Engineered Polypeptides

Abstract

Provided herein are engineered polypeptides (e.g., Fc polypeptides, Fc polypeptide fragments, Fc fusion proteins, antibodies, etc.) that include variants of IgG Fc polypeptides (or portions or fragments thereof), which variants (and polypeptides that include these variants) have one or more improved characteristics compared to known Fc polypeptides.

Description

[Technical Field]

[0001] Array list declarations The Sequence Listing accompanying this application has been submitted in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing this Sequence Listing is named 930585_422WO_SEQUENCE_LISTING.txt. This text file is 71.6 kB, was created on May 20, 2022, and is being submitted electronically via EFS-Web. [Background technology]

[0002] Therapies involving antibodies and other molecules containing the immunoglobulin Fc domain are being developed. Fc can interact with proteins of the immune system (e.g., FcγR and complement C1q), and the nature of such interactions can provide different outcomes (e.g., activation or suppression of the host immune response against pathogens). Summary of the Invention

[0003] Provided herein are engineered polypeptides (e.g., Fc polypeptides, Fc polypeptide fragments, Fc fusion proteins, antibodies, etc.) comprising variants of IgG Fc polypeptides (or portions or fragments thereof), which variants (and polypeptides comprising these variants) have one or more improved characteristics compared to known Fc polypeptides (e.g., reference wild-type Fc polypeptides and / or known variant Fc polypeptides) or polypeptides comprising known Fc polypeptides. Polypeptides of the disclosure possess, for example, increased binding to one or more human FcγRAs (e.g., increased binding to FcγRIIA and / or FcγRIIIA; decreased / reduced binding to human FcγRIIB; increased binding to one or more human FcγRAs relative to binding to human FcγRIIB; increased thermal stability relative to known Fc polypeptides; increased binding to human C1q; increased human FcγRIIIA signaling in host cells expressing FcγRIIIA relative to FcγRIIB, increased human FcγRIIIA signaling in host cells expressing FcγRIIA, decreased human FcγRIIB signaling in host cells expressing FcγRIIB, relatively increased binding to FcγRAs, improved production quality relative to known Fc polypeptides; and combinations of such features.

[0004] In certain embodiments, antibodies comprising variant Fc polypeptides of the present disclosure provide surprising advantages, such as any one or more of the following: increased binding affinity to one or more human FcγRAs (e.g., as determined by surface plasmon resonance (e.g., using a Biacore instrument) and / or electrochemiluminescence assays (e.g., meso scale discovery (MSD) assays)) compared to antibodies comprising a reference Fc polypeptide that does not contain the mutations and / or fucosylation state, and / or increased signaling induced by one or more human FcγRAs (e.g., as determined using (1) an Fc variant antibody, (2) a target cell expressing the antigen, and (3) a reporter cell expressing one or more human FcγRAs and optionally driving expression of a reporter gene (e.g., GFP or luciferase); decreased binding affinity to human FcγRIIB and / or increased signaling induced by human FcγRIIB compared to antibodies comprising a reference Fc polypeptide that does not contain the mutations and / or fucosylation state. a unique and optionally improved binding profile to all of human FcγRIIA-H, human FcγRIIA-R, human FcγRIIB, human FcγRIIIA-F, and human FcγRIIIA-V compared to an antibody comprising a reference Fc polypeptide that does not contain the mutation and / or fucosylation state (wherein improved binding includes an overall increase in binding to FcγRA and / or activation of FcγRA signaling compared to binding to inhibitory FcγRs and / or activation of inhibitory FcγR signaling); an increased binding affinity to human C1q compared to an antibody comprising a reference Fc polypeptide that does not contain the mutation and / or fucosylation state;the variant Fc polypeptide or fragment thereof that does not contain the mutations and / or fucosylation state has no or substantially no detrimental effect on thermal stability, or a reduced negative effect on thermal stability (e.g., a human IgG1 Fc comprising the mutations G236A, A330L, and I332E (e.g., a smaller or no reduction in melting point compared to an antibody comprising a human IgG1 Fc comprising the mutations G236A, A330L, and I332E), or has a higher melting point than an antibody comprising a human IgG1 Fc comprising the mutations G236A, A330L, and I332E); the variant Fc polypeptide or fragment thereof that does not contain the mutations and / or fucosylation state has no or substantially no detrimental effect on thermal stability, or has a reduced negative effect on thermal stability (e.g., a human IgG1 Fc comprising the mutations G236A, A330L, and I332E (e.g., a smaller or no reduction in melting point compared to an antibody comprising a human IgG1 Fc comprising the mutations G236A, A330L, and I332E)); the variant Fc polypeptide or fragment thereof that does not contain the mutations and / or fucosylation state has a higher melting point compared to an antibody comprising a reference Fc polypeptide that does not contain the mutations and / or fucosylation state (e.g., a human IgG1 Fc comprising the mutations G236A, A330L, and I332E); and / or FcγRIIIA) specifically lyses (e.g., via ADCC) antigen-expressing target cells by PBMCs (e.g., antibodies comprising a human IgG1 Fc comprising the mutations G236A, A330L, and I332E); or specifically lyses (e.g., via ADCC) antigen-expressing target cells by monocytes (e.g., optionally expressing FcγRIIIA and / or FcγRIIIA) compared to antibodies comprising a reference Fc polypeptide that does not contain the mutations and / or fucosylation state. increased ADCP by CD14+ monocytes expressing FcγRIIA against target cells expressing the antigen; when provided in combination with the antigen, increased percentage of CD83+ cells (e.g., moDCs) and / or CD83 expression by moDCs in a sample compared to an antibody comprising a reference Fc polypeptide that does not have the mutation and / or fucosylation state when provided in combination with the antigen; when provided in combination with the antigen, increased production of one or more cytokines (optionally selected from the group consisting of IL-1β, IFN-γ, IL-6, and TNF-α) by moDCs in a sample compared to an antibody comprising a reference Fc polypeptide that does not have the mutation and / or fucosylation state when provided in combination with the antigen;and / or when provided in combination with an antigen, the ability of the moDCs to stimulate antigen-specific CD4+ T cells is increased compared to an antibody comprising a reference Fc polypeptide that does not comprise the mutation and / or fucosylation state when provided in combination with the antigen to the moDCs (where optionally (1) the moDCs and CD4+ T cells are derived from the same subject (optionally inoculated with the antigen), and / or (2) stimulation of the antigen-specific CD4+ T cells is determined by increased CD25 expression by the antigen-specific CD4+ T cells, and / or increased proliferation (e.g., as determined by decreased CFSE staining over time), and / or increased expression of CD69, and / or increased expression of NFAT, and / or increased expression of CD44);

[0005] In some embodiments, an engineered Fc or Fc fragment (or polypeptide comprising same) of the present disclosure comprises two or more substitution mutations relative to a reference wild-type Fc or Fc fragment, wherein the combined effect of said two or more substitutions is different from, and possibly greater than, that predicted based on the effect of the individual substitution mutations and / or the effect of a subset of said two or more substitution mutations. In other words, in some embodiments, the combined mutations comprise a non-additive or synergistic effect relative to the individual mutations and / or subsets thereof.

[0006] In some embodiments, variants and polypeptides of the present disclosure comprising same have characteristics such as effector function, ability to bind human C1q, ability to induce cell signaling via FcγRA, ability to bind human FcRn, ability to promote ADCP, ability to promote ADCC, ability to promote CD4+ T cell activation, etc. In some embodiments, engineered polypeptides of the present disclosure comprise antibodies, Fc fusion proteins, or complexes comprising same. Antibodies comprising a variant IgG Fc according to the present disclosure are also provided.

[0007] In certain embodiments, the polypeptides and antibodies of the disclosure have one or more altered characteristics (e.g., increased binding to human FcγRa, decreased binding to human FcγRIIb, increased binding to human FcγRa compared to binding to FcγRIIb, increased binding to human C1q, increased binding to human FcRn, increased Tm, increased binding to FcγRIIIa, or any combination thereof, compared to a reference polypeptide or antibody comprising a variant Fc comprising the following mutations: G236A; G236S; G236A / A330L / I332E; G236A / A330L / I332E / M428L / N434S; G236A / A330L / I332E / M428L / N434A; G236A / S239D / A330L / I332E; or A330L / I332E).

[0008] Related polynucleotides, vectors, host cells, and compositions are also provided.

[0009] The compositions and methods of the present disclosure are useful in various embodiments for the treatment and / or prevention of disease. In some embodiments, the compositions of the present disclosure can be administered at any stage of disease (e.g., during the early stages of infection, during the later stages of infection, when infection is established, or at any other time during infection) to protect against and / or neutralize infection, promote elimination of infected cells, prevent the spread of infection, stimulate host adaptive anti-infection immunity, etc. [Brief explanation of the drawings]

[0010] [Figure 1A]Figures 1A–1E demonstrate that antibodies with certain Fc modifications offer improved protection against influenza A. Figure 1A illustrates the study design. Mice engineered to express human FcγRs were intranasally infected with a lethal dose of H1N1 PR8 and intravenously administered antibodies with variant Fc. Serum IgG levels were assessed at the time of infection (day 0), and mouse weight and survival were assessed over a 14-day period. Figure 1B shows the maximum change in body weight in mice pretreated with the anti-FluA IgG1 antibody "F18" carrying the G236A / A330L / I332E / M428L / N434S ("F18-LS-GAALIE") Fc mutations compared to mice pretreated with the F18 antibody ("F18-LS") carrying only the M428L / N434S mutations in the Fc. Weight change (Figure 1C) and survival (Figure 1D) were also assessed in mice treated with antibodies harboring the following Fc mutations: G236A ("GA"), A330L / I332E ("ALIE"), G236A / A330L / I332E ("GAALIE"), or G237D / H268D / P271G / A330R ("V11"). Mice treated with antibodies harboring wild-type or defucosylated wild-type Fc, or PBS, served as controls. The effects of Fc mutations on antibody binding to FcγRI, FcγRIIa, and FcγRIIb, as well as the ratio of FcγRIIIa to FcγRIIIb binding, are shown in the legend to Figure 1C. Figure 1E summarizes the results for binding to various FcγRs by the indicated Fc variants. [Figure 1B]Figures 1A–1E demonstrate that antibodies with certain Fc modifications offer improved protection against influenza A. Figure 1A illustrates the study design. Mice engineered to express human FcγRs were intranasally infected with a lethal dose of H1N1 PR8 and intravenously administered antibodies with variant Fc. Serum IgG levels were assessed at the time of infection (day 0), and mouse weight and survival were assessed over a 14-day period. Figure 1B shows the maximum change in body weight in mice pretreated with the anti-FluA IgG1 antibody "F18" carrying the G236A / A330L / I332E / M428L / N434S ("F18-LS-GAALIE") Fc mutations compared to mice pretreated with the F18 antibody ("F18-LS") carrying only the M428L / N434S mutations in the Fc. Weight change (Figure 1C) and survival (Figure 1D) were also assessed in mice treated with antibodies harboring the following Fc mutations: G236A ("GA"), A330L / I332E ("ALIE"), G236A / A330L / I332E ("GAALIE"), or G237D / H268D / P271G / A330R ("V11"). Mice treated with antibodies harboring wild-type or defucosylated wild-type Fc, or PBS, served as controls. The effects of Fc mutations on antibody binding to FcγRI, FcγRIIa, and FcγRIIb, as well as the ratio of FcγRIIIa to FcγRIIIb binding, are shown in the legend to Figure 1C. Figure 1E summarizes the results for binding to various FcγRs by the indicated Fc variants. [Figure 1C]Figures 1A–1E demonstrate that antibodies with certain Fc modifications offer improved protection against influenza A. Figure 1A illustrates the study design. Mice engineered to express human FcγRs were intranasally infected with a lethal dose of H1N1 PR8 and intravenously administered antibodies with variant Fc. Serum IgG levels were assessed at the time of infection (day 0), and mouse weight and survival were assessed over a 14-day period. Figure 1B shows the maximum change in body weight in mice pretreated with the anti-FluA IgG1 antibody "F18" carrying the G236A / A330L / I332E / M428L / N434S ("F18-LS-GAALIE") Fc mutations compared to mice pretreated with the F18 antibody ("F18-LS") carrying only the M428L / N434S mutations in the Fc. Weight change (Figure 1C) and survival (Figure 1D) were also assessed in mice treated with antibodies harboring the following Fc mutations: G236A ("GA"), A330L / I332E ("ALIE"), G236A / A330L / I332E ("GAALIE"), or G237D / H268D / P271G / A330R ("V11"). Mice treated with antibodies harboring wild-type or defucosylated wild-type Fc, or PBS, served as controls. The effects of Fc mutations on antibody binding to FcγRI, FcγRIIa, and FcγRIIb, as well as the ratio of FcγRIIIa to FcγRIIIb binding, are shown in the legend to Figure 1C. Figure 1E summarizes the results for binding to various FcγRs by the indicated Fc variants. [Figure 1D]Figures 1A–1E demonstrate that antibodies with certain Fc modifications offer improved protection against influenza A. Figure 1A illustrates the study design. Mice engineered to express human FcγRs were intranasally infected with a lethal dose of H1N1 PR8 and intravenously administered antibodies with variant Fc. Serum IgG levels were assessed at the time of infection (day 0), and mouse weight and survival were assessed over a 14-day period. Figure 1B shows the maximum change in body weight in mice pretreated with the anti-FluA IgG1 antibody "F18" carrying the G236A / A330L / I332E / M428L / N434S ("F18-LS-GAALIE") Fc mutations compared to mice pretreated with the F18 antibody ("F18-LS") carrying only the M428L / N434S mutations in the Fc. Weight change (Figure 1C) and survival (Figure 1D) were also assessed in mice treated with antibodies harboring the following Fc mutations: G236A ("GA"), A330L / I332E ("ALIE"), G236A / A330L / I332E ("GAALIE"), or G237D / H268D / P271G / A330R ("V11"). Mice treated with antibodies harboring wild-type or defucosylated wild-type Fc, or PBS, served as controls. The effects of Fc mutations on antibody binding to FcγRI, FcγRIIa, and FcγRIIb, as well as the ratio of FcγRIIIa to FcγRIIIb binding, are shown in the legend to Figure 1C. Figure 1E summarizes the results for binding to various FcγRs by the indicated Fc variants. [Figure 1E]Figures 1A–1E demonstrate that antibodies with certain Fc modifications offer improved protection against influenza A. Figure 1A illustrates the study design. Mice engineered to express human FcγRs were intranasally infected with a lethal dose of H1N1 PR8 and intravenously administered antibodies with variant Fc. Serum IgG levels were assessed at the time of infection (day 0), and mouse weight and survival were assessed over a 14-day period. Figure 1B shows the maximum change in body weight in mice pretreated with the anti-FluA IgG1 antibody "F18" carrying the G236A / A330L / I332E / M428L / N434S ("F18-LS-GAALIE") Fc mutations compared to mice pretreated with the F18 antibody ("F18-LS") carrying only the M428L / N434S mutations in the Fc. Weight change (Figure 1C) and survival (Figure 1D) were also assessed in mice treated with antibodies harboring the following Fc mutations: G236A ("GA"), A330L / I332E ("ALIE"), G236A / A330L / I332E ("GAALIE"), or G237D / H268D / P271G / A330R ("V11"). Mice treated with antibodies harboring wild-type or defucosylated wild-type Fc, or PBS, served as controls. The effects of Fc mutations on antibody binding to FcγRI, FcγRIIa, and FcγRIIb, as well as the ratio of FcγRIIIa to FcγRIIIb binding, are shown in the legend to Figure 1C. Figure 1E summarizes the results for binding to various FcγRs by the indicated Fc variants.

[0011] [Figure 2] FIG. 2 shows predicted binding affinities of certain Fc variant antibodies to FcγRIIa (R131 allele) and FcγRIIb.

[0012] [Figure 3]Figure 3 shows the binding affinity (measured by a meso-scale discovery-based assay (MSD; using electrochemiluminescence)) of certain IgG1 Fc variant antibodies to FcγR and C1q, as well as other characteristics. The Fc variants shown in the third row and following ("G236A_E272Y_S298N" and following) were identified using an iterative discovery workflow. The G236A_A330L_I332E variant was used for comparison. Binding of the Fc variant antibodies to FcγRIIA-H (high-affinity H158 allele), FcγRIIB, FcγRIIA-R (low-affinity R131 allele), FcγRIIIA-V (high-affinity V158 allele), FcγRIIIA-F (low-affinity F158 allele), FcγRIIIB, and FcRn was examined. Data are reported as fold-change in binding compared to wild-type IgG1. The ratio of FcγRIIA / FcγRIIB binding is shown, as well as the titer (mg / mL) and Tm (°C) produced relative to wild-type IgG1.

[0013] [Figure 4A] Figures 4A-4C show the effect of fucosylation on the production and purification of 20 Fc variant antibodies. The variants were expressed in the absence ("without 2FF") or presence ("with 2FF") of 2-deoxy-2-fluoro-L-fucose (2FF); 2FF reduces fucosylation. Figure 4A shows the antibody titers determined using a Protein A column. Figure 4B shows the yields obtained from two replicate purifications. The table in Figure 4C summarizes the theoretical maximum yield and average yield (both measured in μg), as well as the calculated average recovery and protein concentration of the second eluent (measured in μg / ml). The Fc variants were purified using the two eluents and combined, after which the yields were determined. [Figure 4B]Figures 4A-4C show the effect of fucosylation on the production and purification of 20 Fc variant antibodies. The variants were expressed in the absence ("without 2FF") or presence ("with 2FF") of 2-deoxy-2-fluoro-L-fucose (2FF); 2FF reduces fucosylation. Figure 4A shows the antibody titers determined using a Protein A column. Figure 4B shows the yields obtained from two replicate purifications. The table in Figure 4C summarizes the theoretical maximum yield and average yield (both measured in μg), as well as the calculated average recovery and protein concentration of the second eluent (measured in μg / ml). The Fc variants were purified using the two eluents and combined, after which the yields were determined. [Figure 4C] Figures 4A-4C show the effect of fucosylation on the production and purification of 20 Fc variant antibodies. The variants were expressed in the absence ("without 2FF") or presence ("with 2FF") of 2-deoxy-2-fluoro-L-fucose (2FF); 2FF reduces fucosylation. Figure 4A shows the antibody titers determined using a Protein A column. Figure 4B shows the yields obtained from two replicate purifications. The table in Figure 4C summarizes the theoretical maximum yield and average yield (both measured in μg), as well as the calculated average recovery and protein concentration of the second eluent (measured in μg / ml). The Fc variants were purified using the two eluents and combined, after which the yields were determined.

[0014] [Figure 5] Figure 5 shows a representative absolute size exclusion chromatography analysis of purified Fc variant antibodies. The single peak on the left was typical of the variants investigated, whereas the double peak on the right indicates a variant in which low molecular weight species (LMWS) were observed.

[0015] [Figure 6A] Figures 6A and 6B show the Tm curves of antibodies with wild-type Fc (6A) or R292P variant Fc (6B). [Figure 6B] Figures 6A and 6B show the Tm curves of antibodies with wild-type Fc (6A) or R292P variant Fc (6B).

[0016] [Figure 7A] Figures 7A and 7B summarize the FcγR binding and other characteristics of the Fc variants compared to wild-type Fc. Bars and numbers indicate the fold change in binding compared to wild-type Fc. The indicated Fc variants were not treated with 2FF. Figure 7A shows binding (at pH 6) to FcγRIIA-H (high affinity), FcγRIIA-R (low affinity), FcγRIIB, FcγRIIIA-V (high affinity), FcγRIIIA-F (low affinity), and FcRn. Figure 7B further shows the ratio of FcγRIIA-H / FcγRIIB binding, as well as C1q binding and complement-dependent cytotoxicity (CDC), with the wild-type "baseline" value indicated by the vertical red dotted line. Binding was measured by a mesoscale discovery-based assay (MSD; utilizing electrochemiluminescence). [Figure 7B] Figures 7A and 7B summarize the FcγR binding and other characteristics of the Fc variants compared to wild-type Fc. Bars and numbers indicate the fold change in binding compared to wild-type Fc. The indicated Fc variants were not treated with 2FF. Figure 7A shows binding (at pH 6) to FcγRIIA-H (high affinity), FcγRIIA-R (low affinity), FcγRIIB, FcγRIIIA-V (high affinity), FcγRIIIA-F (low affinity), and FcRn. Figure 7B further shows the ratio of FcγRIIA-H / FcγRIIB binding, as well as C1q binding and complement-dependent cytotoxicity (CDC), with the wild-type "baseline" value indicated by the vertical red dotted line. Binding was measured by a mesoscale discovery-based assay (MSD; utilizing electrochemiluminescence).

[0017] [Figure 8] Figure 8 shows binding of certain Fc variants to FcγRIIA-H (high affinity) and FcγRIIB. Plots connected by lines represent the same variant. The variants shown were not treated with 2FF.

[0018] [Figure 9A] Figures 9A-9B show FcγR signaling through different FcγRs as measured using a reporter cell assay (Promega; cells examined expressed one type / allele of FcγR as indicated). The Fc variants shown are fucosylated ("fuc"; 9A / 9B) or defucosylated ("afuc"; 9B) as indicated in the figures. Values ​​were calculated from the average of three experiments and represent the fold change (shown linearly) in the area under the curve (plotted logarithmically) compared to wild-type Fc. [Figure 9B] Figures 9A-9B show FcγR signaling through different FcγRs as measured using a reporter cell assay (Promega; cells examined expressed one type / allele of FcγR as indicated). The Fc variants shown are fucosylated ("fuc"; 9A / 9B) or defucosylated ("afuc"; 9B) as indicated in the figures. Values ​​were calculated from the average of three experiments and represent the fold change (shown linearly) in the area under the curve (plotted logarithmically) compared to wild-type Fc.

[0019] [Figure 10A] Figures 10A-1 to 10C summarize the characteristics of certain variant Fc. Antibodies containing the indicated Fc were expressed as recombinant human IgG1. The variants shown in Figures 10B-1 to 10B-4 are defucosylated. Binding was measured by a meso-scale discovery-based assay (MSD; using electrochemiluminescence). Values ​​represent the fold change compared to antibodies containing wild-type fucosylated human IgG1 Fc. Figures 10A-3, 10A-4, 10B-3, and 10B-4 also show the fold change in FcγR signaling measured using a reporter cell assay. [Figure 10B]Figures 10A-1 to 10C summarize the characteristics of certain variant Fc. Antibodies containing the indicated Fc were expressed as recombinant human IgG1. The variants shown in Figures 10B-1 to 10B-4 are defucosylated. Binding was measured by a meso-scale discovery-based assay (MSD; using electrochemiluminescence). Values ​​represent the fold change compared to antibodies containing wild-type fucosylated human IgG1 Fc. Figures 10A-3, 10A-4, 10B-3, and 10B-4 also show the fold change in FcγR signaling measured using a reporter cell assay. [Figure 10C] Figures 10A-1 to 10C summarize the characteristics of certain variant Fc. Antibodies containing the indicated Fc were expressed as recombinant human IgG1. The variants shown in Figures 10B-1 to 10B-4 are defucosylated. Binding was measured by a meso-scale discovery-based assay (MSD; using electrochemiluminescence). Values ​​represent the fold change compared to antibodies containing wild-type fucosylated human IgG1 Fc. Figures 10A-3, 10A-4, 10B-3, and 10B-4 also show the fold change in FcγR signaling measured using a reporter cell assay.

[0020] [Figure 11] Figure 11 shows (left) a schematic of the Meso Scale Discovery binding assay for assessing binding of Fc variant antibodies to FcγRs and (right) a schematic of the cellular reporter assay for measuring Fc variant antibody-induced FcγR-mediated cell signaling.

[0021] [Figure 12A] Figures 12A-12B show FcγR signaling by the Fc variant "G236A_R292P_Y300L" through FcγRIIA-H (high affinity, Figure 12A) and FcγRIIB (Figure 12B) as measured using a reporter cell assay. [Figure 12B]Figures 12A-12B show FcγR signaling by the Fc variant "G236A_R292P_Y300L" through FcγRIIA-H (high affinity, Figure 12A) and FcγRIIB (Figure 12B) as measured using a reporter cell assay.

[0022] [Figure 13A] Figures 13A-13B show the relationship between Fc variant binding to FcγR and signaling through FcγRIIA-H (high affinity, Figure 13A) and FcγRIIB (Figure 13B). Binding to FcγR was measured using a Meso Scale Discovery binding assay, and FcγR signaling was measured using a reporter cell assay (Promega). [Figure 13B] Figures 13A-13B show the relationship between Fc variant binding to FcγR and signaling through FcγRIIA-H (high affinity, Figure 13A) and FcγRIIB (Figure 13B). Binding to FcγR was measured using a Meso Scale Discovery binding assay, and FcγR signaling was measured using a reporter cell assay (Promega).

[0023] [Figure 14A] Figures 14A-14B show activation of Jurkat cells expressing human FcγRIIA (H131) (A) or FcγRIIIA (F158) (B) by the variant Fc-containing anti-flu HA antibody FM08 using the A549-CA cell line stably expressing influenza CA-2009-H1N1 HA. Figure 14C shows NK cell-mediated ADCC by the antibody against target A549-CA cells stably expressing influenza CA-2009-H1N1 HA in an LDH release assay, using isolated effector NK cells (HM_WB014_FF) at an E:T ratio of 6:1. [Figure 14B]Figures 14A-14B show activation of Jurkat cells expressing human FcγRIIA (H131) (A) or FcγRIIIA (F158) (B) by the variant Fc-containing anti-flu HA antibody FM08 using the A549-CA cell line stably expressing influenza CA-2009-H1N1 HA. Figure 14C shows NK cell-mediated ADCC by the antibody against target A549-CA cells stably expressing influenza CA-2009-H1N1 HA in an LDH release assay, using isolated effector NK cells (HM_WB014_FF) at an E:T ratio of 6:1. [Figure 14C] Figures 14A-14B show activation of Jurkat cells expressing human FcγRIIA (H131) (A) or FcγRIIIA (F158) (B) by the variant Fc-containing anti-flu HA antibody FM08 using the A549-CA cell line stably expressing influenza CA-2009-H1N1 HA. Figure 14C shows NK cell-mediated ADCC by the antibody against target A549-CA cells stably expressing influenza CA-2009-H1N1 HA in an LDH release assay, using isolated effector NK cells (HM_WB014_FF) at an E:T ratio of 6:1.

[0024] [Figure 15] Figure 15 summarizes results from a C1q binding assay using the indicated FY1 (anti-flu hemagglutinin ("HA") stem; Kallewaard et al. Cell 166(3):596-608 (2016)) Fc variant antibodies. Assay: Binding of antibody Fc to human C1q in solution (randomly captured on the surface of an Octet sensor).

[0025] [Figure 16A]Figures 16A-16B show activation of Jurkat cells expressing human FcγRIIA (H131) (A) or FcγRIIIA (F158) (B) by the variant Fc-containing anti-HBsAg antibody HBC34v35 using a target cell line stably expressing HBsAg. [Figure 16B] Figures 16A-16B show activation of Jurkat cells expressing human FcγRIIA (H131) (A) or FcγRIIIA (F158) (B) by the variant Fc-containing anti-HBsAg antibody HBC34v35 using a target cell line stably expressing HBsAg.

[0026] [Figure 17A] Figures 17A-17D show results from replicate experiments of the experiments in Figures 16A and 16B. [Figure 17B] Figures 17A-17D show results from replicate experiments of the experiments in Figures 16A and 16B. [Figure 17C] Figures 17A-17D show results from replicate experiments of the experiments in Figures 16A and 16B. [Figure 17D] Figures 17A-17D show results from replicate experiments of the experiments in Figures 16A and 16B.

[0027] In Figures 18-20, the following abbreviations were used for the Fc variants: GA = G236A; GALVQE = G236A_L328V_Q295E; GAYL = G236A_Y300L; GARPYL = G236A_R292P_Y300L; and GARPIN = G236A_R292P_I377N; GAALIE = G236A_A330L_I332E; GRLR = G236R_L328R.

[0028] [Figure 18]Figure 18 shows results from experiments measuring binding to FcγRs; the ratio of FcγRIIA allele binding to FcγRIIB; C1q binding; melting temperature; and FcRn binding by certain Fc variant antibodies. The anti-influenza antibody FY1 was expressed as recombinant IgG1m3 with the mutations M428L and N434S in CH3 and the indicated combination mutations elsewhere in the Fc. Binding (in one study) was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Binding data are presented as fold change relative to FY1 rIgG1m3-MLNS without other Fc mutations. Binding to FcγRs was confirmed by FcγR signaling using a reporter cell assay (luciferase-driven NFAT) (Promega).

[0029] [Figure 19] Figure 19 shows results from additional experiments in which antibody characteristics were measured as in Figure 18. In these experiments, FY1 was expressed as recombinant IgG1m3 (i.e., wild-type IgG1m3 CH1-CH3 or with the mutations indicated in the table) lacking the mutations M428L and N434S. FY1-rIgG1m3 and FY1-rIgG1m3-GAALIE antibodies were generated and assayed twice independently on the first plate; average data are shown. FY1-rIgG1m3-GA antibodies were generated twice independently on the first and second plates. Only single experiments were performed for other variants. Binding was measured using a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Binding data are presented as fold change relative to FY1 rIgG1m3 with wild-type Fc. FcγR-binding / activation was measured using a reporter cell assay (luciferase-driven NFAT) (Promega).

[0030] [Figure 20]Figure 20 shows results from additional experiments using defucosylated Fc variant antibodies characterized as in Figure 19. Antibodies were produced in the presence of 2FF to obtain defucosylated glycans. In these experiments, FY1 was expressed as recombinant IgG1m3 (i.e., wild-type IgG1m3 CH1-CH3 or with the mutations indicated in the table) lacking the mutations M428L and N434S. FY1-rIgG1m3 and FY1-rIgG1m3-GAALIE antibodies were produced and assayed twice independently on the first plate; average data are shown. FY1-rIgG1m3-GA antibodies were produced twice independently on the first and second plates. Only one experiment was performed for the other variants. Binding was measured by a meso-scale discovery-based assay (MSD; using electrochemiluminescence).

[0031] [Figure 21] Figure 21 shows FcγRIIA activation / signaling by anti-influenza FY1 antibodies with variant Fc as indicated in the legend. Target cells were A549 cells expressing FluA H1N1 HA, and reporter cells were Jurkat cells expressing FcγRIIA (H131 allele) and luciferase under the control of the NFAT promoter.

[0032] [Figure 22A] Figures 22A and 22B show FcγRIIIA activation / signaling by anti-influenza FY1 antibodies with variant Fc as indicated in the legend. Target cells were A549 cells expressing FluA H1N1 HA, and reporter cells were Jurkat cells expressing FcγRIIIA (the lower affinity F158 allele (A) or the higher affinity V158 allele (B)) and luciferase under the control of the NFAT promoter. [Figure 22B]Figures 22A and 22B show FcγRIIIA activation / signaling by anti-influenza FY1 antibodies with variant Fc as indicated in the legend. Target cells were A549 cells expressing FluA H1N1 HA, and reporter cells were Jurkat cells expressing FcγRIIIA (the lower affinity F158 allele (A) or the higher affinity V158 allele (B)) and luciferase under the control of the NFAT promoter.

[0033] [Figure 23] Figure 23 shows a schematic diagram illustrating a surface plasmon resonance assay to measure the binding kinetics of FY1 Fc variants (expressed as recombinant rIgG1m3 with M428L and N434S but no other Fc mutations or with additional Fc mutations as indicated) to human FcγR. Briefly, biotinylated FcγR was captured by streptavidin using a CAP chip. FY1 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM. Injections were performed consecutively without regeneration between the same sample at different concentrations. Injection: 600 s. Dissociation time: 100 s between each injection.

[0034] [Figure 24] Figure 24 provides a table showing fold change results (compared to the reference FY1-rIgG1m3-LS antibody) from SPR binding data. N=1. Fold change was calculated by dividing the affinity value determined for FY1-rIgG1m3-LS by the value determined for the Fc variant. A larger fold change represents a lower KD and increased affinity. "-" = no or weak binding measurable.

[0035] [Figure 25A]Figures 25A-25B show FcγR activation / signaling by the anti-SARS-CoV-2 antibody S309, which has a variant Fc, as indicated in the legend. All antibodies contained the Fc mutations M428L and N434S, except for the negative control "S309-GRLR" (containing G236R and L328R Fc mutations). Activation / signaling was measured using CHO cells expressing the SARS-CoV-2 S protein and luciferase reporter cells (Promega) expressing FcγRIIIA (A) or FcγRIIA (B). Figure 25C shows NK cell-mediated ADCC by the S309 Fc variant antibody. Donor PBMCs and S-CHO-HiBit cells were used, as indicated. The curves in Figure 25A, when data points were acquired at an antibody concentration of 104 ng / mL, are in the following order from top to bottom: S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GAPAQE-afuc; S309-LS-GALVQE-afuc to S309-LS-GAALIE to S309-LS-GARPYL; S309-LS; S309-LS-GAYL; S309-LS-GA; S309-LS-GAPAQE to S309-LS-GALVQE to S309-GRLR. In Figure 25B, at an antibody concentration of 104 ng / mL, the bottom curve is S309-GRLR, the second-lowest curve is S309-LS, and the third-lowest curve is S309-LS-GAALIE. In Figure 25C, several Fc variants (S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GARPYL; S309-LS-GALVQE-afuc; S309-LS-GAALIE) were not titratable for ADCC because the signal reached near the maximum / plateau of the assay. For other Fc variants, the ranking order was: S309-LS-GAPAQE-afuc > S309-LS > S309-LS-GA > S309-LS-GAYL > S309-LS-GALVQE > S309-LS-GAPAQE > S309-GRLR. [Figure 25B]Figures 25A-25B show FcγR activation / signaling by the anti-SARS-CoV-2 antibody S309, which has a variant Fc, as indicated in the legend. All antibodies contained the Fc mutations M428L and N434S, except for the negative control "S309-GRLR" (containing G236R and L328R Fc mutations). Activation / signaling was measured using CHO cells expressing the SARS-CoV-2 S protein and luciferase reporter cells (Promega) expressing FcγRIIIA (A) or FcγRIIA (B). Figure 25C shows NK cell-mediated ADCC by the S309 Fc variant antibody. Donor PBMCs and S-CHO-HiBit cells were used, as indicated. The curves in Figure 25A, when data points were acquired at an antibody concentration of 104 ng / mL, are in the following order from top to bottom: S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GAPAQE-afuc; S309-LS-GALVQE-afuc to S309-LS-GAALIE to S309-LS-GARPYL; S309-LS; S309-LS-GAYL; S309-LS-GA; S309-LS-GAPAQE to S309-LS-GALVQE to S309-GRLR. In Figure 25B, at an antibody concentration of 104 ng / mL, the bottom curve is S309-GRLR, the second-lowest curve is S309-LS, and the third-lowest curve is S309-LS-GAALIE. In Figure 25C, several Fc variants (S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GARPYL; S309-LS-GALVQE-afuc; S309-LS-GAALIE) were not titratable for ADCC because the signal reached near the maximum / plateau of the assay. For other Fc variants, the ranking order was: S309-LS-GAPAQE-afuc > S309-LS > S309-LS-GA > S309-LS-GAYL > S309-LS-GALVQE > S309-LS-GAPAQE > S309-GRLR. [Figure 25C]Figures 25A-25B show FcγR activation / signaling by the anti-SARS-CoV-2 antibody S309, which has a variant Fc, as indicated in the legend. All antibodies contained the Fc mutations M428L and N434S, except for the negative control "S309-GRLR" (containing G236R and L328R Fc mutations). Activation / signaling was measured using CHO cells expressing the SARS-CoV-2 S protein and luciferase reporter cells (Promega) expressing FcγRIIIA (A) or FcγRIIA (B). Figure 25C shows NK cell-mediated ADCC by the S309 Fc variant antibody. Donor PBMCs and S-CHO-HiBit cells were used, as indicated. The curves in Figure 25A, when data points were acquired at an antibody concentration of 104 ng / mL, are in the following order from top to bottom: S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GAPAQE-afuc; S309-LS-GALVQE-afuc to S309-LS-GAALIE to S309-LS-GARPYL; S309-LS; S309-LS-GAYL; S309-LS-GA; S309-LS-GAPAQE to S309-LS-GALVQE to S309-GRLR. In Figure 25B, at an antibody concentration of 104 ng / mL, the bottom curve is S309-GRLR, the second-lowest curve is S309-LS, and the third-lowest curve is S309-LS-GAALIE. In Figure 25C, several Fc variants (S309-LS-afuc; S309-LS-GA-afuc; S309-LS-GARPYL; S309-LS-GALVQE-afuc; S309-LS-GAALIE) were not titratable for ADCC because the signal reached near the maximum / plateau of the assay. For other Fc variants, the ranking order was: S309-LS-GAPAQE-afuc > S309-LS > S309-LS-GA > S309-LS-GAYL > S309-LS-GALVQE > S309-LS-GAPAQE > S309-GRLR.

[0036] [Figure 26A]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS. [Figure 26B]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS. [Figure 26C]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS. [Figure 26D]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS. [Figure 26E]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS. [Figure 26F]Figures 26A-26F show FcγR signaling (A-D) and NK cell-mediated killing (E-F) induced by the S309 Fc variant. (A) and (B) show FcγRIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIa (H131 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (A) = fucosylated antibody; (B) = defucosylated antibody. (C) and (D) show FcγRIIIa activation / signaling using Jurkat reporter cells (Promega) expressing FcγRIIIa (V158 allele) driving luciferase expression and CHO cells expressing SARS-CoV-2 spike protein (target cells). (C) = Fucosylated antibodies; (D) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. (E) and (F) show NK cell-mediated ADCC. PBMC / NK donor cells expressing FcγRIIIa (heterozygous V158 / F158) and CHO cells expressing the SARS-CoV-2 spike protein (target cells) were used. (E) = Fucosylated antibodies; (F) = Defucosylated antibodies, excluding the comparator antibodies S309-LS and S309-GRLR. Some Fc variants could not be titrated for ADCC because the signal reached near the maximum / plateau of the assay. In Figure 26E, the lowest curve corresponds to S309-GRLR. In Figure 26F, the second-lowest data point at 104 ng / mL antibody corresponds to S309-LS.

[0037] [Figure 27A]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27B]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27C]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27D]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27E]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27F]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27G]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27H]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27I]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR. [Figure 27J]Figures 27A-27J relate to an anti-SARS-CoV-2 antibody containing a variant Fc. (A)-(C): FcγRIIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. In (A), the top curve corresponds to S309; ​​in (B), Wuhan-Hu-1 spike protein was used, which has a stabilizing mutation that prevents S protein export from target cells. (D)-(F): FcγRIIA activation / signaling measured by luminescence at 23 hours using reporter cells expressing human FcγRIIA to drive luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein, as indicated. (G)–(H): NK cell-mediated ADCC using S-CHO-HiBiT cells (expressing the wild-type SARS-CoV-2 Wuhan-Hu-1 spike sequence) as target cells, as indicated (two NK cell donors, one expressing FcγRIIIA F158 / V158 (G) and the other expressing V158 / V158 (H)). (I)–(J) Monocyte-mediated ADCP using CHO cells expressing the SARS-CoV-2 Wuhan spike protein, as indicated (two monocyte donors, one expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / F158 (I) and the other expressing FcγRIIA R131 / H131 and FcγRIIIA F158 / V158 (J)). The horizontal dotted line at / near the bottom of each graph indicates the lysis value for target cells + effector cells without antibody. In Figure 27B, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259-LS-GA-afuc; S2X259-v5 GAALIE; S2X259-LS-GARPYL; S309; ​​S2X259-LS; S2X259-LS-GA; S2X259-LS-GALVQE ~ S2X259-LS-GALVQE-afuc ~ S309-GRLR ~ S2X259-GRLR.In Figure 27C, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S309; ​​S2X259-LS-GALVQE-afuc; S2X259-LS-GRLR; S2X259-LS-GARPYL; S2X259-LS-GA; S2X259-v5-GAALIE; S2X259-LS; S2X259-LS-GALVQE; S309-GRLR. In Figure 27E, at an antibody concentration of 104 ng / mL, the top five curves are, from top to bottom: S2X259-LS-GALVQE; S2X259-LS-GA; S2X259-LS-GARPYL; S2X259-v5-GAALIE; S2X259-LS-GA-afuc. In Figure 27G, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259-GAALIE; S2X259.1-LS; S2X259.1-LS-GA; S2X259.1-LS-GALVQE; S2X259.1-LS-GRLR. In Figure 27H, at an antibody concentration of 104 ng / mL, the curves are, from top to bottom: S2X259.1-LS-GARPYL; S2X259.1-LS-GALVQE-afuc; S2X259.1-LS-GA-afuc; S2X259.1-LS; S2X259-GAALIE; S2X259.1-LS-GA; S2X259.1-LS-GRLR; S2X259.1-LS-GALVQE. In Figure 27I, at an antibody concentration of 104 ng / mL, the top curve corresponds to S309-DEA and the bottom curve corresponds to S2X259.1-LS-GRLR. In Figure 27J, at an antibody concentration of 104 ng / mL, the top curve corresponds to S2X259-GAALIE and the bottom curve corresponds to S2X259.1-LS-GRLR.

[0038] [Figure 28A]Figures 28A-28D relate to certain anti-SARS-CoV-2 antibodies containing variant Fc. (A)-(B) FcγRIIIA activation / signaling determined by luminescence using reporter cells (Promega) expressing human FcγRIIIA driving luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein. (A) Wuhan-Hu-1 spike protein; (B) Wuhan-Hu-1 spike protein with a stabilized mutation that prevents S protein export from target cells. (C)-(D) NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Donor PBMCs / primary NK cells expressing FcγRIIIA F158 / V158 (C) or V158 / V158 (D). Some Fc variants could not be titrated because the signal was near the maximum / plateau of the assay. [Figure 28B] Figures 28A-28D relate to certain anti-SARS-CoV-2 antibodies containing variant Fc. (A)-(B) FcγRIIIA activation / signaling determined by luminescence using reporter cells (Promega) expressing human FcγRIIIA driving luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein. (A) Wuhan-Hu-1 spike protein; (B) Wuhan-Hu-1 spike protein with a stabilized mutation that prevents S protein export from target cells. (C)-(D) NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Donor PBMCs / primary NK cells expressing FcγRIIIA F158 / V158 (C) or V158 / V158 (D). Some Fc variants could not be titrated because the signal was near the maximum / plateau of the assay. [Figure 28C]Figures 28A-28D relate to certain anti-SARS-CoV-2 antibodies containing variant Fc. (A)-(B) FcγRIIIA activation / signaling determined by luminescence using reporter cells (Promega) expressing human FcγRIIIA driving luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein. (A) Wuhan-Hu-1 spike protein; (B) Wuhan-Hu-1 spike protein with a stabilized mutation that prevents S protein export from target cells. (C)-(D) NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Donor PBMCs / primary NK cells expressing FcγRIIIA F158 / V158 (C) or V158 / V158 (D). Some Fc variants could not be titrated because the signal was near the maximum / plateau of the assay. [Figure 28D] Figures 28A-28D relate to certain anti-SARS-CoV-2 antibodies containing variant Fc. (A)-(B) FcγRIIIA activation / signaling determined by luminescence using reporter cells (Promega) expressing human FcγRIIIA driving luciferase expression and ExpiCHO (target cells) transfected with SARS-CoV-2 spike protein. (A) Wuhan-Hu-1 spike protein; (B) Wuhan-Hu-1 spike protein with a stabilized mutation that prevents S protein export from target cells. (C)-(D) NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Donor PBMCs / primary NK cells expressing FcγRIIIA F158 / V158 (C) or V158 / V158 (D). Some Fc variants could not be titrated because the signal was near the maximum / plateau of the assay.

[0039] [Figure 29A]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29B]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29C]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29D]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29E]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29F]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29G]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29H]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29I]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29J]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29K]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29L]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29M]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29N]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29O]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29P]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (N) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. [Figure 29Q]Figures 29A-29Q relate to an anti-HBV ("HBC34-v40") Fc variant antibody. (A)-(B) CD83+ cells in ex vivo HBV+ patient serum (assessed by flow cytometry) using the Fc-variant HBC34-v40 antibody and HBsAg, as indicated. In (B), each condition (30 HBsAg IU / mL, 100 HBsAg IU / mL, 300 HBsAg IU / mL, 1000 HBsAg IU / mL) shows four clusters of vertically dispersed data points. Under each HBsAg concentration condition: the leftmost cluster corresponds to HBC34-v40-rIgG1-GRLR; the second cluster from the left corresponds to HBC34-v40-rIgG1-LS; the second cluster from the right corresponds to HBC34-v40-rIgG1-LS-GAALIE; and the rightmost cluster corresponds to HBC34-v40-rIgG1-LS-GA. (C) Schematic diagram showing the design of the MSD MULTI-SPOT® 96-well 10-spot plate for measuring cytokine production. (D) Cytokine production by donor monocyte-derived dendritic cells (moDCs, 3 donors) in response to HBV+ sera (5 donors) and the indicated Fc variant antibodies. Vertically scattered data points cluster at each HBsAg concentration as follows: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. (E) Flow cytometry showing CD83 expression on moDCs (expressing the indicated FcγR) in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and 30 IU / mL of HBsAg from an HBV+ patient serum. (F) Flow cytometry showing CD83 expression on moDCs in the presence of the indicated HBC34-v40 Fc variant antibody (50 μg / mL) and the indicated concentrations of HBsAg from an HBV+ patient serum (BioIVT).The graph on the left is from a single experiment using the first method of pipetting / generating antibody:HBsAg immune complexes; the graph on the right is from a single experiment using the second method of pipetting / generating antibody:HBsAg immune complexes. (G) Surface CD25 expression (a marker of activation) and CFSE (proliferation) when autologous CD4+ memory T cells (from an HBV vaccine recipient) were incubated with moDCs from the same donor for 5 days. moDCs were first activated overnight with 100 IU / mL HBsAg (from two patient sera) and 50 μg / mL HBC34-v40 Fc variant antibodies. The LS-GAYL variant was compared in a single experiment. (H) Percentage of CFSE-low CD25+ human CD4+ memory T cells from an HBV vaccine recipient using the indicated Fc variant antibodies and HBV+ patient sera. Vertically scattered data points are clustered as follows in the leftmost graph: leftmost cluster = HBC34-v40-GRLR; second-to-left cluster = HBC34-v40-rIgG1-LS; second-to-right cluster = HBC34-v40-rIgG1-LS-GAALIE; right cluster = HBC34-v40rIgG1-LS-GA. The clustering is the same in the middle and right graphs, except that HBC34-v40-rIgG1m3-LS-GAYL is added as the rightmost cluster. (I)–(J) CD14+ monocytes were stimulated with IL-4 and GM-CSF for 6 days. MoDCs were treated overnight with antigen and HBC34-v40 Fc variant antibody (50 μg / mL) and then cocultured with HLA-matched (HLA-DR-restricted) transgenic Jurkat cells expressing an HBsAg-specific human TCR. The readout was a GFP-NFAT reporter in Jurkat cells. (K) Comparison of Jurkat TCR reporter assays for three independent replicate experiments with antibody at 0.125 μg / mL. (L) Summary of data from the different assays. (M) Scheme showing the experimental set-up for assessing the in vitro proliferation of T cells from FcγR-expressing mice immunized with HBsAg vaccine followed by a boost; memory CD44+ CD4+ T cells were sorted, labeled with CFSE, and cultured with BMDCs supplemented with immune complexes (antibody:HBsAg antigen), and proliferation was assessed on day 6.SEB = Staphylococcal enterotoxin B from Staphylococcus aureus / CD4 expression and CFSE staining on (500,000) CD4+ memory T cells as in (N)(M), except BMDCs (50,000) were stimulated with immune complexes containing the indicated HBC34-v40 Fc variant antibody (20 μg / mL) and HBsAg (1000 IU / mL). SEB = 1 μg / mL; Mann-Whitney test. (O) (Left) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg alone, antibody alone, or SEB; (Right) Frequency of CFSE low CD4+ CD44+ T cells after incubation with moDCs pre-treated with HBsAg and the indicated HBC34-v40 Fc variant antibody at the indicated concentrations. moDCs were derived from mice transgenic for human FcγR, and T cells were derived from HuFcγR mice (n = 4 independent experiments) or C57Bl / 6 mice (n = 1 experiment). 50,000 moDCs plus 500,000 T cells were tested. SEB = 1 μg / mL; Mann-Whitney test. (P) Left: Schematic diagram showing the SPR assay setup to study binding of HBC34-v40 Fc variants to FcγR (biotinylated FcγR proteins were captured by streptavidin using a CAP chip; HBC34-v40-rIgG1m3 Fc variants were injected at concentrations of 819, 273, 91, 30.3, and 10.1 nM; injections were performed consecutively without regeneration between the same sample at different concentrations; injection time: 600 s; dissociation time for each injection: 100 s). Right: Example SPR curves showing binding to FcγRIIIA. (Q) Fold change results for Fc variant antibodies, calculated by dividing the respective values ​​for the control (HBC34-v40 rIgG1m3-LS) by the values ​​determined for each variant. Binding was measured by a mesoscale discovery-based assay (MSD; using electrochemiluminescence). Higher numbers reflect lower KD and increased binding affinity. "-" = no binding. DETAILED DESCRIPTION OF THE INVENTION

[0040] As used herein, it will be understood that "FcγRIIA" can be expressed as "FcγRIIa," "FcγRIIIA" can be expressed as "FcγRIIIa," "FcγRIIB" can be expressed as "FcγRIIb," and "FcγRIIIB" can be expressed as "FcγRIIIb."

[0041] Before setting forth this disclosure in more detail, it will be helpful to provide definitions of some terms used herein to aid in its understanding. Additional definitions are set forth throughout this disclosure.

[0042] As used herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the stated range, and fractions thereof, where appropriate (such as 1 / 10 and 1 / 100 of an integer), unless otherwise indicated. Also, any numerical ranges described herein with respect to any physical characteristic (such as polymer subunits, size, or thickness) are understood to include any integer within the stated range, unless otherwise indicated. As used herein, the term "about" means ±20% of the stated range, value, or structure, unless otherwise indicated. For example, in some embodiments, the term "about" can mean ±15%, ±10%, or ±5% of the stated range, value, or structure. The terms "a" and "an" are understood herein to mean "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously and it is intended that these terms and variants thereof be interpreted as open-ended.

[0043] "Optional" or "optionally" means that the element, component, event, or circumstance described after it may or may not occur, and the term includes instances where the element, component, event, or circumstance occurs or does not occur.

[0044] Additionally, it should be understood that each individual construct or group of constructs derived from the various combinations of structures and subunits described herein is disclosed herein to the same extent as if each construct or group of constructs were set forth individually, and thus selection of a particular structure or particular subunit is within the scope of this disclosure.

[0045] The phrase "consisting essentially of" is not equivalent to "comprising" and means materials or steps specified in a claim or materials or steps that do not materially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, Fc, CH2, CH3, CH2-CH3, or CH1-CH3) or protein "consists primarily of" a particular amino acid sequence when the amino acid sequence of the domain, region, module, or protein contains extensions, deletions, mutations, or combinations thereof (e.g., amino acids located at the amino or carboxy terminus, or between domains) that, in combination, contribute to up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein, but do not substantially affect (i.e., reduce) the activity of the domain, region, module, or protein (e.g., target binding affinity of a binding protein) by more than 50%, e.g., by not reducing activity by more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%).

[0046] In certain embodiments, variants of CH2, CH3, CH1-CH3, or Fc polypeptides comprise one or more amino acid substitutions relative to the wild-type or parent CH2, CH3, CH1-CH3, or Fc polypeptide, respectively, which one or more amino acid substitutions comprise, consist essentially of, or consist of the specifically described amino acid substitutions. In some embodiments, variants of CH2, CH3, CH1-CH3, or Fc polypeptides comprise only the specifically described substitution mutations relative to the wild-type or parent CH2, CH3, CH1-CH3, or Fc polypeptide, respectively. In other embodiments, variants of CH2, CH3, CH1-CH3, or Fc polypeptides include the specifically recited substitution mutations and one or more additional amino acid substitution mutations (e.g., in some embodiments, one or more conservative amino acid substitutions and / or one or more amino acid substitution mutations that are physically separated from the specifically recited amino acid substitution mutation(s) in the tertiary structure of the Fc polypeptide or fragment thereof), provided that when presented in an immune complex with an antigen, they retain or substantially retain one or more characteristics of the claimed subject matter, e.g., do not significantly alter binding to and / or activation of one or more FcγRs, binding to FcRn, melting temperature, binding to C1q, promotion of ADCC, promotion of ADCP, promotion of CDC, immune complex formation, activation of dendritic cells (e.g., monocyte-derived dendritic cells), etc. In some embodiments, claimed subject matter that includes one or more amino acid substitutions that consist primarily of the recited amino acid substitutions is a functional variant of the claimed subject matter, wherein the amino acid substitutions consist of the recited amino acid substitution(s).

[0047] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0048] As used herein, "mutation" means that the sequence of a nucleic acid molecule or polypeptide molecule is altered compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of changes in the sequence, including nucleotide or amino acid substitutions, insertions, or deletions. Examples of substitution mutations in Fc polypeptides and Fc sequences containing them are shown in Table 1 and the Sequence Listing.

[0049] "Conservative substitution" refers to an amino acid substitution that does not significantly affect or change the binding characteristics of a particular protein. Generally, a conservative substitution is one in which the substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include those found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into multiple conservative substitution groups by similarity in function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, aliphatic groups may include, for substitution purposes, Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0050] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Protein refers to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, and to unnatural amino acid polymers. Variants of the proteins, peptides, and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined or reference amino acid sequence described herein.

[0051] "Nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound containing covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA) (including mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA) and polydeoxyribonucleic acid (DNA) (including cDNA, genomic DNA, and synthetic DNA), both of which can be single-stranded or double-stranded. When single-stranded, a nucleic acid molecule can be the coding strand or the non-coding (antisense) strand. A nucleic acid molecule encoding a given amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of a nucleotide sequence may also contain introns, which may be removed through co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy in the genetic code, or due to splicing.

[0052] In some embodiments, the polynucleotide (e.g., mRNA) comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises pseudouridine, N6-methyladenosine, 5-methylcytidine, 2-thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N1-methylpseudouridine. These features are known in the art and are discussed, for example, in Zhang et al. Front. Immunol., DOI=10.3389 / fimmu.2019.00594 (2019); Eyler et al. PNAS 116(46): 23068-23071; DOI: 10.1073 / pnas.1821754116 (2019); Nance and Meier, ACS Cent. Sci. 2021, 7, 5, 748-756; doi.org / 10.1021 / acscentsci.1c00197 (2021), and van Hoecke and Roose, J. Translational Med 17:54 (2019); https: / / doi.org / 10.1186 / s12967-019-1804-8, the modified nucleosides and mRNA characteristics of which are incorporated herein by reference.

[0053] Variants of the nucleic acid molecules of the present disclosure are also contemplated. A variant nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a defined or reference nucleic acid molecule described herein, or hybridizes to a polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the ability to encode a corresponding binding domain with a function (e.g., binding to a target molecule) as described herein.

[0054] "Percent sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. Preferred methods for determining sequence identity are designed to obtain the best match between the sequences being compared. For example, sequences are aligned for optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences can be ignored for comparison purposes. Percent sequence identity referred to herein is calculated over the length of the reference sequence, unless otherwise indicated. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using BLAST programs (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. In the context of the present disclosure, when sequence analysis software is used for an analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program, which refers to any set of values ​​or parameters that are originally loaded into the software when it is first initialized.

[0055] The term "isolated" means that a material is removed from its original environment (e.g., the natural environment if the material is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a nucleic acid can be part of a vector, and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate), which is still isolated in the sense that such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide. "Isolated" can also describe, in some embodiments, an antibody, antigen-binding fragment, polypeptide, polynucleotide, vector, host cell, or composition outside of the human body.

[0056] The term "gene" refers to a segment of DNA or RNA involved in producing a polypeptide chain; in some contexts, it includes regions preceding and following the coding segment (e.g., the 5' untranslated region (UTR) and 3' UTR), as well as intervening sequences (introns) between individual coding segments (exons).

[0057] "Functional variant" means a polypeptide or polynucleotide that is similar or substantially similar in structure to a parent or reference compound of the present disclosure, but differs in composition slightly (e.g., a single base, atom, or functional group is different, added, or removed), such that the polypeptide, or the encoded polypeptide, is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide. In other words, a polypeptide of the present disclosure or a functional variant of the encoded polypeptide has "similar binding," "similar affinity," or "similar activity" in a selected assay (e.g., an assay for measuring binding affinity, e.g., an association constant (Ka) or dissociation constant (K DA functional variant is one that exhibits no more than a 50% decrease in performance compared to the parent or reference polypeptide in a method such as Biacore® or tetramer staining, which measures the activity of the functional variant.

[0058] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion, or fragment of a parent or reference compound, where the polypeptide or encoded polypeptide retains at least 50% of the activity associated with that domain, portion, or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" if it exhibits no more than a 50% decrease in performance in a selected assay compared to the parent or reference polypeptide (preferably no more than a 20% or 10% decrease in affinity compared to the parent or reference, or no more than an order of magnitude difference).

[0059] As used herein, the terms "engineered," "recombinant," or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic change or that has been modified by the introduction of a foreign or heterologous nucleic acid molecule, where such change or modification is introduced by genetic engineering (i.e., human intervention). Genetic changes include, for example, modifications that introduce expressible nucleic acid molecules that encode functional RNA, proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or otherwise functional disruption of other nucleic acid molecules in the genetic material of a cell. Additional modifications include, for example, non-coding regulatory regions in which the modification alters expression of a polynucleotide, gene, or operon.

[0060] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to a host cell or subject that has been altered. Included in heterologous, non-endogenous, or exogenous is a gene, protein, compound, or nucleic acid molecule that has been mutated or otherwise altered such that one or both of the structure and activity differ between the native and altered states of the gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterologous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule (e.g., a receptor, ligand, etc.) may not be endogenous to a host cell or subject; instead, a nucleic acid encoding such a gene, protein, or nucleic acid molecule can be added to a host cell by conjugation, transformation, transfection, electroporation, etc., and the added nucleic acid molecule can be integrated into the genome of the host cell or can exist as extrachromosomal genetic material (e.g., a plasmid or other self-replicating vector, etc.). The terms "homologous" or "homolog" refer to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or foreign polynucleotide or gene encoding a polypeptide may be homologous to a naturally occurring polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may be altered in structure, sequence, expression level, or any combination thereof. The non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be derived from the same species, a different species, or a combination thereof.

[0061] In certain embodiments, a nucleic acid molecule or portion thereof native to a host cell is considered heterologous to the host cell if it is altered or mutated; a nucleic acid molecule native to a host cell can be considered heterologous if it is altered with heterologous expression control sequences or if it is altered with endogenous expression control sequences not normally associated with the nucleic acid molecule native to the host cell. Additionally, the term "heterologous" can refer to a different, altered, or biological activity not inherent to the host cell. As described herein, two or more heterologous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple, individually regulated genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.

[0062] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

[0063] The term "expression," as used herein, refers to the process by which a polypeptide is produced based on a coding sequence in a nucleic acid molecule (such as a gene). This process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational regulation, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0064] The phrase "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely related to each other so that the function of one does not affect the function of the other.

[0065] As described herein, two or more heterologous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple, individually regulated genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., the heavy chain of an antibody), or any combination thereof. It is understood that when two or more heterologous nucleic acid molecules are introduced into a host cell, the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a host chromosome at a single site or multiple sites, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of encoding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0066] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure). A (polynucleotide) construct can be present in a vector (e.g., a bacterial vector, a viral vector) or can be integrated into a genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector can be, for example, a plasmid, cosmid, virus, RNA vector, or linear or circular DNA or RNA molecule, which can contain chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon-based vectors (e.g., Sleeping Beauty; see, e.g., Geurts et al., Mol. Ther. 8:108, 2003; Mates et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vectors), those capable of delivering polynucleotides into a cellular genome (e.g., viral vectors), or those capable of expressing linked nucleic acid molecules (expression vectors).

[0067] As used herein, "expression vector" or "vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of effecting expression of the nucleic acid molecule in a suitable host. Included in such control sequences are a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once introduced into a suitable host for transformation, a vector can replicate and function independently of the host genome; in some cases, it can integrate into the genome itself or deliver a polynucleotide contained in the vector into the genome free of vector sequences. As used herein, the terms "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

[0068] The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be integrated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0069] In certain embodiments, the polynucleotides of the present disclosure can be operably linked to certain elements of a vector. For example, polynucleotide sequences necessary for the expression and processing of the linked coding sequence can be operably linked. Expression control sequences can include appropriate transcription initiation, termination, promoter, and enhancer sequences; signals for efficient RNA processing (such as splicing and polyadenylation signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences can be operably linked when they are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to regulate the gene of interest.

[0070] In some embodiments, the vector includes a plasmid vector or a viral vector (e.g., a lentivirus vector or a gamma-retrovirus vector). Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), and paramyxoviruses (e.g., measles and Sendai viruses)), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses (e.g., adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis-sarcoma viruses, mammalian type C, type B, and type D viruses, the TLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, in Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0071] A "retrovirus" is a virus with an RNA genome that is reverse transcribed into DNA using reverse transcriptase, and the reverse-transcribed DNA is then integrated into the genome of the host cell. "Gammaretrovirus" refers to a genus in the Retroviridae family. Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0072] "Lentiviral vectors" include HIV-based lentiviral vectors for gene delivery, which can be integrating or non-integrating, have a relatively large packaging capacity, and can transform a range of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more plasmids (packaging, envelope, and transfer) into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Once inside, viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0073] In certain embodiments, the viral vector can be a vector derived from a gammaretrovirus, such as Moloney murine leukemia (MLV). In other embodiments, the viral vector can be a vector derived from a more complex retrovirus, such as a lentivirus. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia, SIV, and Maedi-Visna virus (ovine lentivirus). Methods for packaging mammalian host cells with retroviral and lentiviral vectors for transduction of the cells with transgene-containing viral particles are known in the art and have been previously described (e.g., U.S. Pat. No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009). Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors can also be used to deliver polynucleotides, including DNA viral vectors (e.g., adenovirus-based vectors and adeno-associated virus (AAV)-based vectors); herpes simplex virus (HSV)-derived vectors, including amplicon vectors, replication-deficient HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0074] Other vectors that can be used in the compositions and methods of the present disclosure include vectors derived from baculoviruses and alpha-viruses (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40, in Friedmann T. ed. The Development of Human Gene Therapy, New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0075] When the genome of a viral vector contains multiple polynucleotides to be expressed in a host cell as separate transcripts, the viral vector can also contain additional sequences between the two (or more) transcripts that allow for bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0076] Plasmid vectors for direct administration to a subject, including plasmid vectors encoding DNA-based antibodies or antigen-binding fragments, are further described herein.

[0077] As used herein, the term "host" refers to a cell or microorganism that is targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure). A host cell can be any individual cell or cell culture capable of accepting a vector or nucleic acid integrant or expressing a protein. The term also encompasses the progeny of the host cell, whether of the same or different genotype or phenotype. Suitable host cells may vary depending on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate vectors or other materials by transformation through calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods, using viral vectors. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989).

[0078] In the context of a disease, "host" can refer to the cells or subject that has the disease. For example, as discussed further herein, a variant of an Fc polypeptide can be administered to improve or alter the host immune response, such as against a pathogen afflicting the host.

[0079] As used herein, "antigen" or "Ag" refers to an immunogenic molecule that elicits an immune response. This immune response may include antibody production, activation of specific immunocompetent cells, complement fixation, antibody-dependent cell-mediated cytotoxicity (also known as antibody-dependent cellular cytotoxicity), antibody-dependent cellular phagocytosis, cytokine production, or any combination thereof. Antigens (immunogenic molecules) can be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is apparent that antigens can be synthesized, recombinantly produced, or derived from biological samples. Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, body fluids, or combinations thereof. Antigens can be produced by cells modified or engineered to express the antigen. Antigens can be produced by cells modified or engineered to express an antigen. Antigens can also be present in a betacoronavirus (e.g., a surface glycoprotein or portion thereof) (e.g., present in a virion) or expressed or present on the surface of a cell infected with a betacoronavirus.

[0080] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized by and specifically bound by a cognate binding molecule (such as an immunoglobulin or other binding molecule, domain, or protein). Epitopic determinants generally contain chemically active surface groupings of molecules (such as amino acids or sugar side chains) and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. When the antigen is or comprises a peptide or protein, the epitope can consist of contiguous amino acids (e.g., a linear epitope), or can consist of amino acids from different portions or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or can consist of non-contiguous amino acids that remain in proximity regardless of protein folding.

[0081] Fc polypeptide variants and polypeptides containing same

[0082] As part of this disclosure, immunoglobulin G (IgG) Fc polypeptides and engineered variants thereof, fragments or portions thereof, and proteins (e.g., antibodies and fusion proteins) comprising the same are provided. By way of background, the Fc region of an antibody (also referred to as an "Fc domain") interacts with Fc receptors and other binding partners (such as complement C1q) and can initiate, participate in, and / or mediate an immune response to, for example, a pathogen or antigen. The Fc variants of the disclosure possess various advantages over native (i.e., wild-type) Fcs and / or known Fc variants, non-limiting examples of which include increased binding to one or more activating Fc receptors (e.g., FcγRIIa) or activating Fc receptors, decreased binding to an inhibitory Fc receptor (e.g., FcγRIIb), providing increased binding to activating Fc receptors relative to binding to inhibitory Fc receptors, binding to complement C1q, facilitating or increasing antibody-dependent cellular phagocytosis (ADCP), facilitating or increasing antibody-dependent cellular cytotoxicity (ADCC), facilitating or increasing complement, facilitating or increasing intracellular signaling through activating Fc receptors, decreasing intracellular signaling through inhibitory Fc receptors, providing increased signaling through activating Fc receptors relative to signaling through inhibitory Fc receptors, and facilitated or increased activation of dendritic cells (e.g., monocyte-derived dendritic cells) when provided in an antibody:antigen complex (having the variant Fc). In certain embodiments, the Fc variants of the disclosure have improved thermal stability (e.g., a higher Tm or a Tm closer to that of the wild-type Fc polypeptide), similar or improved capabilities for purification and / or purification, and / or preferential binding to FcRn, e.g., compared to a wild-type reference Fc polypeptide or a variant Fc polypeptide that does not contain the specified mutation and / or fucosylation state.

[0083] As discussed further herein, immunoglobulins typically comprise two heavy chain polypeptides. Immunoglobulin heavy chains typically comprise a variable region (also referred to as a variable domain) and a constant region (also referred to as a constant domain). For example, in the case of an IgG isotype, the constant region typically comprises a CH1 region, a hinge, a CH2, and a CH3. Heavy chain polypeptide monomers can associate and be held together by shared disulfide bonds to form dimers; the CH2-CH3 portion of an immunoglobulin heavy chain dimer comprises the Fc (fragment crystallizable) portion or domain of an immunoglobulin (e.g., an IgG1 antibody). An example of a wild-type human IgG1 CH1-CH3 amino acid sequence is set forth in SEQ ID NO: 1. An example of a wild-type human IgG1 hinge CH2-CH3 is set forth in SEQ ID NO: 2. An example of a wild-type human IgG1 CH2 is set forth in SEQ ID NO: 3. An example of a wild-type human IgG1 CH3 amino acid sequence is set forth in SEQ ID NO: 4. An example of a wild-type human IgG1 hinge-CH2 amino acid sequence is set forth in SEQ ID NO: 5. It will be understood that the hinge of a hinge-CH2 polypeptide or a hinge-Fc polypeptide can include one or more modifications (e.g., mutations) compared to a wild-type hinge sequence, and that the one or more modifications can further be, for example, a P230A or S219Y mutation, as disclosed herein.

[0084] As used herein, unless otherwise specified, "Fc polypeptide" refers to a CH2-CH3 polypeptide. An Fc polypeptide fragment can include CH2, a portion of CH2, CH3, and / or a portion of CH3, but does not include the complete, full-length CH2-CH3. In certain embodiments, Fc polypeptide fragments are provided that include specific amino acid positions and variations within CH2 and / or CH3, and in some embodiments, include portions of sufficient length to possess one or more of the functions referenced.

[0085] Included among the polypeptides of the present disclosure are polypeptides that include variants of an IgG Fc polypeptide or fragment thereof, which variants contain one or more modifications compared to the IgG Fc polypeptide or fragment thereof. Unless otherwise specified, it will be understood that a "reference" polypeptide or antibody (e.g., a reference IgG Fc polypeptide or fragment thereof, a reference antibody, a reference CH2 polypeptide, a reference IgG hinge-CH2, a reference IgG hinge-Fc polypeptide, a reference CH3 polypeptide) is preferably the same as the molecule to which it refers (e.g., a variant of an Fc polypeptide or fragment thereof; a polypeptide comprising such a variant; an antibody comprising a variant of an Fc polypeptide) except for the noted difference or differences.

[0086] For example, with respect to a variant IgG1 Fc polypeptide comprising an alanine (A) amino acid at EU position 236, it will be understood that a reference Fc polypeptide includes an IgG1 Fc polypeptide that is identical to said variant except for the naturally occurring glycine (G) amino acid found at EU position 236. As another example, with respect to a variant of an Fc polypeptide fragment (e.g., comprising CH2 and part of CH3), the reference Fc polypeptide fragment preferably has the same length as said variant and preferably differs from said variant only in a noted characteristic (e.g., one or more amino acid mutations present in said variant). In some embodiments, the reference Fc polypeptide, Fc polypeptide fragment, or antibody comprises a wild-type amino acid sequence (e.g., wild-type human IgG1). The reference Fc polypeptide, Fc polypeptide fragment, or antibody will be of the same isotype, preferably the same allotype, as said variant except for the noted difference present in said variant. In the case of a reference antibody, the Fab or other antigen-binding domain is preferably the same as that present in the particular antibody comprising the variant Fc polypeptide or fragment thereof.

[0087] In some embodiments, a variant of an IgG Fc polypeptide or fragment thereof comprises one or more amino acid substitutions compared to a reference (e.g., wild-type) IgG Fc polypeptide or fragment thereof. As used herein, amino acid positions in a variant IgG Fc polypeptide or fragment may be described with reference to an "EU position"; it will be understood that an "EU position" is according to the EU numbering system set forth in Kabat. For example, in the exemplary human IgG1 CH1-CH3 amino acid sequence shown in SEQ ID NO: 1, it will be understood that the first amino acid (A) corresponds to EU position 118 and the last amino acid (K) corresponds to EU position 447: ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 1)

[0088] Thus, unless otherwise specified, referenced amino acid positions will be understood to be according to EU numbering for human IgG1, even when a complete antibody heavy chain, complete CH1-CH3, complete CH2-CH3, etc., is not present or explicitly mentioned. In other words, where, for example, only hinge CH2 is described and CH3 and / or CH1 may not be present, amino acid positions within hinge CH2 will be described with reference to EU numbering unless otherwise specified. The correspondence between EU numbering, Kabat numbering, IMGT exon numbering, and IMGT proprietary numbering for immunoglobulin G heavy chain constant domains is known in the art and is shown, for example, in the IMGT Scientific chart (www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html; created May 17, 2001, accessed May 23, 2021, last updated January 20, 2020).

[0089] In the Examples herein, certain Fc variants were generated (expressed in fucosylated and defucosylated human IgG1 antibodies of various allotypes) and various properties were investigated. Certain embodiments of the Fc variants of the present disclosure (which are fucosylated unless otherwise noted) and their non-limiting properties are summarized in Table 1; see also Figures 10A-1-10C.

[0090] [Table 1-1] [Table 1-2]

[0091] Additional features of the disclosed Fc variant-containing antibodies are shown in the Examples and Figures of this disclosure and described herein, e.g., Figures 10B-1 through 10B-4 show certain properties of antibodies containing certain defucosylated variant Fcs.

[0092] It will be understood that two or more amino acid substitutions present in a variant can be expressed in various ways (e.g., as G236A_Y300L or G236A / Y300L). Furthermore, single mutations or combination mutations can be referred to using abbreviated forms that include the original amino acid and the amino acid resulting from the mutation. For example, G236A can be expressed as "GA" or "236A"; G236A_Y300L can be expressed as "GAYL"; G236A_L328V_Q295E can be expressed as "GALVQE"; G236A_R292P_Y300L can be expressed as "GARPYL" and G236A_R292P_I377N can be expressed as "GARPIN", and so on.

[0093] In any embodiment of the present disclosure, a variant of an Fc polypeptide or fragment thereof can be derived from or comprise a human Fc polypeptide or fragment thereof and / or can be derived from or comprise a human IgG1, human IgG2, human IgG3, or human IgG4 isotype. In this context, "derived from" means that the variant is the same as the referenced polypeptide or isotype except for the specified modification (e.g., amino acid substitution). For example, a variant Fc polypeptide comprising a wild-type human IgG1 Fc amino acid sequence except for the amino acid substitution mutation G236A_L328V_Q295E (and optionally other amino acid substitutions) can be said to be "derived from" wild-type human IgG1 Fc. In any embodiment of the present disclosure, the polypeptide, CH2, Fc, Fc fragment, or antibody can comprise a human Ig sequence (e.g., a human IgG1 sequence). In some embodiments, the polypeptide, CH2, Fc, Fc fragment, or antibody can comprise a native or wild-type human Ig sequence except for the mutations described, or can comprise a human Ig (e.g., IgG) sequence containing one or more additional mutations.

[0094] In certain embodiments, the polypeptide contains only the designated or referenced amino acid mutations (e.g., substitutions), e.g., compared to a reference polypeptide (e.g., a wild-type Fc polypeptide or fragment thereof), and does not contain any additional amino acid substitutions or mutations. For example, in some embodiments, a variant Fc polypeptide containing the amino acid substitution G236A_Y300L does not contain any other amino acid substitutions, i.e., it contains an amino acid sequence that is wild-type except for G236A and Y300L.

[0095] In some embodiments, the polypeptide can include one or more additional amino acid mutations (e.g., substitutions), which can be designated (e.g., M428L_N434S; M428L_N434A). In some embodiments, the additional amino acid mutation(s) are physically distant from the referenced amino acid position in the tertiary structure and / or are of a specific nature (e.g., conservative substitutions) such that they do not reduce, or reduce by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, or no more than 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or 1.5-fold. In some embodiments, the polypeptide includes the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation (including those described herein) that enhances binding to human FcRn.

[0096] In some embodiments, a polypeptide is provided that comprises at least a portion of (e.g., human) IgG1 CH2-CH3 or hinge-CH2-CH3 or CH1-CH3, including the amino acid mutations set forth in any one of (i) to (xviii): (i) G236A, L328V, and Q295E; (ii) G236A, P230A, and Q295E; (iii) G236A, R292P, and I377N; (iv) G236A, K334A, and Q295E; (v) G236S, R292P, and Y300L; (vi) G236A and Y300L; (vii) G236A, R292P, and Y300L; (viii) G236S, G420V, G446E, and (ix) G236A and R292P; (x) R292P and Y300L; (xi) G236A and R292P; (xii) Y300L; (xiii) E345K, G236S, L235Y, and S267E; (xiv) E272R, L309T, S219Y, and S267E; (xv) G236Y; (xvi) G236W; (xvii) F243L, G446E, P396L, and S267E; (xviii) G236A, S239D, and H268E (wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat). In one embodiment, the polypeptide is defucosylated. In some embodiments, the IgG1 CH2-CH3 or hinge-CH3-CH3 or heavy chain is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to wild-type human IgG1 CH2-CH3 or hinge-CH2-CH3 or CH1-CH3, respectively.In certain embodiments, polypeptides of the present disclosure include Fc variants comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5 and 36-38.

[0097] In some embodiments, the antibody is provided as an antibody (as further described herein) that includes any one of the amino acid mutations (i) to (xviii) in the (e.g., human) IgG1 heavy chain: (i) G236A, L328V, and Q295E; (ii) G236A, P230A, and Q295E; (iii) G236A, R292P, and I377N; (iv) G236A, K334A, and Q295E; (v) G236S, R292P, and Y300L; (vi) G236A and Y300L; (vii) G236A, R292P, and Y300L; (viii) G236A, R292P, and Y300L; (ix) G236A and R292P; (x) R292P and Y300L; (xi) G236A and R292P; (xii) Y300L; (xiii) E345K, G236S, L235Y, and S267E; (xiv) E272R, L309T, S219Y, and S267E; (xv) G236Y; (xvi) G236W; (xvii) F243L, G446E, P396L, and S267E; (xviii) G236A, S239D, and H268E (wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat). In one embodiment, the polypeptide is defucosylated. In some embodiments, the polypeptide or antibody further comprises one or more mutations that enhance binding to human FcRn (such as the mutations M428L and N434S, or the mutations M428L and N434A (EU numbering)), or any other mutation that enhances binding to human FcRn (such as a mutation described herein). In one embodiment, the polypeptide is defucosylated.In some embodiments, the IgG1 heavy chain comprises a CH1-CH3 or CH2-CH3 or hinge-CH2-CH3, and said CH1-CH3 or CH2-CH3 or hinge-CH2-CH3 is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to wild-type human IgG1 CH1-CH3 or CH2-CH3 or hinge-CH2-CH3, respectively. In certain embodiments, antibodies of the present disclosure include Fc variants comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-5 and 36-38.

[0098] In some embodiments, the polypeptide or antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 6-23 and 45, or a variant thereof (e.g., a variant further comprising one or more mutations that enhance binding to human FcRn, such as the mutations M428L and N434S, or the mutations M428L and N434A (EU numbering)), or any other mutation that enhances binding to human FcRn, including those described herein). In some embodiments, the polypeptide or antibody comprises an amino acid sequence that differs from the amino acid sequence set forth in any one of SEQ ID NOs: 6-23 and 45 only by the presence of one or more IgG1 allotype-specific mutations, and / or the mutations M428L and N434S, or the mutations M428L and N434A, or other mutations that enhance binding to human FcRn.

[0099] Polypeptides of the disclosure can be fucosylated (e.g., contain one or more fucosyl moieties, typically a native (wild-type) fucosylation pattern or a fucosylation pattern containing one or more additional fucosyl moieties or fewer fucosyl moieties compared to native) or defucosylated. In particular, native IgG1 antibodies have a glycan site at N297, which is typically the only site where a core fucose moiety may be found in an antibody, although several glycan sites may arise through mutation (e.g., in the variable domain) during antibody development. Fucosylation of an Fc polypeptide or fragment thereof, or fucosylation of an antibody, can be achieved by introducing an amino acid mutation that introduces or destroys a fucosylation site (e.g., a mutation at position N297 (e.g., N297Q or N297A) that prevents the formation of glycans that can contain a core fucose moiety), but typically it is preferred to maintain N297 and its glycans, such as by expressing the polypeptide in a host cell that has been genetically engineered to lack (or have an inhibited or impaired ability to) fucosylate the polypeptide; or by expressing the polypeptide under conditions in which the ability of the host cell to fucosylate the polypeptide is impaired (e.g., in the presence of 2-fluoro-L-fucose (2FF)). A defucosylated polypeptide can be free of fucose moieties or substantially free of fucose moieties, and / or expressed by a host cell genetically engineered to lack (or have an inhibited or impaired ability to) fucosylate the polypeptide, and / or expressed under conditions (e.g., in the presence of 2-fluoro-L-fucose (2FF)) in which the ability of the host cell to fucosylate the polypeptide is impaired. In some embodiments, the polypeptide does not contain a core fucose moiety at Asn297. In some embodiments, the defucosylated polypeptide has increased binding to FcγRIIIA. In some contexts, addition of 2FF to culture medium containing host cells expressing the antibody results in about 85% or more of the antibody lacking fucose moieties.Thus, antibodies can be described as "defucosylated" when they are produced in the presence of 2FF or a similar reagent. In some contexts, polypeptides or antibodies can be described as, for example, defucosylated, meaning that about 85% or more of the single polypeptide or antibody molecules of the plurality do not contain a fucosyl moiety. In a preferred embodiment, a defucosylated antibody or polypeptide, or a population thereof, or a plurality of defucosylated antibodies or polypeptides, comprises an asparagine (N) at EU position 297. Fucosylation, or lack thereof, can be assessed using, for example, mass spectrometry (e.g., electrospray ionization-mass spectrometry (ESI-MS)). In some embodiments, a composition comprising a plurality of any one or more of the polypeptides of the present disclosure is provided, where the composition comprises a defucosylated polypeptide.

[0100] Also in the examples of the present disclosure, variant Fc's, including those containing the mutations shown in Table 1 above, were expressed in defucosylated human IgG1 antibodies to test various properties (including by comparison with fucosylated wild-type human IgG1 antibodies). See, e.g., Figure 10B; in some contexts, defucosylated polypeptides with Fc variants have similar or even improved properties when fucosylated.

[0101] In certain embodiments, the disclosed variants of IgG Fc polypeptides or fragments thereof have one or more functions that differ from (e.g., are improved compared to) the corresponding function of a reference Fc polypeptide that comprises one or more of the following mutations: G236A; G236S; G236A_A330L_I332E; G236A_A330L_I332E_M428L_N434S; A330L_I332E; or G236A_S239D_A330L_I332E. For example, in certain embodiments, the disclosed variants of IgG Fc polypeptides or fragments thereof have one or more of the following properties relative to a reference Fc polypeptide comprising one or more of the following mutations: G236A; G236S; G236A_A330L_I332E; G236A_A330L_I332E_M428L_N434S; A330L_I332E; or G236A_S239D_A330L_I332E: increased binding (e.g., affinity) to and / or signaling through human FcγRIIa H131; increased binding (e.g., affinity) to and / or signaling through human FcγRIIa R131; increased signaling through R131; decreased binding (e.g., affinity) to human FcγRIIb and / or decreased signaling through human FcγRIIb; increased binding (e.g., affinity) to human FcγRIIa (H131, R131, or both) to the ratio of binding to human FcγRIIb and / or increased ratio of signaling through human FcγRIIa (H131, R131, or both) to signaling through human FcγRIIb (respectively); human FcγRIIIa (V158, F158, or both) increased binding (e.g., affinity) to and / or signaling through human FcγRIIIa (V158, F158, or both); increased binding (e.g., affinity) to human C1q; higher Tm; improved production titer; improved signaling through FcγRIIa (H131, R131, or both) in host cells; increased facilitation of ADCP and / or ADCC by human NK cells and / or human PBMCs in the presence of antigen-presenting cells; and improved ability to stimulate moDCs when in an immune complex with antigen.

[0102] In the present disclosure, binding of a variant Fc polypeptide or fragment can be described as increased (e.g., "greater") or decreased (e.g., "reduced" or "lesser") compared to binding of a comparison antibody to the same binding partner (e.g., binding to a reference wild-type IgG1 Fc, or binding to a reference IgG1 Fc that is wild-type but for the mutations M428L and N434S, or the mutations M428L and N434A, or binding to a variant IgG1 Fc containing the G236A_A330L_I332E mutation). The binding interaction between a variant Fc polypeptide or fragment (or an antibody or polypeptide comprising it) and a binding partner (e.g., human FcγR, FcRn, or C1q) can preferably be determined using an electrochemiluminescence assay, and more preferably using the Meso Scale Discovery ("MSD"; mesoscale.com) platform. The MSD binding assay is similar to an ELISA, except that MSD utilizes electrochemiluminescence as the detection technique, as opposed to colorimetric analysis. Other techniques for measuring binding interactions are known, including, for example, ELISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), and the like.

[0103] In some embodiments, binding includes affinity, avidity, or both. Affinity refers to the strength of binding between a binding molecule and its corresponding binding partner. In some contexts, binding can include affinity and / or avidity. Unless otherwise specified, avidity refers to the overall binding strength of a molecule to a binding partner, reflecting binding affinity, the valency of the binding site (e.g., whether an Fc polypeptide contains one, two, or more binding sites), and the presence of additional agents that may affect binding (e.g., non-competitive inhibitors of the Fc polypeptide).

[0104] The binding interaction between a variant molecule of the present disclosure and a binding partner can be expressed as a fold change compared to the binding interaction between a reference molecule and a binding partner. For example, the binding of a variant Fc-containing antibody of the present disclosure to human FcγRIIa can be stronger than the binding of a wild-type Fc-containing antibody to human FcγRIIa, and the relative increased strength of the variant can be expressed as a fold change (e.g., a linear scale of the area under the curve) relative to the binding of the reference molecule using the same assay. For example, a variant Fc polypeptide or fragment can bind to FcγRIIa with 2-fold, 3-fold, 4-fold, or 5-fold greater binding strength than the reference Fc polypeptide or fragment binds to FcγRIIa. As another example, a variant Fc polypeptide or fragment thereof can bind weaker to FcγRIIb compared to a reference Fc or fragment thereof; e.g., 0.9-fold binding, 0.8-fold binding, 0.7-fold binding, 0.6-fold binding, etc., compared to the reference Fc polypeptide or fragment thereof. For example, the phrase "two-fold greater binding than the binding of a reference" will be understood to mean a two-fold increase in binding compared to the reference.

[0105] Furthermore, binding of a variant molecule of the present disclosure to two different partner molecules can be described as a ratio, which can be compared to a similar ratio obtained using a reference molecule in the same assay. For example, a variant Fc polypeptide may bind to human FcγRIIa H131 five times more strongly than it binds to human FcγRIIb, whereas a reference wild-type Fc polypeptide binds to FcγRIIa H131 as strongly as it binds to human FcγRIIb. In this example, the variant Fc polypeptide can be said to have a binding ratio of 5:1 (binding to FcγRIIIa H131:binding to FcγRIIb), which can be compared to the binding ratio of 1:1 (binding to FcγRIIIa H131:binding to FcγRIIb) of the reference wild-type Fc polypeptide.

[0106] The variant molecules of the disclosure can also be described by their ability to induce signaling in host cells that express or overexpress one or more FcγRs (e.g., FcγRIIa H131, FcγRIIa R131, FcγRIIb, FcγRIIIa F158, or FcγRIIIa V158), and signaling is induced by binding of the variant molecule to the FcγR. Useful reporter cells for determining signaling include, for example, cells in which NFAT drives expression of a luciferase reporter (available, for example, from Promega®).

[0107] Unless otherwise specified, the FcγR, FcRn, and C1q described herein are human.

[0108] In some embodiments, antibodies comprising a variant Fc polypeptide or fragment are preferably capable of inducing one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP); and complement-dependent cytotoxicity. Assays for measuring these functions are known.

[0109] In some embodiments, the variant Fc polypeptide or fragment (or polypeptide or antibody comprising same) preferably has equivalent binding to human FcRn (e.g., at pH 6.0) and / or equivalent in vivo half-life in a mammal compared to the reference Fc polypeptide fragment or antibody, respectively.

[0110] In some embodiments, the variant Fc polypeptide or fragment (or polypeptide or antibody comprising same) preferably has increased binding to human FcRn (e.g., at pH 6.0) and / or increased in vivo half-life in a mammal compared to the reference Fc polypeptide fragment or antibody, respectively.

[0111] In some embodiments, it is preferred that the variant Fc polypeptide or fragment (or polypeptide or antibody comprising same) has a melting point (Tm) that is 12°C, 11°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, or 1°C lower than the melting point (Tm) of the reference Fc polypeptide or fragment (or polypeptide or antibody comprising same), or has a Tm that is higher than the Tm of the reference Fc polypeptide or fragment (or polypeptide or antibody comprising same). In some embodiments, the reference polypeptide or fragment is or comprises a wild-type human Fc polypeptide (or antibody comprising same).

[0112] In some embodiments, the variant Fc polypeptide or fragment (or a polypeptide or antibody comprising same) has a melting temperature that is higher than the melting temperature of a reference Fc polypeptide or fragment (or a polypeptide or antibody comprising same) that comprises the mutations G236A, A330L, I332E, and optionally M428L and N434S.

[0113] In some embodiments, the variant Fc polypeptide or fragment (or polypeptide or antibody comprising same) can preferably be produced in a host cell line (e.g., a CHO cell line) with at least about the same efficiency as (e.g., at least about the same potency and / or with a decrease of less than 0.1-fold, less than 0.09-fold, less than 0.08-fold, less than 0.07-fold, less than 0.06-fold, less than 0.05-fold, less than 0.04-fold, less than 0.03-fold, less than 0.02-fold, or within less than 0.02-fold) of the reference Fc polypeptide or fragment (or polypeptide or antibody comprising same).

[0114] In certain embodiments, provided is a polypeptide comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GALVQE"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0115] In certain other embodiments, provided herein are polypeptides comprising: (i) a variant of an IgG hinge-CH2 polypeptide; or (ii) a variant of an IgG hinge-Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 230, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GAPAQE"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0116] In certain other embodiments, the polypeptide comprises a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GARPIN"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0117] In certain other embodiments, provided is a polypeptide comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 334, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GAKAQE"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0118] In certain other embodiments, provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises serine (S) at EU position 236, proline (P) at EU position 292, and leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GSRPYL"). In some embodiments, the polypeptide further comprises mutations M428L and N434S, or mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0119] In certain other embodiments, provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GARPYL"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0120] In certain other embodiments, provided is a polypeptide comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GAYL"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0121] In certain other embodiments, provided are polypeptides comprising: (i) a variant of an IgG CH2 polypeptide; or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, and a glutamic acid (E) at EU position 268. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GASDHE"). In some embodiments, the polypeptide further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In certain embodiments, the polypeptide is defucosylated.

[0122] In some embodiments, the variant, and optionally the polypeptide (e.g., an antibody or Fc fusion comprising the variant), exhibits increased binding to human FcγRIIa and / or decreased binding to human FcγRIIb compared to binding of a reference polypeptide to human FcγRIIa or human FcγRIIb, respectively, and optionally binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery.

[0123] In certain embodiments, increased binding to human FcγRIIa includes binding to human FcγRIIa that is greater than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or fragment thereof) to human FcγRIIa.

[0124] In some embodiments, the human FcγRIIa comprises H131, and optionally the increased binding to human FcγRIIa H131 comprises binding to human FcγRIIa H131 that is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or a fragment thereof) to human FcγRIIa H131.

[0125] In some embodiments, the human FcγRIIa comprises R131, and optionally the increased binding to human FcγRIIa R131 comprises binding to human FcγRIIa R131 that is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or a fragment thereof) to human FcγRIIa R131.

[0126] In some embodiments, reduced binding to human FcγRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, or between 0.5-fold and 0.9-fold the binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or fragment thereof) to human FcγRIIb.

[0127] In any embodiment of the present disclosure, (1) the ratio of (i) binding of the variant or polypeptide to human FcγRIIa to (ii) binding of the variant or polypeptide, respectively, to human FcγRIIb is greater than the ratio of (2)(iii) binding of a reference polypeptide to human FcγRIIa to (iv) binding of the reference polypeptide to human FcγRIIb, where the reference polypeptide optionally comprises a wild-type human IgG (e.g., IgG1) Fc polypeptide or fragment thereof, optionally wherein binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, the human FcγRIIa comprises H131, R131, or both. In some embodiments, the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than the ratio in (2).

[0128] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GAYL"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0129] In some embodiments, the variant, and optionally the polypeptide, has increased binding to human FcγRIIa compared to binding of a reference polypeptide to human FcγRIIa, and optionally binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery.

[0130] In some embodiments, increased binding to human FcγRIIa includes at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater binding to human FcγRIIa compared to binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or fragment thereof) to human FcγRIIa.

[0131] In some embodiments, the human FcγRIIa comprises H131, and optionally the increased binding to human FcγRIIa H131 comprises at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater binding to human FcγRIIa H131 compared to binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or a fragment thereof) to human FcγRIIa H131.

[0132] In some embodiments, the human FcγRIIa comprises R131, and optionally the increased binding to human FcγRIIa R131 comprises binding to human FcγRIIa R131 that is at least 4-fold greater than binding of a reference polypeptide (optionally comprising a wild-type human IgG (e.g., IgG1) Fc polypeptide or a fragment thereof) to human FcγRIIa R131.

[0133] In one embodiment, (1) (i) the ratio of binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb is greater than (2) (iii) the ratio of binding of a reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to human FcγRIIb, where the reference polypeptide optionally comprises a wild-type human IgG (e.g., IgG1) Fc polypeptide or fragment thereof. In one embodiment, the human FcγRIIa comprises H131, R131, or both. In a further embodiment, the ratio in (1) is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, or at least 17-fold greater than the ratio in (2).

[0134] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GARPYL"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein), are present. In certain embodiments, the polypeptide is defucosylated.

[0135] In one embodiment, the variant, and optionally the polypeptide, has increased binding to human FcγRIIIa compared to binding of a reference polypeptide to human FcγRIIIa, and optionally binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery.

[0136] In some embodiments, increased binding to human FcγRIIa includes binding to human FcγRIIa that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than binding to human FcγRIIa of a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof.

[0137] In some embodiments, the human FcγRIIa comprises H131, and optionally the increased binding to human FcγRIIa H131 comprises binding to human FcγRIIa H131 that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than binding to human FcγRIIa H131 of a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof.

[0138] In some embodiments, the human FcγRIIa comprises R131, and optionally the increased binding to human FcγRIIa H131 comprises binding to human FcγRIIa R131 that is at least two-fold greater than binding to human FcγRIIa R131 compared to binding of a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof, to human FcγRIIa R131.

[0139] In some embodiments, (1) (i) the ratio of binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb is greater than (2) (iii) the ratio of binding of a reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to human FcγRIIb, where the reference polypeptide optionally comprises a wild-type human IgGFc polypeptide or a fragment thereof, optionally wherein binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, the human FcγRIIa comprises H131, R131, or both. In some embodiments, the ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold greater than the ratio in (2).

[0140] In some embodiments, the variants have increased binding to human FcγRIIIa compared to binding of a reference polypeptide to human FcγRIIIa, where binding is optionally determined by electrochemiluminescence assay and optionally by Meso Scale Discovery. In some embodiments, the human FcγRIII comprises V158, F158, or both. In some further embodiments, increased binding to human FcγRIIIa comprises greater than 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, or at least 3.7-fold greater binding to human FcγRIIIa compared to binding of a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or fragment thereof, to human FcγRIIIa.

[0141] In one embodiment, the variant, and optionally the polypeptide, is capable of binding to human complement component 1q (C1q), optionally wherein binding is determined using an electrochemiluminescence assay, and optionally further using Meso Scale Discovery.

[0142] Also provided are polypeptides comprising variants of IgG Fc polypeptides, wherein the variants comprise a serine (S) at EU position 236, a valine (V) at EU position 420, a glutamic acid (E) at EU position 446, and a threonine (T) at EU position 309. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GSGVGELT"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein), are present. In certain embodiments, the polypeptide is defucosylated.

[0143] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide, wherein the variant comprises an alanine (A) at EU position 236 and a proline (P) at EU position 292. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GARP"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0144] In some embodiments, the variant, and optionally the polypeptide, has reduced binding to human FcγRIIb relative to binding of a reference polypeptide to human FcγRIIb, optionally wherein binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, reduced binding to human FcγRIIb comprises less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, less than 0.5-fold, or less than 0.4-fold relative to binding of a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or fragment thereof, to human FcγRIIb.

[0145] In a further embodiment, the variant, and optionally the polypeptide, has increased binding to human FcγRIIa compared to binding of a reference polypeptide to human FcγRIIa, and optionally binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery.

[0146] In some embodiments, increased binding to human FcγRIIa includes binding to human FcγRIIa that is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold greater than binding to human FcγRIIa of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof.

[0147] In one embodiment, the human FcγRIIa comprises H131, R131, or both.

[0148] In some embodiments, (1) (i) the ratio of binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb is greater than (2) (iii) the ratio of binding of a reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to human FcγRIIb, where the reference polypeptide optionally comprises a wild-type human IgGFc polypeptide or a fragment thereof, optionally wherein binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, the human FcγRIIa comprises H131, R131, or both. In certain embodiments, the ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 11-fold, or at least 12-fold greater than the ratio in (2).

[0149] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide, wherein the variant comprises a proline (P) at EU position 292 and a leucine (L) at EU position 300, optionally wherein the variant, and optionally the polypeptide, has increased binding to human FcγRIIIa compared to binding of a reference polypeptide to human FcγRIIIa, optionally wherein the binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, the IgG CH2 polypeptide or IgG Fc polypeptide comprises an (e.g., otherwise wild-type) IgG1 CH2 polypeptide or IgG Fc polypeptide ("RPYL"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0150] In certain embodiments, the human FcγRIIIa comprises V158, F158, or both, and the increased binding to human FcγRIIIa comprises at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.1-fold, or at least 5.2-fold greater binding to human FcγRIIa compared to a reference polypeptide, optionally comprising a wild-type human IgG Fc polypeptide or a fragment thereof.

[0151] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises a leucine (L) at EU position 300. In some embodiments, the IgG CH2 polypeptide, or IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("YL"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0152] Also provided are polypeptides comprising variants of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises a lysine (K) at EU position 345, a serine (S) at EU position 236, a tyrosine (Y) at EU position 235, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GSEKLYSE"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0153] Also provided are polypeptides comprising (i) a variant of an IgG hinge-CH2 polypeptide, or (ii) a variant of an IgG hinge-Fc polypeptide or fragment thereof, wherein the variant comprises an arginine (R) at EU position 272, a threonine (T) at EU position 309, a tyrosine (Y) at EU position 219, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG hinge-CH2 polypeptide, or IgG hinge-Fc polypeptide, or fragment thereof, comprises an (e.g., otherwise wild-type) IgG1 hinge-CH2 polypeptide, or IgG hinge-Fc polypeptide, or fragment thereof ("SYSEERLT"). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein), are present. In certain embodiments, the polypeptide is defucosylated.

[0154] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises a tyrosine (Y) at EU position 236. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GY"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0155] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises a tryptophan (W) at EU position 236. In some embodiments, the IgG CH2 polypeptide, or (ii) the IgG Fc polypeptide or fragment thereof, comprises an (e.g., an otherwise wild-type) IgG1 CH2 polypeptide, or Fc polypeptide or fragment thereof ("GW"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present. In certain embodiments, the polypeptide is defucosylated.

[0156] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, which variant comprises an alanine (A) at EU position 236, wherein said IgG Fc polypeptide or fragment thereof, optionally said polypeptide is defucosylated, and further optionally said variant comprises a leucine (L) at EU position 330 and a glutamic acid (E) at EU position 332, and further optionally said variant does not comprise an aspartic acid (D) at EU position 239, and further optionally comprises a serine (S) at EU position 239. In some embodiments, said IgG CH2 polypeptide, or (ii) IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgG1 CH2 polypeptide or Fc polypeptide or fragment thereof ("GA-afuc" or "GAALIE-afuc", respectively). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0157] Also provided are polypeptides comprising variants of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises a leucine (L) at EU position 243, a glutamic acid (E) at EU position 446, a leucine (L) at EU position 396, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("FLSEPLGE"). In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein), are present. In certain embodiments, the polypeptide is defucosylated.

[0158] Also provided are polypeptides comprising (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, a glutamic acid (E) at EU position 332, a leucine (L) at EU position 428, and a serine (S) or alanine (A) at EU position 434. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., an otherwise wild-type) IgG1 Fc polypeptide or fragment thereof ("GASDIEMLNS" or "GASDIEMLNA"). In certain embodiments, the polypeptide has increased binding to human C1q compared to binding of a reference polypeptide to human C1q, optionally wherein binding is determined using an electrochemiluminescence assay, and optionally using Meso Scale Discovery. In some embodiments, increased binding to human C1q comprises greater than 1-fold, at least 1.5-fold, at least 1.75-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, at least 3.7-fold, at least 3.8-fold, at least 3.9-fold, at least 4.0-fold, at least 4.1-fold, or at least 4.15-fold greater binding to human C1q compared to binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or fragment thereof to human C1q.

[0159] In some embodiments of the disclosure, the polypeptide (i) can bind to human FcγRIIIa, wherein the human FcγRIIIa comprises V158, F158, or both; (ii) can bind to human FcγRIIIb; (iii) can bind to human FcRn, optionally at pH 6; (iv) can bind to human complement component 1q (C1q); (v) (1) compared to a reference polypeptide comprising a human IgG1 Fc polypeptide comprising the amino acid substitutions G236A, S239D, A330L, and I330E (EU numbering), and optionally no other amino acid substitutions compared to a wild-type human IgG1 Fc polypeptide, (2) comprises the amino acid substitutions G236A, A330L, I330E (EU numbering), and optionally further comprises the mutations M428L and N434S, and / or the mutations M428L and N434A, and / or the wild-type human IgG1 Fc polypeptide. (3) compared to a reference polypeptide comprising a human IgG1 Fc polypeptide comprising the amino acid substitutions G236A or G236S (EU numbering), optionally without any other amino acid substitutions compared to wild-type human IgG1 Fc polypeptide, and / or without S239D; (4) compared to a reference polypeptide comprising a human IgG1 Fc polypeptide comprising the amino acid substitutions A330L and I332E (EU numbering), optionally without any other amino acid substitutions compared to wild-type human IgG1 Fc polypeptide; (vi) capable of promoting signaling through FcγRa in a host cell, optionally wherein (a) signaling is increased compared to signaling promoted by the reference polypeptide, and / or (b) said FcγRa is selected from the group consisting of FcγRIIa H131, FcγRIIa R131, FcγRIIIa V158, FcγRIIIa F158, or any combination thereof); (vii) capable of promoting antibody-dependent cellular cytotoxicity (ADCC) at least when included in an antibody;(viii) capable of promoting antibody-dependent phagocytosis (ADCP) at least when contained in an antibody; (ix) capable of promoting complement-dependent cytotoxicity (CDC) at least when contained in an antibody; (x) capable of forming an immune complex at least when contained in an antibody; or (xi) any combination of (i) through (x);

[0160] In any embodiment of the present disclosure, the variant may further comprise one or more modifications that enhance or further enhance binding to human FcRn compared to (1) a reference polypeptide comprising a wild-type human IgG1 Fc polypeptide, and / or (2) a polypeptide lacking the one or more modifications. In some embodiments, the one or more modifications that enhance binding to human FcRn comprise amino acid substitutions: (i) M428L / N434S; (ii) M252Y / S254T / T256E; (iii) T250Q / M428L; (iv) P257I / Q311I; (v) P257I / N434H; (vi) D376V / N434H; (vii) T307A / E380A / N434A; (viii) M428L / N434A; or (ix) any combination of (i)-(viii).

[0161] In any embodiment of the present disclosure, the variant may not contain any additional mutations compared to a reference IgG Fc polypeptide or fragment thereof, IgG hinge-CH2 polypeptide, or IgG hinge-Fc polypeptide or fragment thereof, respectively. In other embodiments, the variant of an IgG Fc polypeptide contains up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acid substitution compared to a wild-type or parent IgG Fc polypeptide, where one or more of the additional amino acid substitutions optionally include conservative amino acid substitutions. In other embodiments, the variant of an IgG Fc polypeptide is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, or at least 97% identical to the wild-type or parent IgG Fc polypeptide.

[0162] In some embodiments, the polypeptide comprises an Fc polypeptide.

[0163] In some embodiments, the polypeptide is a monomer comprised in a polypeptide dimer (e.g., an Fc dimer). In some embodiments, the polypeptide is a monomer comprised in a polypeptide homodimer (e.g., an Fc homodimer). In some embodiments, the polypeptide is a monomer comprised in a polypeptide heterodimer (e.g., an Fc heterodimer, optionally comprising a protrusion in a first Fc of the heterodimer and a corresponding recess in a second Fc of the heterodimer, and / or comprising one or more mutations that provide or contribute to opposite charges in each of the two Fc monomers (e.g., a positive charge in one region of the first monomer and a negative charge in a corresponding region of the second monomer), and / or comprising a heterologous amino acid sequence in one or both monomers to promote dimerization of the two Fc monomers).

[0164] In some embodiments, the variant Fc polypeptide or fragment is comprised in an antibody. Antibodies comprising any of the disclosed variants of the disclosed Fc polypeptides or fragments are also provided. Terms understood by those of ordinary skill in the art of antibody technology are given their art-acquired meanings, unless expressly defined differently herein. For example, the term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody (e.g., an scFv, Fab, or Fab2 fragment) that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, provided that the antibody comprises a variant Fc polypeptide or fragment provided herein. Thus, the term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof that include an Fc polypeptide or fragments (e.g., an Fc polypeptide and fragment antigen-binding (Fab) fragments, F(ab)2 fragments, Fab fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (scFv)), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments). For example, non-limiting examples of contemplated embodiments include intact antibodies; scFv:Fc fusions; scFab:Fc fusions, sdAb:Fc fusions, sdFv:Fc fusions, tri-Fab, DART-Fc, DVD-Ig, di-diabody, scFv-Fc, taFv-Fc, scFv-CH3 fusions, scFv-CH2 fusions, CH3 charge pair antibodies, duobodies, half antibodies, IgG(HA-Tf-Fv), and the like.The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins (such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies), multispecific (e.g., bispecific) antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, and tandem tri-scFvs, provided that a disclosed variant of the Fc polypeptide or fragment thereof is present. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof, provided that a disclosed variant of the Fc polypeptide or fragment thereof is present. The term also encompasses intact, i.e., full-length, antibodies, including antibodies of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0165] "V L " or "VL" and "V HThe terms "VH" and "VH" refer to the variable binding regions from antibody light chains and heavy chains, respectively. In one embodiment, the VL is of the kappa (κ) class (also referred to herein as "VK"). In one embodiment, the VL is of the lambda (λ) class. The variable binding regions comprise discrete, well-defined subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining regions" and "CDRs" are synonyms for "hypervariable regions" or "HVRs" and refer to the sequences of amino acids within antibody variable regions that generally confers antigen specificity and / or binding affinity to the antibody. Contiguous CDRs in an antibody (i.e., CDR1 and CDR2, and CDR2 and CDR3) are separated from each other by framework regions in the primary structure. There are three CDRs in each variable region (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRH and CDRL, respectively). In one embodiment, an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. Generally, the VH and VL combine through their respective CDRs to form an antigen-binding site. Numbering of the CDRs and framework regions can follow any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 thed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pluckthun, J. Mol. Bio. 309:657-670 (2001)).

[0166] In certain embodiments, a polypeptide or antibody of the present disclosure comprises an antigen-binding domain comprising a VH and a VL. In some embodiments, the VH and VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 26 and 27, respectively; (ii) 28 and 29, respectively; (iii) 30 and 31, respectively; (iv) 30 and 33, respectively; (v) 32 and 31, respectively; (vi) 32 and 33, respectively; (vii) 34 and 35, respectively; (viii) 43 and 44, respectively; (ix) 32 and 46, respectively; (x) 41 and 42, respectively; or (xi) 47 and 48, respectively. In some embodiments, the polypeptide or antibody further comprises a kappa light chain constant domain or a lambda light chain constant domain. In some embodiments, the polypeptide or antibody further comprises a CH1.

[0167] In some embodiments, a polypeptide or antibody of the disclosure comprises an antigen-binding domain comprising a VH and a VL, wherein said VH and said VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 28 and 29, respectively, and said polypeptide or antibody further comprises an IgG (e.g., IgG1) and variants of the Fc polypeptide, the variants comprising the following mutations according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; and (v) M428L, N434S, G236A, L328V, and Q295E (provided that the above (vi) M428L, N434S, G236A, R292P, and I377N (provided that the polypeptide or antibody is defucosylated); (vii) M428L, N434S, G236A, and Y300L (provided that the polypeptide or antibody is defucosylated); or (viii) M428L, N434S, G236A, R292P, and Y300L (provided that the polypeptide or antibody is defucosylated). In some embodiments, the variant of an IgG Fc polypeptide (e.g., IgG1) comprises amino acid substitutions consisting essentially of the substitution mutations (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

[0168] In some embodiments, a polypeptide or antibody of the disclosure comprises an antigen-binding domain comprising a VH and a VL, wherein said VH and said VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 43 and 44, respectively, and said polypeptide or antibody further comprises an IgG (e.g., IgG1) and variants of the Fc polypeptide, the variants comprising the following mutations according to EU numbering: (i) M428L, N434A, G236A, L328V, and Q295E; (ii) M428L, N434A, G236A, R292P, and I377N; (iii) M428L, N434A, G236A, and Y300L; (iv) M428L, N434A, G236A, R292P, and Y300L; and (v) M428L, N434A, G236A, L328V, and Q295E (provided that the above (vi) M428L, N434A, G236A, R292P, and I377N, wherein the polypeptide or antibody is defucosylated; (vii) M428L, N434A, G236A, and Y300L, wherein the polypeptide or antibody is defucosylated; or (viii) M428L, N434A, G236A, R292P, and Y300L, wherein the polypeptide or antibody is defucosylated. In some embodiments, the IgG Fc polypeptide variant comprises amino acid substitutions consisting essentially of the substitution mutations (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

[0169] In some embodiments, a polypeptide or antibody of the disclosure comprises an antigen-binding domain comprising a VH and a VL, wherein said VH and said VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 43 and 44, respectively, and said polypeptide or antibody further comprises an IgG (e.g., IgG1) and variants of the Fc polypeptide, the variants comprising the following mutations according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; and (v) M428L, N434S, G236A, L328V, and Q295E (provided that the above (vi) M428L, N434S, G236A, R292P, and I377N (provided that said polypeptide or antibody is defucosylated); (vii) M428L, N434S, G236A, and Y300L (provided that said polypeptide or antibody is defucosylated); or (viii) M428L, N434S, G236A, R292P, and Y300L (provided that said polypeptide or antibody is defucosylated). In some embodiments, the variant of an IgG Fc polypeptide comprises amino acid substitutions consisting essentially of the substitution mutations (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

[0170] In some embodiments, a polypeptide or antibody of the disclosure comprises an antigen-binding domain comprising a VH and a VL, wherein said VH and said VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 43 and 44, respectively, and said polypeptide or antibody further comprises an IgG (e.g., IgG1) and variants of the Fc polypeptide, the variants comprising the following mutations according to EU numbering: (i) M428L, N434A, G236A, L328V, and Q295E; (ii) M428L, N434A, G236A, R292P, and I377N; (iii) M428L, N434A, G236A, and Y300L; (iv) M428L, N434A, G236A, R292P, and Y300L; and (v) M428L, N434A, G236A, L328V, and Q295E (provided that the above (vi) M428L, N434A, G236A, R292P, and I377N (provided that the polypeptide or antibody is defucosylated); (vii) M428L, N434A, G236A, and Y300L (provided that the polypeptide or antibody is defucosylated); or (viii) M428L, N434A, G236A, R292P, and Y300L (provided that the polypeptide or antibody is defucosylated). In some embodiments, the variant of an IgG Fc polypeptide (e.g., IgG1) comprises amino acid substitutions consisting essentially of the substitution mutations (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody comprises a kappa light chain.

[0171] In certain embodiments, the antibodies of the present disclosure include an antigen-binding domain from any of the following non-limiting antibodies: 3F8, 8H9, abagovomab, abiciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, amiflurazine ... Lucitumomab, asclinbacumab, acelizumab, atezolizumab, atinumab, atlizumab, atolizumab, avelumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, berinumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, bro Lucizumab, brontixituzumab, burosumab, cabilalizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, sergituzumab amnaleukin, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakizumab, clinoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, and concizumab CR6261, crenezumab, cloteduumab, dacetuzumab, daclizumab, darotuzumab, dapirolizumab pegol, daratumumab, dectrecumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, dinutuximab, zilidabumab, domagurozumab, dorlimomab alitoxin, drozitumab, durigotumab, dupilumab, durvalumab, dusigitumab, ecloneximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab,Eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, emicizumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, encituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezaki Numab, Fivatuzumab, Ficlatuzumab, Figitumumab, Filibumab, Framvotumab, Fretikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Furlanumab, Futuximab, Galcanezumab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Idalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imal Mabs, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipirinumab, iratumumab, isatuximab, itolizumab, ixexizumab, keliximab, labetuzumab, lampalizumab, lanadelumab, landgrozumab, laprituximab emtansine, lebrikizumab, remaresomab, lendalizumab, lendilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab Buvedotin, lifelizumab, rilotomab satetraxetan, lintuzumab, lirilumab, roderucizumab, loxivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, lumiliximab, lumuletuzumab, MABpl, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mirvetuximab soravtanisin, mitumomab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab, pasudotox, muromonab-CD3,Nacolomab butafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narutumab, natalizumab, nabicixizumab, navivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, occaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, olticumab , otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pasivaximab, palivizumab, pamrevumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placuramab, prosalizumab, pogalizumab, polatuzumab vedotin, ponezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140 Also known as leronlimab, kirisumab, racotumumab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinukumab, risankizumab, rituximab, rivabazumab pegol, lotatumumab, loredumab, romosozumab, lontalizumab, rovalpituzumab tesirin, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sapelizumab, sarilumab, satumomab pendetide, secukinumab Mab, seribantumab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, sotrovimab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tamtubetomab, tanezumab, taplitumomab paptox, talexuzumab, tefibazumab,Terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, ticilimumab, tigatuzumab, tildrakizumab, timolumab, tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevoglumab, tucotuzumab celmoleukin, tuvilumab, ublitz Ximab, urocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab butarilin, bundletuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, bisilizumab, bovalilizumab, volociximab, borsetuzumab mafodotin, votumumab, xentuzumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitoxin, and combinations thereof.

[0172] In some embodiments, the polypeptide or antibody comprises an IgG1 isotype. In certain embodiments, the polypeptide or antibody comprises an IgG1m17 allotype, an IgG1m17,1 allotype, an IgG1m3 allotype, or an IgG1m3,1 allotype.

[0173] In some embodiments, the variant IgG Fc polypeptide does not comprise any other amino acid substitution mutations compared to the wild-type or parent IgG Fc polypeptide. In other embodiments, the variant IgG Fc polypeptide comprises up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acid substitution compared to the wild-type or parent IgG Fc polypeptide, where one or more of the additional amino acid substitutions optionally comprise conservative amino acid substitutions. In other embodiments, the variant IgG Fc polypeptide is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, or at least 97% identical to the wild-type or parent IgG Fc polypeptide.

[0174] In one embodiment, the VH of the IgG Fc polypeptide and the variants are comprised in a heavy chain, which heavy chain comprises VH-CH1-CH2-CH3. In one embodiment, the VL is comprised in a light chain that further comprises a kappa light chain (e.g., IgG1). In another embodiment, the VL is comprised in a light chain that further comprises a lambda light chain (e.g., IgG1).

[0175] "Fab" (Fragment Antigen Binding) is the antigen-binding portion of an antibody and comprises the variable region and CH1 of the heavy chain linked to the light chain by an interchain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding (i.e., has a single antigen-binding site). Pepsin treatment of an antibody generates a single large F(ab)2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. Both Fab and F(ab)2 are examples of "antigen-binding fragments." Fab fragments differ from Fab fragments by having several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab-SH, as used herein, refers to Fab in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab)2 antibody fragments are originally produced as pairs of Fab fragments, with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0176] An "Fv" is a small antibody fragment that contains one complete antigen-recognition and antigen-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. However, even a single variable domain (i.e., half of an Fv, containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although typically with lower affinity than the entire binding site.

[0177] "Single-chain Fv" is also abbreviated as "sFv" or "scFv" and is H Antibody domains and V L In some embodiments, an scFv polypeptide is an antibody fragment in which antibody domains are joined together into a single polypeptide chain. H Domains and V LThe scFv may contain a polypeptide linker between the domains, allowing the scFv to maintain or form the desired structure for antigen binding. Such peptide linkers can be incorporated into fusion polypeptides using standard techniques known in the art. Additionally or alternatively, the Fv may contain a disulfide bond between the VH and VL domains, stabilizing them. For a review of scFvs, see Plückthun; Borrebaeck 1995, infra, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). In one embodiment, the antibody or antigen-binding fragment comprises an scFv comprising a VH domain, a VL domain, and a peptide linker connecting the VH domain to the VL domain. In particular embodiments, an scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL or VL-linker-VH orientation. Any scFv of the disclosure can be engineered so that the C-terminus of the VL domain is linked to the N-terminus of the VH domain by a short peptide sequence, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker can be attached to the N-terminal portion, or to the end of the VH domain, the VL domain, or both. An scFv can be included in a fusion with, linked to, or complexed with an Fc variant or antibody of the disclosure.

[0178] In some embodiments, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295. In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0179] In other embodiments, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 230, and a glutamic acid (E) at EU position 295. In certain further embodiments, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0180] In yet another embodiment, the antibody is provided comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0181] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 334, and a glutamic acid (E) at EU position 295. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0182] In yet another embodiment, the antibody is provided comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a serine (S) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0183] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0184] In yet another embodiment, the antibody is provided comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0185] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a serine (S) at EU position 236, a valine (V) at EU position 420, a glutamic acid (E) at EU position 446, and a threonine (T) at EU position 309. In a further embodiment, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein), are present.

[0186] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0187] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a proline (P) at EU position 292 and a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0188] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a leucine (L) at EU position 300. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0189] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a lysine (K) at EU position 345, a serine (S) at EU position 236, a tyrosine (Y) at EU position 235, and a glutamic acid (E) at EU position 267. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0190] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an arginine (R) at EU position 272, a threonine (T) at EU position 309, a tyrosine (Y) at EU position 219, and a glutamic acid (E) at EU position 267. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0191] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a tyrosine (Y) at EU position 236. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0192] In yet another embodiment, the antibody is provided as comprising a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a tryptophan (W) at EU position 236. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0193] In yet another embodiment, an antibody is provided that comprises an IgG (e.g., IgG1) Fc variant, the variant comprising an alanine (A) at EU position 236, wherein said IgG Fc polypeptide or fragment thereof, optionally said polypeptide is defucosylated, and further optionally said variant comprises a leucine (L) at EU position 330 and a glutamic acid (E) at EU position 332, and further optionally said variant does not comprise an aspartic acid (D) at EU position 239, and further optionally comprises a serine (S) at EU position 239. In a further embodiment, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0194] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises a leucine (L) at EU position 243, a glutamic acid (E) at EU position 446, a leucine (L) at EU position 396, and a glutamic acid (E) at EU position 267. In a further embodiment, the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein) are present.

[0195] In yet another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, the variant comprising alanine (A) at EU position 236, aspartic acid (D) at EU position 239, glutamic acid (E) at EU position 332, leucine (L) at EU position 428, and serine (S) or alanine (A) at EU position 434.

[0196] In another embodiment, the antibody comprises a variant of an IgG (e.g., IgG1) Fc, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, and a glutamic acid (E) at EU position 268.

[0197] In some embodiments, the antibody further comprises the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein).

[0198] In one embodiment, the antibody is defucosylated.

[0199] In any polypeptide or antibody of the present disclosure, the variant Fc or fragment thereof can be derived from the IgG1 isotype, IgG2 isotype, IgG3 isotype, or IgG4 isotype. In certain embodiments, the variant is derived from a human Fc or fragment thereof, or from a human antibody heavy chain or fragment thereof. In further embodiments, the variant is derived from the human IgG1 isotype, human IgG2 isotype, human IgG3 isotype, or human IgG3 isotype. In particular embodiments, the variant is derived from the human IgG1 isotype.

[0200] A polypeptide, CH2, Fc, CH3, Fc fragment or portion, or antibody can be of any allotype or combination of allotypes. "Allotype" refers to the allelic variations found in an IgG subclass. For example, one allotype can include G1m1 (or G1m(a)), G1m2 (or G1m(x)), G1m3 (or G1m(f)), G1m17 (or Gm(z)), G1m27, and / or G1m28 (G1m27 and G1m28 have been described as "alloallotypes").

[0201] The G1m3 and G1m17 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering). G1m3 contains R214(EU), whereas G1m17 contains K214(EU). The G1m1 allotype is located in the CH3 domain (positions 356 and 358(EU)) and represents the substitutions E356D and M358L. The G1m2 allotype represents the replacement of alanine with glycine at position 431(EU). G1m allotypes, alloallotypes, and their characteristics are known in the art and are described, for example, in www.imgt.org / IMGTrepertoire / Proteins / allotypes / human / IGH / IGHC / G1m_allotypes.html and in Lefranc, M.-P. and Lefranc, G. Human Gm, Km, and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism, Methods Mol. Biol. 2012; 882, 635-680. PMID: 22665258, LIGM: 406, in B. Tait, F. Christiansen (Eds.), Immunogenetics, chap. 34, Humana Press, Springer, New York, USA (the contents and allotypes and allotype information of which are incorporated herein by reference).

[0202] The G1m1 allotype can be combined with, for example, G1m3, G1m17, G1m27, G1m2, and / or G1m28 allotypes. In some embodiments, the allotype is G1m3 without G1m1 (G1m3,-1). In some embodiments, the allotype is G1m17,1 allotype. In some embodiments, the allotype is G1m3,1. In some embodiments, the allotype is G1m17 without G1m1 (G1m17,-1). Optionally, these allotypes may be combined (or not) with G1m2, G1m27, or G1m28 allotypes. For example, the allotype can be G1m17,1,2.

[0203] In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the present disclosure comprises the G1m3 allotype or the G1m3,1 allotype. In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the present disclosure comprises the G1m3 allotype and includes the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the present disclosure comprises the G1m3,1 allotype and includes the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn (such as those described herein). In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the present disclosure comprises the G1m17,1 allotype. In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the disclosure comprises the G1m17,1 allotype and includes the mutations M428L and N434S, or M428L and N434A, or any other mutation that enhances binding to human FcRn, as further described herein.

[0204] In some embodiments, a polypeptide, CH2, Fc, Fc fragment or portion, or antibody of the disclosure (i) can bind to human FcγRIIIa, wherein said human FcγRIIIa comprises V158, F158, or both; (ii) can bind to human FcγRIIIb; (iii) can bind to human FcRn, optionally at pH 6; (iv) can bind to human complement component 1q (C1q) with more than 1-fold, at least 2-fold, at least 3-fold, or at least 4-fold increased binding compared to the binding of an antibody comprising a reference Fc polypeptide (or compared to the binding of a reference polypeptide, CH2, Fc, Fc fragment or portion);(v) (1) a human IgG1 Fc comprising the amino acid substitutions G236A, S239D, A330L, and 1330E (EU numbering), and optionally compared to a reference antibody (or reference polypeptide, CH2, Fc, Fc fragment or portion) that does not comprise any other amino acid substitutions compared to wild-type human IgG1 Fc; (2) a human IgG1 Fc comprising the amino acid substitutions G236A, A330L, 1330E (EU numbering), and optionally further comprising: (a) the mutations M428L and N434S, or the mutations M428L and N434A, or any o...

Claims

1. (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or a fragment thereof, wherein the variant is a polypeptide comprising alanine (A) at EU position 236 and leucine (L) at EU position 300.

2. The polypeptide according to claim 1, wherein the variant and optionally the polypeptide have increased binding to human FcγRIIa as compared to the binding of a reference polypeptide thereto, wherein optionally the binding is determined using an electrochemiluminescence assay and further optionally using Meso Scale Discovery, polypeptide.

3. The polypeptide according to claim 2, wherein the increased binding to human FcγRIIa comprises binding to human FcγRIIa that is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to human FcγRIIa.

4. The polypeptide according to claim 2, wherein the human FcγRIIa comprises H131, and optionally the increased binding to human FcγRIIa H131 comprises binding to human FcγRIIa H131 that is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, or at least 18-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to human FcγRIIa H131.

5. The polypeptide according to claim 2, wherein the human FcγRIIa comprises R131, and optionally the increased binding to human FcγRIIa R131 comprises binding to human FcγRIIa R131 that is at least 4-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to human FcγRIIa R131.

6. The polypeptide according to claim 2, wherein (1) (i) the ratio of the binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb respectively is (2) greater than the ratio of (iii) the binding of the reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to human FcγRIIb, provided that the reference polypeptide comprises a wild-type human IgG Fc polypeptide or a fragment thereof, a polypeptide.

7. The polypeptide according to claim 6, wherein the human FcγRIIa comprises H131.

8. The polypeptide according to claim 6, wherein the human FcγRIIa comprises R131.

9. The ratio in (1) is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, or at least 17-fold greater than the ratio in (2), the polypeptide according to claim 6.

10. The polypeptide according to claim 1, further comprising proline (P) at EU position 292.

11. In the polypeptide according to claim 1, wherein (1) (i) the ratio of the binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb respectively is (2) greater than the ratio of (iii) the binding of the reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to human FcγRIIb, provided that the reference polypeptide comprises a wild-type human IgG Fc polypeptide or a fragment thereof, optionally the binding is determined using an electrochemiluminescence assay and optionally using Meso Scale Discovery, a polypeptide.

12. The polypeptide according to claim 11, wherein the human FcγRIIa comprises H131.

13. The polypeptide according to claim 11, wherein the human FcγRIIa comprises R131.

14. The polypeptide according to claim 11, wherein the ratio in (1) is more than 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than the ratio in (2).

15. (i) a variant of an IgG CH2 polypeptide, or (ii) a variant of an IgG Fc polypeptide or a fragment thereof, wherein the variant is a polypeptide comprising alanine (A) at EU position 236, proline (P) at EU position 292, and leucine (L) at EU position 300.

16. In the polypeptide according to claim 15, the variant and optionally the polypeptide have an increased binding to human FcγRIIIa, compared to the binding of a reference polypeptide to the human FcγRIIIa, wherein optionally the binding is determined using an electrochemiluminescence assay and further optionally using Meso Scale Discovery, polypeptide.

17. The polypeptide according to claim 16, wherein the increased binding to human FcγRIIa comprises a binding to human FcγRIIa that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcγRIIa.

18. The polypeptide according to claim 16, wherein the human FcγRIIa comprises H131, and optionally the increased binding to the human FcγRIIa H131 comprises a binding to the human FcγRIIa H131 that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcγRIIa H131.

19. The human FcγRIIa contains R131, and optionally, the increased binding of the human FcγRIIa to H131 is at least 2-fold greater than the binding of a reference polypeptide containing a wild-type human IgG Fc polypeptide or a fragment thereof to the human FcγRIIa R131, the polypeptide according to claim 15.

20. In the polypeptide according to claim 15, (1) (i) the ratio of the binding of the variant or polypeptide to human FcγRIIa to (ii) the binding of the variant or polypeptide to human FcγRIIb respectively is (2) greater than the ratio of (iii) the binding of the reference polypeptide to human FcγRIIa to (iv) the binding of the reference polypeptide to the human FcγRIIb, provided that the reference polypeptide contains a wild-type human IgG Fc polypeptide or a fragment thereof, Optionally, the binding is determined using an electrochemiluminescence assay, and further optionally using Meso Scale Discovery, a polypeptide.

21. The polypeptide according to claim 20, wherein the human FcγRIIa contains H131.

22. The polypeptide according to claim 20, wherein the human FcγRIIa contains R131.

23. The ratio in (1) is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold greater than the ratio in (2), the polypeptide according to claim 20.

24. The variant has increased binding to human FcγRIIIa, compared to the binding of a reference polypeptide to the human FcγRIIIa, Optionally, the binding is determined using an electrochemiluminescence assay, and further optionally using Meso Scale Discovery, the polypeptide according to claim 15.

25. The polypeptide according to claim 24, wherein the human FcγRIIIa contains V158, F158, or both.

26. The binding to human FcγRIIIA of the polypeptide according to claim 24, wherein the increased binding to human FcγRIIIA is more than 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3.0-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, or at least 3.7-fold greater than the binding of a reference polypeptide comprising a wild-type human IgG Fc polypeptide or a fragment thereof to human FcγRIIIA.

27. The polypeptide according to claim 15, wherein the variant and optionally the polypeptide can bind to human complement component 1q (C1q), and optionally the binding is determined using an electrochemiluminescence assay and further optionally using Meso Scale Discovery, the polypeptide.

28. (i) capable of binding to human FcγRIIIA, provided that the human FcγRIIIA comprises V158, F158, or both; (ii) capable of binding to human FcγRIIIB; (iii) capable of binding to human FcRn, optionally at pH 6; (iv) capable of binding to human complement component 1q (C1q); (v) (1) a reference polypeptide comprising a human IgG1 Fc polypeptide containing the amino acid substitutions G236A, S239D, A330L, and I330E (EU numbering) and optionally containing no other amino acid substitutions as compared to a wild-type human IgG1 Fc polypeptide, (2) a reference polypeptide comprising a human IgG1 Fc polypeptide containing the amino acid substitutions G236A, A330L, I330E (EU numbering) and optionally further containing the mutations M428L and N434S, or the mutations M428L and N434A, and / or containing no other amino acid substitutions as compared to a wild-type human IgG1 Fc polypeptide, and / or not containing S239D, (3) a reference polypeptide comprising a human IgG1 Fc polypeptide containing the amino acid substitution G236A or G236S (EU numbering) and optionally containing no other amino acid substitutions as compared to a wild-type human IgG1 Fc polypeptide, and / or A reference polypeptide comprising a human IgG1 Fc polypeptide containing the amino acid substitutions A330L and I332E (EU numbering) and optionally no other amino acid substitutions compared to the wild-type human IgG1 Fc polypeptide having a higher Tm and / or being producible at a greater titer compared to; (vi) capable of promoting signal transduction through FcγRa in a host cell (provided that optionally (a) the signal transduction is optionally increased compared to the signal transduction promoted by the reference polypeptide, and / or (b) the FcγRa comprises FcγRIIa H131, FcγRIIa R131, FcγRIIIa V158, FcγRIIIa F158, or any combination thereof); (vii) capable of promoting antibody-dependent cell-mediated cytotoxicity (ADCC) when contained at least in an antibody; (viii) capable of promoting antibody-dependent phagocytosis (ADCP) when contained at least in an antibody; (ix) capable of promoting complement-dependent cytotoxicity (CDC) when contained at least in an antibody; (x) capable of forming an immune complex when contained at least in an antibody; or (xi) A polypeptide according to any one of claims 1 to 27, which is any combination of (i) to (x). **Claim 29** An antibody, wherein the antibody (i) increases the specific lysis of target cells expressing an antigen by natural killer cells and / or PBMCs (e.g., expressing F158 / V158 or V158 / V158 FcγRIIIA) (e.g., through ADCC) compared to an antibody containing a reference Fc polypeptide free of mutations and / or fucosylation status (e.g., an antibody containing a human IgG1 Fc containing the mutations G236A, A330L, and I332E); (ii) increases ADCP by monocytes (e.g., CD14+ monocytes optionally expressing F158 / V158 FcγRIIA and R131 / H131 FcγRIIA, or F158 / F158 FcγRIIA and R131 / H131 FcγRIIA) of target cells expressing an antigen compared to an antibody containing a reference Fc polypeptide free of mutations and / or fucosylation status; (iii) when provided in combination with said antigen, increasing the proportion of CD83+ cells (e.g., moDC) in a sample and / or the expression of CD83 by moDC as compared to an antibody comprising a reference Fc polypeptide that does not contain a mutation and / or fucosylation state when provided in combination with said antigen; (iv) when provided in combination with said antigen, increasing the production of one or more cytokines (optionally selected from the group consisting of IL-1β, IFN-γ, IL-6, and TNF-α) by moDC in a sample as compared to an antibody comprising a reference Fc polypeptide that does not contain a mutation and / or fucosylation state when provided in combination with said antigen; and (v) increasing the ability of moDC to stimulate antigen-specific CD4+ T cells when provided to said moDC in combination with an antigen as compared to an antibody comprising a reference Fc polypeptide that does not contain a mutation and / or fucosylation state when provided to said moDC in combination with said antigen, wherein optionally (1) said moDC and said CD4+ T cells are derived from the same (optionally antigen-inoculated) subject, and / or (2) the stimulation of antigen-specific CD4+ T cells is determined by an increase in the expression of CD25 by said antigen-specific CD4+ T cells, and / or an increase in proliferation (e.g., as determined by a decrease in CFSE staining over time), and / or an increase in the expression of CD69, and / or an increase in the expression of NFAT, and / or an increase in the expression of CD44, a polypeptide according to any one of claims 1 to 27. [

30. ] wherein said variant further comprises one or more modifications that enhance or further enhance binding to human FcRn, as compared to (1) a reference polypeptide comprising a wild-type human IgG Fc polypeptide, and / or (2) a polypeptide according to any one of claims 1 to 27 that does not have said one or more modifications, a polypeptide according to any one of claims 1 to 27. [

31. ] wherein said one or more modifications that enhance binding to human FcRn are amino acid substitutions at EU positions: (i) M428L / N434S; (ii) M252Y / S254T / T256E; (iii) T250Q / M428L; (iv) P257I / Q311I; (v) P257I / N434H; (vi) D376V / N434H; (vii) T307A / E380A / N434A; (viii) N434A; (ix) M428L / N434A; or (x) Any combination of (i) to (ix) The polypeptide according to claim 30, comprising

32. The polypeptide according to any one of claims 1 to 27, wherein the variant does not contain any additional mutations compared to each of the reference IgG Fc polypeptide or a fragment thereof, the IgG CH2 polypeptide, the IgG hinge-CH2 polypeptide, or the IgG hinge-Fc polypeptide or a fragment thereof.

33. The polypeptide according to any one of claims 1 to 27, comprising an Fc polypeptide.

34. The polypeptide according to any one of claims 1 to 27, which is a monomer contained in a polypeptide dimer (for example, an Fc dimer).

35. The polypeptide according to any one of claims 1 to 27, which is a monomer contained in a polypeptide homodimer (for example, an Fc homodimer).

36. The polypeptide according to any one of claims 1 to 27, which is a monomer contained in a polypeptide heterodimer (for example, an Fc heterodimer, optionally containing a protrusion in the first Fc of the heterodimer and a corresponding depression in the second Fc of the heterodimer, and / or providing opposite charges (for example, a positive charge in a certain region of the first monomer and a negative charge in the corresponding region of the second monomer) or containing one or more mutations contributing to the opposite charges in each of the two Fc monomers, and / or containing a heterologous amino acid sequence in one or both monomers to promote dimerization of the two Fc monomers).

37. The polypeptide according to any one of claims 1 to 27, which is contained in an antibody.

38. An antibody comprising the polypeptide according to any one of claims 1 to 27.

39. An antibody comprising a variant of IgG Fc, wherein the variant contains alanine (A) at EU position 236 and leucine (L) at EU position 300.

40. An antibody comprising a variant of IgG Fc, wherein the variant contains alanine (A) at EU position 236, proline (P) at EU position 292, and leucine (L) at EU position 300.

41. The polypeptide according to any one of claims 1 to 27, the antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the variant is derived from or comprises an IgG1 isotype, an IgG2 isotype, an IgG3 isotype, or an IgG4 isotype.

42. The polypeptide according to any one of claims 1 to 27, the antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the variant is derived from or comprises human Fc or a fragment thereof, or is derived from a human antibody heavy chain or a fragment thereof.

43. The polypeptide according to any one of claims 1 to 27, the antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the variant is derived from or comprises a human IgG1 isotype, a human IgG2 isotype, a human IgG3 isotype, or a human IgG4 isotype.

44. The polypeptide according to any one of claims 1 to 27, the antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the variant is derived from or comprises a human IgG1 isotype optionally comprising an allotype G1m3, G1m17, G1m3,1, or G1m17,1.

45. (i) capable of binding to human FcγRIIIa, wherein the human FcγRIIIa comprises V158, F158, or both; (ii) capable of binding to human FcγRIIIb; (iii) optionally capable of binding to human FcRn at pH 6; (iv) capable of binding to human complement component 1q (C1q), and optionally the binding is increased by more than 1-fold, at least 2-fold, at least 3-fold, or at least 4-fold compared to the binding of an antibody comprising a reference Fc polypeptide; (v) (1) a reference antibody comprising a human IgG1 Fc comprising the amino acid substitutions G236A, S239D, A330L, and I330E (EU numbering), provided that the reference antibody optionally does not contain any other amino acid substitutions in the Fc compared to wild-type human IgG1 Fc; (2) A reference antibody comprising a human IgG1 Fc containing the amino acid substitutions G236A, A330L, and I330E (EU numbering), provided that said reference antibody optionally further comprises (a) the mutations M428L and N434S, or M428L and N434A, and / or (b) contains no other amino acid substitutions in the Fc compared to wild-type human IgG1 Fc, and / or (c) does not contain the mutation S239D), (3) A reference antibody comprising a human IgG1 Fc containing the amino acid substitution G236A or G236S (EU numbering) and optionally containing no other amino acid substitutions in the Fc compared to wild-type human IgG1 Fc, (4) A reference antibody comprising a human IgG1 Fc containing the amino acid substitutions A330L and I332E (EU numbering), provided that said reference antibody optionally contains no other amino acid substitutions in the Fc compared to wild-type human IgG1 Fc; and / or (5) A reference antibody comprising wild-type human IgG1 Fc having a higher Tm and / or being able to be produced at a greater titer and / or binding to human FcγRIIa (optionally H131 and / or R131) with greater affinity and / or avidity and / or binding to human FcγRIIb with lesser affinity and / or avidity compared to (vi) being able to promote signal transduction through FcγRa in a host cell, provided that optionally (a) the signal transduction is increased compared to the signal transduction promoted by a reference antibody, and / or (b) said FcγRa comprises FcγRIIa H131, FcγRIIa R131, FcγRIIIa V158, FcγRIIIa F158, or any combination thereof); (vii) being able to promote antibody-dependent cell-mediated cytotoxicity (ADCC); (viii) being able to promote antibody-dependent phagocytosis (ADCP); (ix) being able to promote complement-dependent cytotoxicity (CDC); (x) being able to form immune complexes; or (xi) An antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40, which is any combination of (i) to (x).

46. (i) increasing specific lysis of target cells expressing an antigen by natural killer cells and / or PBMCs (e.g., expressing F158 / V158 or V158 / V158 FcγRIIIA) (e.g., through ADCC) compared to an antibody comprising a reference Fc polypeptide that does not contain mutations and / or fucosylation states (e.g., an antibody comprising human IgG1 Fc containing mutations G236A, A330L, and I332E); (ii) increasing ADCP by monocytes (e.g., CD14+ monocytes optionally expressing F158 / V158 FcγRIIA and R131 / H131 FcγRIIA, or F158 / F158 FcγRIIA and R131 / H131 FcγRIIA) of target cells expressing an antigen compared to an antibody comprising a reference Fc polypeptide that does not contain mutations and / or fucosylation states; (iii) increasing the proportion of CD83+ cells (e.g., moDC) and / or the expression of CD83 by moDC in a sample when provided in combination with the antigen compared to an antibody comprising a reference Fc polypeptide that does not contain mutations and / or fucosylation states when provided in combination with the antigen; (iv) increasing the production of one or more cytokines (optionally selected from the group consisting of IL-1β, IFN-γ, IL-6, and TNF-α) by moDC in a sample when provided in combination with the antigen compared to an antibody comprising a reference Fc polypeptide that does not contain mutations and / or fucosylation states when provided in combination with the antigen; and (v) When the ability of moDCs to stimulate antigen-specific CD4+ T cells is provided to the moDCs in combination with an antigen, it can achieve any one or more of increasing compared to an antibody containing a reference Fc polypeptide that does not include a mutation and / or fucosylation state when provided to the moDCs in combination with the antigen. Optionally, (1) the moDCs and the CD4+ T cells are derived from the same (optionally antigen-inoculated) subject, and / or (2) the stimulation of antigen-specific CD4+ T cells is determined by an increase in the expression of CD25 by the antigen-specific CD4+ T cells, and / or an increase in proliferation (e.g., determined by a decrease in CFSE staining over time), and / or an increase in the expression of CD69, and / or an increase in the expression of NFAT, and / or an increase in the expression of CD44. An antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40. [

47. ] The variant further comprises one or more modifications that enhance binding to human FcRn, (1) a reference polypeptide comprising a wild-type human IgG Fc polypeptide, and / or (2) compared to an antibody comprising the polypeptide according to any one of claims 1 to 27, or an antibody according to any one of claims 39 or 40, that does not have the one or more modifications. An antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40. [

48. ] The one or more modifications that enhance binding to human FcRn are amino acid substitutions at EU positions: (i) M428L / N434S; (ii) M252Y / S254T / T256E; (iii) T250Q / M428L; (iv) P257I / Q311I; (v) P257I / N434H; (vi) D376V / N434H; (vii) T307A / E380A / N434A; (viii) M428L / N434A; or (ix) any combination of (i) to (viii) An antibody according to claim 47. [

49. ] The variant does not include any additional mutations compared to the reference wild-type IgG Fc. An antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40. [

50. ] The antibody can specifically bind to (i) A pathogen (e.g., a virus, bacterium, parasite, fungus), or a target (e.g., an antigen) expressed or produced by a cell infected with the pathogen (provided that, in some cases, the pathogen includes a virus, and the virus includes: coronavirus; beta-coronavirus; sarbecovirus; embecovirus; novel coronavirus; merbecovirus; metanidovirus; hibecovirus; SARS-CoV-2; hepatitis B virus; hepatitis D virus; influenza A virus; cytomegalovirus; rhinovirus; hepatitis C virus; influenza B virus; human immunodeficiency virus; respiratory virus; respiratory syncytial virus; Zika virus; rabies virus; dengue virus; flavivirus; Ebola virus; or any combination thereof); (ii) A target (e.g., an antigen) expressed by tumor cells, optionally cancer cells, or cells of a proliferative disorder or hyperproliferative disorder, and / or expressed on the cell surface of said cells; (iii) A target (e.g., an antigen) associated with an autoimmune disease; (iv) A target (e.g., an antigen) associated with a neurodegenerative disease (v) Immune system signaling molecules (such as cytokines); (vi) A target (e.g., an antigen) associated with inflammation; (vii) A target (e.g., an antigen) associated with a non-infectious disease; or (viii) Any combination of (i) to (vii) The polypeptide according to claim 37, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, which is

51. The polypeptide according to claim 37, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the antibody can specifically bind to the human immunodeficiency virus.

52. The polypeptide according to claim 37, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, comprising a chimeric antibody, a humanized antibody, a neutralizing antibody, a human antibody, IgNAR, a camel antibody, or any combination thereof.

53. The polypeptide according to claim 37, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the antibody is a multispecific antibody, such as a bispecific antibody, a trispecific antibody, or a tetravalent antibody.

54. The polypeptide according to claim 37, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the antibody is included in an antibody complex.

55. The polypeptide according to any one of claims 1 to 27, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, wherein the polypeptide or the Fc polypeptide comprises (1) an Fc fusion protein and / or (2) an Fcab.

56. The Fc fusion protein further comprises (i) a receptor domain (e.g., the ectodomain of a receptor protein or its ligand-binding portion); (ii) a ligand; (iii) a replacement protein, or (iv) any combination of (i) to (iii) The polypeptide or antibody according to claim 55.

57. The polypeptide according to any one of claims 1 to 27, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40, which is bound, linked, or fused to a payload portion.

58. The payload portion comprises an antibody or its antigen-binding fragment; a cytotoxic agent (e.g., a chemotherapeutic agent); a detectable compound or detectable label; an oligonucleotide (e.g., an antisense oligonucleotide, siRNA, etc.); a vector; an agent that stimulates an immune response; a growth factor; or any combination thereof. The polypeptide or antibody according to claim 57.

59. It is defucosylated; produced in a host cell that cannot be fucosylated or has its ability to fucosylate a polypeptide inhibited; produced by a host cell under conditions that inhibit fucosylation; or any combination thereof. The polypeptide according to any one of claims 1 to 27, an antibody comprising the polypeptide according to any one of claims 1 to 27, or the antibody according to claim 39 or 40.

60. A polypeptide according to any one of claims 1 to 27, an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40, which inhibits fucosylation compared to each of the reference polypeptide or antibody, and / or comprises an amino acid mutation that inactivates a fucosylation site present in each of the reference polypeptide or antibody.

61. A polypeptide according to any one of claims 1 to 9 and 11 to 14, an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40, which comprises the amino acid sequence shown in SEQ ID NO:

11.

62. A polypeptide according to any one of claims 10, 11 to 14 and 15 to 27, an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40, which comprises the amino acid sequence shown in SEQ ID NO:

12.

63. A polynucleotide that encodes a polypeptide according to any one of claims 1 to 27, an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40.

64. The polynucleotide according to claim 63, wherein the codons are optimized for expression in a host cell.

65. An (e.g., expression) vector comprising the polynucleotide according to claim 63.

66. A host cell comprising the polynucleotide according to claim 63, or the vector according to claim 65.

67. A host cell that expresses a polypeptide according to any one of claims 1 to 27, and / or an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40.

68. In a composition, (i) a polypeptide according to any one of claims 1 to 27; and / or (ii) an antibody comprising a polypeptide according to any one of claims 1 to 27, or an antibody according to claim 39 or 40; and / or (iii) the polynucleotide according to claim 63; and / or (iv) the vector according to claim 65; and / or (v) the host cell according to claim 66, together with a pharmaceutically acceptable base, excipient, or diluent.

69. In a pharmaceutical composition for the treatment or prevention of a disease or disorder, the polypeptide according to any one of claims 1 to 27, an antibody comprising the polypeptide according to any one of claims 1 to 27, the antibody according to claim 39 or 40, the polynucleotide according to claim 63, the vector according to claim 65, the host cell according to claim 66, and / or the composition according to claim 68, and a pharmaceutically acceptable excipient, base, or diluent. A pharmaceutical composition.

70. The pharmaceutical composition according to claim 69, wherein the disease includes an infectious disease (optionally caused by viral, bacterial, fungal, or parasitic infection), cancer, a proliferative disorder, a neurodegenerative disease, an autoimmune disease, or any combination thereof.

71. The infectious diseases included are coronavirus infection, beta coronavirus infection, sarbecovirus infection, embecovirus infection, novel coronavirus infection, merbecovirus infection, metapneumovirus infection, hibecovirus infection, SARS-CoV-2 infection, hepatitis B virus infection, hepatitis D virus infection, hepatitis C virus infection, cytomegalovirus infection, influenza A virus infection, influenza B virus infection, human immunodeficiency virus infection, respiratory virus infection, respiratory syncytial virus infection, Zika virus infection, rabies virus infection, dengue virus infection, flavivirus infection, Ebola virus infection, or any combination thereof. The pharmaceutical composition according to claim 70.

72. The pharmaceutical composition according to claim 71, wherein the infectious disease is human immunodeficiency virus infection.

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