Antibodies and their use

Abstract

This specification provides antibodies, such as human antibodies or humanized antibodies, having a modified heavy chain variable (VH) domain, thereby conferring agonist activity upon engagement with an antigen to which the antibody binds by adopting a constrained three-dimensional structure (e.g., an i-shaped format). In some cases, the antibody is a full-length antibody (e.g., an IgG antibody). In some cases, the antibody is a monovalent antibody (e.g., Fab). In some cases, the antibody targets a receptor that requires clustering for activation (e.g., TNFR superfamily receptors). In some cases, the antibody targets two antigens on a molecule having two or more subunits (e.g., an IL-2 receptor). Methods for constructing and using such antibodies, as well as libraries for discovering or screening such antibodies, are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 509,744, filed on 22 June 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML file format, the entirety of which is incorporated herein by reference. The XML copy, created on May 20, 2024, is named P38276-US_SL.xml and has a size of 14,095 bytes.

[0003] This disclosure relates to antibodies that adopt a constrained three-dimensional structure (e.g., an i-shape) when they engage with an antigen to which they bind. [Background technology]

[0004] background Activation of target receptors by biologics can be a highly influential pharmacological mechanism for disease treatment. For example, protein drugs that activate erythropoietin, growth hormone, insulin, and the incretin pathway benefit from direct agonism of cell surface receptors, and in these cases, from their natural corresponding ligands (Thilaka, GK and Kumar, SVApollo Medicine 2016.13, 80-85). Correspondingly, the clinical success of these specific examples contributes to the potential for development of the biologic ligands themselves as pharmaceuticals. However, this is not always the case, and there are still many highly therapeutic receptor targets for which natural ligands are good as research reagents but insufficient as pharmaceuticals. Hurdles may include poor protein stability and / or solubility, complex glycosylation, unfavorable pharmacokinetics (PK) or distribution, and the risk of immunogenicity and consequently the risk of cross-reactivity with endogenous proteins.

[0005] Monoclonal antibodies are the most common and clinically successful class of biotherapeutic drugs and are generally not subject to the same limitations as other protein-based drugs. Despite their high molecular weight complexity, antibody drugs typically possess favorable stability and solution properties, limited, well-controlled, and defined carbohydrate modifications, favorable PKs, and relatively low immunogenicity with little evidence of endogenous cross-reactivity. Furthermore, decades of drug development experience have resulted in extensive research capabilities for drug discovery and optimization, as well as process capabilities for downstream production, purification, formulation, and delivery. Mechanistically, antibodies have demonstrated potent success as competitive inhibitors, mediators of immunoeffector function, delivery of toxic agents, and more recently, immune redirection.

[0006] However, in some cases, antibodies are not as clinically mature as targeted activators, so-called receptor agonists or ligand mimes. The main challenge in this class is that the mechanisms by which native ligands activate receptors are diverse and not always well understood enough to enable the primary design of active agonists. For example, most TNFRSF member ligands induce receptor homotrimerization when expressed in soluble form and higher-order clustering when tethered to the membrane (Wajant, H. Cell Death Differ, 2015.22, 1727-1741), and strong agonistic activity has been observed for divalent aptamers. On the other hand, many cytokine receptors require heterodimerization of two receptors present on the same cell in a conformationally specific manner (Waldmann, TACsh Perspect Biol., 2018.10, a028472). There is a need for therapeutic antibodies against such targets with potent agonist activity.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in whole, as if each individual reference were specifically and individually indicated to be incorporated by reference. [Overview of the project]

[0008] Brief summary of the application In one aspect, this application provides a human antibody or humanized antibody comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first VH and first VL bind to a first target, and the VH domain is 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering, or 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, and the antibody does not bind to HIV. In some embodiments, the VH domain of a human antibody or humanized antibody includes 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the VH of a human antibody or humanized antibody includes at least one substitution at a position selected from 19, 21, 70, 79, and 81. In some embodiments, the VH domain of a human antibody or humanized antibody includes 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the VH of a human antibody or humanized antibody includes at least one substitution at a position selected from 19, 68, 70, and 81. In some embodiments, the VH domain of a human antibody or humanized antibody includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the VH of a human antibody or humanized antibody includes at least one substitution at a position selected from 14, 19, 39, 43, 74, 77, 82a, and 82b.

[0009] In one embodiment, this application relates to a human antibody or humanized antibody derived from a reference antibody, wherein both the antibody and the reference antibody contain a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the VH domain of the human antibody or humanized antibody contains at least one amino acid substitution selected from the group consisting of 1) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering, and 2) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or the VH domain of the human antibody or humanized antibody contains at least one selected from the group consisting of 1) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. The amino acid substitutions include 2) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V and 82aS, or the VH domain of a human antibody or humanized antibody is selected from the group consisting of 1) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V and 113P according to Kabat numbering. The present invention provides a human antibody or humanized antibody comprising at least one amino acid substitution, and 2) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V and 113P, wherein the substitutions in a), b) and c) are substitutions compared to a reference antibody, and optionally, the human antibody or humanized antibody has increased agonist activity compared to a reference antibody. In some embodiments, the antibody (e.g., human antibody or humanized antibody) does not bind to HIV. In some embodiments, the VH domain of the human antibody or humanized antibody comprises 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I and 82aT according to Kabat numbering. In some embodiments, the VH of the human antibody or humanized antibody comprises at least one substitution at a position selected from 19, 21, 70, 79 and 81. In some embodiments, the VH domain of the human antibody or humanized antibody includes 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering.In some embodiments, the VH domain of a human antibody or humanized antibody includes at least one substitution at a position selected from 19, 68, 70, and 81. In some embodiments, the VH domain of a human antibody or humanized antibody includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the VH domain of a human antibody or humanized antibody includes at least one substitution at a position selected from 14, 19, 39, 43, 74, 77, 82a, and 82b.

[0010] In some embodiments described above, using either human antibodies or humanized antibodies, the antibody is a monovalent antibody. In some embodiments, the monovalent antibody is Fab.

[0011] In some embodiments described above, using either a human antibody or a humanized antibody, the antibody is F(ab')2.

[0012] In some embodiments of the above-mentioned human antibody or humanized antibody, the antibody does not have an Fc domain.

[0013] In some embodiments using human antibodies or humanized antibodies described above, the antibody has an Fc domain. In some embodiments, the antibody is an IgG antibody.

[0014] In some embodiments of the above-described human antibody or humanized antibody, the antibody has a modified hinge, which further restricts the flexibility of the hinge.

[0015] In some embodiments of the above-described human antibody or humanized antibody, the human antibody or humanized antibody is a monospecific antibody.

[0016] In some embodiments described above involving either human antibodies or humanized antibodies, the human antibody or humanized antibody binds to cell surface receptors.

[0017] In some embodiments of the above-described human antibody or humanized antibody, the human antibody or humanized antibody activates the target via receptor clustering.

[0018] In some embodiments, the antibody (e.g., a human antibody or humanized antibody) binds to one or more TNFRSF members. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) binds to OX40, CD40, 4-1BB, DR4, or DR5. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) binds to CD40, and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of lavagalimab, dasetuzumab, gilorarimab, and sotigolimab. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) binds to OX40, and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, and 3G8.

[0019] In some embodiments using some of the human antibodies or humanized antibodies described above, the human antibody or humanized antibody binds to the cytokine receptor. In some embodiments, the cytokine can form a complex with at least two different receptors in nature, thereby inducing the cytokine's downstream activity. In some embodiments, the antibody (e.g., human antibody or humanized antibody) binds to the IL-2 receptor. In some embodiments, the IL-2 receptor is IL-2RG or IL-2RB.

[0020] In some embodiments of the human antibody or humanized antibody described above, the human antibody or humanized antibody is a bivalent antibody comprising a second VH domain and a second VL domain that bind to a second target. In some embodiments, the second VH domain has Kabat numbering as 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the second target is different from the first target. In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) binds to both IL-2RG and IL-2RB. In some embodiments, the human VH domain of the humanized antibody contains three VH CDR sequences of B10, and the VL domain contains three VL CDR sequences of B10. In some embodiments, one of the two VH domains contains three VH CDR sequences of B10, one of the two VL domains contains three VL CDR sequences of B10, the other of the two VH domains contains three VH CDR sequences of G25 or G28, and the other of the two VL domains contains three VL CDR sequences of G25 or G28.

[0021] In another embodiment, this application provides a pharmaceutical composition comprising either the human antibody or a humanized antibody described above and a pharmaceutical carrier.

[0022] In another aspect, this application provides isolated nucleic acids encoding either a human antibody or a humanized antibody, or a fragment thereof.

[0023] In another embodiment, this application provides a host cell containing any of the above-mentioned nucleic acids.

[0024] In another embodiment, the present application provides a method for producing any of the above-mentioned human antibodies or humanized antibodies or fragments thereof, comprising culturing any of the above-mentioned host cells under conditions suitable for the expression of the antibody or fragment thereof. In some embodiments, the method further comprises recovering the antibody or fragment thereof from the host cells.

[0025] In another aspect, the present application provides a method for producing an agonist antibody from a reference antibody, comprising substituting one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody in order to promote the i-shaped antibody format.

[0026] In another aspect, this application relates to a method for producing an agonist antibody from a reference antibody, wherein one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody are substituted at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, such that the agonist antibody has a VH domain containing 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT after the substitution, or one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody are substituted at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering. The present invention provides a method comprising substituting an amino acid residue such that the agonist antibody has a VH domain containing 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS after substitution, or substituting one or more amino acid residues on the heavy chain variable (VH) domain of a reference antibody at a position selected from 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113 according to Kabat numbering, such that the agonist antibody has a VH domain containing 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P after substitution.

[0027] In another aspect, this application provides an agonist antibody prepared by any of the methods described above.

[0028] In another aspect, this application relates to a method for enhancing the agonist activity of an antibody, comprising substituting one or more amino acid residues on the heavy chain variable (VH) domain of the antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, wherein the substituted VH domain has amino acid residues such that 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or substituting one or more amino acid residues on the heavy chain variable (VH) domain of the antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering. The present invention provides a method comprising substituting amino acid residues such that the substituted VH domain has 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or substituting one or more amino acid residues on the heavy chain variable (VH) domain of an antibody at a position selected from 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113 according to Kabat numbering, such that the substituted VH domain has 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

[0029] In another aspect, this application provides either the above-mentioned antibody or pharmaceutical composition for use as a pharmaceutical.

[0030] In another aspect, this application provides either of the above-mentioned antibodies or pharmaceutical compositions for use in treating a disease or pathological condition.

[0031] In another aspect, this application provides the use of either an antibody or the above-mentioned pharmaceutical composition in the manufacture of a pharmaceutical for treating a disease or pathological condition.

[0032] In another aspect, the present application provides a method for treating an individual having a disease or pathological condition, comprising administering to the individual an effective amount of either an antibody or the above-mentioned pharmaceutical composition.

[0033] In another embodiment, the present application provides a library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH and VL binding to a target, and the VH domain comprises, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, 82aS, or c) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

[0034] In another embodiment, the present application provides a library of antibodies comprising at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL bind to a target, and the VH domain comprises, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, 82aS, or c) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the antibodies in the library are expressed or planned to be expressed on the surface of one or more phage or yeast cells.

[0035] In another embodiment, the present application provides a method for screening agonist antibodies, comprising contacting an antibody from any of the above libraries with a target or a cell expressing a target. In some embodiments, the method further includes evaluating the agonist activity of the antibody. [Brief explanation of the drawing]

[0036] The drawings illustrate specific features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0037] [Figure 1A] Exemplary structural representations of the domain-exchange iAb 2G12 (left, PDB: 2OQJ) and two iAbs with affinity interfaces, DH851.3 (center, PDB: 7LU9) and DH898.1 (right, PDB: 7L6M), are shown. The light chain (LC) and heavy chain (HC) from each Fab are labeled within each structure. The inset highlights the interface between the heavy chain variable (VH) domains of the two Fabs, with the involved residues shown as bars.

[0038] [Figure 1B] A representative table of amino acid residues in the VH domain predicted to contribute to the iAb three-dimensional structure is shown. VH domain residues are indicated using Kabat numbering. The sequence logos below the table are based on the distribution of amino acids at a given residue across all human antibody sequences in the abYsis database. Residues enclosed in dashed lines are rare mutations present in less than 1% of all deposited human sequences. Asterisks indicate additional non-native hydrophobic substitutions already known to enhance affinity interfaces from the DH851 and DH898 lineages.

[0039] [Figure 2A-2C] Examples of iAb modification are shown. Sequence alignments of previously described iAbs [2G12 (Figure 2A), DH851.3 (Figure 2B), or DH898.1 (Figure 2C)], WT anti-OX40 clones (1A7 and 3C8), and their respective iAb mutation set transfer formats. Residues enclosed in black squares indicate the positions where the anti-OX40 clone sequence was modified to that of a given iAb in order to manipulate iAb formation. Boxes with asterisks indicate additional non-native hydrophobic substitutions that have been previously known to enhance affinity interfaces derived from the DH851 and DH898 strains. In some cases, the residues enclosed in squares were the same between the original iAb and the anti-OX40 antibody, and no sequence modification was required. Figure 2A discloses sequence numbers 4-8 in order of appearance. Figure 2B discloses sequence numbers 9, 5, 10, 7, and 11 in order of appearance. Figure 2C displays 12, 5, 13, 7, and 14 in the order of their appearance.

[0040] [Figure 2D] This figure shows exemplary sketches of WT, Contorsbody, iAbdx, iAbaff1, and iAbaff2 formats, along with corresponding representative negative stained electron microscope 2D classification images.

[0041] [Figure 2E] The analysis of iAbaff2 anti-OX40 clones shows that the SEC chromatogram exhibits a range of elution times.

[0042] [Figure 2F] The table below shows SEC-MALS data that quantitatively characterize each iAbaff2 clone as a monomer, dimer, or a mixture of the two.

[0043] [Figure 2G] This shows the concentration dependence of the SEC-MALS molecular weight of iAbaff2 3C8. As the sample is diluted, it becomes more monomeric, indicating that the iAb interaction is in equilibrium.

[0044] [Figure 3A] This shows the OX40 agonism activity from a panel of 10 anti-OX40 antibodies for a given antibody format. Each symbol represents a unique clone. Data are shown as a magnification change relative to the untreated control.

[0045] [Figure 3B-3C] This shows OX40 agonism activity across three iAb-induced mutation sets. Individual titers were measured for 10 anti-OX40 clones transplanted with either the iAbdx (Figure 3B) or affinity-based (Figure 3C) iAb-induced mutation sets. Data are shown as a fold change relative to the untreated control. 2A3 was not expressed in the iAbdx mutation set, while 1A7 was not expressed in either affinity-based mutation set.

[0046] [Figure 3D] Surface plasmon resonance (SPR) affinity data comparing the KD value (left) and normalized Rmax value (nRmax, right) for each anti-OX40 clone, which is either iAbaff1 or WT IgG. The gray dotted line has a slope of 1, indicating no change between the two formats.

[0047] [Figure 3E]Exemplary cell surface binding to OX40+ Jurkat cells for each anti-OX40 clone, comparing EC50 values ​​in iAbaff1 and WT IgG formats. The gray dotted line has a slope of 1, indicating no change between the two formats.

[0048] [Figure 3F] This section describes the measurement of anti-OX40 cell binding titer. Binding of each anti-OX40 clone—WT IgG, contrast bodies, and iAbaff1—to OX40+ Jurkat cells was detected by FACS using fluorescently labeled anti-human IgG Fab.

[0049] [Figure 4A] The effect of antibody fragmentation on OX40 agonism activity with and without iAbaff1 mutation set implantation is shown for a single anti-OX40 clone 3C8. TNFRSF agonism activity of clones in various formats against F-I)CD40(F, 4nM), 4-1BB(G, 22.2nM), DR4(H, 100nM), and DR5(I, 100nM). Data are shown at single concentrations (shown in parentheses above) obtained from titration curves in Figure S5. CD40 and 4-1BB agonisms are shown as a multiplier change relative to the untreated control, while DR4 and DR5 agonisms are shown as % mortality relative to the untreated control.

[0050] [Figure 5A] The TNFRSF agonism activity of WT IgG1, IgG2 C131S, and iAbaff1 IgG1 clones against CD40 (4nM) is shown. The data are shown at single concentrations (shown in parentheses above) obtained from the titration curve in Figure 5E.

[0051] [Figure 5B] The TNFRSF agonism activity of WT clones and iAbaff1 clones against 4-1BB (22.2 nM) is shown. The data are shown at single concentrations (shown in parentheses above) obtained from the titration curve in Figure 5F.

[0052] [Figure 5C] The TNFRSF agonism activity of WT clones and iAbaff1 clones against DR4 (100 nM) is shown. The data are shown at single concentrations (shown in parentheses above) obtained from the titration curve in Figure 5G.

[0053] [Figure 5D] The TNFRSF agonism activity of WT clones and iAbaff1 clones against DR5 (100 nM) is shown. The data are shown at single concentrations (indicated in parentheses above) obtained from the titration curve in Figure 5H.

[0054] [Figure 5E-5H] This shows iAb-induced agonism across four TNFRSF members. Individual activity titer measurements in various formats of antibody clones against CD40 (Figure 5E), 4-1BB (Figure 5F), DR4 (Figure 5G), and DR5 (Figure 5H) are shown. CD40 and 4-1BB agonisms are shown as fold change relative to the untreated control, while DR4 and DR5 agonisms are shown as % mortality relative to the untreated control. Each anti-CD40 clone was prepared as WT human IgG1, human IgG2 C131S, and iAbaff1, while all clones against other targets were prepared only as WT IgG1 and iAbaff1.

[0055] [Figure 6A] Exemplary sketches and legends illustrating each format manipulated for anti-OX40 3C8 clones are shown. Figures 3B–3F are colored according to the legend.

[0056] [Figure 6B] This shows the OX40 agonism activity for each format.

[0057] [Figure 6C] The receptor-mediated internalization of each format, indicated as MFIX104, is shown. Antibodies against unrelated viral antigens (gD) labeled with the same pH-sensitive dye are shown in black as a control.

[0058] [Figure 6D] The image shows the maximum projection (grayscale) of a representative Jurkat T cell expressing OX40-mNeonGreen and treated with a specified antibody at 13.3 nM in solution, obtained over 12.5 seconds using TIRF microscopy. The inset shows a representative single-molecule track as a gray line within a 2.5 × 2.5 μm area.

[0059] [Figure 6E] The mean squares mean displacement (MSD) plots are shown for all analyzed tracks for each treatment condition. The untreated control is shown in black.

[0060] [Figure 6F] The distribution of average background subtraction molecular track intensity for each treatment condition is shown. The untreated control is shown in black.

[0061] [Figure 7A] An exemplary schematic diagram illustrating the selection and screening process for binding factors to IL-2RG and IL-2RB is shown.

[0062] [Figure 7B] An overview of yeast selection is provided. The schematic diagram shows the selection of IL-2RG binding factors (left column) and IL-2RB binding factors (right column) from an autologous scFv library presented on yeast. For each antigen, selection was either magnetic-based or FACS-based under progressively stricter conditions in terms of both valence and concentration, as shown. The FACS plot at 37 nM for each antigen shows the enrichment of the binding factors after each selection round.

[0063] [Figure 7C]This plot compares affinity and cell surface binding tendency, as determined by SPR, for 34 anti-IL-2RG clones (top) and 61 anti-IL-2RB clones (bottom). Clones that block IL-2 signaling in the Jurkat reporter assay are shown as squares, and non-blocking clones are shown as circles. Gray symbols indicate lead clones selected for IL-2RG and IL-2RB. All other non-lead clones are shown in black.

[0064] [Figure 7D-7E] Characterization of anti-IL-2RB and anti-IL-2RG antibody clones discovered by yeast display is shown. Cell-binding analysis (Figure 7D) and IL-2 blocking analysis (Figure 7E) are performed using IL-2RB and IL-2RG expressing Jurkat cells with STAT5-luc reporters for anti-IL-2RG (top) and anti-IL-2RB (bottom) clones discovered by yeast display. For the IL-2 blocking experiment, cells were first coated with 1 μM of each monospecific anti-IL-2RG or anti-IL-2RB clone for 1 hour, and then serial dilutions of IL-2 were added. For each analysis, trastuzumab, an anti-HER2 antibody, was used as a negative control and labeled with "c". Read clones selected for bispecific reformatting are indicated with an asterisk.

[0065] [Figure 7F] Surface plasmon resonance analysis of anti-IL-2RB antibody clones and anti-IL-2RG antibody clones is shown.

[0066] [Figure 8A]The epitope mapping of the lead anti-IL-2RG clone (blue box) and the lead anti-IL-2RB clone (red box) is shown. Epitopes on IL-2RG and IL-2RB are shown in dark gray. For reference, the IL-2 binding site is highlighted based on the ternary complex (black box, PDB:2ERJ). In each image, the IL-2RG structure is shown on the left and the IL-2RB structure on the right. A 90° rotated image of the crystal structure is shown to show the epitope residues in more detail.

[0067] [Figure 9A] This heatmap shows the fold changes of IL-2 pathway agonism in Jurkat-STAT5 luciferase reporter cell lines expressing IL-2RG and IL-2RB. Bispecific WT IgG (left), contrast bodies (center), and iAbs (right) were tested at 100 nM concentrations using predetermined anti-IL-2RG and anti-IL-2RB clone combinations. Recombinant IL-2 activity is shown for comparison. X in G23 / B65 iAb indicates non-expression.

[0068] [Figure 9B] This shows the concentration-dependent activity of lead-constrained IL-2 pathway agonists and their corresponding WT IgG controls in the Jurkat reporter assay. A legend indicating the symbols for each strain is shown below, and antibodies against unrelated viral antigens (gD) are shown as controls.

[0069] [Figure 9C] An illustrative schematic diagram (left) and plot (right) of an IL-2RG / IL-2RB crosslinked ELISA are shown. A legend indicating the symbols for each strain is shown below, and antibodies against unrelated viral antigens (gD) are shown as controls.

[0070] [Figure 10A] The activity of lead-constrained IL-2 pathway agonists and corresponding WT IgG controls in primary NK cells (left) and primary CD8 T cells (right) is shown. A legend indicating the symbols for each strain is shown below, and antibodies against unrelated viral antigens (gD) are shown as controls.

[0071] [Figure 10B] This shows hierarchical clustering of read-constrained IL-2 pathway agonists and corresponding WT IgG controls based on changes in gene expression in primary CD8 T cells, as determined by RNA sequencing. The heatmap shows changes in gene expression under each condition compared to gD controls for the 40 genes most significantly downregulated by IL-2 (left) and the 40 genes most significantly upregulated by IL-2 (right). [Modes for carrying out the invention]

[0072] Detailed explanation This application provides antibodies having agonist activity and methods for preparing such antibodies. While not theoretically bound, this application is at least partially based on the inventors' insightful finding that one or more antibodies can engage with one or more antigens and activate downstream signaling by modifying the geometric shape of one or more antibodies such that the two antigen-binding domains of one or more antibodies targeting one or more antigens (e.g., receptors requiring homomultimerization or heteromultimerization for activation) are constrained in conformation via non-covalent association (e.g., via an affinity interface between two VH domains). In some embodiments, one or more antibodies adopt an i-shape when engaged with one or more antigens. For an example image of a compact i-shape of an antibody, see, for example, Figure 2D (iAb compared to WT). dx iAb aff1 and iAb aff2 (See representative electron microscope images). Surprisingly, the inventors have found a classical IgG antibody having two Fab arms (for example, an iAb as shown in Figure 2D). dx iAb aff1 and iAb aff2 We found that not only monovalent formats with a single antigen-binding domain (e.g., monomeric Fab) can also enable endogenous agonist activity. See, for example, Example 4.

[0073] In some embodiments, the application relates to a human antibody or humanized antibody comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first VH and first VL bind to a first target, and the VH domain has Kabat numbering as 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the antibody does not bind to HIV. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) is derived from a reference antibody, and the humanized antibody includes at least one amino acid substitution selected from a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, 82aS, or c) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, where the substitutions in a), b), and c) are substitutions compared to the reference antibody. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) has increased agonist activity compared to the reference antibody. In some embodiments, the antibody is a monovalent antibody (e.g., Fab). In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) binds to a cell surface receptor (e.g., a TNFRSF member, e.g., a cytokine receptor).

[0074] Pharmaceutical compositions containing antibodies, isolated nucleic acids encoding antibodies, vectors and host cells containing isolated nucleic acids, methods for producing antibodies, methods for enhancing the agonist activity of antibodies, and methods for treatment by administering antibodies are envisioned. Libraries containing such antibodies or polynucleotides encoding such antibodies, and methods for screening such antibodies are also envisioned.

[0075] I. General Techniques The techniques and procedures described or referenced herein are generally well understood and are conventional methods by those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)); Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RIFreshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), and The The widely used method described in *Antibodies* (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995) is commonly employed.

[0076] II. Definition "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D Affinity can be expressed by ( ). Affinity can be measured by methods common in the art, including those described herein.

[0077] An "affinity matured" antibody refers to an antibody having one or more alterations in one or more complementarity determining regions (CDRs), which alterations result in an improvement in the affinity of the antibody for an antigen as compared to the parental antibody that does not have such alterations.

[0078] As used herein, the terms "agonist", "agonistic", "agonism" or "act on" generally refer to a binding molecule (e.g., an antigen-binding polypeptide or antigen-binding complex) that binds to a receptor on the surface of a cell and is capable of eliciting / mimicking / stimulating a reaction or activity that is the same as or similar to the reaction or activity elicited / mimicked / stimulated by the native ligand of the receptor. In an exemplary embodiment, the agonist described herein is capable of inducing / enhancing / potentiating / stimulating the activation of a signal transduction pathway associated with the receptor.

[0079] The term "antibody that binds to a target" refers to an antibody that can bind to the target with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when the antibody targets the target. In one aspect, the degree of binding of the antibody to an irrelevant non-target protein is less than about 10% of the binding of the antibody to the target, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to the target has a dissociation constant (K -8 ) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M). When an antibody has a K D of 1 μM or less, the antibody is said to "specifically bind" to the target. In certain embodiments, the antibody binds to an epitope of the target that is conserved among targets from different species.

[0080] ​​The term "antibody" as used herein is used in its broadest sense and is not limited to any specific definition, but includes molecules having various antibody structures that exhibit desired antigen-binding activity, such as monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and fusion proteins containing antibody fragments.

[0081] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies (dAb), and multispecific antibodies formed from antibody fragments. For an overview of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0082] The term "epitope" refers to a site on an antigen, either proteinaceous or nonproteinaceous, to which an antibody binds. Epitopes can be formed from continuous amino acid stretch sites (linear epitopes) or from discontinuous amino acids (structural epitopes), and are formed spatially close together, for example, due to the folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound to antibodies even after exposure of proteinaceous antigens to denaturants, while structural epitopes are typically destroyed by treatment with denaturants. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0083] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using, for example, but not limited to, alanine scanning, peptide blotting (see Meth.Mol.Biol.248(2004)443-463), peptide cleavage analysis, epitope cleavage analysis, epitope extraction, chemical modification of antigens (see Prot.Sci.9(2000)487-496), and cross-blocking (see "Antibodies," Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0084] Competitive binding can be used to determine whether an antibody competes for binding with a reference antibody that binds to the same target. For example, an antibody that "competes for binding" with a reference antibody is one that blocks the reference antibody's binding to its antigen by 50% or more in a competitive assay, and conversely, a reference antibody blocks the antibody's binding to its antigen by 50% or more in a competitive assay. Alternatively, to determine whether an antibody competes for binding with a reference antibody, for example, the reference antibody is bound to the target under saturated conditions. After removing the excess reference antibody, the ability of the antibody in question to bind to the target is evaluated. If the antibody can bind to the target after saturated binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to the target after saturated binding of the reference antibody, it may bind to the same epitope to which the reference antibody binds. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation, or binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, namely, setups where both antibodies are saturated antibodies. If, in both setups, only the first (saturated) antibody can bind to the target, it can be concluded that the antibody in question and the reference antibody are competing for binding to the target.

[0085] In some embodiments, if a competitive binding assay measures that a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even more than 99%, then the two antibodies are considered to bind to the same or overlapping epitopes. (See, for example, Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0086] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies are considered to have a “duplicate epitope” if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0087] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, and the remainder of the heavy chain and / or light chain originates from a different source or species.

[0088] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 i-type and has the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0089] As used in this application, the terms “human-derived constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa region or constant light chain lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218, and Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system (also known as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0090] "Effector function" refers to the biological activity caused by the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0091] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0092] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the natural sequence Fc region and the mutant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or may contain cleaved variants of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc region is shown herein without the C-terminal glycine-lysine dipeptide. In one embodiment, the heavy chain containing the Fc region as specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, the heavy chain containing the Fc region as specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine residue (G446, EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0093] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) as the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0094] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody, or antibodies having a heavy chain containing an Fc region as defined herein.

[0095] "Fv" is the minimal antibody fragment containing the complete antigen recognition and binding sites. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain, tightly bonded non-covalently. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific hypervariable regions) has the ability to recognize and bind to the antigen, but with lower affinity than the full binding site.

[0096] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and cells including the progeny of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. The progeny do not have to have nucleic acid content that is exactly the same as that of the parent cells and may contain mutations. The present invention includes progeny of mutants that have the same function or biological activity as those screened or selected for the original transformed cells.

[0097] A "human antibody" is an antibody produced by a human or human cells, or an antibody that has an amino acid sequence corresponding to the amino acid sequence of a non-human antibody that utilizes a sequence encoding a human antibody, such as the human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0098] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. above. In another embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al. above.

[0099] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains substantially at least one, usually two, variable domains in all or nearly all CDRs corresponding to the variable domains of the non-human antibody, and in all or nearly all FRs corresponding to the variable domains of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0100] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of an antibody variable domain whose sequence is hypervariable and which determines antigen-binding specificity, such as “complementarity-determining region” (CDR).

[0101] An "immune conjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0102] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0103] "Isolated" antibodies are those separated from components of their natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0104] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by their base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. Here, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain natural or non-natural nucleotides. Examples of nucleotides not found in nature, including derivatized sugar or phosphate backbone links or chemically modified residues, include modified nucleotide bases. The nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of this application in vitro and / or in vivo in a host or patient, for example. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a target in vivo to produce an antibody (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0105] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within cells, but these nucleic acid molecules exist outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0106] "Isolated nucleic acids encoding an agonist antibody or a fragment thereof" means one or more nucleic acid molecules encoding one or more polypeptides of an agonist antibody or a fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations within a host cell.

[0107] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for, for example, naturally occurring mutations or mutant antibodies that may occur during the preparation of a monoclonal antibody preparation, such mutations are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0108] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light chain (CL) domain.

[0109] The term “package insert” is used to refer to the instructions typically included on the market packaging of a therapeutic product, including information relating to indications, use, dosage, administration, combination therapy, contraindications, and / or warnings for such therapeutic product.

[0110] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the biological activity of the active ingredient contained herein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the pharmaceutical composition may be administered.

[0111] "Pharmacologically acceptable carriers" refer to components in a pharmaceutical composition or preparation other than the active ingredient that are non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0112] A "single-stranded Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, which are located within a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure desirable for antigen binding. For an overview of scFv, see, for example, Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0113] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention that seeks to alter the natural course of a disease in the treated individual, and may be carried out for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological outcomes of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and achieving remission or improving prognosis. In some embodiments, the antibodies of this application are used to delay the onset of the disease or to slow the progression of the disease.

[0114] A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated using the VH or VL domain from the antibody that binds that antigen, respectively, to screen for a library of complementary VL or VH domains. For example, see Portolano et al., J.Immunol. 150:880-887 (1993) and Clarkson et al., Nature 352:624-628 (1991).

[0115] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid it is linked to. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which they are introduced. Certain types of vectors can direct the expression of a operably linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0116] It should be noted that the singular forms "a," "an," and "the" used herein and in the appended claims refer to multiple subjects unless the context clearly indicates otherwise.

[0117] III. Compositions and Methods A.Antibodies This application provides antibodies (e.g., agonist antibodies, e.g., agonist human antibodies or agonist human-humanized antibodies) having a conformationally constrained association between two antigen-binding domains, for example via non-covalent bonding, for example via an affinity interface between two VH domains, for example via two Fab or disulfide bonds between two VH domains. In some embodiments, one or more antibodies take an i-shaped format when engaged with one or more antigens. See, for example, Cell. 2021 May 27;184(11):2955-2972.e25. In some embodiments, the two antigen-binding domains originate from one antibody (e.g., two Fab arms in an IgG antibody). In some embodiments, the two antigen-binding domains originate from two antibodies (e.g., two separate Fab molecules). In some embodiments, the two antigen-binding domains bind to the same antigen (e.g., a TNFRSF member). In some embodiments, the two antigen-binding domains bind to two different antigens (e.g., two antigens associated with a receptor containing two or more different subunits).

[0118] In some embodiments, the human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first antigen-binding domain for a first target includes 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody is a monovalent antibody (e.g., Fab). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, the modified hinge further restricts the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from an IgG2 hinge domain. In some embodiments, the human antibody or humanized antibody binds to cell surface receptors. In some embodiments, the human antibody or humanized antibody activates a target through clustering or polymerization of the target. In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody. In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0119] In some embodiments, the human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the VH domain, which is the first antigen-binding domain for a first target, includes 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody is a monovalent antibody (e.g., Fab). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from an IgG2 hinge domain. In some embodiments, the human antibody or humanized antibody binds to cell surface receptors. In some embodiments, the human antibody or humanized antibody activates a target through clustering or polymerization of the target. In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody. In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0120] In some embodiments, the human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the VH domain, which is the first antigen-binding domain for a first target, includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody is a monovalent antibody (e.g., Fab). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally includes L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, the modified hinge further restricts the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from the IgG2 hinge domain. In some embodiments, the human antibody or humanized antibody binds to a cell surface receptor. In some embodiments, the human antibody or humanized antibody activates a target through clustering or multimerization of the target. In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody. In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0121] In some embodiments, a human antibody or humanized antibody is provided, comprising a) a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and b) a second antigen-binding domain comprising a second VH domain and a second VL domain, wherein the first antigen-binding domain binds to a first target and the second antigen-binding domain binds to a second target, and both the first and second VH domains comprise Kabat numbering 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the first and second targets are the same target. In some embodiments, the first and second targets bind to the same epitope of the target. In some embodiments, the first and second targets bind to two different epitopes of the target. In some embodiments, the first and second targets bind to two different targets. In some embodiments, the two different targets are two subunits of a molecule that requires or involves clustering or multimerization of two subunits for activation of downstream signaling (e.g., cytokine receptors, e.g., IL-2 receptors). In some embodiments, the two different targets are two molecules involved in a complex (e.g., a complex on the cell surface, e.g., a T cell receptor complex) whose formation confers activation of a signaling pathway. In some embodiments, the two different targets include one or more members of the tumor necrosis factor receptor superfamily (TNFRSF). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, the modified hinge further restricting the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from the IgG2 hinge domain.

[0122] In some embodiments, a human antibody or humanized antibody is provided, comprising a) a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and b) a second antigen-binding domain comprising a second VH domain and a second VL domain, wherein the first antigen-binding domain binds to a first target and the second antigen-binding domain binds to a second target, and both the first and second VH domains comprise 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the first target and the second target are the same target. In some embodiments, the first target and the second target bind to the same epitope of the target. In some embodiments, the first target and the second target bind to two different epitopes of the target. In some embodiments, the first and second targets bind to two different targets. In some embodiments, the two different targets are two subunits of a molecule that requires or involves clustering or multimerization of two subunits for activation of downstream signaling (e.g., cytokine receptors, e.g., IL-2 receptors). In some embodiments, the two different targets are two molecules involved in a complex (e.g., a complex on the cell surface, e.g., a T cell receptor complex) whose formation confers activation of a signaling pathway. In some embodiments, the two different targets include one or more members of the tumor necrosis factor receptor superfamily (TNFRSF). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, the modified hinge further restricting the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from the IgG2 hinge domain.

[0123] In some embodiments, a human antibody or humanized antibody is provided, comprising a) a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and b) a second antigen-binding domain comprising a second VH domain and a second VL domain, wherein the first antigen-binding domain binds to a first target and the second antigen-binding domain binds to a second target, and both the first and second VH domains comprise 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering, and the human antibody or humanized antibody does not bind to HIV. In some embodiments, the first target and the second target are the same target. In some embodiments, the first target and the second target bind to the same epitope of the target. In some embodiments, the first and second targets bind to two different epitopes of the target. In some embodiments, the first and second targets bind to two different targets. In some embodiments, the two different targets are two subunits of a molecule (e.g., cytokine receptors, e.g., IL-2 receptors) that require or involve clustering or multimerization of two subunits for activation of downstream signaling. In some embodiments, the two different targets are two molecules involved in a complex (e.g., a complex on the cell surface, e.g., a T cell receptor complex) whose formation confers activation of a signaling pathway. In some embodiments, the two different targets include one or more members of the tumor necrosis factor receptor superfamily (TNFRSF). In some embodiments, the antibody is a bivalent antibody (e.g., F(ab')2). In some embodiments, the antibody has an Fc domain, optionally an IgG Fc domain, and further optionally includes L234A, L235A, and / or P329G. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has a hinge derived from the IgG2 hinge domain.

[0124] In some embodiments, a human antibody or humanized antibody comprising a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first antigen-binding domain for members of the tumor necrosis factor receptor superfamily (TNFRSF) includes 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the TNFRSF member is selected from the group consisting of OX40, CD40, 4-1BB, DR4, or DR5. In some embodiments, the human antibody or humanized antibody is bound to CD40 (e.g., CD40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of CP-870, 893 (RO70099789), SGN-40, sericrelumab, dacetuzumab, Chi Lob 7 / 4, APX005M, ADC-1013, CDX-1140, SEA-CD40, lavagalimab, gyrolarimab, and sotigolimab. In some embodiments, the human antibody or humanized antibody is bound to OX40 (e.g., human OX40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, 3G8, HFB301001, FS120, INBRX-106, BGB-A445, PF-04518600, MEDI6469, MEDI0562, ABBV-368, FS120, INCAGN01949, BMS986178, PF04518600, GSK3174998, and SL-279252. In some embodiments, the human antibody or humanized antibody is bound to DR4 (e.g., human DR4), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of HLX56, mapatumumab, m921 / 922, 4H6, 4G7, AY4, and TR1-mAbs.In some embodiments, the human antibody or humanized antibody binds to DR4 (e.g., human DR5), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of conatumumab, droditumab, DS-8273a, KTRM2, lexatumumab, tigatuzumab, zaptuzumab, inbrx-109, LaDR5, LBy135, mDRA6, WD1, zaptuximab, HMCAZ5, and AD5.10. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the second antigen-binding domain binds to the same target. In some embodiments, the second antigen-binding domain binds to a distinct target (e.g., a checkpoint inhibitor, e.g., PD-1, PD-L1, or CTLA-4; e.g., a T cell receptor, e.g., CD3, CD4, or CD8). In some embodiments, the second antigen-binding domain binds to a second member of TNFRSF. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally derived from an IgG Fc domain, and further optionally containing L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region.

[0125] In some embodiments, a human antibody or humanized antibody comprising a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first antigen-binding domain for members of the tumor necrosis factor receptor superfamily (TNFRSF) includes 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the TNFRSF member is selected from the group consisting of OX40, CD40, 4-1BB, DR4, or DR5. In some embodiments, the human antibody or humanized antibody is bound to CD40 (e.g., CD40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of CP-870, 893 (RO70099789), SGN-40, sericrelumab, dacetuzumab, Chi Lob 7 / 4, APX005M, ADC-1013, CDX-1140, SEA-CD40, lavagalimab, gyrolarimab, and sotigolimab. In some embodiments, the human antibody or humanized antibody is bound to OX40 (e.g., human OX40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, 3G8, HFB301001, FS120, INBRX-106, BGB-A445, PF-04518600, MEDI6469, MEDI0562, ABBV-368, FS120, INCAGN01949, BMS986178, PF04518600, GSK3174998, and SL-279252. In some embodiments, the human antibody or humanized antibody is bound to DR4 (e.g., human DR4), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of HLX56, mapatumumab, m921 / 922, 4H6, 4G7, AY4, and TR1-mAbs.In some embodiments, the human antibody or humanized antibody binds to DR4 (e.g., human DR5), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of conatumumab, droditumab, DS-8273a, KTRM2, lexatumumab, tigatuzumab, zaptuzumab, inbrx-109, LaDR5, LBy135, mDRA6, WD1, zaptuximab, HMCAZ5, and AD5.10. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the second antigen-binding domain binds to the same target. In some embodiments, the second antigen-binding domain binds to a distinct target (e.g., a checkpoint inhibitor, e.g., PD-1, PD-L1, or CTLA-4; e.g., a T cell receptor, e.g., CD3, CD4, or CD8). In some embodiments, the second antigen-binding domain binds to a second member of TNFRSF. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally derived from an IgG Fc domain, and further optionally containing L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region.

[0126] In some embodiments, a human antibody or humanized antibody comprising a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first antigen-binding domain for members of the tumor necrosis factor receptor superfamily (TNFRSF) includes, according to Kabat numbering, 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the TNFRSF member is selected from the group consisting of OX40, CD40, 4-1BB, DR4, or DR5. In some embodiments, the human antibody or humanized antibody is bound to CD40 (e.g., CD40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of CP-870, 893 (RO70099789), SGN-40, sericrelumab, dacetuzumab, Chi Lob 7 / 4, APX005M, ADC-1013, CDX-1140, SEA-CD40, lavagalimab, gyrolarimab, and sotigolimab. In some embodiments, the human antibody or humanized antibody is bound to OX40 (e.g., human OX40), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, 3G8, HFB301001, FS120, INBRX-106, BGB-A445, PF-04518600, MEDI6469, MEDI0562, ABBV-368, FS120, INCAGN01949, BMS986178, PF04518600, GSK3174998, and SL-279252. In some embodiments, the human antibody or humanized antibody is bound to DR4 (e.g., human DR4), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of HLX56, mapatumumab, m921 / 922, 4H6, 4G7, AY4, and TR1-mAbs.In some embodiments, the human antibody or humanized antibody binds to DR4 (e.g., human DR5), and optionally, the human antibody or humanized antibody is derived from an antibody selected from the group consisting of conatumumab, droditumab, DS-8273a, KTRM2, lexatumumab, tigatuzumab, zaptuzumab, inbrx-109, LaDR5, LBy135, mDRA6, WD1, zaptuximab, HMCAZ5, and AD5.10. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the second antigen-binding domain binds to the same target. In some embodiments, the second antigen-binding domain binds to a distinct target (e.g., checkpoint inhibitors, e.g., PD-1, PD-L1, or CTLA-4; e.g., T cell receptors, e.g., CD3, CD4, or CD8). In some embodiments, the second antigen-binding domain binds to a second member of TNFRSF. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally derived from an IgG Fc domain, and further optionally containing L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region.

[0127] In some embodiments, a human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the VH domain, which is the first antigen-binding domain for the cytokine receptor, includes 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the cytokine can spontaneously form complexes with at least two different receptors or two subunits of receptors, which induces downstream activity of the cytokine. In some embodiments, the human antibody or humanized antibody binds to an IL-2 receptor. In some embodiments, the IL-2 receptor is IL-2RG or IL-2RB. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the first antigen-binding domain and the second antigen-binding domain bind to IL-2RG and IL-2RB, respectively. In some embodiments, the human VH domain of the humanized antibody includes three VH CDR sequences of B10, and the VL domain includes three VL CDR sequences of B10. In some embodiments, one of the two VH domains contains three VH CDR sequences of B10, one of the two VL domains contains three VL CDR sequences of B10, the other of the two VH domains contains three VH CDR sequences of G25 or G28, and the other of the two VL domains contains three VL CDR sequences of G25 or G28. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally the Fc domain is derived from an IgG Fc domain, and further optionally the IgG Fc domain contains L234A, L235A and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge.In some embodiments, the antibody has an IgG2 hinge region.

[0128] In some embodiments, a human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the VH domain, which is the first antigen-binding domain for the cytokine receptor, includes 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the cytokine can spontaneously form complexes with at least two different receptors or two subunits of receptors, which induces downstream activity of the cytokine. In some embodiments, the human antibody or humanized antibody binds to an IL-2 receptor. In some embodiments, the IL-2 receptor is IL-2RG or IL-2RB. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the first and second antigen-binding domains bind to IL-2RG and IL-2RB, respectively. In some embodiments, the human VH domain of the humanized antibody includes three VH CDR sequences of B10, and the VL domain includes three VL CDR sequences of B10. In some embodiments, one of the two VH domains contains three VH CDR sequences of B10, one of the two VL domains contains three VL CDR sequences of B10, the other of the two VH domains contains three VH CDR sequences of G25 or G28, and the other of the two VL domains contains three VL CDR sequences of G25 or G28. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally the Fc domain is derived from an IgG Fc domain, and further optionally the IgG Fc domain contains L234A, L235A and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge.In some embodiments, the antibody has an IgG2 hinge region.

[0129] In some embodiments, a human antibody or humanized antibody comprises a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the VH domain, which is the first antigen-binding domain for the cytokine receptor, includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the cytokine can form a complex innately with at least two different receptors or two subunits of a receptor, which induces downstream activity of the cytokine. In some embodiments, the human antibody or humanized antibody binds to an IL-2 receptor. In some embodiments, the IL-2 receptor is IL-2RG or IL-2RB. In some embodiments, the antibody further comprises a second antigen-binding domain including a second VH domain and a second VL domain, the second VH domain also including 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the first and second antigen-binding domains bind to IL-2RG and IL-2RB, respectively. In some embodiments, the human VH domain of the humanized antibody includes three VH CDR sequences of B10, and the VL domain includes three VL CDR sequences of B10. In some embodiments, one of the two VH domains contains three VH CDR sequences of B10, one of the two VL domains contains three VL CDR sequences of B10, the other of the two VH domains contains three VH CDR sequences of G25 or G28, and the other of the two VL domains contains three VL CDR sequences of G25 or G28. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally the Fc domain is derived from an IgG Fc domain, and further optionally the IgG Fc domain contains L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody.In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region.

[0130] In some embodiments, a human antibody or humanized antibody derived from a reference antibody is provided, wherein both the antibody and the reference antibody include a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and the VH domain of the human antibody or humanized antibody, compared to the reference antibody, includes at least one amino acid substitution selected from the group consisting of 1) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering, and 2) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, and optionally, the human antibody or humanized antibody has increased agonist activity compared to the reference antibody. In some embodiments, the antibody includes or is a monovalent antibody (e.g., Fab). In some embodiments, the antibody comprises a second antigen-binding domain including a second VH domain and a second VL domain, and optionally the second VH also comprises, compared to a reference antibody, at least one amino acid substitution selected from the group consisting of 1) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, according to Kabat numbering, and 2) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, and optionally the Fc domain is derived from an IgG Fc domain, and further optionally the IgG Fc domain includes L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region. In some embodiments, the human antibody or humanized antibody binds to a cell surface receptor. In some embodiments, the human antibody or humanized antibody activates a target through clustering or multimerization of the target. In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody.In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0131] In some embodiments, a human antibody or humanized antibody derived from a reference antibody is provided, wherein both the antibody and the reference antibody include a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and the VH domain of the human antibody or humanized antibody includes at least one amino acid substitution selected from the group consisting of 1) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering, and 2) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, and optionally, the human antibody or humanized antibody has increased agonist activity compared to the reference antibody. In some embodiments, the antibody includes or is a monovalent antibody (e.g., Fab). In some embodiments, the antibody comprises a second antigen-binding domain including a second VH domain and a second VL domain, and optionally the second VH also comprises at least one amino acid substitution selected from the group consisting of 1) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, as defined by Kabat numbering, and 2) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, and optionally the Fc domain is derived from an IgG Fc domain, and further optionally the IgG Fc domain includes L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region. In some embodiments, the human antibody or humanized antibody binds to a cell surface receptor. In some embodiments, the human antibody or humanized antibody activates a target through clustering or multimerization of the target. In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody.In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0132] In some embodiments, a human antibody or humanized antibody derived from a reference antibody is provided, wherein both the antibody and the reference antibody include a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, and the VH domain of the human antibody or humanized antibody includes at least one amino acid substitution selected from the group consisting of 1) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering, and 2) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, and optionally, the human antibody or humanized antibody has increased agonist activity compared to the reference antibody. In some embodiments, the antibody comprises or is a monovalent antibody (e.g., Fab). In some embodiments, the antibody comprises a second antigen-binding domain comprising a second VH domain and a second VL domain, and optionally the second VH also comprises at least one amino acid substitution selected from the group consisting of 1) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, according to Kabat numbering, and 2) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the antibody is F(ab')2. In some embodiments, the antibody does not have an Fc domain. In some embodiments, the antibody has an Fc domain, optionally derived from an IgG Fc domain, and further optionally the IgG Fc domain includes L234A, L235A, and P329G. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody has a modified hinge, which further restricts the flexibility of the hinge. In some embodiments, the antibody has an IgG2 hinge region. In some embodiments, the human antibody or humanized antibody binds to a cell surface receptor. In some embodiments, the human antibody or humanized antibody activates the target through clustering or multimerization of the target.In some embodiments, the human antibody or humanized antibody is a monospecific antibody or comprises a monospecific antibody. In some embodiments, the human antibody or humanized antibody is a multispecific antibody or comprises a multispecific antibody.

[0133] The antibodies described herein may be monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity (e.g., binding to a target). In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a polyvalent (e.g., bivalent) antibody.

[0134] In some embodiments, the antibody is a full-length antibody or comprises a full-length antibody. In some embodiments, the antibody is an intact IgA, IgG, IgM, IgD, IgE antibody or another antibody class or isotype as defined herein.

[0135] In some embodiments, the antibody is chimeric, human, partially humanized, fully humanized, or semi-synthetic. The described antibodies and / or antibody fragments may originate from mouse antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0136] In some embodiments, the antibody comprises an Fc fragment. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments derived from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG.

[0137] In some embodiments, the target antigen is a molecule (e.g., a receptor) that requires clustering or polymerization for activation.

[0138] 1. Constraint structure (e.g., i-shape format) The antibodies described herein are regulated such that at least a portion of the antigen-binding domains of the antibody are constrained in their conformation to allow for a more rigid geometric shape between the two antigen-binding domains. In some embodiments, the antibody comprises two antigen-binding domains (in the same antibody or two different antibodies) that are parallel to each other or form an angle of less than a certain degree when engaged with a target antigen. In some embodiments, the antibody comprises two antigen-binding domains (in the same antibody or two different antibodies) that are less than or equal to a certain distance from each other when engaged with a target antigen. In some embodiments, the antibody comprises two antigen-binding domains (in the same antibody or two different antibodies).

[0139] In some embodiments, antibodies are provided herein designed to adopt a unique interface between two antigen-binding domains that bind to one or more target antigens (e.g., receptors requiring clustering or multimerization for activation). In some embodiments, the interface between the two antigen-binding domains is tuned so that the two antigen-binding domains engage with their target receptors in the same way that the receptor's native ligands engage with the target receptors via clustering or multimerization. For example, TNFR is activated by ligands of the TNF superfamily. TNFSF ligands (TNFLs) form a structurally relatively homogeneous protein family, characterized by a C-terminal TNF homologous domain (THD) that promotes assembly into homotrimers, and possibly into a heterotrimeric molecular superfamily. See, for example, Front Cell Dev Biol. 2021 Feb 11;8:615141. For example, for a computational simulation of the space-time process of binding between TNF ligands and their receptors, see Comput Struct Biotechnol J. 2020 Jan 18;18:258-270. In some embodiments, the interface between the two antigen-binding domains is adjusted so that the geometric shape through which the two antigen-binding domains engage with their target receptors includes a compact i-shape.

[0140] In some embodiments, the angle between two antigen-binding domains that bind to the same or adjacent antigens (e.g., clustered or multimerized receptors) is measured by electron microscopy. See, for example, Figure 2D. In some embodiments, the two antigen-binding domains are parallel to each other. In some embodiments, the two antigen-binding domains form an angle of about 90 degrees or less between the two domains (e.g., less than about 75 degrees, less than 70 degrees, less than 65 degrees, less than 60 degrees, less than 50 degrees, less than 45 degrees, less than 40 degrees, or less than 35 degrees). In some embodiments, the angle between the two antigen-binding domains is about 30 degrees or less, about 25 degrees or less, about 20 degrees or less, about 15 degrees or less, or about 10 degrees or less. In some embodiments, the angle between the two antigen-binding domains is about 5 degrees.

[0141] In some embodiments, the “i-shaped” format described herein refers to an antibody that, when engaging with an antigen, adopts a format such that the two antigen-binding domains (of a single antibody or two antibodies) that target the antigen form an angle of about 45 degrees or less (e.g., about 40 degrees or less, 35 degrees or less, 30 degrees or less, 25 degrees or less, 20 degrees or less, 15 degrees or less, 10 degrees or less, or 5 degrees or less). In some embodiments, the “i-shaped” format described herein refers to an antibody that, when engaging with an antigen, adopts a format such that the two antigen-binding domains (of a single antibody or two antibodies) that target the antigen form an angle of 5 degrees or less. In some embodiments, the “i-shaped” format described herein refers to an antibody that, when engaging with an antigen, adopts a format such that the two antigen-binding domains (of a single antibody or two antibodies) that target the antigen are parallel to each other.

[0142] In some embodiments, two adjacent antigen-binding domains that bind to one or more target antigens (e.g., receptor molecules requiring clustering or multimerization for activation) have a distance (e.g., average distance) approximately between the two adjacent receptors or within that range when the two adjacent receptors cluster or multimerize upon binding by the receptor's native ligand. In some embodiments, the two adjacent antigen-binding domains have a distance (e.g., average distance) of about 12 nm or less, 11 nm or less, 10 nm or less, 9 nm or less, 8 nm or less, 7 nm or less, 6 nm or less, 5 nm or less, 4 nm or less, 3 nm or less, 2 nm or less, or 1 nm or less when engaged. In some embodiments, the target antigen is a cytokine receptor (e.g., an IL-2 receptor), and the two adjacent antigen-binding domains have a distance (e.g., average distance) of about 3 to 8 nm (e.g., 3 to 5 nm or 5 to 8 nm). In some embodiments, the antibody is an IgG antibody having two Fab arms and having an average paratope distance of approximately ≤12 nm, ≤11 nm, ≤10 nm, ≤9 nm, ≤8 nm, ≤7 nm, ≤6 nm, ≤5 nm, ≤4 nm, ≤3 nm, ≤2 nm, or ≤1 nm. In some embodiments, the distance (e.g., average distance) between two adjacent antigen bindings that bind to one or more target antigens (e.g., clustered or multimerized receptors) is also measured by electron microscopy.

[0143] In some embodiments, the antibody is an IgG antibody and adopts a constrained structure (e.g., i-shaped format) that is in dynamic equilibrium with the Y-shaped structure exhibited by typical IgG antibodies. In some embodiments, one or more antibodies adopt the i-shaped structure in most particles when evaluated by negative staining electron microscopy. In some embodiments, the i-shaped structure is observed in at least about 15%, 20%, 30%, 40%, or 50% of the particles. In some embodiments, the i-shaped structure is observed in approximately or at least about 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, and 90% to 100% of the particles. In some embodiments, at least more than about 50% or more than about 60% of the particles adopt the iAb structure.

[0144] In some embodiments, the i-shaped format is induced in the antibody of interest by transplantation of a specific set of mutations. In some embodiments, the i-shaped format is induced in the antibody of interest via an affinity interface. In some embodiments, the affinity interface utilizes a hydrophobic patch on the surface of the heavy chain variable domain (VH domain) to facilitate intramolecular or intermolecular association between the two antigen-binding domains, thereby promoting the i-shaped format.

[0145] 2. Example VH domain In some embodiments, the antibodies described herein have one or more specific residues on the VH domain that promotes a VH-VH affinity interface or domain exchange, thereby adopting a constrained three-dimensional structure (e.g., an i-shaped format). See, for example, Cell. 2021 May 27;184(11):2955-2972.e25;J Virol. 2010 Oct;84(20):10700-10707. In some embodiments, one or more specific residues promote a hydrophobic patch between two VH domains. In some embodiments, one or more specific residues are located in the framework sequence of the VH domain of a human antibody or humanized antibody.

[0146] In some embodiments, the antibody comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, the first VH and first VL binding to a first target, and the VH domain comprises one or more of the following Kabat numbering: 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT. In some embodiments, the VH domain comprises the following Kabat numbering: 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT. In some embodiments, the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody contains a human consensus framework or a substantially similar framework (e.g., having at least 90%, 95%, 98%, and 99% sequence identity) in the VH domain, except for residues at 7, 17, 19, 21, 68, 70, 77, 79, 81, and 82.

[0147] In some embodiments, the antibody comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, the first VH and first VL binding to a first target, and the first VH domain comprises one or more of the following Kabat numbering: 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS. In some embodiments, the first VH domain comprises the following Kabat numbering: 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS. In some embodiments, the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody contains a human consensus framework or a substantially similar framework (e.g., having at least 90%, 95%, 98%, and 99% sequence identity) in the VH domain, except for residues at 7, 17, 19, 21, 68, 70, 77, 79, 81, and 82a.

[0148] In some embodiments, the antibody comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, the first VH and first VL binding to a first target, and the first VH domain comprises one or more of the following Kabat numbering: 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the first VH domain comprises the following Kabat numbering: 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. In some embodiments, the human antibody or humanized antibody does not bind to HIV. In some embodiments, the antibody contains a human consensus framework or a substantially similar framework (e.g., having at least 90%, 95%, 98%, and 99% sequence identity) in the VH domain, except for residues at 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113.

[0149] In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) is derived from a reference antibody, and both the antibody and the reference antibody contain a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain of the antibody contains at least one substitution at a position selected from 19, 21, 70, 79, and 81. In some embodiments, the VH domain contains at least two, at least three, at least four, or at least five substitutions at a position selected from 19, 21, 70, 79, and 81. In some embodiments, the VH domain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine substitutions at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and 82. In some embodiments, the VH domain contains at least one (e.g., at least two, at least three, at least four, or at least five) amino acid substitutions selected from the group consisting of 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering. In some embodiments, the amino acid substitutions are substitutions compared to a reference antibody. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) has increased agonist activity compared to a reference antibody. In some embodiments, the human antibody or humanized antibody does not bind to HIV.

[0150] In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) is derived from a reference antibody, and both the antibody and the reference antibody contain a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH of the antibody contains at least one substitution at a position selected from 19, 68, 70, and 81. In some embodiments, the VH of the human antibody or humanized antibody contains at least one, at least two, at least three, or at least four substitutions at a position selected from 19, 68, 70, and 81. In some embodiments, the VH domain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine substitutions at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and 82a. In some embodiments, the VH domain contains at least one (e.g., at least two, at least three, at least four, or at least five) amino acid substitutions selected from the group consisting of 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the VH domain contains 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering. In some embodiments, the amino acid substitutions are substitutions compared to a reference antibody. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) has increased agonist activity compared to a reference antibody. In some embodiments, the human antibody or humanized antibody does not bind to HIV.

[0151] In some embodiments, the antibody (e.g., a human antibody or humanized antibody) is derived from a reference antibody, and both the antibody and the reference antibody contain a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH of the human antibody or humanized antibody contains at least one substitution at a position selected from 14, 19, 39, 43, 74, 77, 82a and 82b. In some embodiments, the VH of the human antibody or humanized antibody contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight substitutions at a position selected from 14, 19, 39, 43, 74, 77, 82a and 82b. In some embodiments, the VH domain includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine substitutions at positions selected from 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113. In some embodiments, the VH domain includes at least one (e.g., at least two, at least three, at least four, or at least five) amino acid substitutions selected from the group consisting of 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the VH domain includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering. In some embodiments, the amino acid substitutions are substitutions compared to a reference antibody. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) has increased agonist activity compared to a reference antibody. In some embodiments, the antibody (e.g., a human antibody or humanized antibody) does not bind to HIV.

[0152] In some embodiments, the reference antibody contains a human consensus framework or a substantially similar framework (e.g., having at least 90%, 95%, 98%, or 99% sequence identity) in its VH domain.

[0153] 3. Exemplary targets of antibodies The antibody targets described herein may be any molecule (e.g., a protein molecule) or a part thereof (e.g., a subunit) whose activation (e.g., activation of its downstream signaling) is desired. In some embodiments, molecular activation involves or requires molecular clustering or polymerization. In some embodiments, molecular activation involves or requires molecular dimerization or trimerization. In some embodiments, molecular activation involves or requires homopolymerization or heteropolymerization (e.g., dimerization or trimerization).

[0154] In some embodiments, the target is a cell surface receptor. In some embodiments, the cell surface receptor is a mammalian cell surface receptor (e.g., human cell surface receptors, non-human primate cell surface receptors, rodent cell surface receptors, etc.). Examples of cell surface receptors include receptors belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, tumor necrosis factor (TNF) receptor family (TNFR), also substituted for the TNF receptor superfamily (TNFRSF), G protein-coupled receptor (GPCR) family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion molecule family, and hormone receptor family.Various references are available regarding receptors belonging to these receptor families and their characteristics, for example: Cooke B A., King RJ B., van der Molen H J. ed. New Comprehensive Biochemistry Vol.18B “Hormones and their Actions Part II” pp.1-46 (1988) Elsevier Science Publishers BV., New York, USA; Patthy L. (1990) Cell, 61:13-14; Ullrich A., et al. (1990) Cell, 61:203-212; Massagul J. (1992) Cell, 69:1067-1070; Miyajima A., et al. (1992) Annu. Rev. Immunol., 10:295-331; Taga T. and Kishimoto T. (1992) FASEB J.,7:3387-3396, Fantl W I.,et al.(1993)Annu.Rev.Biochem.,62:453-481,Smith C A.,et al.(1994)Cell,76:959-962,Flower D R. (1999) Biochim. Biophys. Acta, 1422:207-234, and M. Miyasaka ed., Cell Technology, supplementary volume, Handbook series, “Handbook for Adhesion Factors” (1994) (Shujunsha, Tokyo, Japan).

[0155] Cell surface receptors include, for example, hormone receptors and cytokine receptors. Exemplary hormone receptors include, for example, estrogen receptors. Exemplary cytokine receptors include, for example, hematopoietic factor receptors, lymphokine receptors, growth factor receptors, differentiation regulatory factor receptors, and the like. Examples of cytokine receptors include erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony-stimulating factor (G-CSF) receptor, macrophage colony-stimulating factor (M-CSF) receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1) receptor, interleukin-2 (IL-2) receptor, interleukin-3 (IL-3) receptor, interleukin-4 (IL-4) receptor, interleukin-5 (IL-5) receptor, interleukin-6 (IL-6) receptor, interleukin-7 (IL-7) receptor, interleukin-9 (IL-9) receptor, interleukin-10 (IL-10) receptor, interleukin-11 (IL-11) receptor, and interleukin-12 (IL- 12) Examples include receptors such as interleukin-13 (IL-13) receptor, interleukin-15 (IL-15) receptor, interferon-alpha (IFN-alpha) receptor, interferon-beta (IFN-beta) receptor, interferon-gamma (IFN-gamma) receptor, growth hormone (GH) receptor, insulin receptor, hematopoietic stem cell growth factor (SCF) receptor, vascular epithelial growth factor (VEGF) receptor, epidermal growth factor (EGF) receptor, nerve growth factor (NGF) receptor, fibroblast growth factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-beta (TGF-beta) receptor, leukocyte migration inhibitor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M (OSM) receptor, and Notch family receptors. An additional, non-limiting example of cytokine receptors is disclosed in Wang et al. (2009) Ann. Rev. Immunol. 27:29-60.

[0156] IL-2 receptor In some embodiments, an antibody (e.g., a human antibody or a humanized antibody) binds to the interleukin-2 (IL-2) receptor. The IL-2 cytokine forms a high-affinity quaternary complex with three receptors: IL-2RA, IL-2RB, and IL-2RG. IL-2RB and IL-2RG are responsible for downstream signaling during heterodimerization, while IL-2RA stabilizes the complex and enhances the potency of IL-2 (see, for example, Spolski, R. et al., Nat Rev Immunol. 2018.18, 648-659). In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) that binds to the IL-2 receptor includes a bispecific antibody. In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) binds to both IL-2RB and IL-2RG (e.g., those disclosed in the examples).

[0157] In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) includes a bispecific antibody that binds to the IL-2 receptor (e.g., via binding to both IL-2RG and IL-2RB).

[0158] In some embodiments, the antibody that binds to IL-2RG is derived from a reference antibody selected from the group consisting of G02, G12, G23, G25, G28, and G33. In some embodiments, the antibody that binds to IL-2RB is derived from a reference antibody selected from the group consisting of B09, B10, B26, B30, B37, B39, B43, and B65. In some embodiments, the antibody includes a VH domain containing three VH CDR sequences of B10. In some embodiments, the antibody includes a VL domain containing three VL CDR sequences of B10.

[0159] In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) comprises a bispecific antibody that binds to the IL-2 receptor, comprising a first Fab and a second Fab, wherein the first Fab is derived from a reference antibody selected from the group consisting of G02, G12, G23, G25, G28, and G33, and the second Fab is derived from a reference antibody selected from the group consisting of B09, B10, B26, B30, B37, B39, B43, and B65. In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) comprises a bispecific antibody that binds to the IL-2 receptor, wherein one of the two VH domains comprises three VH CDR sequences of B10, one of the two VL domains comprises three VL CDR sequences of B10, the other of the two VH domains comprises three VH CDR sequences of G25 or G28, and the other of the two VL domains comprises three VL CDR sequences of G25 or G28.

[0160] Tumor necrosis factor receptor superfamily (TNFRSF) In certain embodiments, the target is a member of the tumor necrosis factor receptor (TNFR) family. TNFRSF receptors are typically activated by molecular clustering induced by corresponding cell membrane tumor necrosis factor superfamily (TNFSF) ligands. In some embodiments, TNFRSF members are targets for triggering their downstream signaling via clustering. In some embodiments, targets that are TNFRSF members share similar ligand-receptor trimer structures for signaling activation. In some embodiments, antibodies adopting an iAb conformation (e.g., human antibodies or humanized antibodies) can induce cell membrane receptor clustering and activation of TNFRSF members. In some embodiments, the formation of an active TNFSF3-TNFRSF3 complex is the minimum unit for TNFRSF signaling activation. In some embodiments, oligomerization of two or more TNFSF3-TNFRSF3 complexes may be required so that the TNFRSF member can fully stimulate the TNFRSF downstream signaling cascade. Non-exclusive examples of TNFRs include TNFR1, TNFR2, lymphotoxin β receptor, OX40, CD40, Fas, decoy receptor 3, CD27, CD70, CD226, CD137, ICOS, 2B4, CD30, 4-1BB, death receptor 3 (DR3), death receptor 4 (DR4), death receptor 5 (DR5), death receptor 6 (DR6), decoy receptor 1, decoy receptor 2, NFκB receptor activator (RANK), and osteoprotegeri. Examples include TNFR receptors (OPG), TWEAK receptor, TACI, BAFF receptor (BAFF-R), HVEM (herpesvirus entry mediator), nerve growth factor receptor, B cell maturation antigen (BCMA), glucocorticoid-induced TNF receptor (GITR), toxic and JNK-inducing factor (TAJ), RELT, TNFRSF22, TNFRSF23, ectodysprascin A2 isoform receptor and ectodysprascin 1, and anhidrotic receptors. Additional non-limiting examples of TNFRs are disclosed in Naismith and Sprang (1998) Trends in Biochemical Sciences 23(2):74-79.In some embodiments, the antibody (e.g., a human antibody or a humanized antibody) binds to OX40, CD40, 4-1BB, DR4, or DR5.

[0161] In some embodiments, the antibody binds to the cysteine-rich domain (CRD) subunit of the TNFR. In some embodiments, the antibody binds to the cysteine-rich domain (CRD) subunit outside the ligand-binding domain (i.e., the domain to which the native ligand that binds to the TNFR binds). In some embodiments, the antibody binds to the distal CRD domain of the TNFR membrane.

[0162] In some embodiments, an antibody (e.g., a human antibody or humanized antibody) binds to OX40 (e.g., human OX40). OX40 is also known as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), or CD134. OX40 is primarily expressed on effector and regulatory T cells. OX40L, the native ligand for OX40, is expressed on activated antigen-presenting cells such as dendritic cells, endothelial cells, macrophages, and activated B cells. In some embodiments, the involvement of OX40-OX40L is key to enhancing the proliferation and prolonged survival of effector T cells by suppressing apoptosis, enhancing T cell effector function (e.g., cytokine production), and generating T helper memory cells. See, for example, Pharmaceuticals. 2022;14(12):2753.

[0163] In certain embodiments, a human antibody or humanized antibody that binds to OX40 is modified to adopt an iAb conformation using the method described herein. In some embodiments, the antibody that binds to OX40 (e.g., a human antibody or humanized antibody) is derived from an OX40 antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, and 3G8. See, for example, Biomolecules. 2022 Sep;12(9):1209, Clin Cancer Res. 2018 Nov 15;24(22):5735-5743, Proc Natl Acad Sci US A. 2022 Jun 7;119(23):e2201562119.

[0164] In some embodiments, the OX40 antibody is derived from a reference antibody selected from HFB301001, FS120, INBRX-106, BGB-A445, PF-04518600, MEDI6469, MEDI0562, ABBV-368, FS120, INCAGN01949, BMS986178, PF04518600, GSK3174998, and SL-279252. For example, see Curr Oncol Rep.2022 Jul;24(7):951-960, Cancer Immunol Res 2020;8:781-93;Journal for ImmunoTherapy of Cancer 2020;8:doi:10.1136 / jitc-2020-SITC2020.0699.

[0165] In some embodiments, an antibody (e.g., a human antibody or humanized antibody) binds to CD40 (e.g., human CD40). CD40 is also known as member 5 (TNFSF5) of the tumor necrosis factor receptor superfamily. CD40 is expressed not only on platelets, B cells, and myeloid cells, but also on non-hematopoietic cells such as endothelial cells, fibroblasts, smooth muscle cells, and certain types of tumor cells. The corresponding ligand for CD40 is CD154 (TNFSF5 / CD40L). CD40 expression on monocytes and their progeny, macrophages and dendritic cells (DCs), as well as on B cells, plays a crucial role in immune cell function. CD40 signaling is a key trigger in the monocyte maturation process, leading to differentiation into macrophages and DCs, primarily in the M1 spectrum.

[0166] In some embodiments, the CD40 antibody is derived from a reference antibody selected from CP-870, 893 (RO70099789), SGN-40, sericrelumab, dacetuzumab, Chi Lob 7 / 4, APX005M, ADC-1013, CDX-1140, SEA-CD40, lavagalimab, gyrolarimab, and sotigolimab. See, for example, Oncol Lett. 2020 Nov;20(5):176.

[0167] In some embodiments, an antibody (e.g., a human antibody or humanized antibody) binds to 4-1BB (e.g., human 4-1BB). 4-1BB is also known as CD137 and TNFRSF9. 4-1BB is expressed on both CD4+ T cells and CD8+ T cells, as well as on natural killer (NK) cells and DCs. 4-1BB delivers costimulatory signals, activating the cytotoxic effect of CD8+ T cells and aiding in the formation of memory T cells. Furthermore, 4-1BB signaling can activate NK cells and dendritic cells. Like other TNFRSF members, three monomeric 4-1BB bind to the trimer CD137L to activate intracellular signaling.

[0168] In some embodiments, the 4-1BB antibody is urelumab (BMS-663513), EU101, citalizumab, LVGN-6051, YH-004, GEN1046, PRS343, ES101, Synlevafsup alfa, HLX-35, IBI309, TJ-033721, ATG101, LBL-024, MCLA-145, ABL-503, P It is derived from a reference antibody selected from M1032, QLF-31907, FS-120, RO7227166, HBM-7008, ND-021, GNC-035, GNC-038, GNC-039, ADG-106, utomirumab, ATOR-1017, AGEN-2373, CTX-471, PRS-344, RO-7122290, and HOT-1030. See, for example, Front Immunol. 2022 Sep 16;13:975926.

[0169] In some embodiments, an antibody (e.g., a human antibody or a humanized antibody) binds to DR4 (e.g., human DR4). DR4 is also known as TRAIL receptor 1 (TRAILR1) and tumor necrosis factor receptor superfamily member 10A (TNFRSF10A). In some embodiments, an antibody (e.g., a human antibody or a humanized antibody) binds to DR5 (e.g., human DR5). DR5 is also known as TRAIL receptor 2 (TRAILR2) and tumor necrosis factor receptor superfamily member 10B (TNFRSF10B). DR4 and DR5 are cell surface receptors of the TNF receptor superfamily that bind to tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) and mediate apoptosis. Engagement of DR4 or DR5 with TRAIL induces apoptosis through the recruitment of the adapter protein Fas-associated death domain (FADD) and the formation of a macromolecular complex called the death-inducing signaling complex (DISC).

[0170] In some embodiments, the DR4 antibody is derived from a reference antibody selected from HLX56, mapatumumab, m921 / 922, 4H6, 4G7, AY4, and TR1-mAbs. See, for example, Antibodies (Basel). 2017 Dec;6(4):16.

[0171] In some embodiments, the DR5 antibody is derived from a reference antibody selected from conatumumab, droditumab, DS-8273a, KTRM2, lexatumumab, tigatuzumab, zaptuzumab, inbrx-109, LaDR5, LBy135, mDRA6, WD1, zaptuximab, HMCAZ5, and AD5.10. See, for example, Antibodies (Basel). 2017 Dec;6(4):16.

[0172] In some embodiments, the target is a member of the low-density lipoprotein receptor (LDLR) family. Non-limiting examples of LDLRs include LDLR, low-density lipoprotein receptor-associated protein (LRP)1, LRP10, LRP1B, LRP2, LRP4, LRP5, LRP5L, LRP6, LRP8, Nidogen (NID)-1, NID2, Sortilin-associated receptor, L(SORL1), and very low-density lipoprotein receptor (VLDLR).

[0173] In some embodiments, the target is a member of the receptor tyrosine kinase (RTK) family. Non-limiting examples of RTKs include leukocyte receptor tyrosine kinase (LTK), receptor tyrosine kinase-like orphan receptors (ROR), ephrin receptor (Ephs), Trk receptor, insulin receptor (IR), and Tie2. Further non-limiting examples of RTKs are disclosed in Alexander et al. (2013) The Concise Guide to Pharmacology 2013 / 14: Enzymes. Br. J. Pharmacol. 170: 1797-1867, Li and Hristova (2010), and Lemmon and Schlessinger (2010) Cell 141(7): 1117-1134.

[0174] In some embodiments, the target is a growth hormone receptor, insulin receptor, leptin receptor, Flt-3 ligand receptor, or insulin-like growth factor (IGF)-I receptor. Examples of these receptors include, for example, hEPOR (Simon, S. et al. (1990) Blood 76, 31-3), mEPOR (D'Andrea, AD et al. (1989) Cell 57, 277-285), hG-CSFR (Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci. USA. 87, 8702-8706), mG-CSFR (Fukunaga, R. et al. (1990) Cell 61, 341-350), hTPOR (Vigon, I. et al. (1992) 89, 5640-5644), mTPOR (Skoda, RC. et al. (1993) 12, 2645-2653), and hInsR (Ullrich, A. et al. Examples include al. (1985) Nature 313, 756-761), hFlt-3 (Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 459-463), hPDGFR (Gronwald, R GK. et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439), and hIFNa / b R (Uze, G. et al. (1990) Cell 60, 225-234, and Novick, D. et al. (1994) Cell 77, 391-400).

[0175] In some embodiments, the target is the nerve growth factor receptor family and / or the neurotrophic factor receptor family. Non-limiting examples of nerve growth factor receptors and neurotrophic factor receptors include p75 (also known as low affinity nerve growth factor receptor (LNGFR)), TrkA, TrkB, and TrkC. Additional non-limiting examples of nerve growth factor receptors and neurotrophic factor receptors are disclosed in Lotz et al. (1996) J. of Leukocyte Biology 60(1):1-7.

[0176] In some embodiments, the target is a member of the growth factor receptor family. For example, but not limited to, growth factor receptors may be receptors that signal via the JAK / STAT, MAP kinase, and PI3 kinase pathways. Non-exclusive examples of growth factor receptors include fibroblast growth factor receptors (FGFRs) and ErbB family receptors (e.g., epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR)).

[0177] In some embodiments, the target may include receptors that form heterodimers or heterotrimers to induce cellular signaling. For example, but not limited to, the target may be a member of the serine / threonine kinase receptor family. Non-exclusive examples of serine / threonine kinase receptors include activin A receptor type II-like I (ALK1), activin A receptor type I (ALK2), osmotic protein receptor type IA (BMPR1A), activin A receptor type I (ALK4), activin A receptor type IC (ALK7), transforming growth factor beta receptor 1 (TGFBR1), osmotic protein receptor type I (BMPR1B), transforming growth factor beta receptor II (TGFBR2), osmotic protein receptor type II (BMPR2), anti-Müllerian hormone receptor type II (MISR2), activin A receptor type IIA (ActR2), activin A receptor type IIB (ActR2B), and transforming growth factor beta receptor III (TGFBR3).

[0178] In some embodiments, an antibody (e.g., a human antibody or a humanized antibody) binds to a cytokine receptor, where the cytokine can form a complex with at least two different receptors in nature, which causes the cytokine's downstream activity. In some embodiments, the cytokine's downstream activity results in activation of signaling pathways, alteration of gene expression, cell proliferation and differentiation, cell survival, cell death, metabolic changes, or cytoskeletal rearrangement. In some embodiments, the cytokine's downstream activity activates signaling pathways such as Akt, AMPK, apoptosis, estrogen, insulin, JAK-STAT, MAPK, mTOR, NF-κB, Notch, p53, TGF-β, Toll-like, VEGF, or Wnt signaling pathways.

[0179] 4. Antibody affinity In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) have a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10⁻⁸ M or less, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M).

[0180] In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) have dissociation constants (KD) of 100 nM to 1 μM, 10 nM to 100 nM, 1 nM to 10 nM, 0.1 nM to 1 nM, 0.01 nM to 0.1 nM, or 0.001 nM to 0.1 nM.

[0181] In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) have one or more modifications that promote a weaker dissociation constant with respect to a target.

[0182] In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) are 10⁻²s -1 Below, 5×10-3s -1 Below, 10-3s -1Below, 5×10-4s -1 Below, 10-4s -1 Below, 5×10-5s -1 The following, or 10-5s -1 The following off-speed constant (k off ) has. In certain embodiments, the antibodies provided herein are 10-2s -1 ~5×10⁻³s -1 , 10-3s -1 ~5×10⁻³s -1 , 5×10-4s -1 ~10-3s -1 , 10-4s -1 ~5×10⁻⁴s -1 , 10-4s -1 ~5×10⁻⁴s -1 , 5x10-5s -1 ~10-4s -1 Or 10-5s -1 ~5×10⁻⁵s -1 Off rate (k off ) has.

[0183] In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) have one or more modifications that facilitate a faster off-rate constant against a target.

[0184] In one aspect, K DThis is measured using the BIACORE® surface plasmon resonance assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C with an immobilized antigen CM5 chip to ~10 response units (RUs). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 10 mM sodium acetate (pH 4.8) to 5 μg / ml (approximately 0.2 μM) and then injected at a flow rate of 5 μl / min to reach approximately 10 response units (RUs) of binding protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at a flow rate of approximately 25 μL / min into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated using a simple one-to-one Langmuir coupled model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. D ) is k off / k on It is calculated as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The ON velocity is 10 by the above surface plasmon resonance assay. 6 M -1 s -1If it exceeds this value, this ON rate can be determined by using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band-passing) of a 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C in the presence of gradually increasing concentrations of antigen, measured with a spectrometer such as an Aviv Instruments stop-flow spectrophotometer equipped with a stirred cuvette or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0185] Alternatively, K D This is measured by radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a titration series of unlabeled antigens at the lowest concentration. 125 I) Fab is equilibrated with labeled antigen and then measured by capturing the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2–5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, but the incubation can be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. Then transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Next, remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that yields less than 20% of the maximum binding for use in competitive binding assays.

[0186] 5. Antibody fragment In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) are antibody fragments.

[0187] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the variable domains of the heavy and light chains (VH and VL, respectively). Therefore, the term "Fab fragment" refers to an antibody fragment containing a light chain fragment containing the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains. A "Fab' fragment" differs from a Fab fragment by the addition of a residue at the carboxyl terminus of the CH1 domain, containing one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domain retain a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0188] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097, International Publication No. 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0189] In a further embodiment, the antibody fragment is a single-stranded Fab fragment. The "single-stranded Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab fragments will be further stabilized by the formation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0190] In another embodiment, the antibody fragment is a single-stranded variable fragment (scFv). A "single-stranded variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, usually rich in glycine for flexibility and rich in serine or threonine for solubility, and capable of linking the N-terminus of VH to the C-terminus of VL, and vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as International Publication No. 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0191] In another embodiment, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In a particular embodiment, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA, e.g., U.S. Patent No. 6,248,516B1).

[0192] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant preparation using recombinant host cells (e.g., Escherichia coli), as described herein.

[0193] 6. Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, e.g., a monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0194] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies usually contain one or more variable domains (CDRs or parts thereof) derived from the non-human antibody, and FRs (or parts thereof) derived from the human antibody sequence. Humanized antibodies also optionally contain at least a portion of the human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0195] Humanized antibodies and their production methods are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409, and Kashmiri et al., Methods. Further details can be found in 36:25-34 (2005) (describes specificity determination region (SDR) graphing), Padlan, Mol.Immunol.28:489-498 (1991) (describes "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describes "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (describes the "guided selection" approach to FR shuffling).

[0196] Framework regions usable for humanization are not limited to, but include framework regions selected using a "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (e.g., Baca et al. See also al., Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0197] 7. Human antibodies In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) are human antibodies. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0198] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or it is located extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology, U.S. Patent No. 5,770,429 describing HUMAB® technology, U.S. Patent No. 7,041,870 describing KM MOUSE® technology, and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions derived from intact antibodies produced from such animals may be further modified, for example, by combining them with different human constant regions.

[0199] Human antibodies can also be made by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0200] Human antibodies can also be generated by isolating variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0201] 8. Multispecific Antibodies In certain embodiments, the antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) are multispecific antibodies, e.g., bispecific antibodies. A “multispecific antibody” is a monoclonal antibody that has binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one binding specificity is for a target and another specificity is for any other antigen. In certain embodiments, a bispecific antibody may bind to two (or more) different epitopes of a target. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells expressing a target. Multispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0202] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain–light-chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and “knob-in-hole” modifications (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be made by modifying the electrostatic steering effect for making antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), making bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), using general light-chain techniques to avoid light-chain mispairing problems (see, e.g., WO 98 / 50431), using “diabody” techniques for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and can also be made by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0203] For example, this also includes modified antibodies having three or more antigen-binding sites, such as "octopus antibody," or DVD-Ig (see, for example, International Publication Nos. 2001 / 77342 and International Publication Nos. 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publication Nos. 2010 / 115589, 2010 / 112193, 2010 / 136172, 2010 / 145792, and 2013 / 026831. A bispecific antibody or its antigen-binding fragment also comprises a “dual-acting FAb” or “DAF” containing antigen-binding sites that bind to a target, as well as to another different antigen or two different epitopes of the target (see, for example, U.S. Patent Application Publication 2008 / 0069820 and International Publication 2015 / 095539).

[0204] Multispecific antibodies may also be provided in an asymmetric form having a domain crossover in one or more binding arms of the same antigen specificity, i.e., by exchanging a VH / VL domain (see, e.g., International Publication No. 2009 / 080252 and International Publication No. 2015 / 150447), a CH1 / CL domain (see, e.g., International Publication No. 2009 / 080253), or a complete Fab arm (see, e.g., International Publication No. 2009 / 080251, International Publication No. 2016 / 016299, and also Schaefer et al, PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). In one embodiment, the multispecific antibody comprises a cross-Fab fragment. The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which either the variable or constant regions of the heavy and light chains are exchanged. A cross-Fab fragment includes a polypeptide chain consisting of a light chain variable region (VL) and heavy chain constant region 1 (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and light chain constant region (CL). Asymmetric Fab arms can also be modified by introducing charged or uncharged amino acid mutations into the domain contact surfaces to direct correct Fab pairing. See, for example, International Publication 2016 / 172485.

[0205] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0206] Certain types of multispecific antibodies, as also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, such as a tumor cell, and to the activating invariant component of the T cell receptor (TCR) complex (such as CD3), thereby retargeting T cells and killing the target cells. Thus, in certain embodiments, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies, where one binding specificity is to a target and the other is to a different target (e.g., another antigen).

[0207] Examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567, Nagarsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb", Kipriyanov et al., J Mol Biol This specification includes, but is not limited to, the following: “DART” (Dual Affinity Retargeting) molecules based on the Diabody format but characterized by a C-terminal disulfide crosslink for stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and so-called triomabs, which are all-hybrid mouse / rat IgG molecules (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T-cell bispecific antibody formats included herein are described in International Publication Nos. 2013 / 026833, 2013 / 026839, 2016 / 020309, and Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0208] In some embodiments, a multispecific or bispecific antibody binds to at least two antigens, which are two subunits of the molecule, and the multimerization of the two subunits promotes the activation of the molecule.

[0209] 9. Antibody variants The amino acid sequence variants of antibodies described herein may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding).

[0210] (i) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are described in further detail below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or reduced or increased effector function. [Table 1]

[0211] Amino acids can be classified according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0212] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0213] One type of substitution mutant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutants selected for further testing have altered (e.g., improved) specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution mutants are affinity-matured antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. In short, one or more CDR residues are mutated, and the mutant antibody displayed on a phage is screened for specific biological activity (e.g., binding affinity).

[0214] To improve antibody affinity, modifications (e.g., substitutions) may be made in the CDR, for example. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that come into contact with the antigen, and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by construction of a secondary library and re-selection therefrom is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutation). A secondary library is then constructed. Next, this library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed methods in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0215] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unchanged or has one, two, or three or fewer amino acid substitutions.

[0216] A useful method for identifying residues or regions of an antibody that can be targets for mutagenesis is what is referred to as "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of residues of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they contain the desired properties.

[0217] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions in the range of lengths from one residue to polypeptides containing more than 100 residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion mutants of antibody molecules include the N-terminal or C-terminal fusions of an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or a polypeptide to the antibody, which increases the serum half-life of the antibody.

[0218] (ii) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0219] If the antibody contains an Fc region, the oligosaccharide attached to the antibody may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides, generally linked by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of this application may be carried out to produce antibody variants having specific improved properties.

[0220] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called "afucosylated" oligosaccharide) is, in particular, an N-linked oligosaccharide lacking a fucose residue bound to the first GlcNAc in the stem of a branched oligosaccharide structure. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the natural antibody or the parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight variations in antibody sequences. Antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. For example, see U.S. Patent Application Publications 2003 / 0157108 and 2004 / 0093621.

[0221] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application Publication No. 2003 / 0157108, and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, such as FUT8 of the alpha-1,6-fucosyltransferase gene, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004), Kanda, Y. et al.). Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or eliminated (see, for example, U.S. Patent Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0222] In a further embodiment, the antibody variant is provided with a bifurcated oligosaccharide, for example, in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999), Ferrara et al., Biotechn Bioeng 93, 851-861 (2006), International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0223] Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087, 1998 / 58964, and 1999 / 22764.

[0224] (iii) Fc region mutant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein to create Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0225] In some embodiments, the antibodies described herein include one or more amino acid substitutions that enable reduced effector function. In some embodiments, the antibody includes IgG Fc having one or more substitutions selected from L234A, L235A, and P329G. In some embodiments, the antibody includes L234A, L235A, and P329G.

[0226] In some embodiments, the antibody contains one or more mutations that promote antibody multimerization. In some embodiments, the antibody contains IgG Fc including E345R, E430G, and / or S440Y.

[0227] In some embodiments, the antibody has a modified Fc that facilitates binding to FcgRIIB. In some embodiments, the antibody has an IgG Fc containing S267E. In some embodiments, the antibody has an IgG Fc containing S267E and L328F.

[0228] In certain embodiments, this application envisions antibody variants that, by possessing some, but not all, effector functions, would be desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), and No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the target molecule can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Furthermore, a C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006, see International Publication No. 2013 / 120929)).

[0229] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant, which has substitutions at residues 265 and 297 of alanine, as well as Fc variants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0230] Specific antibody variants exhibiting improved or reduced binding to FcR have been described. (See, for example, U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0231] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0232] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 (residues in EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) within the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) within the Fc region derived from the human IgG1 Fc region.

[0233] In some embodiments, modifications are made in the Fc region that result in alterations (e.g., reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as disclosed, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0234] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions in one or more of the Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 (e.g., U.S. Patent No. 7,371,826, Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0235] The Fc region residues crucial to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J.Immunol 169(2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering of the residues) are involved in the interaction (Medesan, C., et al., Eur.J.Immunol.26(1996) 2533, Firan, M., et al., Int.Immunol.13(2001) 993, Kim, JK, et al., Eur.J.Immunol.24(1994) 542). Residues I253, H310, and H435 have been found to be critical for the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are important for this interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993, Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J.Immunol.182(2009)7667-7671) reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0236] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at 253 and / or 310 and / or 435 (EU numbering of residues) in the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A., et al., J.Immunol. 194 (2015) 5497-5508.

[0237] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310 and / or 433 and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460).

[0238] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. 94 / 29351.

[0239] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce self-recognition, for example, substitutions at the R355, E356, K414, E438, K439, and S440 positions (EU numbering of residues) in the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. 94 / 29351.

[0240] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In some embodiments, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, EU index numbering of amino acid position).

[0241] (iv) Cysteine-modified antibody variant In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as THIOMAB®, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By substituting these residues with cysteine, the reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to conjugate the antibody to other sites, such as a drug site or a linker drug site, to produce an immunoconjugate, as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication No. 2016040856.

[0242] (v) Modification of the hinge In certain embodiments, the antibodies provided herein include a modified hinge (e.g., compared to a reference antibody). In some embodiments, the antibody includes an IgG2 hinge or a substantially similar hinge. In some embodiments, the antibody includes an IgG4 hinge or a substantially similar hinge. In some embodiments, the antibody includes an IgG1 hinge or a substantially similar hinge. In some embodiments, the antibody includes an IgG3 hinge or a substantially similar hinge. Structural analysis has revealed that the IgG2 isotype has the most rigid hinge region, and the least active isotype (IgG3) is the most flexible. See, for example, Trends Mol Med. 2023 Jan;29(1):48-60.

[0243] In some embodiments, the antibody does not contain the IgG3 hinge.

[0244] In some embodiments, the modified hinge further restricts the flexibility of the hinge. In some embodiments, the modified hinge restricts the flexibility of the hinge by at least 10% compared to the corresponding reference antibody. In some embodiments, the modified hinge restricts the flexibility of the hinge by about 10% to about 30%, about 30% to about 50%, about 50% to about 70%, about 70% to about 90%, or about 90% to about 100% compared to the corresponding reference antibody. In some embodiments, the modified hinge restricts the flexibility of the hinge by 50% compared to the corresponding reference antibody. Flexibility can be evaluated, for example, using a computational model. See, for example, J Pharm Sci. 2019 May;108(5):1663-1674.

[0245] 10. Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties that are known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to this antibody may vary, and when two or more polymers are bound, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions, although this is not limiting.

[0246] B. Recombination methods and compositions The antibodies described herein (e.g., the antibodies provided by this application, e.g., the reference antibody) may be prepared using recombinant methods and recombinant compositions, for example, as described in the Examples. For these methods, one or more isolated nucleic acids encoding a given antibody are provided. If two or more isolated nucleic acids are used, these nucleic acids may be on the same expression vector or different expression vectors, and typically these nucleic acids are located on two or three expression vectors, i.e., one vector may contain more than one of these nucleic acids. An example of such bispecific antibodies is CrossMab (see, e.g., Schaefer, W. et al, PNAS, 108(2011)11187-1191). For example, one heterozygous monoweight chain may contain a so-called "knob mutation" (T366W and optionally one of S354C or Y349C), while the other may contain a so-called "hole mutation" (T366S, L368A, and Y407V and optionally Y349C or S354C) (according to EU index numbering) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73).

[0247] In one embodiment, an isolated nucleic acid encoding an antibody reported herein is provided.

[0248] In some embodiments, nucleic acids encoding antibodies (e.g., human antibodies or humanized antibodies) are provided herein. In some embodiments, the nucleic acid encodes a human antibody or humanized antibody adopting an iAb three-dimensional structure. In some embodiments, the nucleic acid encodes a human antibody or humanized antibody adopting an iAb three-dimensional structure via a domain exchange mechanism. In some embodiments, the nucleic acid encodes a human antibody or humanized antibody adopting an iAb three-dimensional structure via an affinity interface mechanism.

[0249] In some embodiments, the nucleic acids provided herein are contained in one or more vectors. For example, in some embodiments, vectors containing nucleic acids encoding human antibodies or humanized antibodies are provided herein. In some embodiments, the vector contains nucleic acids encoding human antibodies or humanized antibodies of the Disclosure.

[0250] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a gamma retroviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adenovirus vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector is a pEF-ENTR A vector.

[0251] In some embodiments, the vector encodes multiple gene products. In some embodiments, the vector is a bicistronic vector. In some embodiments, the vector contains a nucleic acid encoding a second protein product, such as a fluorescent protein, like green fluorescent protein (GFP).

[0252] In some embodiments, the vector is a transposase vector. In some embodiments, the vector is a piggyBac vector.

[0253] In some embodiments, the vector includes a promoter. In some embodiments, a nucleic acid encoding a human antibody or a humanized antibody is operably ligated to the promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a ubiquitously expressed promoter. In some embodiments, the vector includes an EF1-a promoter. In some embodiments, a nucleic acid encoding a chimeric receptor is operably ligated to the EF1-a promoter.

[0254] In one embodiment, a method for producing an antibody as described herein is provided, comprising culturing host cells containing nucleic acids encoding an antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture).

[0255] In the case of recombinant antibody production, for example, the nucleic acid encoding the antibody described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody), or they can be produced by recombinant methods or obtained by chemical synthesis.

[0256] Host cells suitable for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of agonist antibodies and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, antibodies may be isolated from bacterial cell paste in a suitable soluble fraction and further purified.

[0257] In addition to prokaryotes, useful eukaryotic microorganisms such as filamentous fungi or yeasts, including fungal and yeast strains whose glycosylation pathways have been "humanized" and which result in the production of antibodies with a partially or completely human glycosylation pattern, are also suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414 and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0258] Furthermore, suitable host cells for expressing (glycosylated) antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified, and these may be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0259] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for producing antibodies in transgenic plants).

[0260] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7), human embryonic kidney cells (e.g., 293 cells or 293T cells as described in Graham et al., J Gen Virol 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical tumor cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather, JP et al., Annals). These include NYAcad.Sci.383(1982)44-68, MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlaub, G. et al., Proc. Natl. Acad.Sci. USA 77(1980)4216-4220), including DHFR-CHO cells, and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol.248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), pp.255-268.

[0261] In one embodiment, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0262] C. Assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0263] Agonist activity and affinity In certain embodiments, antibodies described herein (e.g., human antibodies or humanized antibodies) are tested for their agonist activity. In some embodiments, agonist activity is measured against untreated control cells or cells exposed to a control antibody (e.g., a reference antibody discussed herein). In some embodiments, the relative agonist activity of a human antibody or humanized antibody against a target receptor is tested in a cell-based assay utilizing a human cell line expressing the target receptor and a luciferase reporter system. See, for example, Figure 3A. In some embodiments, agonist activity is evaluated by a multiplicative change in the reporter expression signal relative to a control (e.g., an untreated control). In some embodiments, a value greater than at least 2x indicates target receptor agonism. In some embodiments, agonism against the target receptor results in at least a 2x, 5x, 10x, 15x, or 20x increase in the expression of a reporter gene, e.g., luciferase. In some embodiments, a corresponding wild-type (WT) IgG antibody or contrast body is tested as a comparison. In some embodiments, human antibodies or humanized antibodies in iAb format exhibit improved agonist activity compared to WT IgG control antibodies and / or contour bodies.

[0264] In some embodiments, an antibody (e.g., a human antibody or a humanized antibody) activates the target via receptor clustering. In some embodiments, the agonist activity of the human antibody or humanized antibody results in an increase in the proportion of activated receptors on the target cells. In some embodiments, the proportion of activated receptors on the target cells increases by at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold compared to untreated target cells. In some embodiments, the proportion of activated receptors on the target cells increases by 2-fold compared to untreated target cells.

[0265] Antibody binding assays in agonist antibodies and other assays In one embodiment, the antibody is tested for its antigen-binding activity by known methods such as ELISA or Western blotting.

[0266] In some embodiments, the binding of a human antibody or humanized antibody to the target receptor described herein is tested by surface plasmon resonance (SPR) to measure the 1:1 affinity of the solution phase. In some embodiments, the binding of a human antibody or humanized antibody to the target receptor described herein is tested by other known methods, such as cell binding based on FACS, ELISA, or Western blotting. In some embodiments, the human antibody or humanized antibody in iAb format is similar to the corresponding WT IgG antibody in K D Values ​​and normalized R max (nR max ) has a value of nR max The value represents the theoretically normalized maximum response for analyte binding. In some embodiments, the iAb structure is the K of the human antibody or humanized antibody compared to the corresponding WT IgG antibody. D Value and nR max The values ​​do not change. In some embodiments, the iAb structure does not alter the avidity of the human antibody or humanized antibody to its target receptors compared to the corresponding WT IgG antibody. In some embodiments, the human antibody or humanized antibody adopting the iAb format has similar EC to the corresponding WT IgG antibody.50 and has a maximum signal value. In some embodiments, the antibody (e.g., human antibody or humanized antibody) has an EC50 value that is up to about 5% to about 20% different from the EC50 value of the corresponding WT IgG antibody. In some embodiments, the antibody (e.g., human antibody or humanized antibody) has an EC50 value that is up to about 20% different from the EC50 value of the corresponding WT IgG antibody. In some embodiments, the antibody (e.g., human antibody or humanized antibody) has a maximum signal value that is up to about 5% to about 20% different from the signal value of the corresponding WT IgG antibody. In some embodiments, the antibody (e.g., human antibody or humanized antibody) has a maximum signal value that is up to about 20% different from the signal value of the corresponding WT IgG antibody.

[0267] In another embodiment, a competitive assay may be used to identify antibodies that compete with a reference antibody for binding to a desired target. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or structural epitope) bound by the reference antibody. Detailed illustrative methods for mapping the epitopes to which antibodies bind are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0268] In an exemplary competition assay, an immobilized target is incubated in a solution containing a first labeled antibody that binds to the target (e.g., a reference antibody) and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to the target. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized target is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the target, excess unbound antibody is removed and the amount of labeling associated with the immobilized target is measured. If the amount of labeling associated with the immobilized target is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the target. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0269] Activity assay In one embodiment, an assay is provided for identifying a human antibody or humanized antibody that has biological activity. Biological activity may include, for example, binding to a target substrate. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0270] In certain embodiments, the antibodies described herein are tested for such biological activity.

[0271] D. Library and Library Generation In certain embodiments, this application further provides a library (for example, a library comprising the VH domain and VL domain of the antibodies described herein) and its use.

[0272] In some embodiments, a library is provided comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, the VH and VL binding to a target, and the VH domain comprises, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, 82aS, or c) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

[0273] In some embodiments, a library is provided comprising at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, where the VH and VL bind to a target, and the VH domain comprises, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

[0274] In some embodiments, the antibody is expressed or planned to be expressed on the surface of one or more phage or yeast cells.

[0275] The antibodies of this application can be isolated by screening a combinatorial library for antibodies having the desired activity(s). Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al.'s Nature Reviews 16:498-508 (2016). Various methods for creating phage display libraries and screening such libraries for antibodies having desired binding characteristics are known in the art. Such methods have been reviewed, for example, in Frenzel et al.'s mAbs 8:1177-1194 (2016), Bazan et al.'s Human Vaccines and Immunotherapeutics 8:1817-1828 (2012), and Zhao et al.'s Critical Reviews in Biotechnology 36:276-289 (2016), as well as in Hoogenboom et al.'s Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and Marks and Bradbury's Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0276] In certain phage display methods, the repertoire of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). The phages typically present antibody fragments as single-stranded Fv (scFv) fragments or Fab fragments. Libraries from immunization sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and autoantigens, as described by Griffiths et al., EMBO Journal 12:725-734 (1993). Furthermore, as described in Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992), naive libraries can also be synthetically created by cloning an unrearranged V gene segment derived from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving in vitro rearrangement. Patent publications describing human antibody phage libraries include, for example, U.S. Patents 5,750,373, 7,985,840, 7,785,903 and 8,679,490, and U.S. Patent Application Publications 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0277] Further examples of known methods in the art for screening combinatorial libraries for antibodies with one or more desired activities include ribosome and mRNA displays, and methods for antibody presentation and selection on bacterial, mammalian, insect, or yeast cells. Methods for yeast surface displays are outlined, for example, in Methods in Molecular Biology 503:135-56 (2012) by Scholler et al., Methods in Molecular Biology 1319:155-175 (2015) by Cherf et al., and in Methods in Molecular Biology 889:73-84 (2012) by Zhao et al. Methods for ribosome displays are described, for example, in Nucleic Acids Research 25:5132-5134 (1997) by He et al., and in PNAS 94:4937-4942 (1997) by Hanes et al.

[0278] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0279] Certain aspects of this disclosure relate to libraries for screening antibodies that adopt a constrained three-dimensional structure for agonist activity (e.g., an iAb structure). In some embodiments, the library comprises a plurality of polynucleotides, each encoding an antibody. In some embodiments, the library comprises a plurality of antibodies. In some embodiments, the antibodies in the library are presented on the cell surface and the phage surface. In some embodiments, the antibodies comprise an antibody heavy chain variable domain (VH) and / or an antibody light chain variable domain (VL). The libraries described herein are useful for screening and / or identifying one or more agonist antibodies.

[0280] In some embodiments, at least 1.6 × 10 9A S. cerevisiae yeast display scFv library containing unique sequence diversity is used to select binding factors that target proteins. In some embodiments, the S. cerevisiae yeast display scFv library is used to select binding factors for cell surface receptors. In some embodiments, the S. cerevisiae yeast display scFv library is used to select binding factors for IL-2RG and IL-2RB. In some embodiments, a plasmid encoding the scFv library is electroporated into yeast and grown in SDCAA medium at 30°C until the logarithmic phase. In some embodiments, yeast exhibiting 10-fold diversity of the library was grown in galactose-containing SGCAA medium at 20°C for 24–48 hours at OD600 of 1.0 before each round of selection. In some embodiments, for the first round of selection using magnetic SA beads, the biotinylated antigen was mixed with the beads, and then the yeast was added to enhance avidity. In some embodiments, for subsequent rounds of selection using tetrameric SA, the yeast was first stained with the biotinylated antigen, washed with PBS containing 1% BSA, and then stained with SA. In some embodiments, each round was checked for enrichment of the binding population by staining the yeast with antigen titration and analyzing the fluorescence.

[0281] In some embodiments of the libraries described herein, each antibody comprises an scFv including a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments of the libraries described herein, each antibody comprises a Fab including a heavy chain variable region (VH) and a light chain variable region (VL).

[0282] In some embodiments, the library of the Disclosure comprises one or more vectors (e.g., expression vectors and / or display vectors) containing one or more polynucleotides (e.g., synthetic polynucleotides) of the Disclosure. In some embodiments, each antibody is fused with all or part of a protein (e.g., a viral coat protein, a bacterial surface protein, a yeast surface protein, an insect cell surface protein, a mammalian cell surface protein) (i.e., to produce an agonist antibody). In some embodiments, the agonist antibody is presented on the surface of a particle or a host cell. In some embodiments, the library of the Disclosure comprises host cells and particles (e.g., phages) presenting the antibodies of the Disclosure.

[0283] For example, the present invention further provides a method for preparing a library by preparing and combining polynucleotide sequences (e.g., synthetic polynucleotides) of the library of this disclosure. The polynucleotides encoding the antibodies described herein can be cloned into any suitable vector for the expression of part or all of the polypeptide sequence. In some embodiments, the polynucleotide is cloned into a vector that enables the production of part or all of the polypeptide fused to all or part of a protein, such as a viral coat protein, bacterial surface protein, yeast surface protein, insect cell surface protein, or mammalian cell surface protein (i.e., the production of an agonist antibody), and presented on the surface of a particle or cell. Several types of vectors, such as phagemide vectors, are available and can be used to carry out the present disclosure. Phagemide vectors generally contain various components such as a promoter, a signal sequence, a phenotypic selection gene, an origin of replication site, and other necessary components known to those skilled in the art. In some embodiments, the polynucleotide encoding the polypeptide region can be cloned into a vector for expression in bacterial cells for bacterial display or yeast cells for yeast display. An exemplary vector is described in U.S. Patent Application Publication No. 20160145604. In some embodiments, the vector is a display vector comprising a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., the surface of a phage, bacterium, yeast, insect, or mammalian cell) from 5' to 3', a restriction site, a second polynucleotide encoding a surface peptide that can be displayed on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast outer wall protein (e.g., Aga2), a bacterial outer membrane protein, a cell surface tether domain or adapter, or a cleavage or derivative thereof. In some embodiments, the surface peptide is for phage display, yeast display, bacterial display, insect display, or mammalian display, or for shuttle display between them.In some embodiments, upon expression, the amino acid sequence and surface peptide are presented on the surface as an agonist antibody. In some embodiments, the vector further comprises a fusion tag 5' for a first restriction site or a fusion tag 3' for a second restriction site.

[0284] Certain aspects of this disclosure relate to a population of cells containing the vectors described herein. Antibodies encoded by polynucleotides produced by the techniques described herein or by any other preferred techniques can be expressed and screened to identify antibodies having a desired structure and / or activity. Protein expression can be carried out, for example, using cell-free extracts (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cells are bacterial cells, yeast cells, insect cells, or mammalian cells (e.g., Chinese hamster ovary (CHO) cells). Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and are described in the references cited herein. The expression of proteins (e.g., from the libraries of this disclosure) in these cell types, and the screening for antibodies of interest, are described in more detail below.

[0285] Alternatively, polynucleotides can be expressed in E. coli expression systems, such as those described in Pluckthun and Skerra (Meth. Enzymol., 1989, 178:476; Biotechnology, 1991, 9:273). Mutant proteins can be expressed in culture medium and / or in the bacterial cytoplasm for secretion, as described in Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, the polypeptide is bound to the 3' end of a sequence encoding a signal sequence, such as ompA, phoA, or pelB signal sequences (Lei et al., J. Bacteriol., 1987, 169:4379). These gene fusions are assembled in a dicistronic construct so that they can be expressed from a single vector, secreted into the periplasmic space of E. coli, where they can be refolded and recovered in an active form (Skerra et al., Biotechnology, 1991, 9:273).

[0286] In other embodiments, the polypeptide sequences of the present disclosure are expressed on the membrane surface of prokaryotes, such as Escherichia coli, using secretory signaling and lipid-forming moieties, as described, for example, in U.S. Patent Publication No. 20040072740, U.S. Patent Publication No. 20030100023, and U.S. Patent Publication No. 20030036092.

[0287] Alternatively, the polypeptide sequences of this disclosure can be expressed and screened by, for example, immobilized periplasmic expression (APEx 2-hybrid surface display) as described in Jeong et al., PNAS, 2007, 104:8247, or by other immobilization methods as described in, for example, Mazor et al., Nature Biotechnology, 2007, 25:563.

[0288] Mammalian cells, such as myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, are also used for the expression of polypeptides of this disclosure. Polypeptides expressed in mammalian cells (e.g., agonist antibodies) may be designed to be secreted into the culture medium or expressed on the cell surface.

[0289] In other embodiments, polypeptides or antibodies (e.g., agonist antibodies) can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103:9637). In some embodiments, as illustrated above and below, polypeptides or antibodies can be fused to all or part of a viral coat protein (i.e., agonist antibody production) and selected after the production of some or all polypeptides or antibodies to be presented on the surface of particles or cells, for example, using phage display.

[0290] In some embodiments, the application provides a polynucleotide library encoding one or more antibodies or antibody libraries, where each antibody in the library includes an antigen-binding domain. In some embodiments, the antigen-binding domain includes an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the antigen-binding domain includes both an antibody light chain variable region and an antibody heavy chain variable region. In some embodiments, the antigen-binding domain includes an antibody heavy chain variable region but does not include an antibody light chain variable region. In some embodiments, the antigen-binding domain of the disclosure includes an antibody light chain variable region and / or an antibody heavy chain variable region having specificity to any target of interest, for example, any of the targets described herein.

[0291] In some embodiments, the antibody comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a kappa light chain or a lambda light chain. The antibody heavy chain may be any class such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is an IgG class, e.g., IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein can be converted from one class or subclass to another using methods known in the art.

[0292] In some embodiments, the antibody in the library includes the heavy and light chains of a full-length antibody. In some embodiments, the antibody in the library includes antibody fragments (e.g., Fab fragments, F(ab')2 fragments, etc.). In some embodiments, at least one antibody or antibody fragment in the library binds to a target with an equilibrium dissociation constant (Kd) of about 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, or 10⁻¹¹ M or less.

[0293] In some embodiments, the library contains at least two antibodies. In some embodiments, the library contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 100, at least 1000, at least ten 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, at least 10 8 pieces, at least 10 9 pieces, at least 10 10 pieces, at least 10 11 pieces, at least 10 12 pieces, at least 10 13 pieces, at least 10 14 pieces, at least 10 15 pieces, at least 10 16 pieces, at least 10 17 pieces, at least 10 18 pieces, at least 1019 one or at least 10 20 antibodies. In some embodiments, the library comprises about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 100, about 1000, about 10 4 , about 10 5 about 10 6 about 10 7 about 10 8 about 10 9 about 10 10 about 10 11 about 10 12 about 10 13 about 10 14 about 10 15 about 10 16 about 10 17 about 10 18 about 10 19 or about 10 20 antibodies.

[0294] In some embodiments, the antibodies in the library comprise at least two unique antibody heavy chain variable regions. In some embodiments, the antibodies in the library comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least one hundred, at least one thousand, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , at least 10 16 , at least 10 17 , at least 10 18 , at least 10 19 or at least 1020 It contains one unique antibody heavy chain variable region. In some embodiments, the number of antibodies in the library is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, and 10 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, at least 10 8 pieces, at least 10 9 pieces, at least 10 10 pieces, at least 10 11 pieces, at least 10 12 pieces, at least 10 13 pieces, at least 10 14 pieces, at least 10 15 pieces, at least 10 16 pieces, at least 10 17 pieces, at least 10 18 pieces, at least 10 19 pieces or at least 10 20 It contains a unique antibody heavy chain variable region.

[0295] In some embodiments, the antibodies in the library contain at least two unique antibody heavy chain variable regions. In some embodiments, the antibodies in the library contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 100, at least 1000, and at least ten 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, at least 10 8 pieces, at least 10 9 pieces, at least 10 10 pieces, at least 10 11 pieces, at least 10 12 pieces, at least 10 13 pieces, at least 10 14 pieces, at least 10 15 pieces, at least 10 16 pieces, at least 1017 pieces, at least 10 18 pieces, at least 10 19 pieces or at least 10 20 The antibody in the library contains approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, or 1020 unique antibody heavy chain variable regions.

[0296] In some embodiments, the antibodies in the library contain at least two unique antibody heavy chain variable regions. In some embodiments, the antibodies in the library contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 100, at least 1000, and at least ten 4 pieces, at least 10 5 pieces, at least 10 6 pieces, at least 10 7 pieces, at least 10 8 pieces, at least 10 9 pieces, at least 10 10 pieces, at least 10 11 pieces, at least 10 12 pieces, at least 10 13 pieces, at least 10 14 pieces, at least 10 15 pieces, at least 10 16 pieces, at least 10 17 pieces, at least 10 18 pieces, at least 10 19 pieces or at least 10 20 It contains one unique antibody heavy chain variable region. In some embodiments, the number of antibodies in the library is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, and 10 4 pieces, at least 105 pieces, at least 10 6 pieces, at least 10 7 pieces, at least 10 8 pieces, at least 10 9 pieces, at least 10 10 pieces, at least 10 11 pieces, at least 10 12 pieces, at least 10 13 pieces, at least 10 14 pieces, at least 10 15 pieces, at least 10 16 pieces, at least 10 17 pieces, at least 10 18 pieces, at least 10 19 pieces or at least 10 20 It contains a unique antibody heavy chain variable region.

[0297] Method for screening F. agonist antibodies In some embodiments, methods for screening the polynucleotide and polypeptide libraries described herein for a desired antibody (e.g., an agonist antibody targeting a specific antigen) are provided herein. Screening for antibodies derived from any of the libraries described herein can be carried out by any suitable method known in the art, including, for example, the use of ELISA, surface plasmon resonance, affinity chromatography, and activity assays.

[0298] In some embodiments, a method is provided for screening agonist antibodies, comprising contacting an antibody from any of the previously examined libraries with a target or cells expressing the target. In some embodiments, the method further includes evaluating the agonist activity of the antibody.

[0299] In some embodiments, methods are provided herein for using a library of polynucleotides described herein for agonist antibodies that bind to a target, comprising: a) contacting the expressed antibody of the library with the target to determine a first binding affinity; and b) isolating one or more expression products that bind to the target. In some embodiments, the method further comprises: c) determining whether one or more isolated expression products function as agonists.

[0300] In some embodiments, a method is provided herein for using a library containing antibodies described herein for agonist antibodies that bind to a target, comprising: a) contacting the expressed antibody in the library with the target to determine a first binding affinity; and b) isolating one or more expression products that bind to the target. In some embodiments, the method further comprises: c) determining whether one or more isolated expression products function as agonists.

[0301] In some embodiments that may be combined with any of the preceding embodiments, the target is a mammalian cell surface receptor protein. In some embodiments, the mammalian cell surface receptor protein is a tumor necrosis factor receptor (TNFR) superfamily member or a G protein-coupled receptor superfamily member. In some embodiments that may be combined with any of the preceding embodiments, the target is selected from the group consisting of OX40, GITR, CD27, CD40, CD137, DR5, 4-1BB, and Tie2. In some embodiments that may be combined with any of the preceding embodiments, the target is a human protein, a non-human primate protein, or a rodent protein.

[0302] G. Pharmaceutical Compositions In further embodiments, pharmaceutical compositions are provided, for example, for use in any of the following therapeutic methods, comprising one of the antibodies provided herein. In one embodiment, the pharmaceutical composition comprises one of the agonist antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the agonist antibodies provided herein and at least one additional therapeutic agent, for example, those described below.

[0303] The agonist antibody pharmaceutical compositions (formulations) described herein can be prepared by combining the agonist antibody with a pharmaceutically acceptable carrier or excipient known to those skilled in the art. See, for example, International Publication No. 2019 / 224842, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1st Ed., Woodhead Publishing (2015), §4 and Falconer RJ, Biotechnology Advances (2019), 37, 107412. Exemplary agonist antibody pharmaceutical compositions described herein include lyophilized, aqueous, and frozen formulations.

[0304] Pharmaceutically acceptable carriers are typically non-toxic to the recipient at the doses and concentrations used, and are not limited to buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); and low molecular weight molecules. Examples include polypeptides and proteins in small quantities (less than approximately 10 residues), hydrophilic polymers such as serum albumin, gelatin or immunoglobulin, polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG).

[0305] The pharmaceutical compositions described herein may also include multiple active ingredients required for the specific indication being treated, preferably those having complementary activities that do not adversely affect one another. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0306] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0307] H. Treatment methods and routes of administration Any of the antibodies provided herein can be used in therapeutic methods.

[0308] In one embodiment, an antibody (e.g., an agonist antibody) for use as a pharmaceutical is provided. In a further embodiment, an antibody (e.g., an agonist antibody) for use in the treatment of a disease or symptom is provided. In a particular embodiment, an antibody (e.g., an agonist antibody) for use in a treatment method is provided. In a particular embodiment, the application provides an antibody (e.g., an agonist antibody) for use in a method of treating an individual having a disease or symptom (e.g., a disease or symptom involving an antigen), which includes administering an effective amount of the antibody to the individual. In one such embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual, as described below, for example. In a further embodiment, the application provides an antibody for use in promoting receptor clustering and activation of downstream signaling pathways of cell surface receptors, for example. In a particular embodiment, the application provides an antibody for use in a method of promoting the iAb conformation in an antibody, thereby promoting the activation of cell surface receptors in an individual. The "individual" in any of the above embodiments is preferably a human being.

[0309] In further embodiments, the present application provides the use of antibodies (e.g., agonist antibodies) in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of a disease or condition accompanied by or caused by abnormal cell surface receptor signaling. In further embodiments, the pharmaceutical is for use in a method of treating a disease, comprising administering an effective amount of the pharmaceutical to an individual having the disease. In such one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., one described below, to the individual. In further embodiments, the pharmaceutical is for use in a method of promoting receptor clustering of cell surface receptors and activation of downstream signaling pathways, comprising administering an effective amount of the pharmaceutical to an individual. The “individual” in any of the above embodiments may be a human.

[0310] In a further embodiment, the present application provides a method for treating a disease or condition (e.g., cancer or tumor). In one embodiment, the method comprises administering an effective amount of the antibody described herein (e.g., an agonist antibody) to an individual having such a disease or condition.

[0311] In some embodiments, a method is provided for treating a disease or condition (e.g., cancer or tumor) comprising administering two or more different antibodies (e.g., two Fabs, e.g., two scFvs) in which the two different antibodies bind to two different antigens. In some embodiments, the two different antigens are two subunits of a molecule that requires or involves clustering or multimerization of two subunits for activation of downstream signaling. In some embodiments, the two different antigens are two molecules involved in a complex (e.g., a cell surface complex, e.g., a T cell receptor complex) or a part thereof, the formation of which confers activation of a signaling pathway. In some embodiments, the two different antigens are two members of TNSFSR.

[0312] In some embodiments, a method is provided for treating a disease or condition (e.g., cancer or tumor) comprising administering a multispecific antibody (e.g., a bispecific antibody) in which the antibody binds to two different antigens. In some embodiments, the two different antigens are two subunits of a molecule that requires or involves clustering or multimerization of two subunits for activation of downstream signaling. In some embodiments, the two different antigens are two molecules involved in a complex (e.g., a complex on a cell surface, e.g., a T cell receptor complex) whose formation confers activation of a signaling pathway. In some embodiments, the two different antigens are two members of TNSFSR.

[0313] In some embodiments, the method further includes administering an effective amount to the individual at least one additional therapeutic agent, such as those described below.

[0314] An "individual" in any of the above-described manner may be a human being.

[0315] In a further embodiment, the present application provides a method for promoting the agonist activity of a human antibody or humanized antibody against an antigen and / or promoting receptor clustering and activation of downstream signaling pathways in the cells of an individual. In one embodiment, the method comprises administering an effective amount of antibody to an individual. In one embodiment, “individual” is a human.

[0316] In further embodiments, the application provides a pharmaceutical composition comprising one of the antibodies provided herein for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises one of the antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the antibodies provided herein and at least one additional therapeutic agent, for example, one of those described below.

[0317] The antibody of this application may be administered alone or used in combination therapy. For example, combination therapy may include administering the antibody of this application and administering at least one additional therapeutic agent (e.g., additional therapeutic agents 1, 2, 3, 4, 5, or 6). In certain embodiments, combination therapy may include administering the antibody of this application and administering at least one additional therapeutic agent.

[0318] Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical composition) and separate administration, in which case the administration of the antibody of this application may be performed simultaneously with and / or prior to the administration of additional therapeutic agents or drugs. In one embodiment, the administration of the antibody and the administration of additional therapeutic agents are performed within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. In one embodiment, the antibody and the additional therapeutic agent are administered to the patient on day one of treatment. The antibody of this application may also be used in combination with radiotherapy.

[0319] The antibodies (and any additional therapeutic agents) of this application may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired for topical treatment, intralesional administration. Parenteral administrations include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., intravenous or subcutaneous injection, depending in part whether the administration is short-term or long-term. Various dosing schedules, including but not limited to single doses, multiple doses at various time points, bolus doses, and pulse infusions, are contemplated herein.

[0320] The antibodies of this application will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The antibodies will be formulated, optionally but not necessarily, with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs will depend on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route that is empirically / clinically deemed appropriate.

[0321] For the prevention or treatment of disease, the appropriate dosage of the antibody of this application (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the present invention is preferably administered to the patient in a single dose or over a series of treatments. In repeated administrations over several days or more, treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. However, other drug regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

[0322] IV.Manufactured products In another aspect of the present invention, a product is provided comprising a material useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label or accompanying documentation on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container holds the composition to be used alone or in combination with another composition effective for treating, preventing and / or diagnosing a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is the antibody of the present invention. The label or accompanying documentation indicates that the composition is used to treat a selected condition. Furthermore, the product may comprise (a) a first container containing a composition comprising the antibody of the present invention, and (b) a second container containing a composition comprising a further cytotoxic agent or other therapeutic agent. The product of this aspect of the present application may further include accompanying documentation indicating that the composition may be used to treat a particular condition. Alternatively, or in addition thereto, the product may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The product may further comprise other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, and syringes. [Examples]

[0323] The following examples are included for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0324] Example 1: Structural determinants of the i-shaped antibody (iAb) described above Recent studies have characterized a subset of broad-spectrum neutralizing HIV antibodies isolated from infected humans and rhesus monkeys that share a unique linear i-shaped three-dimensional structure distinct from the conventional Y-shape. These antibodies possess shortened paratope distances resulting from intramolecular association between Fab domains. Physiologically, this proximity simultaneously increases the avidity of interaction with viral surface glycans, generating additional paratopes at the Fab-Fab interface.

[0325] Thorough studies of these i-shaped antibodies (iAbs) have revealed distinct, independently evolved mechanisms that determine their unique three-dimensional structure. A single-human antibody isolated from an HIV patient, called 2G12, achieves its iAb structure through the exchange of heavy chain variable (VH) domains between Fabs (Figure 1A, left) (Calarese, DA, et al., Science, 2003.300, 2065-2071 and Trkola, A., et al., J Virol., 1996.70, 1100-1108). In SHIV-infected rhesus monkeys, two antibody lines, called DH851 and DH898, were identified, possessing affinity-driven intramolecular Fab-Fab homotype interactions between VH domain β chains A, B, D, and E (Figure 1A, center and right) (Williams, WB, et al., Cell, 2021.184, 2955-2972.e25). Both of these mechanisms involve non-covalent Fab-Fab associations mediated through different but topologically similar VH interfaces (Figure 1).

[0326] Consistent with structural studies, the specific residues contributing to domain exchange are exclusively located in the VH domain and have little to no effect on antigen binding. The iAb assumed to be most important for inducing the domain-exchanged iAb three-dimensional structure dx A set of VH residues called iAb was selected from 2G12 (Figure 1B). Similarly, a set of residues was designed based on structural analysis and predicted to mediate Fab-Fab affinity interactions in the DH851 and DH898 lines, respectively, and iAb was selected. aff1 and iAb aff2This was named as such (Figure 1B). Some of the amino acids found in these residues are significantly under-presented in known antibody sequences (Figure 1B), suggesting that they are not encoded in the germline and arose into the repertoire from somatic mutations.

[0327] Example 2: The manipulated residue graft induces the iAb three-dimensional structure. Since antibodies share a high degree of sequence and structural homology, it was hypothesized that mutating the sites identified above within a given antibody of interest might be sufficient to induce iAb formation. To test this hypothesis, each putative iAb induction residue set (iAb dx iAb aff1 , and iAb aff2 The ) was transplanted into a panel of 10 different anti-OX40 antibody clones possessing diverse sequences, germline precursors, epitopes, and affinities (Figures 2A-2C) (Leonard, B., et al., Proc National Acad Sci, 2022.119, e2201562119). OX40 is a therapeutically relevant TNFRSF member, and receptor clustering is known to play a role in its activation. For comparison, all 10 anti-OX40 antibodies were also constructed as contrast bodies, a recently described structure-constrained format in which the heavy and light chains of Fab are fused to the N-terminus and C-terminus of the Fc domain, respectively, using a gene linker (Georges, GJ, et al., Comput Struct Biotechnology J., 2020.18, 1210-1220).

[0328] The effect of transplanted iAb residues on the antibody three-dimensional structure was evaluated by negative staining electron microscopy. The 2D classes from representative anti-OX40 antibody clones possessing each residue set were compared to those from wild-type (WT) IgG and representative contrast body clones (Figure 2D). Each iAb clearly showed two parallel interacting Fabs, resulting in 2D classes similar to those previously reported for 2G12 and rhesus monkey broad-spectrum neutralizing HIV antibody (Wu, Y. et al., Cell Reports, 2013.5, 1443-1455). dx The clone images showed a structural distribution in which 29% of the particles were i-shaped antibodies and the remaining 71% were standard Y-shaped antibodies. aff1 and iAb aff2 For both residue sets, approximately 64% of the particles adopted the iAb three-dimensional structure, while the remaining particles showed the standard Y-shaped IgG three-dimensional structure. Furthermore, iAb aff2 The images revealed an iAb dimer structure in which Fab molecules associate with their tips facing each other (Figure 2D, rightmost image). This result is consistent with the results of size exclusion chromatography (Figures 2E and 2F). Concentration-dependent studies of the monomer:dimer ratio support the non-covalent nature of the affinity-based iAb interface, suggesting that the iAb interaction is in dynamic equilibrium (Figure 2G). As expected, none of the wild-type IgG images were found to have a unique iAb shape, and the contrast bodies adopted a barrel-shaped structure with Fab fixed next to the Fc molecules (Figure 2D).

[0329] Example 3: iAb reformatting enables endogenous OX40 agonist activity. Like many TNFRSF members, standard antibodies against OX40 generally do not intrinsically promote signaling, but rather rely on exogenous crosslinking to induce Fc binding to the cell surface Fc gamma receptor (FcgR), crosslinking using secondary antibodies, or receptor clustering via coatings on beads or plates. To determine whether iAb formation can enhance the activity of transplanted anti-OX40 antibodies, their activity was tested in cell-based assays using Jurkat cell lines engineered to express OX40 and nuclear factor κB (NF-κB) luciferase reporters. Corresponding WT IgG and contour bodies were tested as control groups. Antibody clones expressed as single-specific WT IgG or contour body formats showed little to no activity (Figure 2H). Conversely, OX40 agonism activity was observed in all iAb formats with affinity interfaces, iAb aff1 and iAb aff2 Observed over time, it showed the strongest and most consistent acquisition of function (Figure 2H and Figures 2I-2J). iAb dx Only 4 / 9 of the transplanted clones exhibited endogenous agonism activity (clone 2A3 did not express it), but iAb aff1 and iAb aff2 All clones transplanted showed some improvement in activity compared to the WT IgG control. iAb dx Various activity and iAb of the panel aff2 Considering the dimeric impurities observed in the sample (Figures 2D-2G), iAb aff1 The graft was used for the remainder of the experiment.

[0330] To determine whether the enhanced activity of the manipulated iAb was due to an effect on antigen binding, surface plasmon resonance (SPR) was performed to measure the 1:1 affinity of the solution phase. WT IgG and iAb aff1 The transplanted clones exhibited similar KD values ​​and normalized R values ​​across the entire anti-OX40 panel tested. max (nR max ) has both values, which means iAb aff1The residue set does not affect affinity or the ability of both antibody Fabs to bind to OX40 simultaneously (Figure 2K). In addition to this solution-based analysis, FACS-based cell binding experiments were also performed to investigate whether avidity is influenced by the iAb conformation during antigen recognition on the cell surface. WT IgG and iAbs of each anti-OX40 clone exhibited similar cell binding in terms of both EC50 and maximum signal (Figures 2L and 2M). Taken together, these data indicate that the endogenous agonism activity in iAb format is not a result of altered affinity or cell surface avidity.

[0331] Example 4: The iAb interface can activate OX40 agonism as an intermolecular interaction. To further characterize the iAb interface, experiments were conducted to investigate the strength of homotype Fab interactions. Since binding measurements can be confounded by intramolecular Fab interactions in the case of divalent IgG, iAb aff1 The transplanted 3C8 anti-OX40 clone was expressed as monomeric Fab, and we tested whether the iAb interface could enable agonist activity in monovalent form via intermolecular interactions (Figure 4A). Although the EC50 was nearly 100-fold lower compared to iAb IgG, the iAb interface still enabled endogenous agonist activity as monomeric Fab. Surprisingly, the maximum activity was nearly twofold higher for iAb Fab compared to iAb IgG. Interestingly, this result led to the creation of an F(ab')2 version of the 3C8 antibody, which also showed a similarly elevated maximum activity level with an EC50 comparable to iAb IgG (Figure 4A). These results suggest that the presence of the Fc region in full-length IgG may sterically limit iAb activity. These results indicate that the iAb structure can enable endogenous agonism via both intramolecular and intermolecular Fab interactions.

[0332] Example 5: iAb enables broad endogenous TNFRSF agonism. After elucidating the ability of the iAb structure to intrinsically agonize OX40, further experiments were conducted to investigate whether the same constrained structure could be generalized to enable agonism against other TNFRSF members. Four panels of publicly available and autologous antibodies with diverse sequences, germline precursors, and affinities against CD40, 4-1BB, DR4, and DR5 were used, along with WT IgG and iAb. aff1 We created the product in both formats. We prepared a constrained h2B isoform as a comparison with the CD40-promoting mutant (C131S) IgG2.

[0333] For the majority of clones across all targets, iAb reformatting showed enhanced agonist activity compared to the corresponding WT IgG format, while the WT IgG format generally showed low agonism or no agonism (Figures 5A–5H). For CD40, different formats (WT IgG, IgG2 C131S, iAb) were used. aff1 There was no clear correlation between the activity of a given antibody in ) and its previously reported epitope (Smith, KE et al., Expert Opin Biol Th, 2021.21, 1635-1646). However, the strongest agonistic activity was observed in iAbs lavagalimab, dacetuzumab, gyrolarimab, and sotigolimab. aff1The format was observed (Figure 5A). The most consistent results were observed for 4-1BB agonism, with 5 / 6 iAb clones enabling at least twice as much endogenous activity as the untreated control, in contrast to the corresponding WT IgG comparator, which was all inactive without crosslinking (Figure 5B). This result may suggest more relaxed epitope and / or Fab binding orientation constraints governing the agonism of this receptor. DR4 and DR5 agonism proved to be more variable, with only 3 / 5 anti-DR4 iAb clones showing twice as much activity as the WT IgG control, and 5 / 12 anti-DR5 iAbs demonstrating the ability to kill at least 25% of cells (Figures 5C and 5D). Overall, these results highlight the broad applicability of the constrained iAb format as a modification tool for generating effective endogenous agonists against TNFRSF members.

[0334] Example 6: Comparison of divalent iAb and high-avidity hexamer IgG Activation of many TNFRSF members has been shown to benefit from higher-order receptor clustering mediated by exogenous crosslinking. For this reason, effective antibody-based agonists have been generated by modified approaches to increase avidity, such as IgG hexamerization. Considering the enhanced activity of iAbs against WT IgG when there is no discernible difference in binding properties, we compared the mechanism of action based on conformation with that of avidity. For this purpose, we introduced a mutation known to induce hexamerization and activate endogenous OX40 agonism into the Fc of a 3C8 antibody (Figure 6A). In an OX40 NF-κB reporter assay, the 3C8 iAb had a similar EC50 with a slightly lower maximum signal compared to the hexameric IgG format (Figure 6B), which is surprising considering that the 3C8 iAb has only 2 valencies compared to the hexameric 3C8 with 12 valencies. In addition to activity, the effect of these formats on receptor-mediated internalization was evaluated by labeling antibodies with pH-sensitive dyes that increase fluorescence under low pH conditions, such as dyes present in acidified lysosomes.

[0335] Fluorescence detection and quantification using flow cytometry revealed that hexameric IgG promotes substantially increased internalization compared to WT IgG, along with its high endogenous activity (Figure 6C). Interestingly, iAb similarly mediates potent endogenous OX40 agonism, albeit slightly less than hexameric IgG, but with a slightly greater associated level of receptor downregulation than inactive WT IgG. These results offer the prospect that different modification approaches to endogenous agonism may result in different activity-versus-internalization profiles, and this consideration is clearly important for the design of biological therapies.

[0336] The mechanistic effects of 3C8 iAb and hexamers were further investigated using total internal reflection fluorescence (TIRF) microscopy to track the clustering patterns and single-particle mobility of fluorescently tagged OX40 after treatment with antibody formats. Maximum projection images of all acquisitions over 12.5 seconds under each treatment condition showed a broadly diffused distribution of OX40 for both untreated and WT 3C8 treated samples, although iAb and hexamer-treated samples had receptor accumulation hotspots with hexamers, exhibiting a more punctate pattern (Figure 6D). Insertion windows with molecular orbitals show how each molecule moves and provide further clarity regarding the confinement of individual molecules. Mean square displacement analysis of the trajectories further highlighted the differences between formats, clearly demonstrating that hexamers restrict the movement of OX40 compared to other formats (Figure 6E). The iAb structure still allowed for more free 2D diffusive movement of OX40, similar to untreated cells and WT 3C8 (Figure 6E), but a more rigorous analysis of individual track intensity values ​​showed a shift in iAb distribution similar to that of the hexamer (Figure 6F). Taken together, these data suggest that both iAbs and hexamers tend to tightly ligate two or more receptors, explaining the increased activity and internalization mediated by these two formats. However, since hexamers induce higher-order, larger clusters of OX40 with significantly reduced intramembrane mobility, there may be mechanistic differences that could explain the greater activity and more pronounced internalization caused by treatment in this format.

[0337] Example 7: Discovery of antibodies against IL-2RG and IL-2RB for application of iAb modification to cytokine mimetic. Given the success in applying iAbs to monospecific agonists across multiple TNFRSF targets, further experiments were conducted to investigate whether the platform could be applied to bispecific antibody agonists of cytokine receptors. The IL-2 pathway was selected for evaluation due to the significant interest in ligand-mimicking agonists in this field. The IL-2 cytokine forms a high-affinity quaternary complex with three receptors: IL-2RA, IL-2RB, and IL-2RG. IL-2RB and IL-2RG are responsible for downstream signaling during heterodimerization, while IL-2RA stabilizes the complex and enhances the potency of IL-2.

[0338] In the first step, antibody-binding factors for IL-2RG and IL-2RB were discovered and characterized from an autologous naive scFv library presented on yeast (Figure 7A). Binding factors were selected using magnetic-based and fluorescence-based sorting techniques under progressively stricter conditions in terms of both antigenic titer and concentration (Figure 7B). After several selections and subsequent sequence analysis, 34 unique anti-IL-2RG and 61 unique anti-IL-2RB clones were selected for IgG reformatting and recombination. These clones were initially characterized by their ability to block cell surface binding, SPR, and IL-2 signaling (Figures 7C-7F). Based on these analyses, eight anti-IL-2RB and six anti-IL-2RG antibodies were further characterized by epitope mapping and selected for bispecific assembly in WT IgG, contrast body, and iAb formats.

[0339] Example 8: Epitope mapping of anti-IL-2RG and anti-IL-2RB reads While several techniques exist for identifying antibody epitopes, we sought finer-grained structural information beyond epitope binning by cross-blocking. Using mutation scanning technology, alanine was introduced into each residue of the his-tagged extracellular domain (ECD) of IL-2RG and IL-2RB, yielding receptor variants 203 and 206, respectively. These variants were then arrayed on an SPR chip, and each read clone was injected onto the chip to identify which alanine variant disrupted binding. Mapping these results to the three-dimensional crystal structure of the receptor (PDB ID: 2ERJ) (Stauber, DJ, et al., Proc National Acad Sci, 2006.103, 2788-2793) revealed the epitopes of each clone through hit clustering.

[0340] Regarding the IL-2 binding site at the interface between IL-2RG and IL-2RB (Figure 8A, black box), the epitopes of anti-IL-2RG clones were dispersed throughout the receptor, and there were some commonalities among the clones (Figure 8A, blue box). For example, clones G02, G25, and G28 overlapped with the IL-2 binding site, but G12 and G23 bound to the proximal C-terminus of the membrane, and G33 was the only clone to bind to the distal N-terminus. Conversely, all anti-IL-2RB clones had a similar binding site proximal to the IL-2 binding site (Figure 8A, red box). Clone B30 differed slightly in that its binding site almost completely overlapped with the IL-2 binding site, which is consistent with its potent IL-2 blocking ability (Figures 8A and 7D-7E).

[0341] Example 9: The constraint format enables IL-2 path agonism. Six anti-IL-2RG and eight anti-IL-2RB lead clones were reformatted in a matrix-based manner as bispecific WT IgG, contour body, and iAb, where all IL-2RG clones were paired with all IL-2RB clones, yielding 48 bispecific combinations for each format. Each bispecific antibody was tested for IL-2 pathway agonism using a STAT5 luciferase reporter and Jurkat cell lines engineered with overexpression of both IL-2RG and IL-2RB. No activity was observed against the WT IgG combinations, but several clone combinations were active against both the contour body and iAb-restricted formats (Figure 9A). Overall, the contour body format had a higher hit rate in 12 / 48 clones, increasing more than twofold compared to the untreated control. There was a clone and epitope activity dependency, with active anti-IL-2RG clones targeting epitopes near the IL-2 binding site (Figure 8A). It remains unclear whether this IL-2RG region is intrinsically advantageous for antibody-mimicking activity, or whether the increased activity of clones binding to this region was biased due to a lack of epitope diversity within anti-IL-2RB clones. In the iAb panel, using the same cutoff, only 3 / 47 clones were active (G23 / B65 was not expressed), and despite the epitope similarities between the IL-2RB clones mentioned above, all three were paired with a single IL-2RB clone, B10. Interestingly, there was no overlap between the active contrast bodies and iAb clone pairings. In summary, these results suggest that iAb formation is most pronounced for the B10 clone and / or that distinctly different conformations accessed in two formats are related to different Fab arm pairings in a non-epitope-based manner.

[0342] Two lead contour bodies (B09 / G02 and B09 / G28) and two lead iAbs (B10 / G25 and B10 / G28) were selected and further characterized. First, the titer of each lead molecule was measured in a Jurkat reporter assay, and its activity was compared to that of both WT IgG control molecules and IL-2 cytokines with the same clonal combination (Figure 9B). Each constraint format showed increased activity compared to its respective WT IgG control, and the activity of the lead-engineered formats was equivalent to that of IL-2. To determine whether the observed increase in agonist activity was simply due to an increased ability to simultaneously bind to both IL-2RG and IL-2RB receptors, we subsequently performed a cross-linking enzyme-linked immunosorbent assay (ELISA). For each clonal combination, the constraint format showed a decreased ability to simultaneously bind to both receptors, suggesting that the enhancement of activity in these formats was due to receptor proximity rather than increased binding (Figure 9C).

[0343] Example 10: Restrictive antibody agonists mimic the IL-2 proliferation activity and transcriptional reprogramming of primary cells. To more rigorously examine the activity of the top bispecific agonist antibodies and compare them to IL-2 cytokines in more physiologically relevant cell types, titrations of the same read clone combinations as above were performed in both primary NK and CD8 T cells endogenously expressing the IL-2 receptor. As seen with the Jurkat reporter, all read formats showed higher activity than controls in both primary cell types (Figure 10A). In contrast to both Jurkat reporter cells and NK cells, recombinant IL-2 activity was significantly potent than that of mimics on CD8 T cells, which is likely due to the upregulation of IL-2RA on the surface of CD8 T cells upon CD3 / CD28 stimulation performed prior to IL-2 treatment.

[0344] To clarify the effect of mimicry formats on transcriptional profiles, RNA sequencing was performed on CD8 T cells treated with read IL-2 pathway agonists and their respective controls (Figure 10B). Hierarchical clustering of normalized differentially expressed genes revealed two major treatment groups: one group containing read-restricted formats closely associated with IL-2, and the other containing WT IgG controls. Heatmaps of the 40 most downregulated and upregulated genes by IL-2 showed strong overlap with the restriction formats but not with the WT IgG controls. In summary, these data indicate that restrictive antibody formats can be used to enable agonism of otherwise inactive bispecific antibody combinations in a manner that mimics native ligands at both the proliferation and transcriptional levels.

[0345] Method of the above embodiment Molecular cloning

[0346] Antibody clones for each target were generated from various sources. Anti-OX40, anti-4-1BB, anti-DR4, and anti-DR5 antibodies were discovered in-vitro through mouse immunization campaigns. The sequences of all anti-CD40 antibodies used in this experiment were obtained from publicly available databases and patent literature. The anti-IL-2RB and anti-IL-2RG antibodies used in this experiment were discovered using yeast display, as described below.

[0347] Gene fragments encoding all antibody constructs were synthesized as gBlocks or eBlocks (IDTs) and cloned into pRK mammalian expression vectors using Gibson assemblies (NEB, catalog number E2611L). The pRK vectors contained a cytomegalovirus (CMV) enhancer and promoter to regulate gene expression, an N-terminal secretory signal (MGWSCIILFLVATATGVHS, SEQ ID NO: 1), a C-terminal Simianvirus 40 (SV40) polyA sequence, and an ampicillin resistance gene for bacterial selection. Unless otherwise noted, all Fc regions were human IgG1 with the effector-less mutant L234A / L234A / P329G (EU numbering). Contose bodies were constructed by fusing the heavy-chain Fab domain and light-chain Fab domain to the N-terminus and C-terminus of the Fc domain via the flexible (G4S)2 linker (SEQ ID NO: 2), respectively (14).

[0348] To construct the hexamer 3C8, the heavy chain variable region was cloned into the hIgG1 backbone containing the E345R / E430G / S440Y(RGY) mutation (13, 17, 26). For anti-CD40 antibodies, the C131S mutation was used to promote the formation of the IgG2 h2B isoform (EU numbering corresponds to the C127S mutation of White and his colleague) (16). The Fab construct consisted of a cleavable heavy chain (1-225, EU numbering) and a light chain paired with a C-terminal TEV protease-cleavable Flag tag. For all bispecific antibodies, a knob-in-hole mutation was introduced into the Fc to enable heterodimerization (48).

[0349] OX40 ECD (L29-D170) was cloned into a pRK mammalian expression vector with a TEV protease-cleavable N-terminal His tag. IL-2RB ECD (A27-T240) and IL-2RG ECD (L23-A262) were cloned into pRK mammalian expression vectors with a C-terminal His tag.

[0350] iAb modification

[0351] In this experiment, the iAb structure was induced to the target antibody by transplanting a specific set of mutations (Figure 1B and Figure S1). The residue set used to induce domain exchange (iAbdx) was inspired by previous structural and mutational studies on 2G12 antibodies (8, 11), and specific mutations with representative examples of the grafting approach can be found in Figure 2B and Figure S1. The affinity interface iAb mechanism promotes intramolecular Fab-Fab association by utilizing hydrophobic patches on the surface of the heavy chain variable domain. The residue sets used to generate these Fab-Fab interactions and promote iAb formation (iAbaff1 and iAbaff2) were inspired by broad-spectrum neutralizing anti-HIV antibody lines discovered in SHIV-infected rhesus monkeys, specifically DH851 and DH898 (9). To transplant each residue set into an "acceptor" antibody clone, the inventors first aligned each antibody sequence and then substituted the amino acids at the given residues in Figure 1B with appropriate iAb-inducing residue sets, with representative examples of two anti-OX40 antibody grafts shown in Figure S1. Based on varying degrees of amino acid conservation at each residue, transplantation of residue sets resulted in 4 to 8 mutations per antibody, and an average of 7 mutations across all antibodies tested in this experiment.

[0352] Protein expression and purification

[0353] Except for anti-DR4 and anti-DR5 antibodies, protein expression was performed by DNA transfection into HEK293 cells. Since anti-DR4 and anti-DR5 antibodies induce apoptosis in HEK293 cells, they were produced in CHO cells. For IgG and iAb, simultaneous transfection of heavy and light chain DNA was performed. Because the contrast body contains a light chain gene fusion to the Fc region, only a single plasmid was required for a single-specific format. OX40 ECD was expressed in Tni insect cells using a baculovirus expression system in the presence of 10 mg / L kyfunesin.

[0354] After expression, affinity chromatography was performed using MabSelect SuRe resin (Cytiva, catalog number 17543803) for Fc-containing proteins, CaptureSelect CH1-XL resin (Thermo, catalog number 194346201L) for Fab, and NiNTA agarose resin (Qiagen, catalog number 30210) for the receptor ECD. The elution buffer for MabSelect SuRe and CaptureSelect CH1-XL resins consisted of 50 mM sodium citrate and 150 mM NaCl at pH 3.0, while the elution buffer for NiNTA resin consisted of 50 mM sodium phosphate, 200 mM NaCl, and 400 mM imidazole at pH 7.4. Size exclusion chromatography was used as the final purification step using a HiLoad 16 / 600 Superdex 200 column. Protein quality was determined by analytical SEC and SDS-PAGE using a Waters xBridge BEH200A SEC 3.5um (7.8x300mm) column (Waters, catalog no. 176003596). All antibody formats were stored in a buffer consisting of 20mM histidine acetate and 150mM NaCl at pH 5.5, and the receptor ECD was stored in 25mM Tris pH 7.5 and 150mM NaCl.

[0355] The preparation of bispecific IgG and iAb was carried out as previously described (49). Briefly, a semi-antibody containing either a knob mutation or a hole mutation was first expressed in a separate cell culture and purified as described above. The two semi-antibodies were assembled into a single bispecific antibody through annealing, reduction, and oxidation steps. The desired heterodimer species was separated from the undesirable homodimer using size exclusion chromatography. Due to light chain gene fusion, a bispecific contrast body was prepared in the single cell culture as described above without any in vitro assembly steps.

[0356] Negative staining electron microscopy

[0357] The antibody sample for negative staining EM analysis was replaced with a buffer consisting of 25 mM Tris and 150 mM NaCl, concentrated to 1 mg / ml, and filtered through a 0.22 μm membrane (Costar, catalog number 8160). The sample was then diluted to 0.01 mg / ml, and 4 μl of the diluted sample was deposited onto an ultrathin carbon-coated 400-mesh copper grid (Electron Microscopy Sciences) that had been immediately glow-discharged (Solarus plasma cleaner, Gatan). After 30 seconds of incubation, the remaining liquid was absorbed with filter paper (Whatman, catalog number WHA1001090), and the grid was washed five times with 30 μl of filtered 2% uranyl acetate (Electron Microscopy Sciences). After 30 seconds, excess uranyl acetate stain was absorbed with filter paper. The grid was imaged at 73,000x magnification (2 Å per pixel) using a Talos 200 C equipped with a 4K Ceta CMOS camera (ThermoFisher). SerialEM was used for all data acquisition, and image processing was performed using cisTEM analysis software to generate 2D class averages. The proportions of i-shaped and Y-shaped antibodies in a given sample were calculated using the number of particles in each 2D class.

[0358] Generation of F(ab')2

[0359] The F(ab')2 construct of 3C8 iAbaff1 was generated by proteolytic cleavage of the lower hinge using modified matrix metalloproteinase 3 (MMP3). The MMP3 protease was fused to the N-terminus of an autologous affinity mature anti-human Fc antibody based on rheumatoid factor RF61 (50). Furthermore, the MMP3 protease was manipulated by adding an enterokinase cleavage site within the prodomain for more efficient activation. Prodomain cleavage and subsequent activation of the MMP3 antibody fusion construct were achieved by incubating 16 units of enterokinase (NEB, P8070L) per 25 μg of protein for 16 hours at room temperature in a buffer containing 25 mM Tris in pH 7.5, 150 mM NaCl, and 10 mM CaCl2. To inactivate the enterokinase, 0.1 mg / ml of soy trypsin inhibitor (Sigma, 17075029) was added to the protein solution. The activated MMP3 antibody fusion was mixed with the 3C8 iAbaff1 construct in a 1:10 molar ratio and incubated overnight at 37°C. After removing all cleaved Fc, unreacted IgG, and MMP3 antibody fusion protein using MabSelect SuRe resin, the supernatant was purified by size exclusion chromatography and analyzed by SDS-PAGE.

[0360] Affinity measurement

[0361] The solution affinity constants of all antibodies were determined using Biacore 8k+ or T200. Antibodies were diluted to 1 μg / ml with HBS-P+ buffer (Cytiva, catalog no. BR100671) and captured on Series S Protein A chips (Cytiva, catalog no. 29127555) according to the manufacturer's protocol. Serial dilutions of appropriate receptor ECDs (as described above, recombinantly produced OX40, CD40, 4-1BB, DR4, DR5, IL-2RB, and IL-2RG) were prepared with HBS-P+. The dilutions were injected for 3 minutes, followed by a 5-minute dissociation step. Affinity constants were determined from kinetic fitting to sensorograms using Biacore evaluation software.

[0362] Cell binding analysis

[0363] A 0.6 μM solution of each anti-IL-2RB or anti-IL-2RG antibody was incubated overnight at 4°C with 2.4 μM Alexa Fluor 488-labeled anti-human IgG goat affinity Fab fragment (Jackson, catalog no. 109-547-008). Serial dilutions of each Fab:antibody mixture in a 4:1 molar ratio were then performed in 20 μL of FACS buffer (1×PBS containing 1% BSA) in a clear 384-well FACS plate. 20 μL of FACS buffer containing 80,000 IL-2RB and IL-2RG overexpressing Jurkat cells was added to each well and incubated at 4°C for 4 hours. Cells were pelleted, washed twice, resuspended in 40 μL of FACS buffer, and analyzed using an iQue3 cytometer (Sartorius).

[0364] Receptor-mediated internalization assay

[0365] WT IgG, iAbaff1, or hexameric versions of anti-OX40 clone 3C8 were labeled with pHAb amine-reactive dyes according to the manufacturer's protocol (Promega, catalog no. G9841). OX40-expressing Jurkat cells were treated with each pHAb-labeled format at a predetermined concentration in RPMI medium containing 10% FBS and 2 mM L-glutamine (cRPMI) at 37°C and 5% CO2 for 1 hour. The cells were then washed twice with PBS containing 1% BSA, and fluorescence was measured using the BL2 channel of a Sartorius iQue3.

[0366] Total internal reflection fluorescence (TIRF) microscopy and single-particle tracking

[0367] Jurkat T cells were transfected with 0.3 μg of OX40-mNeongreen plasmid in a pRK vector backbone 48–72 hours prior to live cell imaging (Amaxa). 48-well glass-bottom plates were coated with 100 μg / mL poly-L-lysine at 37°C for 30 minutes, washed, and dried overnight. Anti-CD3ε antibody (10 μg / mL OKT3) was then added to stimulate and enhance T cell spread. All imaging was performed on a Nikon TIRF system with a 100 × 1.49 NA objective lens, a Hamatsu Orca FusionBT SCMOS camera, and an iLas2 laser system for elliptical illumination, with a field of view homogenized at an imaging depth of 75–100 nm. After cell adhesion to the surface, pre-processed datasets were acquired at 20 Hz for 12.5–25 seconds (250–500 frames). Next, a predetermined anti-OX40 antibody format was added to the imaging wells at 2 ug / mL (13.3 nM), and the same pre-treated cells and additional cells were acquired for the next 10 minutes at the same frame rate. At least six cells per condition were analyzed from two independent experiments, yielding over 20,000 OX40 trajectories per condition. Tracking was performed using the DiaTrack 3.0 MatLab runtime application, and mean square displacement plots were generated with a custom-written Igor track analysis code as previously described (51). Representative maximum projection images were created using imageJ, track inserts were created using Igor, and registered in representative fields in Adobe Illustrator.

[0368] Yeast display

[0369] Using an autologous S. cerevisiae yeast display scFv library containing 1.6 × 10⁹ unique sequence diversity, binding factors for IL-2RG and IL-2RB were selected using a combination of magnetically activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), as shown in Figure S6. Briefly, plasmids encoding the scFv library were electroporated into yeast and grown in SDCAA medium at 30°C until the logarithmic phase. Yeast exhibiting 10-fold diversity from the library was grown in galactose-containing SGCAA medium at 20°C for 24–48 hours at an OD600 of 1.0 before each selection. Between each selection, scFv expression on the yeast surface was confirmed using Alexa488-labeled anti-cMyc antibody (Cell Signaling). Binding to IL-2RG or IL-2RB was measured by adding a predetermined concentration of in-house-derived IL-2RB-biotin or commercially available IL-2RG-biotin (Acro biosystems) and Alexa647-labeled streptavidin (SA) beads (MACS) or tetramers. For the selection of the first round using magnetic SA beads, the biotinylated antigen was mixed with the beads, and then yeast was added to enhance avidity. For subsequent rounds using tetramer SA, the yeast was first stained with the biotinylated antigen, washed with PBS containing 1% BSA, and then stained with SA. Each round was checked for enrichment of the binding population by staining the yeast by antigen titration and analyzing fluorescence using iQue3 (Sartorius). The results for 37 nM are shown in Figure S6.

[0370] Epitope Mapping

[0371] Epitope mapping of anti-IL-2RB and anti-IL-2RG clones was performed using Carterra LSA. First, alanine substitution was introduced into each residue of 6× histidine (SEQ ID NO: 3)-tagged IL-2RB and IL-2RG ECD cells using PCR-based mutagenesis (n=206 and 203, respectively). If alanine was already present, the residue was mutated to glycine. Cystine was not mutated. All mutants were expressed in 293 cells and purified using NiNTA agarose resin as described above. The purified IL-2RB mutants were then arrayed and captured on a NiHC200M sensor chip for 5 minutes. A bispecific format with only one IL-2RB-specific arm was flowed on the chip for 5 minutes, and buffer was flowed for 5 minutes to allow dissociation. The chip was then regenerated twice with 350 mM EDTA for 5 minutes each and prepared with 5 mM NiCl for 5 minutes. This process was repeated for each anti-IL-2RB lead clone, and similarly for IL-2RG mutants and combinations with anti-IL-2RG lead clones. The mutant receptor capture level was calculated for each mutant in each cycle, and response unit measurements were performed at the end of the association period for each antibody. Ligand levels were plotted against antibody binding to identify alanine mutations that affected antibody binding, and these positions were highlighted on the previously reported crystal structures of the corresponding receptors (33).

[0372] Cross-linked ELISA

[0373] Recombinant human IL-2RB was coated overnight at 4°C with a 1 μg / ml PBS solution onto a Maxisorp 96-well plate (Thermo, catalog no. 44-2404-21). The wells were then blocked at room temperature for 1 hour with a solution of 0.5% BSA and 2 mM EDTA in PBS. A triple serial dilution series of the lead antibody was prepared in PBS with a maximum concentration of 60 μg / ml, and 100 μl of the antibody dilution was added to the wells. The plate was incubated at room temperature for 1 hour, and then washed three times with PBS. During antibody incubation, solutions of 10 μg / ml biotinylated human IL-2RG (Acro Biosystems, catalog no. ILB-H82E3) and 100 μg / ml streptavidin-HRP (SouthernBiotech, catalog no. 7100-05) were prepared in PBS and incubated at 37°C for 30 minutes. IL-2RG and streptavidin-HRP solution were diluted 10-fold, and 100 μl was added to each washed Maxisorp well. After incubation at 37°C for 30 minutes, the plate was washed three times with PBS. 100 μl of TMB substrate (Thermo, catalog number N301) was added to each well, and the absorbance at 650 nm was measured after 10 minutes. The absorbance signal was recorded as a magnification change relative to the control well without antibody.

[0374] Cellular reporter assay

[0375] The OX40 assay, 4-1BB assay, DR4 assay, and DR5 assay were performed as previously described (13, 17). For the CD40 bioassay, reporter cells were purchased from Promega (catalog no. JA2151) and used to evaluate CD40 agonist activity as follows: The cells were thawed and 10,000 cells were seeded in 20 μL of cRPMI in each well of a black-walled 384-well tissue culture plate (Corning, catalog no. 3764). The cells were adhered at 37°C and 5% CO2 for 6 hours. Then, 20 μL of serial dilutions of the specified antibody in cRPMI were added to the cells and incubated overnight under the same conditions. The following day, 40 μL of Bright-Glo reagent (Promega, catalog no. E2650) was added to each well and the luciferase signal was read using a Perkin Elmer Envision plate reader.

[0376] For the IL-2 reporter assay, Jurkat cells were engineered to express IL-2RB, IL-2RG, and STAT5-luciferase reporter constructs. 20,000 cells in 20 μL of cRPMI were added to 20 μL of cRPMI containing serial dilutions of antibody or recombinant IL-2, and incubated overnight at 37°C and 5% CO2. The following day, 40 μL of Bright-Glo reagent (Promega, catalog no. E2650) was added to each well, and the luciferase signal was read using a Perkin Elmer Envision plate reader. For the IL-2 blocking experiment, cells were first coated with 1 μM of each single-specific anti-IL-2RG clone or anti-IL-2RB clone for 1 hour, and then serial dilutions of IL-2 were added.

[0377] Primary cell assay

[0378] Purified primary human CD8+ T cells (catalog no. 70027) or NK cells (catalog no. 70036) were purchased from STEMCELL technologies. CD8+ T cells were pre-stimulated with 1 × 10⁶ cells / mL of CD3 / CD28 T-Activator Dynabeads (Gibco, catalog no. 11131D) in cRPMI at 37°C and 5% CO₂. After 48 hours, the Dynabeads were magnetically separated from the cells, and the cells were left to stand overnight in cRPMI at 37°C and 5% CO₂. Then, 50 μl of cRPMI containing 25,000 cells was added to 50 μl of cRPMI containing a predetermined mimetic antibody format or serial dilutions of recombinant IL-2, and incubated in white 96-well plates (Corning, catalog no. 3917) at 37°C and 5% CO₂. After 48 hours, 100 μL of CellTiter-Glo 2.0 (Promega, catalog number G9242) was added to each well, and the luciferase signal was read using a Perkin Elmer Envision plate reader. The same protocol was followed for NK cells, except for the pre-stimulation step.

[0379] RNA-seq

[0380] Purified primary human CD8+ T cells (STEMCELL technologies, catalog number 70027) were pre-stimulated as described above and allowed to stand. 2 × 10⁶ cells were plated in 2 mL of cRPMI in a 6-well plate. Each well was treated with 100 nM of a specified mimetic antibody format or recombinant IL-2 in 3 replicates and incubated at 37°C and 5% CO₂ for 24 hours. The cells were pelleted and RNA was extracted using the RNeasy mini-kit (Qiagen, catalog number 74014).

[0381] Total RNA was quantified using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific), and its quality was evaluated using RNA ScreenTape on an Agilent 4200 TapeStation. For sequencing library preparation, a Trueq Stranded mRNA Kit (Illumina) was used with 100 ng of total RNA. The libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and the average library size was determined using D1000 ScreenTape on an Agilent 4200 TapeStation. The libraries were pooled and sequenced using a NovaSeq 6000 (Illumina), generating 30 million 50-base-length single-ended reads for each sample.

[0382] RNA sequencing data were analyzed using HTSeqGenie(52) in BioConductor(53) as follows: First, low-nucleotide quality reads (70% of bases with less than 23 quality) or matches with rRNA and adapter sequences were removed. The remaining reads were aligned to the human reference genome (human:GRCh38.p10) using GSNAP(54,55) version "2013-10-10-v2", allowing a maximum of two mismatches per 75 base sequence (parameter: "-M2-n10-B2-i1-N1-w200000-E1-pairmax-rna=200000-clip-overlap"). Transcriptional annotation was based on the Gencode gene database (human:GENCODE27). To quantify gene expression levels, the number of reads clearly mapped to the exons of each gene was calculated.

[0383] The replicates for each condition were grouped compared to the negative control condition (gD), and differential expression was calculated using EdgeR(56) (version 3.40.1). Differentially expressed genes were identified using a Benjamini-Hochberg false detection rate of 1%. After normalizing the log2 factor change of differentially expressed genes within each condition using sklearn.preprocessing.normalize(57) (scikit-learn version 1.0.1), hierarchical clustering was performed using scipy.cluster.hierarchy(58) (SciPy version 1.7.3, parameter: method="ward").

Claims

1. A human antibody or humanized antibody comprising a first antigen-binding domain including a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, wherein the first VH and the first VL bind to a first target, and the VH domain is, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) Includes 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, A human antibody or humanized antibody that does not bind to HIV.

2. A human antibody or humanized antibody derived from a reference antibody, wherein both the antibody and the reference antibody include a first antigen-binding domain comprising a first heavy chain variable (VH) domain and a first light chain variable (VL) domain, a) The VH domain of the human antibody or humanized antibody contains, according to Kabat numbering, 1) at least one amino acid substitution selected from the group consisting of 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, and 2) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) The VH domain of the human antibody or humanized antibody contains, according to Kabat numbering, 1) at least one amino acid substitution selected from the group consisting of 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, and 2) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) The VH domain of the human antibody or humanized antibody comprises, according to Kabat numbering, 1) at least one amino acid substitution selected from the group consisting of 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, and 2) 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P. The substitutions in a), b), and c) are substitutions compared to the reference antibody, and optionally, the human antibody or humanized antibody has increased agonist activity compared to the reference antibody.

3. A human antibody or humanized antibody according to claim 2, which does not bind to HIV.

4. The human antibody or humanized antibody according to any one of claims 1 to 3, wherein the first VH domain of the human antibody or humanized antibody comprises 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT according to Kabat numbering.

5. The human antibody or humanized antibody according to any one of claims 1 to 3, wherein the first VH domain of the human antibody or humanized antibody comprises 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS according to Kabat numbering.

6. The human antibody or humanized antibody according to any one of claims 1 to 3, wherein the first VH domain of the human antibody or humanized antibody comprises 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P according to Kabat numbering.

7. The human antibody or humanized antibody according to any one of claims 2 to 4, wherein the first VH of the human antibody or humanized antibody includes at least one substitution at a position selected from 19, 21, 70, 79, and 81.

8. The human antibody or humanized antibody according to any one of claims 2 to 3 and 5, wherein the first VH of the human antibody or humanized antibody includes at least one substitution at a position selected from 19, 68, 70, and 81.

9. The human antibody or humanized antibody according to any one of claims 2 to 3 and 6, wherein the first VH of the human antibody or humanized antibody includes at least one substitution at a position selected from 14, 19, 39, 43, 74, 77, 82a and 82b.

10. The human antibody or humanized antibody according to any one of claims 1 to 9, wherein the antibody is a monovalent antibody.

11. The human antibody or humanized antibody according to claim 10, wherein the monovalent antibody is Fab.

12. The aforementioned antibody is F(ab'). ２ A human antibody or humanized antibody according to any one of claims 1 to 9.

13. The human antibody or humanized antibody according to any one of claims 1 to 12, wherein the antibody does not have an Fc domain.

14. The human antibody or humanized antibody according to any one of claims 1 to 10, wherein the antibody has an Fc domain.

15. The human antibody or humanized antibody according to any one of claims 1 to 10 and 14, wherein the antibody is an IgG antibody.

16. The human antibody or humanized antibody according to any one of claims 1 to 16, wherein the antibody has a modified hinge, and the modified hinge further restricts the flexibility of the hinge.

17. A human antibody or humanized antibody according to any one of claims 1 to 16, which is a single-specific antibody.

18. A human antibody or humanized antibody according to any one of claims 1 to 17, which binds to a cell surface receptor.

19. A human antibody or humanized antibody according to claim 18, which activates a target via receptor clustering.

20. A human antibody or humanized antibody according to any one of claims 1 to 19, which is bound to one or more TNFRSF members.

21. A human antibody or humanized antibody according to claim 20, which binds to OX40, CD40, 4-1BB, DR4, or DR5.

22. A human antibody or humanized antibody according to claim 21, which is bound to CD40 and optionally derived from an antibody selected from the group consisting of lavagalimab, dacetuzumab, gyrolarimab, and sotigolimab.

23. A human antibody or humanized antibody according to claim 21, which is bound to OX40 and optionally derived from an antibody selected from the group consisting of 3C8, 1A7, 2A3, 2B5, 2F10, 2G7, 2H5, 3F5, 3G5, and 3G8.

24. A human antibody or humanized antibody according to any one of claims 1 to 17, which binds to a cytokine receptor.

25. The human antibody or humanized antibody according to claim 24, wherein the cytokine can innately form a complex with at least two different receptors that induce downstream activity of the cytokine.

26. A human antibody or humanized antibody according to claim 24 or claim 25, which binds to the IL-2 receptor.

27. The human antibody or humanized antibody according to claim 26, wherein the IL-2 receptor is IL-2RG or IL-2RB.

28. A human antibody or humanized antibody according to any one of claims 1 to 9 and 12 to 27, comprising a bivalent antibody having a second antigen-binding domain including a second VH domain and a second VL domain that binds to a second target.

29. The aforementioned second VH domain, according to Kabat numbering, a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) A human antibody or humanized antibody according to claim 28, comprising 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

30. The human antibody or humanized antibody according to claim 28 or claim 29, wherein the second target is different from the first target.

31. A human antibody or humanized antibody according to any one of claims 27 to 30, which binds to both IL-2RG and IL-2RB.

32. The human antibody or humanized antibody according to any one of claims 27 to 31, wherein the VH domain of the human antibody or humanized antibody comprises three VH CDR sequences of B10, and the VL domain comprises three VL CDR sequences of B10.

33. The human antibody or humanized antibody according to claim 31 or 32, wherein one of the two VH domains comprises three VH CDR sequences of B10, one of the two VL domains comprises three VL CDR sequences of B10, the other of the two VH domains comprises three VH CDR sequences of G25 or G28, and the other of the two VL domains comprises three VL CDR sequences of G25 or G28.

34. A pharmaceutical composition comprising an antibody and a pharmaceutical carrier according to any one of claims 1 to 33.

35. Isolated nucleic acid encoding an antibody or fragment thereof according to any one of claims 1 to 33.

36. A host cell containing the nucleic acid described in claim 35.

37. A method for producing an antibody or fragment thereof according to any one of claims 1 to 33, comprising culturing the host cells described in claim 36 under conditions suitable for the expression of the antibody or fragment thereof.

38. The method according to claim 37, further comprising recovering the antibody or a fragment thereof from the host cell.

39. A method for producing an agonist antibody from a reference antibody, comprising substituting one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody in order to promote the i-shaped antibody format.

40. A method for producing an agonist antibody from a reference antibody, a) Substituting one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, wherein the agonist antibody, after substitution, has a VH domain containing 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) Substituting one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, wherein the agonist antibody, after substitution, has a VH domain containing 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) A method comprising substituting one or more amino acid residues on the heavy chain variable (VH) domain of the reference antibody at a position selected from 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113 according to Kabat numbering, wherein the agonist antibody, after substitution, has a VH domain comprising 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P, wherein the substituted amino acid residues are such that the agonist antibody, after substitution, has a VH domain comprising 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

41. An agonist antibody prepared by the method described in any one of claims 37 to 40.

42. A method for promoting the agonist activity of an antibody, a) Substituting one or more amino acid residues on the heavy chain variable (VH) domain of the antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, wherein the substituted VH domain contains amino acid residues including 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) Substituting one or more amino acid residues on the heavy chain variable (VH) domain of the antibody at a position selected from 7, 17, 19, 21, 68, 70, 77, 79, 81, and / or 82a according to Kabat numbering, wherein the substituted VH domain contains amino acid residues including 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) A method comprising substituting one or more amino acid residues on the heavy chain variable (VH) domain of the antibody at positions selected from 14, 19, 39, 43, 57, 74, 75, 77, 82a, 82b, 82c, 84, and 113 according to Kabat numbering, wherein the substituted VH domain contains amino acid residues comprising 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

43. An antibody according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 34, for use as a pharmaceutical.

44. An antibody according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 34, for use in treating a disease or symptom.

45. Use of an antibody according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 34 in the manufacture of a pharmaceutical for treating a disease or symptom.

46. A method for treating an individual having a disease or symptoms, comprising administering to the individual an effective amount of an antibody according to any one of claims 1 to 33 or a pharmaceutical composition according to claim 34.

47. A library comprising polynucleotides, wherein the polynucleotides in the library encode at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL domains bind to a target, and the VH domain is numbered according to Kabat numbering. a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) A library including 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

48. A library of antibodies comprising at least two, at least three, at least four, at least five, or at least ten unique antibodies, each of which comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL domains bind to a target, and the VH domain is numbered according to Kabat numbering. a) 7S, 17T, 19V, 21L, 68T, 70F, 77Q, 79I, 81I, and 82aT, or b) 7S, 17S, 19I, 21S, 68F, 70F, 77T, 79Y, 81V, and 82aS, or c) A library including 14A, 19I, 39R, 43G, 57R, 74L, 75E, 77F, 82aH, 82bK, 82cM, 84V, and 113P.

49. The library according to claim 47 or 48, wherein the antibody is expressed or scheduled to be expressed on the surface of one or more phage or yeast cells.

50. A method for screening agonist antibodies, comprising contacting an antibody in a library according to claim 48 or claim 49 with a target or cells expressing a target.

51. The method according to claim 50, further comprising evaluating the agonist activity of the antibody.

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