH, with a minimal change at physiological pH

[0127] The inverse folding model discussed in Example 1 was used to predict mutations in an IgGFc region having mutations M252Y / S254I7T256E (“YTE background”) that enhance binding to the FcRn at acidic pH. Antibody variants with the predicted mutations were generated in Fc WT background and in Fc YTE background, and their binding to FcRn (KD) at pH 6.0, as well as their retention binding (koff) at pH 7.4, was assessed using BLI.

[0128] The tested variants and the binding data are listed in Table 4. All data are shown relative to YTE variant. Many variants on YTE background showed increased binding to FcRn at pH 6.0, as measured by BLI, while showing minimal changes in koff at pH 7.4. The variants with the YTE background showed a different and greater impact (enhanced binding) on FcRn binding at pH 6.0 (FIG. 5 gray bars) compared to variants with the same mutation in the WT background (FIG. 5 black bars). There are multiple approaches to determine the koff value, such as analyzing the dissociation during the initial 5 seconds, or over the entire dissociation phase 63US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(200 seconds). We compared both methods and selected the koff determined from the initial 5 seconds (5 sec), as it most accurately captures rapid dissociation and represents the observed dissociation kinetics. Surprisingly, many of the identified Fc mutations that increased binding to FcRn at pH 6.0 with a minimal change in dissociation rate at pH 7.4 were distant from the binding interface to FcRn. As discussed above, Fc mutations that were shown to alter antibody half-life were located at the site of or near the interface to FcRn (see FIG. 1 and Table 2).Table 4, Relative changes in FcRn binding, as determined by BLI, of IgG Fc variants in YTE background, as compared to YTE IgG Fc variant.Variant IgG Fv Fold change Fold change Fold Distance of (KD) to (koff) to change WT Residue YTE at pH YTE at pH (koff) to to FcRn (A) 6.0 7.4 (200 YTE atseconds) pH 7.4 (5seconds)V266L IgGl Anti-RSV 0.79 1.01 0.83 30.87 H268K IgGl Anti-RSV 0.89 1.24 0.90 33.87 H268S IgGl Anti-RSV 1.28 1.00 0.93 33.87 N276S IgGl Anti-RSV 0.94 1 08 0.90 16.95 A287L IgGl / Xnti-RSV 1.17 0.98 1.00 14.97 A287M IgGl Anti-RSV 0.79 0.76 0.97 14.97 V302L IgGl Anti-RSV 0.94 078 1.05 24.80 S304 / \ IgGl / Xnti-RSV 0.95 1.30 0.99 17.88 T307N IgGl Anti-RSV 1.16 1.02 0.94 7.96 L309G IgGl Anti-RSV 1.97 1.17 1 33 3.04 K320T IgGl Anti-RSV 1.13 0.97 1.02 16.61 K320S IgGl Anti-RSV 1.00 092 1.08 16.61 N325S IgGl Anti-RSV 1.04 1.03 0.84 25.84 Q342P IgGl Anti-RSV 1.04 1.16 0.90 16,66 Y349T IgGl Anti-RSV 0.91 1.25 0.88 17.66 Y349Q IgGl Anti-RSV 0.79 1.16 0.90 17.66 R416T IgGl Anti-RSV 0.97 0.94 0.91 21.68 R416V IgGl Anti-RSV 0.93 095 0.95 21.68 N421E IgGl / Xnti-RSV 0.94 1.23 0.91 19 52 N421V IgGl Anti-RSV 0.88 1.14 0.94 19.52 S426V IgGl Anti-RSV 2.31 1 14 1.24 10.41 S426RIgGl / Xnti-RSV 1.87 1.21 1.30 10.4164US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355E430R IgGl Anti-RSV 0.54 0.97 0.83 7.63 L432S IgGl Anti-RSV 0.79 0.77 0.82 5.42 L432C IgGl Anti-RSV 0.62 1.17 0.81 5.42 H433K IgGl Anti-RSV 1.56 1.92 1.88 3.69 N434G IgGl Anti-RSV 3.84 1.01 1.92 1.930.81 1.06 0.94 17.781.441 C IgGl Anti-RSVExample 4: Fc variants in the YTE background with increased binding to FcRn at acidic pH were combined, resulting in a further increase in FcRn binding

[0129] Antibody variants were made with combinations of Fc mutations that had been found to increase binding to FcRn at pH 6.0 in the YTE background as described m Example 3, The antibody variants were produced in both anti-respiratory syncytial virus (nirsevimab / Beyfortus™; “anti-RSV”) and in the anti-plasmodium falciparum circumsporozoite protein (L9; “anti-Pf-CSP”) antibody backbones, both IgGl subclass and having the YTE mutations, to assess generalizability across antibodies with different antigen specificities.Antibody variant binding to FcRn (KD) at pH 6.0 and retention binding (koff) at pH 7.4 was assessed as described in Example 3. The tested variants and the binding data are listed in Table 5 and FIGS. 6A-6B. All data are shown relative to YTE variant. The combinations further improved binding at acidic pH, while minimally altering binding at physiological pH.Table 5. Relative changes in FcRn binding of IgG Fc variants combined with YTE (M252Y / S254T / T256E), normalized to YTE, as determined by BLIFold change Fold change Variant IgG Fv (KD) to YTE at (koff) to YTE at pH 6,0 pH 7,4 (5 sec) YTE L309G S426V IgGl Anti-RSV 2.12 1.04 YTE L309G S426R IgGl Anti-RSV 3.26 1.72 YTE L309G N434G IgGl Anti-RSV 2.97 1.48 YTE S426V N434G IgGl Anti-RSV 3.46 1.80 YTE S426R N434G IgGl Anti-RSV 3.37 1.80 YTE L309G S426V N434G IgGl Anti-RSV 3.83 1.57 YTE L309G S426R N434G IgGl Anti-RSV 3.51 1.49YTE L309G S426V IgGl Anti-Pf-CSP 2.24 1.6565US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355YTE L309G S426R IgGI Anti-Pf-CSP 2.53 1.70 YTE L309GN434G IgGI Anti-Pf-CSP 2.06 1.21 YTE S426V N434G IgGI Anti-Pf-CSP 2.64 1.81 YTE S426R N434G IgGI Anti-Pf-CSP 2.61 1.79 YTE L309G S426V N434G IgGI Anti-Pf-CSP 2.57 1.26YTE L309G S426R N434G IgGI Anti-Pf-CSP 2.36 1.19Example 5: Fc variants were combined in LS, LA, or LG backgrounds, resulting in a further increase in FcRn binding

[0130] Additional antibody variants with Fc mutations that had been found to increase binding to FeRn at pH 6.0 in the YTE background were made. These variants did not include the YTE mutation but instead had the LS (M428L / N434S) or LA (M428L / N434A) mutations. The antibody variants were produced in both the anti-RSV and anti-Pf-CSP antibody backbones (as described in Example 4). Variants with individual Fc mutations and combinations of these Fc mutations were made. Antibody variant binding to FcRn (KD) at pH 6.0 and retention binding (koff) at pH 7.4 was assessed as described in Example 3.

[0131] The tested variants and the binding data are listed in Tables 6 and 7 (see also FIGS. 7A-7B). All data are shown relative to LS variant. Both approaches enhanced binding at acidic pH compared to LS, while minimally affecting binding at physiological pH.Table 6, Relative changes in FcRn binding of IgG Fc variants combined with LS (M428L / N434S), normalized to LS, as determined by BL1.Fold change Fold change Variant IgG Fv (KD) to LS at (koff) to LS at pH 6.0 pH 7.4 (5 sec) LS L309G IgGI Anti-RSV 1.90 1.40LS S426V IgGI Anti-RSV 1.80 1.21LS S426R IgGI Anti-RSV 0.99 0.67LS L309G S426V IgGI Anti-RSV 2.81 1.51LS L309G S426R IgGI Anti-RSV 1.32 1.01LS L309G IgGI Anti-Pf-CSP 1.32 1.21LS S426V IgGI Anti-Pf-CSP 1.26 1.0966US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355LS S426R IgGl Anti-Pf-CSP 0.52 0.67LS L309G S426V IgGl Anti-Pf-CSP 1.71 1.26LS L309G S426R IgGl Anti-Pf-CSP 1.11 0.94Table 7. Relative changes in FcRn binding of IgG Fc variants combined with LA (M428L / N434A), normalized to LS (M428L / N434S), as determined by BLI.Fold change Fold change Variant IgG Fv (KD) to LS at (koff) to LS at pH 6.0 pH 7.4 (5 sec) LA IgGl Anti-RSV 1.36 1.08LA L309G IgGl Anti-RSV 2.03 1.54LA S426V IgGl Anti-RSV 1.48 1.39LA S426R IgGl Anti-RSV 0.95 0.75LA L309G S426V IgGl Anti-RSV 2.88 1.52LA L309G S426R IgGl Anti-RSV 1.51 1.11LA IgGl Anti-Pf-CSP 0.99 1.16 LAL309G IgGl Anti-Pf-CSP 1.36 1.15LA S426V IgGl Anti-Pf-CSP 1.03 1 20LA S426R IgGl Anti-Pf-CSP 1.22 1.15LA L309G S426V IgGl Anti-Pf-CSP 1.83 1.66LA L309G S426R IgGl Anti-Pf-CSP 1.35 1.13

[0132] N434G, which improves FcRn binding as shown from the antibody variant with YTE, occurs at the same site as S in LS and A in LA, As such, the Fc mutation LG (M428L / N434G) was generated and combined with both the individual Fc mutations identified m the YTE background and their combinations in additional variant antibodies in both anti-RSV and anti-Pf-CSP antibody backbones. Antibody variant binding to FcRn (KD) at pH 6.0 and retention binding (koff) at pH 7.4 was assessed as described in Example 3. The tested variants and the binding data are listed in Table 8 (see also FIGS. 7A-7B). All data are shown relative to LS variant. The variant antibodies having the LG mutation had further improved dissociation at physiological pH (7.4) compared to LS, with a minimal impact on its binding at the acidic pH (6).67US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355Table 8, Relative changes in FcRn binding oflgG Fc variants combined with LG (M428L / N434G), normalized to LS (M428L / N434S), as determined by BLI.Fold change Fold change Variant IgG Fv (KD) to LS at (koff) to LS at pH 6.0 pH 7.4 (5 sec) LG IgGl Anti-RSV 1.98 1.55LG L309G IgGl Anti-RSV 1.68 0.90LG S426V IgGl Anti-RSV N / A N / ALG S426R IgGl Anti-RSV 0.70 0.47 LGL309GS426V IgGl Anti-RSV 1.14 0.88 LGL309G S426R IgGl Anti-RSV 1.14 0.70LG IgGl Anti-Pf-CSP 0.76 0.6LG L309G IgGl Anti-Pf-CSP 1.17 0.89LG S426V IgGl Anti-Pf-CSP 0.79 0.58LG S426R IgGl Anti-Pf-CSP 0.54 0.47LG L309G S426V IgGl Anti-Pf-CSP 1.21 0.85LGL309G S426R IgGl Anti-Pf-CSP 0.95 0.62Example 6: Fc variants in the YTE background with increased binding to FcRn at acidic pH generalize across monoclonal antibody IgG subclasses, resulting in a further increase in FcRn binding

[0133] Representative individual Fc variants tested in the YTE background of IgGl antibodies and found to increase binding to FcRn at pH 6.0 were also validated m alternate non-IgGl antibodies to establish generalizability across other monoclonal antibody subclasses. Individual and combination amino acid substitutions were similarly combined with the YTE set of mutations and characterized in the IgG2 subclass with anti -proprotein convertase subtilism / kexin type 9 (Evolocumab / Repatha®; “anti-PCSK9”) and in the IgG4 subclass with anti -programmed cell death protein 1 (Pembrolizumab / Keytruda®; “anti-PD-1”) antibody backbone. Fold change in KD and koff was determined as described in the prior examples. All data are shown relative to YTE variants. Data for the anti-PCSK9 antibody variants are shown in Table 9 and FIG. 8A, and data for the anti-PD-1 antibody variants are shown m Table 10 and FIG, 8B. The combinations68US2008 32326720 1Attorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355further improved binding at acidic pH beyond YTE, while minimally altering binding at physiological pH.Table 9, Relative changes in FcRn binding of!gG2 Fc variants normalized to WT, as determined by BLI.Fold change Fold change Variant IgG Fv (KD) to WT at (koff) to WT at pH 6.0 pH 7.4 (5 sec) YTE IgG2 Anti-PCSK9 4.01 2.51 YTE L309G IgG2 Anti-PCSK9 5.67 3.77 YTE L309G S426R IgG2 Anti-PCSK9 7.31 3.70 YTE S426R N434G IgG2 Anti-PCSK9 10.45 3.46Anti-PCSK9 7.65 4.00YTE S426V N434G IgG2Table 10. Relative changes in FcRn binding of IgG4 Fc variants normalized to WT, as determined by BLI.Fold change Fold change Variant IgG Fv (KD) to WT at (koff) to WT at pH 6.0 pH 7.4 (5 sec) YTE IgG4 Anti-PDl 5.40 1.55 YTE N434G IgG4 Anti-PDl 10.38 3.28 YTE S426V IgG4 Anti-PDl 8.04 2.20 YTE S426R N434G IgG4 Anti-PDl 11.45 3.23IgG4 Anti-PDl 12.63 3.17YTE S426V N434GExample 7: Fc variants were combined in LS background, resulting in a further increase in FcRn binding

[0134] Additional antibody variants with Fc mutations that had been found to increase binding to FcRn at pH 6.0 in the YTE background were made. These variants did not include the YTE mutation but instead had the LS (M428L / N434S) mutations. The antibody variants were produced in both the anti-RSV antibody backbone (as described in Example 5). Variants with individual Fc mutations and combinations of these Fc mutations were made. Antibody variant binding to FcRn (KD) at pH 6.0 and retention binding (koff) at pH 7,4 was assessed as described in Example 3.69US2008 32326720 IAttorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355

[0135] The tested variants and the binding data are listed in Table 11 (see also FIG, 9). All data are shown relative to LS variant. Both approaches enhanced binding at acidic pH compared to LS, while minimally affecting binding at physiological pH. Some of the variants had faster dissociation than the LS variant at pH 7.4, indicating more efficient release from FcRn.Table 11, Relative changes in FcRn binding of IgG Fc variants combined with LS (M428L / N434S), normalized to LS, as determined by BLI.Fold change Fold change(koff) to LS at Variant IgG Fv (KD) to LS atpH 7.4 (5 pH 6.0seconds) LS L251 V IgGl Anti -RS V 0.79 0.84LS L251Y IgGl Anti-RSV 0.86 0.39LS S254T IgGl Anti-RSV 1.33 0.74LS V266L IgGl Anti-RSV 1.39 0.98LS H268K IgGl Anti-RSV 1.67 0.74LS H268S IgGl Anti-RSV 1.69 0.95LS N276S IgGl Anti-RSV 1.86 1.49LS A287L IgGl Anti-RSV 1.73 0.87LS A287M IgGl Anti-RSV 1.18 0.95LS V302L IgGl Anti-RSV 1.65 0.97LS S304A IgGl Anti-RSV 1.45 0.76LS T307N IgGl Anti-RSV 1.55 0.89LS K320T IgGl Anti-RSV 1.88 1.02LS K320S IgGl Anti-RSV 1.79 0.95LS N325S IgGl Anti-RSV 1.73 1.08LS Q342P IgGl Anti-RSV 1.58 0.83LS Y349T IgGl Anti-RSV 1.64 1.23LS Y349Q IgGl Anti-RSV 1.42 1 08LS R416T IgGl Anti-RSV 1.88 1.38 LS R416V IgGl Anti-RSV 1.33 0.96LS N421E IgGl Anti-RSV 1.59 1.38 LS N421V IgGl Anti-RSV 1.67 1.00LS E430R IgGl Anti-RSV 1.09 1.12LS L432S IgGl Anti-RSV 1.47 1 27LS L432C IgGl Anti-RSV 0.73 0.73LS H433K IgGl Anti-RSV 1.34 1 4370US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355LS L441C IgGl Anti-RSV 1.38 1.03Example 8: Fc variants were combined with LG (M428L / N434G), resulting in a further increase in FcRn binding

[0136] Additional antibody variants with Fc mutations that had been found to increase binding to FcRn at pH 6.0 in the YTE background were made. These variants did not include the YTE mutation but instead had the LG (M428L / N434G) mutations. The antibody variants were produced in the anti-RSV antibody backbone (as described in Example 5). Variants with individual Fc mutations were made. Antibody variant binding to FcRn (KD) at pH 6.0 and retention binding (koff) at pH 7.4 was assessed as described in Example 3.

[0137] The tested variants and the binding data are listed in Table 12 (see also FIG. 10). All data are shown relative to LS variant. Most variants showed enhanced binding at acidic pH compared to LS, while resulting in dramatically faster dissociation at physiological pH. This indicates more efficient release from FcRn at physiological pH (7.4).Table 12. Relative changes in FcRn binding of IgGFc variants combined with LG (M428L / N434G), normalized to LS, as determined by BLI.Fold change Fold change(Koff) to LS at Variant IgG Fv (KD) to LS atpH 7.4 (5 pH 6.0seconds) LGL251V IgGl Anti-RSV 0.90 0.46 LGL251Y IgGl Anti-RSV 0.59 0.24LG S254T IgGl Anti-RSV 1.16 0.51 LGV266L IgGl Anti-RSV 1.24 0.61 LGH268K IgGl Anti-RSV 1.34 0.62 LGH268S IgGl Anti-RSV 1.31 064 LGN276S IgGl Anti-RSV 1.47 0.68 LGA287L IgGl Anti-RSV 1.25 0.67LG A287M IgGl Anti-RSV 1.30 0.64LG V302L IgGl Anti-RSV 1.16 0.63LG S304A IgGl Anti-RSV 1.16 0.56LG T307N IgGl Anti-RSV 1.17 0.5871US2008 32326720 1Attorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355LG K320T IgGl Anti - RS V 1.14 068 LGK320S IgGl Anti-RSV 1.51 0.68LG N325S IgGl Anti-RSV 1.18 0.62LG Q342P IgGl Anti-RSV 1.24 062LG Y349T IgGl Anti-RSV 1.22 0.62 LGY349Q IgGl Anti-RSV 1.31 0.61 LGR416T IgGl Anti-RSV 1.17 059 LGR416V IgGl Anti-RSV 1.26 0.64 LGN421E IgGl Anti-RSV 1.26 0.82 LGN421V IgGl Anti-RSV 1.44 0.62LG E430R IgGl Anti-RSV 1.63 0.50 LGL432S IgGl Anti-RSV 1.00 0.56 LGL432C IgGl Anti-RSV 1.07 0.55 LGH433K IgGl Anti-RSV 0.96 0.74LGL441C IgGl Anti-RSV 1.31 0.66Example 9: Expression enhancement

[0138] Antibodies containing Fc variants according to the present disclosure are expressed and purified. Their expression is measured as described in Example 1. Some of the antibodies containing Fc variants exhibit enhanced expression in comparison to the corresponding WT antibodies.

[0139] It is understood that the examples and embodiments described in the present disclosure are for illustrative purposes only and that various modifications or changes m light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited in the present disclosure are hereby incorporated by reference in their entirety for all purposes.72US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355SELECTED REFERENCES CITED IN THE DISCLOSURE(1) Raghavan, M.; Bjorkman, P. J. Fc Receptors and Their Interactions with Immunoglobulins. Anmi. Rev. Cell Dev. Biol. 1996, 12 (1), 181-220.doi. org / 10.1146 / annurev. cellbio.12.1.181.(2) Yeung, Y. A.; et al. Engineering Human IgGl Affinity to Human Neonatal Fc Receptor: Impact, of Affinity Improvement on Pharmacokinetics in Primates. The Journal of Immunology’ 2009, 182 (12), 7663-7671, doi, org / 10.4049 / jimmunol.0804182,(3) Vaccaro, C.; et al. Divergent Activities of an Engineered Antibody in Murine and Human Systems Have Implications for Therapeutic Antibodies. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (49), 18709-18714. doi. org / 10.1073 / pnas.0606304103.(4) DalFAcqua, W. F.; et al. Properties of Human IgGls Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn). Journal of Biological Chemistry 2006, 281 (33), 23514-23524. https: / / doi.org / 10.1074 / jbc. M604292200.(5) Grevys, A.; et al. Fc Engineering of Human IgGl for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions. The Journal of Immunology 2015, 194 (11), 5497-5508. https: / 7doi. org 10.4049 / j immunol.1401218.(6) Shanker, V. R.; et al. Unsupervised Evolution of Protein and Antibody Complexes with a Structure-Informed Language Model. Science 2024, 385 (6704), 46-53.https: / / doi.org / 10.1126 / science.adk8946.(7) DalFAcqua, W. F; et al. Increasing the Affinity of a Human IgGl for the Neonatal Fc Receptor: Biological Consequences. The Journal of Immunology 2002, 169 (9), 5171-5180. https: / / doi. org 0.4049 / j immunol.169.9.5171.(8) Zalevsky, J.; et al. Enhanced Antibody Half-Life Improves in vivo Activity. Nat Biotechnol 2010, 28 (2), 157-159. https: / / doi.org / 10.1038 / nbt.160L73US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(9) Lee, C.-H.; et al. An Engineered Human Fc Domain That Behaves like a pH-Toggle Switch for Ultra-Long Circulation Persistence. Nat Commun 2019, 10 (1), 5031,https: / / dot. org / 10.1038 / s41467-019-13108-2.(10) Ko, S.; et al. An Fc Variant with Two Mutations Confers Prolonged Serum Half-Life and Enhanced Effector Functions on IgG Antibodies. Exp Mol Med 2022, 54 (11), 1850-1861. https: / / doi.org / 10.1038 / sl2276-022-00870-5.(11) Foss, S.; et al. Human IgG Fc-Engineering for Enhanced Plasma Half-Life, Mucosal Distribution and Killing of Cancer Cells and Bacteria. Nat Commun 2024, 15 (1), 2007, https: / / doi.org / 10.1038 / s41467-024-46321-9.(12) Doniachowske, J. B.; et al. Safety, Tolerability and Pharmacokinetics of MEDI8897, an Extended Half-Life Single-Dose Respiratory Syncytial Virus Prefusion F-Targeting Monoclonal Antibody Administered as a Single Dose to Healthy Preterm Infants. Pediatric Infectious Disease Journal 2018, 37 (9), 886-892, https: / / doi.org / 10.1097 / INF.0000000000001916.(13) Keam, S. J. Tixagevimab + Cilgavimab: First Approval. Drugs 2022, 82 (9), 1001-1010. https: / / doi.org / ! 0.1007 / s40265 -022-01731 - 1.(14) Kulasekararaj, A. G.; etal. Ravulizumab (ALXN1210) vs Eculizumab in C5-Inhibitor-Experienced Adult Patients with PNH: The 302 Study. Blood 2019, 133 (6), 540-549. https: / / doi.org / 10.1182 / blood-2018-09-876805.(15) Pinto, D.; et al. Cross-Neutralization of SARS-CoV -2 by a Human Monoclonal SARS-CoV Antibody. Nature 2020, 583 (7815), 290-295. https: / / doi.org / 10.1038 / s41586-020-2349-y.(16) Foss, S., et al. Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria. Nat Commun 15, Article No. 2007 (2024). https: / / doi.org / T0.1038 / s41467-024-46321-9.(17) Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcgammaR: current models. Immunol Lett. 2002 Jun 3;82(l-2):57-65. doi: 10.1016 / s0165-2478(02)00019-6.74US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(18) Pinto S, et al. Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide. J Control Release. 2024 Feb; 366:621-636. doi:10.1016 / j. j conrel.2024.01.015.(19) Fieux M, etal. FcRn as a Transporter for Nasal Delivery of Biologies: A Systematic Review. IntJMolSci. 2021 Jun 17;22(12):6475. dot: 10.3390 / ijms22126475.(20) Tien J, et al. Modifying antibody-FcRn interactions to increase the transport of antibodies through the blood-brain barrier, MAbs. 2023 Jan-Dec;! 5(1 ):2229098. doi:10.1080 / 19420862.2023.2229098.(21) Jung, S. T., et al. Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcyRIIa affinity and selectivity. AGS' Chem Biol. 2013 Feb 15;8(2):368-75. doi: 10.1021 / cb300455f.(22) Nimmerjahn, F., Ravetch, J. V. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008 Jan; 8(l):34-47. doi: 10.1038 / nri2206. PMID: 18064051.(23) Shen, L., etal. The functional properties of Fc gamma RI, II and III on myeloid cells: a comparative study of killing of erythrocytes and tumor cells mediated through the different Fc receptors. Mol Immunol. 1989 Oct;26(10):959-69. doi: 10.1016 / 0161-5890(89)90114-4.(24) Richards, J O., et al. (2014). Correlations between changes in conformational dynamics and physical stability in a mutant IgGl mAb engineered for extended serum half-life. mAbs, 6(5): 1235-1247.(25) Robbie, G. J., etal., A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults. Antimicrob Agents Chemother. 2013 Dec;57(12):6147-53. doi: 10.1128 / AAC.01285-13.(26) Ison, M. G., et al., for the EVADE Study Group, Prevention of COVED- 19 Following a Single Intramuscular Administration of Adintrevimab: Results From a Phase 2 / 3 Randomized, Double-Blind, Placebo-Controlled Trial (EVADE), Open Forum Infectious Diseases, Volume 10, Issue 7, July 2023, ofad314, doi.org / 10.1093 / ofid / ofad31475US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(27) Fukuzawa T, el al., Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases. Sei Rep. 2017 Apr 24;7(l):1080. doi: 10.1038 / s41598-017-01087-7(28) Schmidt P, el al. Antibody-mediated protection against symptomatic COVID-19 can be achieved at low serum neutralizing titers. Sei Transl Med. 2023 Mar 22;15(688):eadg2783. doi: 10.1126 / scitranslmed.adg2783, Epub 2023 Mar 22.(29) Lefranc MP, Lefrane G. Using IMGT unique numbering for IG allotypes and Fc-engineered variants of effector properti es and half-life of therapeutic antibodies. Immunol Rev, 2024 Nov; 328(l):473-506. doi: 10.1111 / imr.13399. Epub 2024 Oct 4.76US2008 32326720 I

Claims

Attorney Docket No.: 11022L1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355WHAT IS CLAIMED IS:

1. A polypeptide comprising a human Fc polypeptide amino acid sequence comprising one or more amino acid substitutions L251 V, L251Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C, wherein the one or more amino acid substitutions are numbered with respect to the human Fc polypeptide ammo acid sequence.

2. The polypeptide of claim 1, wherein the human Fc polypeptide ammo acid sequence is a human IgGFc ammo acid sequence,3. The polypeptide of claim 1 or 2, wherein the one or more amino acid substitutions comprise one or more of V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C.

4. The polypeptide of any one of claims 1 -4, wherein the one or more amino acid substitutions comprise L309G; S426V; S426R; S426R; L309Gand S426V; L309Gand S426R; L309G and N434G; S426V and N434G; S426R and N434G; L309G, S426V, and N434G; or L309G, S426R, and N434G.

5. The polypeptide of any one of claims 1 to 4, wherein the one or more amino acid substitutions further comprise at least one of (a) M252Y, S254T, and T256E, or (b) (i) M428L and (ii) N434S, N434A, or N434G.

6. The polypeptide of any one of claims 1 to 5, wherein the human Fc polypeptide amino acid sequence has at least 90% sequence similarity to SEQ ID NO:1,7. The polypeptide any one of claims 1 to 6, wherein(i) the polypeptide has increased binding affinity’ for human FcRn at pH about 5.5 to about 6.5, as compared to the polypeptide without the one or more amino acid substitutions;(ii) the polypeptide has decreased binding affinity7for human FcRn at pH about 5.5 to about 6.5, as compared to the polypeptide without the one or more amino acid substitutions; or 77US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(iti) the polypeptide has substantially unaltered binding affinity for human FcRn at. pH about 5.5 to about 6.5, as compared to the polypeptide without the one or more amino acid substitutions.

8. The polypeptide any one of claims 1 to 7, wherein:(i) the polypeptide has a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions;(ii) the polypeptide has a slower dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions; or (iii) the polypeptide has a substantially unaltered dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions.

9. The polypeptide any one of claims 1 to 8, wherein:(i) polypeptide has increased binding affinity for human FcRn at pH about 5.5 to about 6.5, as compared to the polypeptide without the one or more amino acid substitutions;(ii) polypeptide has a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more ammo acid substitution;(iii) polypeptide has increased binding affinity for human FcRn at pH about 5.5 to about 6.5, and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions;(iv) the polypeptide has increased binding affinity for human FcRn at pH about 5.5 to about 6.5, and a substantially unaltered dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions; or (v) the polypeptide has a substantially unaltered binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide has altered binding to one or more human FcyRs, as compared to the polypeptide without the one or more amino acid substitutions.78US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-35511. The polypeptide of any one of claims 1 to 10, wherein the polypepti de is linked to at least one other polypeptide and / or to at least one non-peptide moiety.

12. The polypeptide of claim 11, wherein the at least one non-peptide moiety is one or more of a radioactive isotope, a molecule comprising a radioactive label, fluorophore, a drug molecule, a toxin, or a nucleic acid.

13. A molecule or a molecular complex comprising the polypeptide of any one of claims 1 to 12.

14. The molecule or the molecular complex of claim 13, wherein the molecule or the molecular complex is an antibody or the antibody fragment.

15. The molecule or the molecular complex of claim 13 or 14, wherein the molecule or the molecular complex is linked to at least one other polypeptide and / or to at least one non-peptide moiety.

16. The molecule or the molecular complex of any one of claims 13 to 15, wherein the at least one non-peptide moiety is one or more of a radioactive isotope, a molecule comprising a radioactive label, fluorophore, a drug molecule, a toxin, or a nucleic acid.

17. A recombinant nucleic acid molecule encoding the polypeptide of any one ofclaims 1 to 12.

18. The recombinant nucleic acid molecule of claim 17, wherein the recombinant nucleic acid molecule is a synthetic sequence designed for expression in a host cell.

19. A DNA construct comprising the recombinant nucleic acid molecule of claim 17 or 18 operably linked to a promoter.

20. A vector comprising the recombinant nucleic acid molecule of claim 17 or 18 or the DN A construct of claim 18.

21. A host cell comprising the recombinant nucleic acid molecule of claim 17 or 18, the DN A construct of claim 19, or the vector of claim 20.79US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-35522. The host, cell of claim 21, wherein said host, cell is a bacterial cell,23. The host cell of claim 22, wherein said host cell is a eukaryotic cell,24. A composition comprising the polypeptide of any one of claims 1 to 12, the molecule or the molecular complex of any one of claims 13 to 15, the recombinant nucleic acid molecule of claim 17 or 18, the DNA construct of claim 19, or the vector of claim 20, and a pharmaceutically acceptable carrier.

25. A method of treating a subject with a disease or a condition, comprising administering to the subject the composition of claim 24 in an amount effective to alleviate the disease or the condition.

26. The method of claim 25, wherein the disease or the condition is one or more of an autoimmune disease or condition, a genetic disease or condition, a cancer, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a gastrointestinal disease or condition, an endocrine disease or condition, a neurological disease or condition, an ophthalmological disease or condition, a musculoskeletal disease or condition, or an infection.

27. A method of modifying a polypeptide comprising a human Fc polypeptide amino acid sequence, the method comprisingintroducing into the human Fc polypeptide amino acid sequence one or more amino acid substitutions L251V, L251 Y, M252D, S254T, V266L, H268K, H268S, N276S, A287L, A287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421V, S426V, S426R, E430R, E432S, L432C, H433K, N434G, or L441C, wherein the one or more amino acid substitutions are numbered with respect to the human Fc polypeptide ammo acid sequence, thereby producing a modified human Fc polypeptide amino acid sequence,wherein a polypeptide comprising the modified human Fc polypeptide ammo acid sequence has at least one of an altered in vivo half-life, binding affinity for human FcRn, or binding affinity for one or more human FcyRs, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions.80US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-35528. The method of claim 27, wherein the altered in vivo half-life is increased half-life.

29. The method of claim 27, wherein the altered in vivo half-life is reduced half-life,30. The method of claim 27, wherein the binding affinity for human FcRn is increased binding affinity.

31. The method of claim 27, wherein the binding affinity for human FcRn is decreased binding affinity.

32. The method of claim 27, wherein the polypeptide comprising the modified human Fc polypeptide amino acid sequence has:(i) increased binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to the polypeptide comprising human Fc polypeptide ammo acid sequence without the one or more amino acid substitutions;(ii) decreased binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions; or(ii) substantially unaltered binding affinity for human FcRn at pH from about 5.5 to about 6.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions.

33. The method of claim 27 wherein the dissociation rate for human FcRn is increased.

34. The method of claim 27 wherein the dissociation rate for human FcRn is decreased.

35. The method of claim 27, wherein the polypeptide comprising the modified human Fc polypeptide amino acid sequence has:(i) an increased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions;(ii) a decreased dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions; or81US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-355(iii) a substantially unaltered dissociation rate at human FcRn at pH from about 7.0 to about 7.5, as compared to the polypeptide comprising human Fc polypeptide amino acid sequence without the one or more amino acid substitutions.

36. The method of claim 27, wherein the polypeptide comprising the modified human Fc polypeptide amino acid sequence has:(i) increased binding affinity for human FcRn at pH about 5.5 to about 6.5, as compared to the polypeptide without the one or more amino acid substitutions;(ii) a faster dissociation rate from human FcRn at pH about 7.0 to about 7,5, as compared to the polypeptide without the one or more amino acid substitution;(iii) increased binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions;(iv) increased binding affinity for human FcRn at pH about 5.5 to about 6.5 and a substantially unaltered dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions; or(v) a substantially unaltered binding affinity for human FcRn at pH about 5.5 to about 6.5 and a faster dissociation rate from human FcRn at pH about 7.0 to about 7.5, as compared to the polypeptide without the one or more amino acid substitutions.

37. The method of any one of claims 27 to 36, wherein the polypeptide comprising the modified human Fc polypeptide amino acid sequence has altered binding to FcyRs, as compared to the polypeptide without the one or more amino acid substitutions.

38. The method of any one of claims 27 to 37, wherein the human Fc polypeptide ammo acid sequence has at least 90% sequence similarity to SEQ ID NO: 1.

39. The method of any one of claims 27 to 38, wherein the one or more amino acid substitutions comprise one or more of V266L, H268K, H268S, N276S, A287L, / X287M, V302L, S304A, T307N, L309G, K320T, K320S, N325S, Q342P, Y349T, Y349Q, R416T, R416V, N421E, N421 V, S426V, S426R, E430R, L432S, L432C, H433K, N434G, or L441C.82US2008 32326720 IAttorney Docket No.: 110221-1521809-012010WO Applicant Ref. Nos: CZB-318S-PC / S24-35540. The method of any one of claims 27 to 39, wherein the one or more amino acid substitutions comprise L309G; S426V; S426R; S426R; L309G and S426V; L309G and S426R; L309G and N434G; S426V and N434G; S426R and N434G; L309G, S426V, and N434G; or L309G, S426R, and N434G.

41. The method of any one of claims 27 to 40, wherein the one or more amino acid substitutions comprise at least one of (a) M252Y, S254T, and T256E, or (b) (i) M428L and (ii) N434S, N434A, or N434G.83US2008 32326720 I

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