Immunotherapy proteins

Abstract

This disclosure relates to an immunotherapy protein, and discloses its use and production method, wherein the immunotherapy protein comprises one or more immunoglobulin heavy chain polypeptides comprising one or more constant heavy chain domains 1 (CH1), 2 (CH2), 3 (CH3), and hinge regions, one or more polypeptides comprising hexamerization mutations, and one or more polypeptides comprising at least one C1 q Includes binding modifications.

Description

[Technical Field]

[0001] This disclosure relates to an immunotherapy protein and discloses its use and production methods, the immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising one or more constant heavy chain domains 1 (CH1), 2 (CH2), 3 (CH3), and hinge regions, the one or more polypeptides comprising hexamerization mutations, and the one or more polypeptides comprising at least one C1q binding modification. [Background technology]

[0002] Human immunoglobulins are produced to recognize foreign antigens through their antigen-binding domains. Such antibodies can be produced with the heavy chain of the immunoglobulin being a different immunoglobulin class, such as human IgG, human IgA, human IgE, human IgM, human IgG, or human IgD.

[0003] In some cases, the heavy chain of immunoglobulin (Ig) may consist of subclasses of the immunoglobulin class; for example, an antibody may contain the heavy chain of human IgG1, or human IgG2, or human IgG3, or human IgG4, which are subclasses of human IgG.

[0004] To mediate effective outcomes, several effector systems may be utilized by immunoglobulins. Such outcomes include immune or innate immune responses to target antigens, such as antibodies induced by infection or vaccination, or resistance to pathogens by monoclonal antibodies targeted at specific targets, such as cancer cells or pathogens. Another outcome is the modulation of inflammatory responses in autoimmune diseases.

[0005] The heavy chain of immunoglobulins is involved in the effector function mediated by immunoglobulins. However, not all classes or subclasses of immunoglobulins can mediate all effector functions. In fact, taking the human IgG class as an example, its subclasses differ in their ability to initiate effector functions.

[0006] The two main effector systems utilized by immunoglobulins are the serum complement system, a multifaceted cascade of serum protein activation that, when activated, can lead to target death by complement-dependent cytotoxicity (CDC)-mediated lysis, or by cellular mechanisms mediated by inflammatory leukocytes or antibodies attracted to the complement-activated region and other leukocytes or tissue cells capable of ingesting or destroying complement-opsonized circulating targets. Not all human Ig classes or subclasses can activate complement.

[0007] The second major system is the Fc receptor, a cell surface protein expressed on the surface of leukocytes and other cells. These are cell surface receptors that specifically bind to immunoglobulins, often in the form of immune complexes (complexes of antigens and their corresponding antibodies). Receptors are characterized by the immunoglobulin class of the immunoglobulin to which they bind. For example, IgG is bound by the IgG Fc receptor-FcγR, IgA is bound by the IgA Fc receptor-FcαR, and so on. The interaction between immunoglobulins and Fc receptors can initiate a variety of cellular effector responses.

[0008] Not all immunoglobulins can utilize one or both effector systems. Human IgG1 or human IgG3 are universal activators of the effector response and are therefore potent activators of both serum complement and cellular FcγR receptors. That is, human IgG1 or human IgG3 activates serum complement and binds to all activated and suppressed FcγR receptors.

[0009] In contrast, it is widely known that human IgG2 or human IgG4 subclasses cannot activate serum complement. Furthermore, they are not universal ligands for FcγR, but rather exhibit specific binding to limited types of FcγR. Specifically, IgG2 cannot bind to all but one activating form of FcγR, binding only to one allele form of human FcγRIIa (FcγRIIa-H131), and cannot bind to the repressive FcγRIIb or other activating forms of FcγR. Human IgG4 binds only to the high-affinity activating receptor FcγRI and the repressive FcγRIIb.

[0010] Given the above, it is perhaps not surprising that considerable research effort has been applied to elucidate the details of Fc interactions (e.g., interactions with FcγR and complement proteins such as C1q and membrane attack complex (MAC) proteins C4-C9) and to identify modifications (e.g., mutations) that may occur to Fc to potentially enhance the potency of therapeutic mAbs. However, such Fc mutations have rarely been found in therapeutic mAbs to date. The best known examples are antibodies in which the heavy chain glycan is engineered to lack fucose, thereby enhancing the FcγRIII interaction, such as obinutuzumab and margetuximab (including 6-point mutations in the heavy chain of IgG1 antibodies), as well as other antibodies in clinical trials but not yet approved, such as triple mutations in the Fc fragment of IgG1, i.e., amino acid substitutions at positions 345 (i.e., E345R), 430 (i.e., E430G), and 440 (i.e., S440Y) (also known as "IgG-RGY" (see International Patent Publication No. 2014 / 006217)), or single mutations at position 430 (i.e., E430G) of the Fc in IgG1 (de Jong et al., PLoS Biol). These include various HexaBody® mAbs (GenMab BV, Copenhagen, Denmark), including 14(1):e1002344,2016). The E430G mutation has been shown to enhance the ability of IgG1 antibodies to form hexamers on target molecules, and IgG1 variants containing the E430G mutation have been reported to exhibit strongly enhanced CDC conditioned on antigen binding in target cells (de Jong et al., 2016, above). Therefore, it will be obvious to those skilled in the art that there is a need in the art for modifications to enhance the therapeutic efficacy of immunotherapy proteins (e.g., mAbs). [Overview of the Initiative]

[0011] In the research leading to this disclosure, the inventors produced a series of immunotherapy proteins (particularly mutant immunoglobulin G (IgG) molecules) containing point mutations at various positions within and around the interface of the CH2 and CH3 domains of heavy chain polypeptides. Several molecules containing mutations at position 429, a “filled” or inaccessible site in the IgG1 structure (see Figure 1) thought to be occupied by a histidine (His / H) residue, were found to exhibit enhanced activation of the complement system in complement function assays, for example, through the classical complement pathway, including C1q binding, membrane attack complex formation, and complement-dependent cytotoxicity. In addition, the inventors obtained evidence that several molecules containing Fc region components with mutations at position 429 can form oligomers either in solution or when bound to relevant targets. Furthermore, the inventors identified a series of C1q binding modifications that regulate the binding of immunotherapy proteins to C1q.

[0012] Accordingly, in one embodiment, the present invention provides an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, wherein one or more polypeptides comprises a hexamerization mutation, and one or more polypeptides comprises at least one C1q binding modification.

[0013] Accordingly, in one embodiment, the present invention provides an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, wherein one or more polypeptides comprises an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain, and one or more polypeptides comprises at least one C1q linkage modification.

[0014] In one embodiment, the present invention provides an oligomer comprising an immunotherapy protein as described herein.

[0015] In one embodiment, the present invention provides nucleic acids that encode the immunotherapy proteins described herein.

[0016] In one embodiment, the present invention provides the use of immunotherapy proteins, oligomers, or nucleic acids described herein for treating or preventing a disease or condition in a subject, the disease or condition being selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative diseases.

[0017] In one embodiment, the present invention provides the use of immunotherapy proteins, oligomers, or nucleic acids described herein in the manufacture of a pharmaceutical product for treating or preventing a disease or condition, the disease or condition being selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative diseases.

[0018] In one embodiment, the present invention provides a method for treating or preventing a disease or condition, the method comprising administering an effective amount of an immunotherapy protein, oligomer, or nucleic acid described herein to a target, the disease or condition being selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative diseases.

[0019] In one embodiment, the present invention provides a pharmaceutical composition or pharmaceutical comprising an immunotherapy protein, oligomer, or nucleic acid described herein and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0020] In one embodiment, the present invention provides a kit comprising at least one immunotherapy protein, oligomer, or nucleic acid described herein.

[0021] In one aspect, the present invention provides a method for producing the immunotherapy protein described herein, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for the expression of the protein, (i) A weakly acidic pH for recovering immunotherapy proteins in monomeric form, or (ii) Recovering the immunotherapy protein in oligomeric form from the culture supernatant under substantially neutral pH conditions.

[0022] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge regions.

[0023] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge regions, and the one or more polypeptides comprise an amino acid substitution with an aromatic, cyclic, or hydrophobic amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0024] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid, wherein one or more polypeptides comprise one or more of a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3), and a hinge region, and one or more polypeptides comprise at least one C1q linkage modification.

[0025] In one embodiment, the present invention provides a method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the polypeptide comprises one or more of the constant heavy chain domain 5 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge regions.

[0026] In one embodiment, the present invention provides a method for producing an immunotherapy protein described herein, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for the expression of the protein, and recovering the protein from the culture supernatant using a method comprising affinity chromatography, which may include, for example, using an elution buffer containing arginine at a concentration of less than 130 mM and with a pH of 5.0 or lower.

[0027] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge regions.

[0028] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, and the one or more polypeptides comprise an amino acid substation with F, Q, E, S, A, Y, L, V, G, W, R, or P at a position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0029] In one embodiment, the present invention provides a method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P, wherein one or more polypeptides comprise one or more of a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3), and a hinge region, and one or more polypeptides comprise at least one C1q linkage modification.

[0030] In one embodiment, the present invention provides a method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the polypeptide comprises one or more of the constant heavy chain domain 5 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge regions.

[0031] In one embodiment, the present invention provides an immunotherapy protein comprising a modified C1q linkage, the immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, the one or more polypeptides comprising an aromatic, cyclic, or hydrophobic amino acid substitution of an amino acid at a position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0032] In one embodiment, the present invention provides a method for producing / modifying C1q bonds in an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid.

[0033] In one embodiment, the present invention provides a method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid.

[0034] In one embodiment, the present invention provides an immunotherapy protein comprising a modified C1q linkage, the immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, the one or more polypeptides comprising amino acid substitutions by F, Q, E, S, A, Y, L, V, G, W, R, or P at the position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0035] In one embodiment, the present invention provides a method for producing / modifying C1q bonds in an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P.

[0036] In one embodiment, the present invention provides a method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, the method comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P. [Brief explanation of the drawing]

[0037] [Figure 1]This diagram provides a structural representation of human IgG1 Fc, showing that the H429 residue is located in an inaccessible region within the Fc fragment. The representation is of human IgG1-Fc (PDB:1Fc1), reproduced from Deisenhofer J., Biochemistry 20:2361-2370, 1981, using a space-filled rendering. (A) In the side view of Fc, the A chain is shown in black, the B chain in light gray, and the N-linked glycan in dark gray. The box region of chain A shows the enlarged region in panels B and C, (B) the enlarged box region of panel A, in gray, shows the solvent-accessible amino acids of chain B labeled according to the Eu numbering of human IgG1: methionine (M428) at position 428, glutamic acid 430 (E430), alanine 431 (A431), leucine 432 (L432), histidine H435, and an obscured histidine 429 (H429) at position Fc, located above histidine 429 (H429), (C) the figure of panel B after the solvent-accessible residues shown in panel B have not been rendered, i.e., removed from the figure. Here for the first time, the buried H429, shown in gray CPK, is revealed beneath the adjacent solvent-accessible residues that overlap these shown in panel B. The accessible surface area (ASA) for the residue calculated using PISA (i.e., the "Protein Interfaces, Surfaces and Assemblies" service PISA, http: / / www.ebi.ac.uk / pdbe / prot_int / pistart.htm) and Krissinel E and K Henrick (J Mol Biol 372:774-797, 2007) shows that H429 is inaccessible, recording a value of 0.0. [Figure 2]This provides a representation of immunoglobulin (antibody) and antibody-like (Ab-like) molecules demonstrating the modularity of antibodies. Left panel: Provides a representation of the immunoglobulin structure of a prototype. Immunoglobulins include a chain of variable (V) and constant (C) domains located on a heavy (H) chain, and optionally a self-assembling light (L) chain. Domains are further specified by the chain (e.g., CH3 is the third constant domain of the heavy chain). The heavy (H) chain typically further includes a separate linkage or hinge sequence between the Fab segment and the Fc segment. Some H chains contain three constant H domains as shown in the panel (having residue H429 shown in the CH3 domain), other H chains contain four constant H domains (e.g., IgM and IgE), and CH4 is the domain equivalent to CH3 in other antibodies (e.g., IgG and IgA). Antibodies from many species include a light (L) chain. In antibodies lacking a light chain, the two H chains dimerize (H2) and usually contain the antibody. On the other hand, in antibodies containing a light (L) chain, the two antibody H chains usually dimerize, and the L chain associates with each H chain (i.e., H2L2). Some antibodies lacking strong H chain interactions (e.g., human IgG4) can sometimes be antibody "halves" containing a monomeric H chain and one L chain (H1L1). The Fc fragment (or Fc) of an immunoglobulin is a dimer formed by covalent and / or non-covalent interactions between portions of each H chain (i.e., two Fc regions of the H chain, each containing the hinge, CH2, and CH3 domains of the heavy chain). Right panel: Provides an indication that the modularity of immunoglobulins allows for flexibility in the production of Ab-like molecules and fusion proteins (containing the immunoglobulin portion). All molecules shown contain an "H-like" chain containing at least a CH3 domain. In particular, each depicted Ab-like molecule consists of a V domain for target antigen recognition, which is conjugated by a linkage sequence to, for example, the CH3 domain of the Fc region from IgG (which in the variant of the shown molecule may be an equivalent CH4 domain from IgE or IgM). The CH3 domain may harbor mutations such as those described herein at position 429 (Eu numbering).The V and CH3 domains may include heterologous or synthetic linking sequences and / or sequences that may include other homologous or heterologous sequences or domains linking the V and CH3 domains (for example, as depicted in the example shown in the figure, the V and CH3 domains may be linked by CH1-CH2 or CH2, with or without a further shorter polypeptide sequence such as a heterologous or synthetic linking sequence or an immunoglobulin hinge sequence), and the CH1, CH2, and hinge sequence modules may be provided in various combinations and orders, not limited to the example shown. Each shown molecule is in monomeric form that can dimerize to form homodimer or heterodimer forms. Similarly, for the fusion proteins shown, each consists of a target recognition region (depicted as "X") that is joined by, for example, a linking sequence to at least the CH3 domain of the Fc region from IgG (or the equivalent CH4 domain of IgE or IgM). Again, the CH3 domain may include mutations such as one at position 429 (Eu numbering), and the target recognition portion and H chain module (e.g., the CH3 domain, CH2-CH3, or CH3-CH2 component) may include heterogeneous or synthetic ligation sequences and / or sequences that link the V domain and the CH3 domain, and / or sequences that include other homologous or heterogeneous sequences or domains (e.g., CH1-CH2 or CH2, with or without a further shorter polypeptide sequence such as a heterogeneous or synthetic ligation sequence or an immunoglobulin hinge sequence). [Figure 3]This paper illustrates the conservation of domain structure and sequence homology in immunoglobulin G (IgG) and immunoglobulin A (IgA) molecules. Sequence comparisons of the hinge and constant domains of human immunoglobulin heavy (H) chain sequences are provided by drawing the domain-based structures of the H chains (i.e., CH1-hinge-CH2-CH3 defined by IgG1; the Eu numbering shown for the first amino acids of each IgG1 domain and hinge is therefore shown as follows: CH1 domain, amino acids 118-215; hinge amino acids 216-230; CH2 domain amino acids 231-340; and CH3 domain amino acids 341-447). Other IgG subclasses IgG2, IgG3, IgG4, and IgA subclasses IgA1 and IgA2 have corresponding domain structures with amino acid sequence homology. The conservation of the histidine residue at position 429 in human IgG1 across other IgG and IgA subclasses is also shown (indicated by the arrow). The amino acid sequences were derived from translations of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). The accessions for the H chain sequences are: IgG1 H chain, J00228-IGHG1; IgG3 H chain, X03604-IGHG3; IgG4 H chain, K01316-IGHG; IgG2 H chain, J00230-IGHG2; IgA1 H chain, J00220-IGHA1; and IgA2 H chain, J00221-IGHA2. Alignment was performed using Clustal, except for manually aligned hinges. [Figure 4]This shows that histidine at position 429 in IgG1 is conserved across all immunoglobulin classes. A comparison of the amino acid sequences of the IgG1 CH3 domain and the corresponding domains in other human immunoglobulin classes shows that histidine 429, indicated by the arrow, is conserved in the CH3 domains of other IgG subclasses IgG2, IgG3, and IgG4, as well as IgA subclasses IgA1 and IgA2, and IgD, and in the equivalent domains (CH4) of IgE and IgM. The IgG1 CH3 domain is defined and numbered according to Eu numbering, and the first amino acid within the CH3 domain is indicated by an inverted triangle above glycine. The amino acid sequences shown are derived from translations of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). The accession numbers for the H chain sequences are: IgG1 CH3 domain, J00228-IGHG1; IgG3 CH3 domain, X03604-IGHG3; IgG4 CH3 domain, K01316-IGHG4; IgG2 CH3 domain, J00230-IGHG2; IgA1 CH3 domain, J00220-IGHA1; IgA2 CH3 domain, J00221-IGHA2; IgE CH4 domain, IGHE-CH4-J00222; IgM CH4 domain, IGHM-CH4-X57331; and IgD CH3 domain, IGHD-K02879. [Figure 5]The results of the purification of the ACE2-Fc H429Y fusion protein in accordance with this disclosure are shown. (A) Anion exchange (IEX) chromatography (flow-through (ft), elution fraction, and wash) of flACE2-Fc-WT with ACE2-Fc-containing peaks highlighted by *, (B) SDS-PAGE of flow-through (ft) and IEX peak fractions with flACE2-Fc H429Y moving above the 250 kDa marker and low molecular weight (mw) impurities marked with †, (C) Size exclusion chromatography (SEC) of the IEX fraction containing flACE2-Fc-WT (comparative) using a Superose 6 column showing oligomer (oli), monomer (mn), and low mw impurities (†), and (D) SEC of the IEX fraction containing flACE2-Fc H429Y showing a high proportion of oligomer species. The monomeric (mn) species is thought to be a single molecule (i.e., a monomeric molecule) containing two copies of each ACE2-Fc fusion polypeptide that self-associated through the Fc domain component. [Figure 6]This disclosure provides graphical results demonstrating the SARS-CoV-2 RBD binding activity of ACE2-Fc fusion proteins. ACE2-Fc fusion protein binding to immobilized RBD-Ig was determined by ELISA for: (A) trACE2-Fc-WT, (B) flACE2-Fc-WT, and (C) and EflACE2-Fc-WT, and their variants: ACE2-Fc protein containing an F-mutated H429F Fc region component; SEC-purified ACE2-Fc protein containing a Yoli-mutated H429Y Fc region component (oligomeric protein); SEC-purified ACE2-Fc protein containing a Ymn-mutated H429Y Fc region component (monomer protein; containing two copies of the ACE2-Fc fusion protein dimerized through the Fc region component); and trACE2-Fc-WT protein produced in the presence of the kif mannosidase inhibitor, kifunensin. (D) Summary of EC50 binding constants for trACE2-Fc-WT, flACE2-Fc-WT, and variant proteins, showing higher apparent RBD binding affinity to flACE2-Fc-WT than trACE2-Fc-WT, and weaker binding affinity to the flACE2-Fc-H429Y monomer (Ymn; i.e., containing two copies of the flACE2-Fc-H429Y fusion protein dimerized through the Fc domain component). Welch's unpaired t-test p=0.0332(*), <0.0001(****). [Figure 7]This disclosure provides results demonstrating that the flACE2-Fc H429Y fusion protein forms pH-dependent oligomers. SEC of flACE2-Fc H429Y purified by IEX was dialyzed against (A) PBS 7.4 or (B) 100 mM citrate, 100 mM NaCl pH 5, and then SEC was performed in the same buffer. SEC at pH 5 yielded a larger proportion of monomer (mn) fusion protein than separation at pH 7.4. (C) Native PAGE (1 μg) of ACE2-Fc H429Y. Lane 1, SEC pH 5 oligomer (oli); Lane 2, SEC pH 5 monomer (mn); Lane 3, SEC pH 7.4 oligomer; Lane 4, SEC pH 7.4 monomer; Lane 5, SEC pH 5 monomer purified by SEC after redialysis against PBS pH 7.4 and mn fraction collected as in Panel D. (D) The SEC chromatogram of the pH 5.0 oligomer was analyzed in lane 1 of panel C, redialysis was performed in PBS pH 7.4, and SEC was performed in PBS pH 7.4. (E) Improved RBD-Ig binding activity of the flACE2-Fc-H429Y monomer (Ymn) prepared at pH 5. The monomer is considered to be a single molecule (i.e., a monomeric molecule) containing two copies of each ACE2-Fc fusion polypeptide dimerized through the Fc domain component. [Figure 8]This disclosure provides results demonstrating that the SARS-CoV-2 neutralizing efficacy of ACE2-Fc fusion proteins is affected by the ACE2 scaffold (i.e., cleaved or full-length) and Fc mutations. The neutralizing efficacy of the ACE2 polypeptide, as well as three groups of ACE2-Fc-WT fusion proteins and variant proteins, was determined by titration to a cytotoxic effect (CPE) endpoint in a microneutralization assay. The fusion proteins are trACE2-Fc, flACE2-Fc, and EflACE2-Fc WT, as well as the Fc variant, H429F, the H429Y oligomer on F;SEC, Yoli, and the H429Y monomer on SEC, Ymn (containing two copies of the fusion protein dimerized through the Fc region component). The trACE2-Fc fusion protein includes the glycan-modified trACE2-Fc-kif. Neutralization endpoint mean ± SEM, ANOVA by Dunnett's multiple comparison test compared to ACE2 and ACE2-Fc WT. p=0.1234(ns), 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). [Figure 9] This disclosure provides the results of an assay to evaluate the interaction of ACE2-Fc fusion proteins with FcγR. Ramos-S cells were opsonized with ACE2-Fc WT fusion protein and variant protein (5 μg / ml). Biotinylated (A) dimer rsFcγRIIa or (B) dimer rsFcγRIIIa probes, followed by streptavidin-APC, were bound to the opsonized cells, and binding, described as median fluorescence intensity (median FI), was determined by flow cytometry. The FcR binding activity of ACE2-Fc fusion proteins was readily detected, with the exception of the ACE2-Fc H429Y fusion protein, which showed significantly reduced activity (3 replicates, mean ± SEM). [Figure 10]We provide the results of an assay to evaluate the ability of ACE2-Fc fusion proteins according to this disclosure to mediate cell activation via FcγRIIIa. The results demonstrate that the flACE2-Fc protein is a potent activator of FcγRIIIa, with the exception of any Fc H429Y variant of any ACE2 form that cannot be stimulated. Defucosylated trACE2-Fc-kif is also a potent activator of FcγRIIIa. Ramos-S target cells were opsonized with (A) trACE2-Fc, (B) flACE2-Fc, and (C) EflACE2-Fc, WT, as well as variant proteins including H429F, F;H429Y oligomer, Yoli;H429Y monomer, Ymn (containing two copies of a fusion protein dimerized through the Fc region component), or trACE2-Fc kif produced from trACE2-Fc WT in Expi293 cells in the presence of the mannosidase inhibitor kifunensin. In some experiments, Ramos-S target cells were separately opsonized with anti-CD20 mAb rituximab, RIT. These opsonized targets were incubated with FcγRIIIa-NF-κB-RE nanoluciferase reporter cells, and FcγRIIIa activation was measured by induction of nanoluciferase (RLU). To estimate EC50, the data were fitted to an agonist response curve. (D) The EC50(nM) values ​​from the curve fit are shown. Mean ± SEM, n≧4, ANOVA with Dunnett's multiple comparison test compared to trACE2-Fc WT. p=0.0021(**), 0.0002(***), <0.0001(****). [Figure 11]The results provided demonstrate that ACE2-Fc fusion proteins according to this disclosure, containing Fc region components with H429F and H429Y mutations, strongly fixate complement and direct complement-dependent cytotoxic death of Ramos-S target cells, determined by ELISA analysis of the complement fixation activity of trACE2-Fc(A, C, E) or flACE2-Fc(B, D, F) bound to plate-bound avidin-captured SARS-CoV-2 RBD-biotin: C1q binding (A, B) to ACE2-Fc fusion protein variants (2.0 μg / ml) in avidin-captured RBD at different concentrations on a plate; titration (mean ± SEM) of C1q (C, D) or C5b-9 (E, F) binding with serially diluted ACE2-Fc fusion protein variants (2.5 μg / ml) bound to avidin-captured RBD-biotin; two independent ELISA experiments. (G) Flow cytometry analysis of complement-dependent cytotoxicity (CDC) (death %) of opsonized Ramos-S cells was performed using a 1 / 3 dilution of normal human serum as a complement source (EC50(nM) values ​​from curve fitting are shown). [Figure 12]This study provides assay results demonstrating that an IgG1 antibody containing an Fc region component with the H429F mutation in the CH3 domain strongly fixes complement and exhibits identical antigen binding. (A, B) TNP-BSA antigen was adsorbed to wells of an ELISA plate at different concentrations (20 μg / ml to 0.625 μg / ml) and reacted with single-concentration (2 μg / ml) chimeric anti-TNP human IgG1 and IgG2 mAbs containing unmodified wild-type (WT) heavy chains, or with a 2 μg / ml anti-TNP mAb (TNP-IgG1-H429F) containing an IgG1 heavy chain with the Fc component mutation H429F. The antibody-opsonized TNP-BSA coated wells were treated with (A) purified human C1q and C1q fixed with anti-C1q rabbit polyclonal antibody, and (B) human serum as a complement source, and the formation of membrane attack complexes (C5b-C9) was detected with anti-C5b-C9 rabbit polyclonal antibody. (C) provides results from ELISA showing the antigen-binding activity of chimeric anti-TNP mAbs used in panels A and B, including unmodified (TNP-IgG1-WT and TNP-IgG2-WT mAbs) and H429-modified mAbs, i.e., TNP-IgG1-H429F. After coating the ELISA plates with TNP-BSA, the anti-TNP mAbs were titrated and antibody binding with HRP-conjugated anti-human IgG was detected. All mAbs showed similar antigen-binding activity. [Figure 13]This study provides results demonstrating C1q fixation and MAC (C5b-9) formation by mAbs containing IgG1 heavy chains with glutamic acid, glutamine, or serine modification at position 429 of the heavy chain. TNP-BSA (20 μg / ml) was adsorbed into wells and reacted with chimeric human anti-TNP mAbs, then titrated over a concentration range of 4 μg / ml to 0.125 μg / ml. The tested mAbs contained unmodified wild-type (WT) heavy chains of IgG1 (TNP-IgG1-WT) (Panels A-D) or IgG2 (TNP-IgG2-WT) (Panels A-C), or IgG1 H chains containing mutant TNP-IgG1-H429Q or TNP-IgG1-H429E (A, C) and TNP-IgG1-H429S (B, D). Wells coated with antibody-opsonized TNP-BSA were treated with human serum as a complement source. C1q fixation (A, B) was detected with anti-C1q rabbit polyclonal antibody, and the formation of the membrane attack complex (C5b-C9) (C, D) was detected with rabbit anti-C5b-C9 polyclonal antibody. [Figure 14] This report provides the results of flow cytometry quantification of SEC-purified mAb binding to target cell surface antigens. The binding activity of unmodified (WT) rituximab, daratumumab-WT, 11B8-WT mAbs, and their CH3-modified mutants possessing mutations H429F or H429Y (SEC (IgG H2L2) peak fraction p1 and IgGoli peak fraction p2) was evaluated by flow cytometry on Ramos lymphoma cells expressing CD20 and CD38. The binding activity of unmodified trastuzumab-WT, pertuzumab-WT, and their CH3 heavy chain mutants possessing mutation H429F was evaluated on HER2-expressing SK-OV-3 cells. [Figure 15]This document provides elution chromatograms of the protein A affinity purification characteristics of rituximab-WT, trastuzumab-WT, and their variants with modifications to the heavy chain CH3. IgG was eluted with sodium citrate buffer at pH 3.0 and then recovered from the column. (A) Elution profile of anti-CD20 rituximab-based mAbs. Rituximab-WT IgG and each variant IgG eluted as a single allogeneic peak. (B) Elution profile of anti-HER2 trastuzumab-based mAbs. Trastuzumab-WT IgG and each variant IgG eluted as a single allogeneic peak. [Figure 16]We provide size exclusion chromatography (SEC) profiles that reveal that the H429 mutation can alter the physical properties of IgG. After protein A affinity purification, mAbs were further purified by SEC at pH 7.2. (A) The SEC profiles of the unmodified rituximab-WT (WT) and rituximab-H429F (H429F) mutants consist of a single homogeneous IgG peak (right side of the vertical dashed line) with minimal oligomeric species (left side of the vertical dashed line). Thus, for example, the SEC profile of unmodified rituximab-WT, as expected, shows a single major species corresponding to IgG(H2L2), as confirmed by SDS-PAGE analysis (Figure 17A), which migrates with the expected mass of approximately 150 kDa of unreduced IgG and, after reduction in DTT, is degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. On the other hand, rituximab-H429Y mAb contained both non-oligomeric IgG and oligomeric IgG, and (B) SEC profiles of protein A purified anti-HER2 trastuzumab wild-type (WT) (i.e., the unmodified form of trastuzumab) and its variants containing amino acid substitutions of H429F or H429Y. A single peak of non-oligomeric IgG to the right of the vertical dashed line was observed for trastuzumab-WT and H429F variant mAb, while trastuzumab-H429Y variant IgG (H429Y) contained both non-oligomeric IgG and oligomeric IgG. The non-oligomeric IgG peak of trastuzumab-H429Y was consistent with the peaks of unmodified trastuzumab-WT or its H429F variant and equivalent rituximab-based mAb in panel (A). The SEC profile of oligomeric IgG in trastuzumab-H429Y was consistent with that of oligomeric IgG in rituximab-H429Y in panel (A). [Figure 17]Images obtained from SDS-PAGE of SEC-purified mAbs are provided. SEC-purified mAbs were analyzed by SDS-PAGE in a 5–15% gradient gel with or without disulfide bond reduction. (A) Under non-reducing conditions, the non-oligomeric IgG peaks of rituximab-WT (WT IgG) and rituximab-H429F (HF IgG) migrated as a single species with the expected molecular size of approximately 150 kDa for IgG (i.e., H2L2). After reduction in DTT, the 150 kDa IgG was, as expected, degraded into a heavy (H) chain of approximately 50 kDa and a light (L) chain of approximately 25 kDa. Before reduction, the non-oligomeric IgG (HY IgG(H2L2)) and oligomeric (HY IgG(oli)) forms of rituximab-H429Y mAb (see Figure 16) both migrated similarly as a single 150 kDa IgG, i.e., the H2L2 species. After reduction, both forms migrated as a heavy (H) chain of approximately 50 kDa and a light (L) chain of approximately 25 kDa, with WT = wild type, HF = H429F, HY = H429Y; M = molecular weight marker, their mass (kD) shown on the left, and (B) under non-reducing conditions, the IgG peaks of trastuzumab-WT (WT IgG) and trastuzumab-H429F (HF IgG) migrated as a single species with the expected molecular size of approximately 150 kD for IgG, i.e., H2L2. After reduction with dithiothreitol (DTT), the 150 kDa IgG was, as expected, broken down into a heavy (H) chain of approximately 50 kDa and a light (L) chain of approximately 25 kDa. Before reduction, both the non-oligomeric IgG (HY IgG(H2L2)) and oligomeric (HY IgG(oli)) forms of trastuzumab-H429Y mAb migrated similarly as a single 150 kDa IgG species under non-reducing conditions, and after reduction, they migrated as the expected heavy (H) chain of approximately 50 kDa and light (L) chain of approximately 25 kDa. WT = wild type, HF = H429F, HY = H429Y; M = molecular weight marker, their masses (kD) are shown on the left. [Figure 18]This paper provides experimental results demonstrating that oligomeric and non-oligomeric forms of H429Y-modified IgG antibody exhibit equivalent CDC efficacy and that their formation is pH-sensitive. (A) SEC of rituximab-H429Y at pH 7.2 showing the presence of two major IgG forms (see also Figure 16A). Non-oligomeric IgG (H2L2) (to the right of the vertical dashed line, indicated as p1) and the oligomeric form of IgG (IgG(oli)) (to the left of the vertical dashed line, indicated as p2) were collected separately for further evaluation of complement-dependent cytotoxicity (CDC) efficacy. (B) Oligomerous (p2) and non-oligomeric (p1) IgG forms from panel A, as well as SEC-purified unmodified rituximab-WT IgG, were titrated, and CDC efficacy was determined by flow cytometry using Ramos lymphoma cells and 1 / 3 diluted normal human serum as a complement source. The degree of opsonization of Ramos cells was determined using non-oligomeric rituximab-H429Y p1 (white square), oligomeric rituximab-H429Y p2 (triangle), or unmodified rituximab-WT (black circle). Complement control background CDC in the absence of mAbs (C' only, no mAb) is shown as a black diamond (C). IgG oligomer formation is pH sensitive. Purified protein A trastuzumab-H429Y was analyzed by size exclusion chromatography (SEC) at either pH 7.2 (left panel) or pH 5.0 (right panel). At pH 7.2, both oligomeric (IgG(oli)) and non-oligomeric IgG were present, while at pH 5.0, only a single peak corresponding to non-oligomeric IgG was present. [Figure 19]The results demonstrate that the H429F mutation in the CH3 domain of the IgG H chain strongly promotes C1q binding and complement-dependent cell-mediated cytotoxicity. Flow cytometry detection of C1q binding (fluorescence intensity) in Ramos lymphoma cells opsonized with unmodified rituximab-WT (A) or modified rituximab-H429F mAb (B), or SK-OV-3 ovarian cancer cells opsonized with trastuzumab-WT (C) or modified trastuzumab-H429F mAb (D) is plotted. Cells opsonized with the mAbs were treated with normal human serum as a complement source, and C1q binding was detected by staining with an anti-C1q rabbit polyclonal antibody. C1q binding to mAb-opsonized cells is shown in the unshadowed histogram, and C1q background binding controls (i.e., cells treated with serum complement in the absence of the mAb) are shown in the gray-shadowed histogram. The median fluorescence intensity (MFI) value for each histogram is shown in parentheses. (E) Complement-dependent cytotoxicity (CDC; i.e., death %) detected using Zombie Green) of Ramos lymphoma cells opsonized with rituximab-WT (black circles) or its mutated variant mAb rituximab-H429F (black squares) was determined by flow cytometry using normal human serum as the complement source. CDC is significantly enhanced by the mutation at position 429 in rituximab-H429F compared to the rituximab-WT mAb. [Figure 20] The results of protein A affinity chromatography of type II anti-CD20 11B8-WT mAbs and mutant mAbs, including the H429 substitution, are shown. The mAb was 11B8-WT mAb (WT) produced on wild-type human IgG monoheavy chains. 11B8-H429F (H429F) was produced on modified IgG monoheavy chains in which the amino acid histidine at position 429 in the CH3 domain of the Ig heavy chain was replaced with phenylalanine. [Figure 21]The results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of 11B8-WT mAbs and 11B8-H429F (H429F) mutants are shown. (A) Chromatogram of mAbs purified by SEC at pH 7.2; monomeric non-oligomeric IgG is shown to the right of the vertical dotted line, and (B) SDS-PAGE analysis of SEC-purified monomeric IgG mAbs from panel A (5-15% gradient gel). Before reduction (non-reduced), all SEC-purified antibodies migrated with the expected molecular size of approximately 150 kDa for IgG, i.e., H2L2. After reduction by DTT (reduced), all antibodies were degraded, as expected, into heavy (H) chains of approximately 50 kDa and light (L) chains of approximately 25 kDa. WT = wild type, HF = H429F, M = molecular weight marker, their mass (kD) is shown on the left. [Figure 22] The results demonstrate that the H429 modification of the CH3 domain confers CDC efficacy to type II anti-CD20 mAbs. CDC by 11B8-WT mAb (filled circle) or CH3 mutant 11B8-H429F mAb (filled square). Complement-dependent lysis was determined using Ramos lymphoma cells opsonized with the mAb at the indicated concentrations, and normal human serum diluted to 1 / 3 as a complement source. CDC (death %) was determined by flow cytometry using Zombie Green. Complement-controlled background CDC in the absence of the mAb (C' only without the mAb) is shown as a black diamond. The 11B8-WT mAb failed to mediate CDC, while the 11B8-H429F mutant mAb mediated potent complement-dependent lysis of lymphoma cells (WT = wild type). [Figure 23] The results of protein A affinity chromatography for anti-CD38 daratumumab-WT mAb and daratumumab-H429F mAb are shown. The elution chromatograms indicate that each antibody eluted as a single, identical homogeneous peak. [Figure 24]The results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of daratumumab-WT mAb and mutant mAb, daratumumab-H429F, are shown. (A) Chromatogram of SEC-purified mAb. In each case, the mAb contained a single non-oligomeric IgG peak (right side of the vertical dashed line) and the absence of oligomeric species (left side of the vertical dashed line). (B) SDS-PAGE analysis of SEC-purified IgG from panel A (5-15% gradient gel). Before reduction (non-reduced), all of the antibody migrated with the expected molecular size of approximately 150 kDa for IgG (i.e., H2L2), and after reduction by DTT (reduced), all of the antibody was degraded, as expected, into heavy (H) chains of approximately 50 kDa and light (L) chains of approximately 25 kDa. WT = wild type, HF = H429F, M = molecular weight marker, their mass (kD) is shown on the left. [Figure 25] The results show that H429F substitution greatly enhances complement-dependent lysis of lymphoma cells by daratumumab-WT. The graph shows the level of CDC (death %) by daratumumab-WT mAb (filled circles) and daratumumab-H429F (filled squares). Background lysis (filled triangles) was determined in the absence of mAb, but in the presence of complement alone. CDC was determined by flow cytometry using Zombie Green. Daratumumab-H429F mAb mediated more potent complement-dependent lysis of lymphoma cells than unmodified daratumumab-WT. [Figure 26]The results demonstrate that H429F substitution confers complement-dependent lysis of myeloma and leukemia cells resistant to lysis with anti-CD38 mAbs. CDC by complement-dependent lysis was determined using: (A) KMS-12-PE myeloma cells opsonized with daratumumab-WT mAb (filled circle) or daratumumab-H429F mutant mAb (filled square) at indicated concentrations. Background lysis (filled triangle) was determined in the absence of mAbs but in the presence of complement alone; and (B) SUP-15 acute lymphoblastic leukemia (ALL) cells opsonized with daratumumab-WT mAb (solid circle) and daratumumab-H429F mutant mAb (solid square) at indicated concentrations. Background lysis (filled triangle) was determined in the absence of mAbs but in the presence of complement alone. CDC (death rate) was determined by flow cytometry using Zombie Green. [Figure 27] The results of protein A affinity chromatography for the anti-HER2 mAb pertuzumab-WT and the CH3 H429F variant are shown. The elution chromatograms indicate that each antibody eluted as a single, identical homogeneous peak. [Figure 28]The results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of the anti-HER2 mAb pertuzumab-WT and CH3 H429F variant are shown. (A) Chromatogram of SEC-purified mAbs. After protein A affinity chromatography, the mAbs were further purified by size exclusion chromatography (SEC) at pH 7.2. Chromatographic profiles are shown for unmodified pertuzumab-WT and for the Fc-modified variant, pertuzumab-H429F (H429F). In each case, the protein A-purified mAbs contained a single non-oligomeric IgG peak (right side of the vertical dashed line) and the absence of oligomeric species (left side of the vertical dashed line). (B) SDS-PAGE analysis of SEC-purified IgG from panel A (5-15% gradient gel). Before reduction (non-reduced), all of the SEC-purified antibodies migrated at the expected molecular size of approximately 150 kDa for IgG (i.e., H2L2). After reduction by DTT (reduction), all of the antibody was degraded, as expected, into a heavy (H) chain of approximately 50 kDa and a light (L) chain of approximately 25 kDa. WT = wild type, HF = H429F, M = kilodalton (kD) is the molecular weight marker. [Figure 29]The results provide evidence of synergistic and functional synergistic effects in a mixture of mAbs containing amino acid substitutions at position H429, and in particular, the results show that combining H429F-modified mAbs can enhance C1q binding to target cells. (A) Flow cytometry histograms of C1q binding to opsonized (unfilled histograms) HER2-expressing SK-OV-3 cells with trastuzumab-H429F alone, pertuzumab-H429F alone, or a 1:1 mixture of trastuzumab-H429F and pertuzumab-H429F. Black histograms indicate background binding of C1q to non-opsonized cells. Median fluorescence intensity (MFI) values ​​for each histogram are also shown. C1q binding was detected using an anti-C1q-specific polyclonal rabbit antibody, and (B) titration of enhanced functional synergy. SK-OV-3 cells were opsonized with titrated individual anti-HER2 mAbs: trastuzumab-WT, pertuzumab-WT, trastuzumab-H429F, pertuzumab-H429F, or a 1:1 mixture of trastuzumab-WT with pertuzumab-WT or trastuzumab-H429F with pertuzumab-H429F. The vertical double arrows illustrate the extensive synergy in the pertuzumab-H429F mixture with trastuzumab-H429F at limited antibody concentrations compared to C1q binding of any mAb alone. [Figure 30]This provides graphical results demonstrating that the synergistic and functional effects of H429-modified mAbs enhance complement-dependent cell death (CDC) of target cells. The indicated rituximab-based mAbs were titrated alone on Ramos cells (light gray column) or in the presence of fixed concentrations of the indicated anti-CD38 or anti-CD20 mAbs (dark column): (A) rituximab-WT titrated in the presence of 0.025 μg / ml daratumumab-WT, (B) rituximab-HF titrated in the presence of 0.025 μg / ml daratumumab-H429F, and (C) rituximab-HF titrated in the presence of 0.5 μg / ml 11B8-H429F. CDC (death %) was determined by flow cytometry using Zombie Green. The dotted horizontal lines in (B, C) indicate the percentage of CDC death obtained in the absence of a rituximab-based mAb (0 μg / ml), but in the presence of (B) 0.025 μg / ml daratumumab-H429F or (C) 0.5 μg / ml 11B8-H429F alone. The black arrows in (B, C) indicate enhanced CDC of a mixture of mAbs over the CDC of individual mAbs (i.e., CDC of rituximab-H429F alone at the indicated concentrations (bright column), or CDC of either 0.025 μg / ml daratumumab-H429F or 0.5 μg / ml 11B8-H429F (dark column, 0 μg / ml rituximab)). [Figure 31]This paper provides the results of flow cytometry analysis of the CDC efficacy of rituximab antibodies containing the H429F mutation against normal peripheral blood CD19+ B lymphocytes. The results show that rituximab-H429F mAbs exhibit more potent CDC death of normal peripheral blood B cells compared to unmodified rituximab-WT. After treating peripheral blood mononuclear cells with the indicated mAbs and human complement, the percentage of live or dead B cells was determined by staining with anti-CD19 to identify B cells and with Zombie Green (ZG) to identify dead cells. Cytological findings are shown as median Zombie Green fluorescence intensity (ZG MFI) (median CD19 fluorescence intensity (CD19 MFI)) of gated CD19 B cells. In each cell finding, the percentage of dead cells (ZG-positive) for rituximab-WT (30.4% ZG-positive), rituximab-H429F (85.6% ZG-positive), and the corresponding negative control mAbs, trastuzumab-WT (3.05% ZG-positive) and trastuzumab-H429F (3.11% ZG-positive), is shown in the upper section Q1, while the percentage of live B cells is shown in the lower section Q2. [Figure 32] This provides results demonstrating that the effect of H429 modification on antibody function is not limited to IgG1 but extends to other immunoglobulin types as well. Here, we evaluated the CDC efficacy of rituximab antibodies formalized with (A) wild-type (WT) or H429F (HF) modified heavy chains of (A) IgG3 or (B) IgG4 subclasses against Ramos lymphoma cells. [Figure 33] The results demonstrate that effective CDC of target cells mediated by mAbs depends on the presence of both monoclonal antibodies and serum complement. CDC (death %) of Ramos cells with rituximab-H429F, daratumumab-H429F, or 11B8-H429F, or SUP-15 cells with daratumumab-H429F is shown. A = death percentage in the presence of both the indicated mAb and human serum as a complement source, B = lysis in the presence of mAb only at the same concentration as in A, and C = lysis in the presence of complement only. [Figure 34]This study provides results demonstrating that H429F substitution confers complement-dependent lysis of leukemia cells resistant to lysis with the unmodified anti-CD38 mAb isatuximab. Complement-dependent lysis-mediated CDC was determined using SUP-15 acute lymphoblastic leukemia (ALL) cells opsonized with isatuximab-WT mAb (dashed line with black circle) and isatuximab-H429F mutant mAb (solid line with black square) at the indicated concentrations. Background lysis (unfilled circle) was determined in the absence of mAb, but also in the presence of complement alone (C' only, no mAb). Background lysis induced by mAb at 5 μg / ml in the absence of complement was determined for isatuximab-WT mAb (unfilled inverted triangle) and isatuximab-H429F mutant mAb (white unfilled square). CDC (death rate) was determined by flow cytometry using Zombie Green. [Figure 35]This report provides flow cytometry quantification results of the binding of purified DR5-specific mAbs to Colo205 colorectal cells, containing either unmodified wild-type (WT) heavy chains of human IgG1 or IgG2 subclasses, or human IgG1 or human IgG2 subclass heavy chains modified by replacing histidine 429 with phenylalanine (H429F). The panel is shown below. (A) Binding of BDR5-1WT containing a wild-type H chain of the human IgG1 subclass, or binding of BDR5-1HF mAb containing an IgG1 heavy chain with H429F modification; (B) Binding of BDR5-2WT containing an unmodified heavy chain of the human IgG2 subclass, or binding of BDR5-2HF mAb containing an IgG2 heavy chain with H429F modification; (C) Binding of TDR5-1WT mAb containing an unmodified heavy chain of the human IgG1 subclass, or binding of TDR5-1HF mAb containing an IgG1 heavy chain with H429F modification; and (D) Binding of TDR5-2WT containing an unmodified heavy chain of the human IgG2 subclass, or binding of TDR5-2HF mAb containing an IgG2 heavy chain with H429F modification. The mAbs were titrated 2-fold consecutively, and the binding activity to Colo205 cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. In all panels, the level of nonspecific binding of the fluorescent conjugate to cells is shown (▲conjugate), and MFI = median fluorescence intensity. [Figure 36]This report provides flow cytometry quantification results of the binding of purified DR5-specific mAbs to target Ramos lymphoma cells, including unmodified wild-type (WT) H chains of human IgG1 or human IgG2 subclasses, or human IgG1 or human IgG2 heavy chains modified by replacing histidine 429 with phenylalanine (H429F). The panel is shown below. (A) Binding of BDR5-1WT containing an unmodified heavy chain of the human IgG1 subclass, or binding of BDR5-1HF mAb containing an IgG1 heavy chain with H429F modification; (B) Binding of BDR5-2WT containing an unmodified heavy chain of the human IgG2 subclass, or binding of BDR5-2HF mAb containing an IgG2 heavy chain with H429F modification; (C) Binding of TDR5-1WT containing an unmodified heavy chain of the human IgG1 subclass, or binding of TDR5-1HF mAb containing an IgG1 heavy chain with H429F modification; and (D) Binding of TDR5-2WT containing an unmodified heavy chain of the human IgG2 subclass, or binding of TDR5-2HF mAb containing an IgG2 heavy chain with H429F modification. The mAbs were titrated 2-fold consecutively, and binding to Ramos cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. In all panels, the level of nonspecific binding to cells was determined using an unrelated IgG antibody (negative IgG), and background binding of the fluorescent anti-IgG conjugate to cells is shown (▲conjugate); MFI = median fluorescence intensity. [Figure 37]This report provides flow cytometry quantification results of the binding of purified DR5-specific mAbs to target KMS-12-PE myeloma cells, containing unmodified wild-type (WT) H chains of human IgG1 or human IgG2 subclasses, or human IgG1 or human IgG2 heavy chains modified by replacing histidine 429 with phenylalanine (H429F). Binding activity is shown for DR5-specific mAbs, BDR5, and TDR5, containing unmodified wild-type H chains of human IgG1 subclasses (BDR5-1WT, TDR5-1WT) or human IgG2 subclasses (BDR5-2WT, TDR5-2WT), or Fc-mutated H chains of IgG1 subclasses (BDR5-1HF, TDR5-1HF) or IgG2 subclasses (BDR5-2HF, TDR5-2HF) containing the H249F mutation. KMS12-PE cells were incubated with mAbs at 5 μg / ml. Binding activity was quantified by flow cytometry using an anti-IgG secondary reagent labeled with FITC goat anti-hIgG Fc. Background control of binding of anti-IgG conjugate only is shown with background fluorescence of cells only (cells) (conjugate). [Figure 38]This study provides results demonstrating the survival of Colo205 colorectal cells in the presence of purified DR5-specific mAbs containing unmodified wild-type H chains of human IgG1 or human IgG2 subclasses, or Fc-mutated H chains of human IgG1 or human IgG2 subclasses containing the H429F mutation. 10,000 cells were cultured in each well of a 96-well plate in the presence of 20 μg / ml of each of the following mAbs: TDR5-1WT mAb containing unmodified wild-type H chains of human IgG1 subclasses, or TDR5-1HF mAb containing H chains of human IgG1 subclasses containing the H429F mutation, or TDR5-2WT mAb containing unmodified wild-type H chains of human IgG2 subclasses, or TDR5-2HF mAb containing H chains of human IgG2 subclasses containing the H429F mutation. Additionally, Colo205 cells were cultured separately in the presence of a mixture of 10 μg / ml TDR5-2HF and 10 μg / ml BDR5-2HF mAbs (TDR5-2HF + BDR5-2HF), both of which contained Fc mutant human IgG2 H chains carrying the H429F mutation. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibody (no antibody). Survival is expressed as follows: [experimentally determined mAb or control treatment absorbance at 450 nm - background absorbance of cell culture medium at 450 nm], mean absorbance at 450 nm, and four repeat values. [Figure 39]This study provides results demonstrating the survival of Ramos lymphoma cells in the presence of purified DR5-specific mAbs containing unmodified wild-type or Fc-mutated heavy chains of human IgG1 or IgG2 subclasses containing the H429F mutation. 10,000 cells were cultured in each well of a 96-well plate in the presence of 20 μg / ml of each of the following mAbs: TDR5-1WT mAb containing an unmodified wild-type heavy chain of human IgG1 subclass, or TDR5-1HF mAb containing an H chain of human IgG1 subclass containing the H429F mutation; TDR5-2WT mAb containing an unmodified wild-type heavy chain of human IgG2 subclass, or TDR5-2HF mAb containing an H chain of human IgG2 subclass containing the H429F mutation. Additionally, Ramos cells were cultured separately in the presence of a mixture of 10 μg / ml TDR5-2HF and 10 μg / ml BDR5-2HF mAbs (TDR5-2HF + BDR5-2HF), both containing Fc mutant human IgG2 H chains carrying the H429F mutation. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibody (no antibody). Survival is expressed as follows: [experimentally determined mAb or control treatment absorbance at 450 nm - background absorbance of cell culture medium at 450 nm], mean absorbance at 450 nm, and four repeat values. [Figure 40]The results demonstrate the viability of Colo205 colorectal cells in the presence of a mixture of purified DR5-specific mAbs, the mAbs containing wild-type or Fc-modified H chains of the same IgG subclass. BDR5 and TDR5 mAbs contained either unmodified wild-type H chains of human IgG1 or human IgG2 subclasses, or heavy chains of IgG1 or IgG2 subclasses containing H429F modification. Colo205 cells, 30,000 cells per well in a 96-well plate, were cultured in the presence of a 1:1 mixture of mAbs containing the same H chains, in a 2-fold serial dilution. A starting concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. Therefore, the mixture used was as follows: BDR5-1WT mAb containing an unmodified wild-type IgG1 H chain mAb mixed with TDR5-1WT containing an unmodified wild-type IgG1 H chain (BDR5-1WT + TDR5-1WT), BDR5-2WT mAb containing an unmodified wild-type IgG2 H chain mAb mixed with TDR5-2WT containing an unmodified wild-type IgG2 H chain (BDR5-2WT + TDR5-2WT), BDR5-1HF mAb containing an H429F-modified IgG1 H chain mixed with TDR5-1HF mAb containing an H429F-modified IgG1 H chain (BDR5-1HF + TDR5-1HF), BDR5-2HF mAb containing an H429F-modified IgG2 H chain mixed with TDR5-2HF mAb containing an H429F-modified IgG2 H chain (BDR5-2HF + TDR5-2HF). Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibody (antibody-free), and maximum mortality was determined by culturing cells with sodium dodecyl sulfate (SDS). Survival is expressed as follows: [experimentally determined mAb or control treatment absorbance at 450 nm - background absorbance of cell culture medium at 450 nm]. [Figure 41]The results demonstrate the survival of Colo205 colorectal cells in the presence of different pair combinations of purified DR5-specific mAbs, the mAbs containing wild-type or Fc-mutated H chains of different IgG subclasses. The mAbs contained H chains of human IgG1 or human IgG2 subclasses, either containing wild-type heavy chains or heavy chains modified by H429F modification. Colo205 cells, 10,000 cells per well in a 96-well tissue culture plate, were cultured in the presence of a 1:1 mixture of mAbs serially diluted 2-fold from 1 μg / ml. The starting concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. The wild-type mAb mixtures used were as follows: As observed in Figure 40, the following were observed: BDR5-2WT containing wild-type human IgG2 H chains mixed with TDR5-1WT containing unmodified wild-type human IgG1 H chains (BDR5-2WT+TDR5-1WT), BDR5-1WT containing wild-type human IgG1 H chains mixed with TDR5-2WT containing unmodified wild-type human IgG2 H chains (BDR5-1WT+TDR5-2WT), and a control mixture of BDR5-2WT containing unmodified wild-type human IgG2 H chains mixed with TDR5-2WT containing unmodified wild-type human IgG2 H chains (BDR5-2WT+TDR5-2WT). The mixture of DR5 mAbs containing H429F Fc mutant H chains was as follows: As observed in Figures 38 and 40, positive cell death control mixtures were obtained: BDR5-2HF containing H429F Fc mutant human IgG2 H chain mixed with TDR5-1HF containing H429F Fc mutant human IgG1 H chain (BDR5-2HF+TDR5-1HF), BDR5-1HF containing H429F Fc mutant human IgG1 H chain mixed with TDR5-2HF containing H429F Fc mutant human IgG2 H chain (BDR5-2HF+TDR5-2HF), and BDR5-2HF containing H429F Fc mutant human IgG2 H chain mixed with TDR5-2HF containing H429F Fc mutant human IgG2 H chain (BDR5-2HF+TDR5-2HF). Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate.Maximum cell viability was determined by culturing cells in the absence of antibody (antibody-free), and maximum mortality was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability is expressed as follows: [Experimentally determined absorbance of mAb or control treatment at 450 nm - background absorbance of cell culture medium at 450 nm]. [Figure 42] This study provides results demonstrating the survival of Colo205 cells in the presence of paired mixtures of BDR5 and TDR5 mAbs in different ratios. 10,000 Colo205 cells were cultured for 48 hours in the presence of individual mAbs containing wild-type IgG1 H chains (BDR5-1WT or TDR5-1WT), individual mAbs containing H chains with H429F modification (BDR5-1HF or TDR5-1HF), or mixtures of BDR5-1HF with TDR5-1HF mAbs in the following ratios: 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixtures shown in the plot, BDR5-1HF mAbs are designated as B-1HF and TDR5-1HF as T-1HF. Controls include maximum cell survival in the absence of mAbs (no antibody) or maximum mortality controls in the presence of sodium dodecyl sulfate (SDS). Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and viability. Survival is expressed as follows: [Experimentally determined mAb or control treatment absorbance at 450 nm - background absorbance of cell culture medium at 450 nm]. [Figure 43]This study provides results demonstrating the survival of Ramos lymphoma cells in the presence of paired mixtures of BDR5 and TDR5 mAbs in different ratios. 10,000 Ramos cells were cultured for 48 hours in the presence of individual mAbs containing wild-type H chains (BDR5-1WT or TDR5-1WT), individual mAbs containing H chains with H429F modification (BDR5-1HF or TDR5-1HF), or mixtures of BDR5-1HF with TDR5-1HF mAbs in the following ratios: 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixtures shown in the plot, BDR5-1HF mAbs are designated as B-1HF and TDR5-1HF as T-1HF. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibody (antibody-free), and maximum mortality was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability is expressed as follows: [Experimentally determined absorbance of mAb or control treatment at 450 nm - background absorbance of cell culture medium at 450 nm]. [Figure 44] This study provides results for detecting different epitopes and quantifying the binding of DR5-specific mAbs, including wild-type or Fc-mutant heavy chains of human IgA2 subclasses, to Colo205 cells. mAbs used as tissue culture supernatants from appropriately transfected Expi293 cells contained unmodified wild-type IgA2 heavy chains (BDR5-A2WT) and (TDR5-A2HF) of human IgA2 subclasses, or Fc-mutant IgA2 H chains (BDR5-A2HF) and (TDR5-A2WT) carrying the H429F mutation. Background fluorescence, determined by measuring binding of the fluorescent conjugate only (without antibody), is shown along with non-specific fluorescence of cells only (cells). [Figure 45]This study provides results demonstrating the survival of Colo205 cells in the presence of two different DR5-specific mAbs containing heavy chains of human IgA2 subclasses. Colo205 cells, 10,000 cells per well in a 96-well plate, were cultured for 48 hours in the presence of serial 2-fold dilutions of tissue culture supernatant from Expi293 cells producing the DR5 IgA mAbs used in Figure 44. The mAbs used were: BDR5-A2WT containing wild-type heavy chains of human IgA2 subclasses; TDR5-A2WT containing wild-type heavy chains of human IgA2 subclasses; BDR5-A2HF containing heavy chains of human IgA2 subclasses with H429F modification; and TDR5-A2HF containing heavy chains of human IgA2 subclasses with H429F modification. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibodies (antibody-free), and maximum mortality was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability is expressed as follows: [Experimentally determined absorbance at 450 nm - background absorbance of cell culture medium at 450 nm]. [Figure 46]The results demonstrate enhanced death of Ramos lymphoma cells with a mixture of H429F-modified mAbs that detect different molecular targets. Ramos cells were cultured separately in the presence of the anti-CD38 mAb isatuximab (Isa-WT) containing an IgG1 WT H chain, or the anti-DR5 mAb BDR5-1 (BDR5-1WT) containing an IgG1 WT H chain, or a 1:1 mixture of both mAbs (Isa-WT+BDR5-1WT), or together with the Fc-modified counterpart, isatuximab (Isa-HF) containing an H chain with H429F modification as described in Example 7, and BDR5-1 (BDR5-1HF) containing an H chain with H429F modification, or a 1:1 mixture of both (Isa-HF+BDR5-1HF). All mAbs were present in the culture at 10 μg / ml, and for mixtures, each mAb was present at 10 μg / ml relative to a total mAb concentration of 20 μg / ml. After 48 hours of incubation, cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the supernatant of CCK8-treated cells (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by cell culture in the absence of antibody (antibody-free) and by maximum cell death by cell incubation with SDS before CCK8 treatment of cells. Survival is expressed as follows: [experimentally determined absorbance at 450 nm - background absorbance of cell culture medium at 450 nm], mean absorbance at 450 nm, and four repeat values. [Figure 47] This study provides results demonstrating that H429F substitution confers complement-dependent lysis (CDC) to leukemia cells resistant to lysis with the unmodified anti-CD38 mAb mezagitamab. Complement-dependent lysis-mediated CDC was determined, in particular, using Ramos lymphoma cells opsonized with mezagitamab-WT mAb or mezagitamab-H429F mutant mAb at the indicated concentrations. Background lysis was determined in the absence of mAbs, but in the presence of complement alone (C' only). CDC (percentage of cells killed) was determined by flow cytometry using Zombie Green. [Figure 48]The results suggest that additional mutations in the Fc region component, such as K439E or S440K, can suppress H429F-modified rituximab-enhanced CDC, and that the combination of rituximab-H429F / K439E with rituximab-H429F / S440K restores efficient CDC death in Ramos cells. (A) Ramos lymphoma cells were opsonized with individual rituximab-WT(WT) or rituximab-H429F(H429F) mAbs, or with rituximab-H429F(H429F / K439E) containing an additional H chain mutation K439E, or with rituximab-H429F(H429F / S440K) containing an additional H chain mutation S440K, or with a pair of rituximab-H429F / K439E and rituximab-H429F / S440K mixtures (H429F / K439E+H429F / S440K). Background lysis was determined in the absence of mAbs, but also in the presence of complement alone (C' only, no mAbs). CDC (death %) of mAbs was measured by flow cytometry using Zombie green in the presence of a 1 / 3 dilution of human serum. Enhanced CDC mediated by the H429F modification of rituximab was suppressed by either the K439E or S440K mutation-inhibiting Fc:Fc interaction, but was found to be completely restored by a mixture of both IgG variants. (B) For binding analysis, the proteins were titrated 2x sequentially, and binding activity (to Ramos cells) was quantified by flow cytometry using a FITC conjugate anti-hIgG-Fc secondary reagent. The level of nonspecific background binding of the fluorescent conjugate to cells is shown (conjugate only); MFI = median fluorescence intensity. All mAbs gave nearly identical binding to CD20. [Figure 49]This report provides the results of flow cytometry analysis of the binding (A, C) and CDC (B, D) of purified flACE2-Fc fusion protein (A, B) and SARS-CoV spike-specific mAbs (C, D) using Ramos-S cells. For binding analysis, the proteins were titrated 2x sequentially, and binding activity (to Ramos-S cells) was quantified by flow cytometry using a FITC conjugate anti-hIgG-Fc secondary reagent. The level of nonspecific background binding of the fluorescent conjugate to cells is shown (conjugate); MFI = median fluorescence intensity. For CDC analysis, the proteins were titrated 2x sequentially, and human serum was used as the complement source. CDC lysis was quantified using Zombie Green. Background lysis by complement in the absence of the antibody was determined and shown (C' only, without mAb). [Figure 50]This provides graphical results from flow cytometry analysis showing that the synergistic and functional effects of H429F-modified flACE2-Fc fusion protein and H429F-modified anti-SARS-CoV-2 mAb further enhance complement-dependent cell death (CDC). (A) mAb S2P6-H429F (S2P6-HF) or S2P6-WT was titrated alone, or in the presence of a fixed concentration of flACE2-Fc-H429 fusion protein (final concentration of 1 μg / ml) (S2P6-HF + flACE2-Fc-HF), which when used alone mediated 23.4% CDC death (indicated by white diamond symbols), or in the presence of a fixed concentration of flACE2-Fc-WT (final concentration of 1 μg / ml) (S2P6-WT + flACE2-Fc-WT), which when used alone mediated 5.0% death (black diamond symbols). (B) The graph shows background lysis by complement (C' only) in the absence of mAb or Fc fusion protein, with arrows indicating examples of maximum synergistic effect. The CDC killing efficacy of H429F-modified flACE2-Fc was evaluated on Ramos-S cells. mAb CC40.8-H429F (CC40.8-HF) and CV3-25-H429F (CV3-25-HF) were used alone (final concentration 2.5 μg / ml) or mixed with flACE2-Fc-H429F (CC40.8-HF + flACE2-Fc-HF; CV3-25-HF + flACE2-Fc-HF; note that the final concentration of mAb was 2.5 μg / ml and the final concentration of flACE2-Fc-H429F was 1 μg / ml). The killing efficacy was evaluated by flow cytometry assay using Zombie Green. The percentage of CDC death mediated by flACE-2-Fc-WT, and background lysis by complement (C' only) in the absence of mAb or Fc fusion protein are also shown. Four replicate values ​​and SEM images are presented. The mean death percentage is shown above each column. [Figure 51]The results demonstrate that ACE2-Fc fusion proteins in three forms, namely trACE2-Fc, flACE2Fc, and EflACE2-Fc, containing either wild-type sequences or Fc region components with the H429F mutation, bind equally to Ramos-S target cells, as determined by flow cytometry analysis using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC: (A) trACE2-Fc (mean ± SEM, n=3), (B) flACE2-Fc (mean ± SEM, n=3), and (C) EflACE2-Fc (WT n=3, H429F n=1). In all panels, the level of nonspecific binding of the fluorescent conjugate to cells is shown (conjugate only); MFI = median fluorescence intensity. EC50 (nM) values ​​from curve fitting are shown. [Figure 52] This study provides results demonstrating that ACE2-Fc fusion proteins in three forms (trACE2-Fc, flACE2-Fc, and EflACE2-Fc) containing either wild-type sequences or Fc region components with the H429F mutation strongly fixate complement and direct complement-dependent cytotoxicity (CDC) in Ramos-S target cells, as determined by flow cytometry analysis of CDC in opsonized Ramos-S cells using diluted normal human serum as the complement source: (A) trACE2-Fc (mean ± SEM, n=3), (B) flACE2-Fc (mean ± SEM, n=3), (C) EflACE2-Fc (WT n=3, H429F n=1). In all panels, the level of nonspecific complement death in the absence of ACE2-Fc fusion proteins is shown for complement only (C' only). EC50 (nM) values ​​from curve fitting for the H429F protein are shown. The compatibility of the WT protein could not be determined (nd). [Figure 53]This provides a representation of immunoglobulins (antibodies) and immunoglobulin (antibody)-like molecules that demonstrate the modularity of antibodies. (A) Left panel: The definitions of immunoglobulin molecular chains and components are as shown and defined in the left panel of Figure 2. Center panel: An example of an Ab-like fusion protein is provided, demonstrating that the modularity of immunoglobulins allows for flexibility in the production of Ab-like molecules (as provided in Figure 2). In a particular drawn Ab-like fusion protein, the target recognition structure (shown as X1) is the same in all chains, as in the case of the Ab-like molecule described in Example 14 (where the EflACE2 polypeptide is separately linked to both the H chain and L chain constant domains in the CH1 domain, allowing for assembly into an H2L2 Ab-like fusion protein). Right panel: Illustrates possible Ab-like fusion proteins, including fusion to different target recognition structures (or enzymes and / or reporter molecules), in any combination of specificity (e.g., "X1 X1 X1 X1", "X1 X1 X1 X2", "X1 X1 X2 X2", "X1 X1 X2 X3", "X1 X2 X3 X4", where "X1", "X2", "X3", and "X4" represent different target recognition structures (or enzymes or reporter molecules)). (B) SDS-PAGE analysis of EflACE2-Ab-like-WT fusion protein eluted from a protein A affinity matrix with 0.4 M arginine (pH 4) (lane 1), showing that a fully disulfide-linked molecule consistent with the H2L2Ab-like configuration was achieved. This molecule includes an EflACE2 polypeptide fusion to an immunoglobulin constant heavy chain (EflACE2-CH) that self-assembles with an equivalent EflACE2 polypeptide fused to the immunoglobulin light chain constant domain (EflACE2-CL). Upon reduction with dithiothreitol, these two chains (i.e., EflACE2-CH(ACE2-CH) and EflACE2-CL(ACE2-CL); lane 2) are degraded separately. (C, D) The results show that the EflACE2-Ab-like-H429F fusion protein (H429F) strongly targets the CDC of Ramos-S target cells, while the corresponding fusion protein with the wild-type sequence (WT) H chain is ineffective.The CDC of opsonized Ramos-S cells was determined using the presence of a 1 / 3 dilution of normal human serum as a complement source. This potency CDC, mediated by the EflACE2-Ab-like-H429F fusion protein, was not attributed to different levels of opsonization compared to the EflACE2-Ab-like-WT fusion protein, as these bindings with Ramos cells expressing the SARS-CoV-2 spike were equivalent for both proteins. Binding was determined using flow cytometry analysis with an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. Background binding of anti-Ig fluorescent conjugates is shown as MFI = median fluorescence intensity; (conjugate only). [Figure 54] The purification results of the EflACE2-Ab-like-Fc-H429F fusion protein using elution with protein A and arginine are shown. (A) Protein A chromatography using a Hitrap® protein A column with gradient elution from 30 mM arginine (pH 4) to 35% 130 mM arginine (pH 4); (B) Size exclusion chromatography (SEC) of the pooled and concentrated protein A fraction containing EflACE2-Ab-like-Fc-H429F using a Superose 6 Increase 10 / 300 column with the indicated oligomeric material (HMW); and (C) SDS-PAGE analysis of the pooled protein A eluate and pooled SEC monomer fraction under non-reducing (without DTT, dithiothreitol) and reducing (with DTT) conditions. [Figure 55]This study provides results demonstrating the viability of Colo205 colorectal cells in the presence of purified DR5-specific mAbs containing IgG1 H chains with a single H429F mutation (HF) or L234A, L235A, and H429F(LA / LA / HF) mutations. The mAbs were titrated individually or mixed (BDR5-1LA / LA / HF + TDR5-1LA / LA / HF) and compared to a mixture of BDR5-1HF + TDR5-1HF, where both mAbs contained IgG1 H chains with only H429F modification. Colo205 cells, 10,000 cells per well in a 96-well plate, were cultured in the presence of serial 2-fold dilutions of the indicated mAbs. Individual mAbs were titrated starting from 1 μg / ml. For the titration of the mixture, a starting concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (absorbance at 450 nm) is a measure of cell viability and survival rate. Maximum cell viability was determined by culturing cells in the absence of antibody (antibody-free), and maximum mortality was determined by culturing cells with sodium dodecyl sulfate (SDS). Survival is expressed as follows: [Experimentally determined absorbance at 450 nm of mAb or control treatment - background absorbance of cell culture medium at 450 nm]. [Figure 56]This document illustrates the sequence comparison of hinge and constant domains in human immunoglobulin heavy (H) chain sequences, showing the domain-based structure of the H chain (i.e., CH1-hinge-CH2-CH3; amino acid position numbering is shown vertically, following Eu numbering; thus, CH1 domain amino acids 118-215, hinge amino acids 216-230, CH2 domain amino acids 231-340 including the lower hinge position, and CH3 domain amino acids 341-447). Other IgG subclasses, IgG2 and IgG4, have corresponding domain structures with amino acid sequence homology. The amino acid sequences were obtained from translations of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser). Alignment was performed using Clustal, except for manually aligned hinges. Gaps represented by dots may exist in the aligned sequences for optimal sequence alignment. Residues 231–237 (including 231 and 237) contain the structural lower hinge of the IgG subclass, which is encoded by the CH2 exon and may also be known in the literature as the proximal region of the CH2 hinge. The accessions for the H chain sequences are J00228-IGHG1 for IgG1 H chain, K01316-IGHG for IgG4 H chain, and J00230-IGHG2 for IgG2 H chain. [Figure 57]This paper provides ELISA results demonstrating the ability of IgG antibodies containing complement-fixing Fc region components, as measured by human C1q binding. Serial dilutions of mAbs, starting at 4 μg / ml, were titrated against TNP-BSA antigen adsorbed to wells of an ELISA plate to determine human C1q binding, and their C1q binding curves are shown. All mAbs exhibited similar antigen-binding activity (not shown). The mAbs recognized the TNP hapten and contained either an unmodified wild-type (WT) heavy chain of the human IgG1 subclass (TNP-IgG1-WT), an unmodified wild-type (WT) heavy chain of the human IgG2 subclass (TNP-IgG2-WT), or a mutated heavy chain of the human IgG2 subclass (indicated as mAb TNP2×4, mAb TNP2×5, mAb TNP2×15, and mAb TNP2×17). OD=OD(450nm). [Figure 58] This paper provides ELISA results demonstrating the ability of IgG antibodies containing complement-fixing Fc region components, as measured by human C1q binding. Serial dilutions of mAbs, starting at 4 μg / ml, were titrated against TNP-BSA antigen adsorbed to wells of an ELISA plate to determine human C1q binding, and their C1q binding curves are shown. All mAbs exhibited similar antigen-binding activity (not shown). The tested mAbs recognized TNP and contained either an unmodified wild-type (WT) heavy chain of the human IgG1 subclass (mAb TNP-IgG1-WT), an unmodified wild-type (WT) heavy chain of the human IgG2 subclass (TNP-IgG2-WT), or a mutant heavy chain of the human IgG1 subclass (shown as TNP1×32 in (A) and as mAb TNP1×1, mAb TNP1×2, mAb TNP1×3, mAb TNP1×5, mAb TNP1×9, mAb TNP1×10, mAb TNP1×12, mAb TNP1×14, and mAb TNP1×15 in (B)). OD=OD(450nm). [Figure 59]This paper provides ELISA results demonstrating the ability of IgG antibodies containing complement-fixing Fc region components, as measured by human C1q binding. Serial dilutions of mAbs, starting at 4 μg / ml, were titrated against TNP-BSA antigen adsorbed to wells of an ELISA plate to determine human C1q binding, and their C1q binding curves are shown. All mAbs exhibited similar antigen-binding activity (not shown). The tested mAbs recognized the TNP hapten and contained either an unmodified wild-type (WT) heavy chain of the human IgG1 subclass (mAb TNP-IgG1-WT), an unmodified wild-type (WT) heavy chain of the human IgG2 subclass (mAb TNP-IgG2-WT), or a mutant heavy chain of the human IgG1 subclass, indicated as mAb TNP1×16, mAb TNP1×22, mAb TNP1×23, mAb TNP1×24, mAb TNP1×25. OD=OD(450nm). [Figure 60] This report provides results of flow cytometry quantification of the binding of SEC-purified mAbs to target cell surface antigens. The mAbs detected CD20, were based on the rituximab variable domain, and contained either an unmodified wild-type heavy chain of the IgG2 or IgG1 subclass, or a mutant IgG2 heavy chain containing one or more mutations. Binding was evaluated by flow cytometry in CD20-expressing Ramos lymphoma cells. The data show that mAbs containing IgG2-based heavy chains bound to each other similarly and to Rit-IgG2-WT, and in all cases, the binding levels of these IgG2-based mAbs were considerably lower than those of rituximab containing an unmodified wild-type heavy chain of the IgG1 subclass (Rit-IgG1-WT). HF was the H429F mutation, EG was the E430G mutation, LLGG was the PVA(233-236)ELLG mutation, and FLGG was the PVA(233-236)EFLG mutation. [Figure 61]The results demonstrate that the H429F modification of the mutant provides superior CDC compared to mAbs carrying the H chain mutation without a Stellabody. Complement-dependent cytotoxicity (CDC) of target cells was determined using Daudi lymphoma cells opsonized with the indicated concentrations of mAbs and normal human serum diluted to 1 / 3 as a complement source. CDC (percentage of cell death) was determined by flow cytometry using Zombie Green. Complement control background CDC (C' only without mAb) in the absence of mAbs is shown. Rit-IgG2-WT containing unmodified wild-type heavy chains did not mediate detectable CDC, but all mAbs containing the specifically mutated IgG2 H chains did. (A) CDC mediated by Rit-IgG2-LLGG or Rit-IgG2-FLGG was superior to CDC mediated by unmodified Rit-IgG2-WT, which did not mediate CDC. (B) CDC mediated by mAbs Rit-IgG2-FLGG HF, Rit-IgG2-LLGG HF, Rit-IgG2-FLGG-EG, Rit-IgG2-LLGG EG, Rit-IgG2-HF, Rit-IgG2-LLGG, or Rit-IgG2-FLGG was superior to CDC mediated by Rit-IgG2-WT mAbs containing unmodified H chains that could not mediate CDC. Background lysis control (C' without mAbs), i.e., CDC in the presence of complement (serum) but without mAbs, is shown in both panels. HF is the H429F mutation, EG is the E430G mutation, LLGG is the PVA(233~236)ELLG mutation, and FLGG is the PVA(233~236)EFLG mutation. [Figure 62]This report provides results from a competitive binding assay of anti-CD20 rituximab mAbs in Daudi cells. mAbs were titrated sequentially 2-fold from an initial concentration of 20 μg / mL, and competition for binding to the cell surface with biotinylated anti-CD20 IgG1-WT (RIT-IgG1-WT-biotin) was measured by flow cytometry. (A) A comparison of RIT-IgG1-WT and RIT-IgG2-WT shows that IgG1 is a better competitor than IgG2, despite having the same VL and VH domains. A control for nonspecific effects was used, where each mAb was titrated in the absence of biotinylated anti-CD20 IgG1-WT antibody and subsequently incubated with a fluorescent conjugate (streptavidin-FITC) (mean ± SEM, n=4). (B) A comparison of IgG1, IgG2, IgG3, and IgG4 human antibody subclasses shows that the IgG2 subclass uniquely exhibits reduced binding ability to the CD20 antigen (mean ± SEM, n=2). All panels show the maximum binding signal of biotinylated anti-CD20 IgG1-WT in the absence of competitors, and nonspecific binding of streptavidin-FITC alone (conjugate only as control). MFI = median fluorescence intensity. [Figure 63]This report provides binding profiles of anti-CD20 rituximab mAbs in target Ramos cells. mAb binding to Ramos cells was detected using goat anti-human IgG Fc FITC and flow cytometry. (A) RIT-IgG1 and RIT-IgG2 containing WT or H429F mutant heavy chains, and rituximab formalized as IgG2, suggest decreased binding to target cells. mAbs were titrated sequentially 2-fold from 2 μg / mL (mean ± SEM, n=2). (B) RIT-IgG2 mAbs containing the lower hinge residue of IgG4 (RIT-IgG2-EFLGG), the lower hinge residue of IgG1 (RIT-IgG2-ELLGG), the H429F mutation of the CH3 domain (RIT-IgG2-H429F), or a combination of the lower hinge and the H429F mutation (RIT-IgG2-EFLGG-H429F and RIT-IgG2-ELLGG-H429F). mAbs were titrated sequentially 2-fold from 20 μg / mL (mean ± SEM, n=2). Binding profiles of IgG1-WT mAbs are shown for comparison. Levels of nonspecific binding of anti-IgG Fc-FITC to cells are shown (▲conjugate only). MFI = median fluorescence intensity. [Figure 64]This study provides results demonstrating the ability of anti-CD38 mAb variants to kill Ramos cells in the presence of serum. Purified mAbs were titrated 2-fold sequentially, and CDC was measured by flow cytometry. Normal human serum diluted to one-third was used as a complement source. (A) Comparison of cytotoxic effects of RIT-IgG1 and RIT-IgG2 containing WT or H429F mutant heavy chains. mAbs were titrated 2-fold sequentially from 2 μg / mL (n=1). (B) Comparison of cytotoxic effects of RIT-IgG2 mAbs containing the lower hinge residue of IgG4 (RIT-IgG2-EFLGG), the lower hinge residue of IgG1 (RIT-IgG2-ELLGG), the H429F mutation of the CH3 domain (RIT-IgG2-H429F), or combinations of the lower hinge and H429F mutation (RIT-IgG2-EFLGG-H429F and RIT-IgG2-ELLGG-H429F). For comparison, IgG2-WT and IgG1-WT mAbs were included. mAbs were titrated sequentially from 40 μg / mL to a 2x decrease (mean ± SEM, n=3). Nonspecific target cell death in the absence of mAbs (C' only) is shown as a filled triangle (▲). [Figure 65]Schematic diagrams of the light chain and M1, M2, and M3 mutant IgG2 H chains are shown, including only the VH, CH1, upper hinge, core hinge, and lower hinge sequences. Each shows the expected disulfide bond between the light chain (LC) and heavy chain (HC) in the upper hinge region of each IgG2 mutant mAb (M1, M2, and M3) (drawn as a black line). The core hinge, CPPC, forms an inter-heavy chain disulfide not shown. Cysteine ​​residues that can form the remaining inter-chain disulfide bonds are shown as black circles, and serine-substituted cysteine ​​residues are shown as black circles with white crosses. (A) The M1 mutant includes the heavy chain upper hinge sequence of IgG2, as well as cysteine-to-serine substitutions at position 131 (C131S) and position 220 (C220S) of the CH1 domain, and is therefore expected to form a disulfide bond between the cysteine ​​at position 214 of the light chain (C214) and the cysteine ​​at position 219 (C219) of the heavy chain upper hinge region. (B) The M2 mutant includes the heavy chain upper hinge sequence of IgG2, as well as cysteine-to-serine substitutions at position 131 (C131S) and position 219 (C219S), and is therefore expected to form a disulfide bond between the cysteine ​​at position 214 of the light chain (C214) and the cysteine ​​at position 220 (C220) of the heavy chain upper hinge region. The (C)M3 mutant contains the heavy chain upper hinge sequence (EPKSCDKTHT) of IgG1 and a cysteine-to-serine substitution at position 131 (C131S), and is therefore expected to form a disulfide bond between the cysteine ​​at position 214 of the light chain (C214) and the cysteine ​​at position 220 of the heavy chain upper hinge region (C220). Because this mutant has the IgG1 upper hinge sequence, it has two additional amino acids. [Figure 66]Each panel provides size exclusion chromatography (SEC) elution profiles of various isatuximab IgG1 and IgG2 mAbs. Protein A affinity purified IgG was further purified by SEC at pH 7.2. (A)Isa-IgG1-WT, (B)Isa-IgG2-WT, (C)Isa-IgG2-M1, (D)Isa-IgG2-M2, (E)Isa-IgG2-M3, (F)Isa-IgG2-C131S-C219S, and (G)Isa-IgG2-EFLGG, each consisting of a single IgG peak with minimal oligomeric species. [Figure 67] (A) The results of unreduced SDS-PAGE analysis of SEC-purified mAbs are shown, indicating that each mAb migrated with a mass of approximately 150 kDa, as expected for IgG(H2L2). (B) Furthermore, the purified mAbs were reduced with dithiothreitol and investigated by SDS-PAGE analysis, showing that each mAb was degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. M = molecular weight marker. [Figure 68]This report provides the results of flow cytometry quantification of anti-CD38 mAb binding to Raji cells. mAbs were titrated 3-fold sequentially from 5 μg / mL, and binding to Raji cells was detected by flow cytometry using the goat (Fab')2 anti-human-IgG Fc FITC secondary reagent. (A) Isa-IgG1-WT and Isa-IgG2-WT, suggesting reduced binding to target cells with isatuximab formalized as IgG2. (B) Isa-IgG2-WT and hinge-modified IgG2 mutants (Isa-IgG1-M1, Isa-IgG2-M2, and Isa-IgG2-M3). (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S, and Isa-IgG2-EFLG, showing that the effect of either upper hinge mutations or lower hinge mutations alone on binding is minor. The level of nonspecific binding of goat anti-human-IgG Fc FITC to cells is shown in all panels (▲conjugate only). MFI = median fluorescence intensity. Mean ± SEM, n=4. P-values ​​were determined by two-way ANOVA compared to Isa-IgG2-WT for the main effect. Dunnett's multiple comparison test was used for B and C. [Figure 69]This report provides results of a flow cytometry competitive binding assay for the binding of anti-CD38 isatuximab mAbs to Raji cells. The mAbs were titrated 3-fold sequentially and used to compete for binding with biotinylated Isa-IgG1-WT. RIT-IgG1-WT was used as a negative control that did not compete with biotinylated Isa-IgG1-WT for binding. (A) Isa-IgG1-WT and Isa-IgG2-WT show that IgG2-WT exhibits reduced binding to target cells (mean ± SEM, n=6). (B) Isa-IgG2-WT, Isa-IgG1-M1, Isa-IgG2-M2, and Isa-IgG2-M3 show that hinge modification enhances binding and competition (mean ± SEM, n=6). (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S, and Isa-IgG2-EFLGG show no effect on binding by either upper hinge mutation or lower hinge mutation alone. In all panels, the level of nonspecific binding of the fluorescent conjugate (streptavidin-FITC) to cells is shown (▲conjugate only), and the maximum binding of biotinylated Isa-IgG1-WT (i.e., in the absence of a competitor mAb) is shown (Isa-IgG1-WT-biotin). MFI = median fluorescence intensity. P values ​​were determined for the main effect compared to Isa-IgG2-WT by two-way ANOVA and Dunnett's multiple comparison test. [Figure 70]This study provides results demonstrating that anti-CD38 mAbs mediate the death of Raji cells in the presence of serum. Purified mAbs were titrated 3-fold sequentially, and CDC was measured by flow cytometry. Normal human serum diluted to 1 / 3 was used as a complement source. (A) Isa-IgG1-WT and Isa-IgG2-WT (mean ± SEM, n=6), (B) Isa-IgG2-WT, Isa-IgG1-M1, Isa-IgG2-M2, and Isa-IgG2-M3 (mean ± SEM, n=6), (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S, Isa-IgG2-EFLGG (mean ± SEM, n=3). P-values ​​for A, B, and C were determined by two-way ANOVA with main effects compared to Isa-IgG2-WT using Dunnett's multiple comparison test for B and C. (D) Cell death in the presence of each mAb at the highest concentration tested (5 μg / mL) was measured with and without human serum as a complement source (+C') (mean ± SEM, n=4). [Figure 71] This study provides results demonstrating C1q binding as a C1 complex to opsonized Ramos cells. Ramos cells were opsonized with 5 μg / mL anti-CD38 mAb, IgG-WT and IgG2 mutants, followed by the addition of normal human serum (NHS, diluted to 1 / 3 in DPBS+BSA) as a source of C1. C1 binding was detected with anti-C1q rabbit polyclonal antibody, followed by a fluorescently labeled anti-rabbit-Fc probe. Gray bars indicate C1 binding to mAb-opsonized cells, and black bars indicate background fluorescence levels from mAb-opsonized cells in the absence of C1q. Background C1 binding to mAb-free cells is shown for the "mAb-free" control. Mean ± SEM, n=4. MFI = median fluorescence intensity. [Figure 72]This study provides size exclusion chromatography (SEC) profiles of various isatuximab IgG1 and IgG2 mAbs generated with and without the H429F mutation. After protein A affinity purification, the mAbs were further purified by SEC at pH 7.2. SEC elution profiles are shown for (A) Isa-IgG1-WT and Isa-IgG1-H429F, (B) Isa-IgG2-WT and Isa-IgG2-H429F, (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F, (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F, (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F, and (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F, each consisting of a single, homogeneous IgG peak with minimal oligomeric species. The fraction containing monomeric IgG is enclosed in a dotted line frame. This fraction was pooled for subsequent analysis of mAb function. [Figure 73] (A) The results of unreduced SDS-PAGE analysis of the SEC-purified mAbs are shown, indicating that each primarily migrated with a mass of approximately 150 kDa, as expected for IgG(H2L2). (B) The sample was also reduced with dithiothreitol and investigated by SDS-PAGE, showing that each mAb was degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. M = molecular weight marker. [Figure 74]This report provides the results of flow cytometry quantification of the binding of anti-CD38 isatuximab mAbs with and without the H429F mutation to Raji cells. The MAbs were titrated 3-fold sequentially, and binding activity was quantified by flow cytometry using goat anti-human IgG Fc-FITC. (A) Isa-IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5), (B) Isa-IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=6), (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=4), (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5), (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5), (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5). The level of nonspecific binding of the goat anti-human IgG Fc FITC conjugate to cells is shown in all panels (▲conjugate). MFI = median fluorescence intensity. The P-values ​​for the nonlinear fit were determined by two-way ANOVA with main effects. [Figure 75]This report provides the results of a competitive binding assay comparing the binding of each anti-CD38 isatuximab mAb to its H429F equivalent. The mAbs were titrated 3-fold sequentially, and competition for binding to the cell surface with biotinylated anti-CD38 IgG1-WT (Isa-IgG1-WT-biotin) was measured by flow cytometry. (A) Isa-IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5), (B) Isa-IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=6), (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=3), (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5), (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5), (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5). In all panels, the level of nonspecific binding of the fluorescent conjugate (streptavidin-FITC) to cells is shown (▲conjugate only), and the maximum binding of biotinylated Isa-IgG1-WT is shown (Isa-IgG1-WT-biotin). MFI = median fluorescence intensity. P-values ​​were determined by two-way ANOVA. [Figure 76]This study provides results demonstrating isatuximab mAbs and their H429F equivalents mediated CDC in Raji cells. SEC-purified mAbs were titrated 3-fold sequentially, and CDC efficacy was determined by flow cytometry using 1 / 3 diluted normal human serum as a complement source. (A) Isa-IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5), (B) Isa-IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=5), (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=3), (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5), (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5), (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5). In all panels, the level of nonspecific target cell death (C' only) in the absence of mAbs is shown as a filled triangle (▲). P-values ​​were determined for the main effects by two-way ANOVA. [Figure 77] The results show the death of Raji target cells in the presence of each mAb at the highest concentration tested (5 μg / mL) with and without human serum as a complement source (+C'), revealing that the observed CDC activity was dependent on the presence of complement. Plots for Isa-IgG variants are shown: (A) IgG1-WT and IgG1-H429F, (B) IgG2-WT and IgG2-H429F, (C) IgG2-EFLGG and IgG2-EFLGG-H429F, (D) IgG2-M1 and IgG2-M1-H429F, (E) IgG2-M2 and IgG2-M2-H429F, (F) IgG2-M3 and IgG2-M3-H429F. Mean ± SEM, n=4. P-values ​​were determined by pairwise t-tests with Holm correction for multiple comparisons. *p<0.05, ns=not significant. [Figure 78]This study provides results indicating that the H429F mutation enhances C1 binding to Ramos cells opsonized with anti-CD38 IgG1-WT or IgG2-WT, or the indicated modified IgG2 variant. Ramos cells were opsonized with 5 μg / mL anti-CD38 mAb, and then normal human serum (diluted to 1 / 3 in DPBS+BSA) was added as a source of C1. C1 binding was detected with anti-C1q rabbit polyclonal antibody, followed by fluorescently labeled anti-rabbit-Fc donkey antiserum. This is expressed as median fluorescence intensity (MFI). Gray bars indicate C1 binding to mAb-opsonized cells in the presence of serum, and black bars indicate background fluorescence levels from mAb-opsonized cells in the absence of serum C1 (mean ± SEM, n=4). Background C1 binding to mAb-free cells is shown for the "mAb-free" control. [Figure 79] This document provides size exclusion chromatography (SEC) profiles of various isatuximab IgG1 and IgG2 mAbs with the indicated mutations. After protein A affinity purification, the mAbs were further purified by SEC at pH 7.2. The SEC elution profiles of the Isa-IgG variants are shown. (A)IgG1-H429F, (B)IgG1-LALA, (C)IgG1-LALA-H429F, (D)IgG1-K274Q, (E)IgG1-K274Q-H429F, (F)IgG1-H268E-A327D-H429F, (G)IgG2-LALA-H429F, and (H)IgG2-Q274K-H429F, each consisting of a single homogeneous IgG peak with minimal oligomeric species. Fractions containing monomeric IgG are enclosed in dotted lines. These fractions were pooled for subsequent analysis of mAb function. [Figure 80] (A) The results of unreduced SDS-PAGE analysis of SEC-purified mAbs are shown, indicating that each primarily migrated with a mass of approximately 150 kDa, as expected for IgG(H2L2). (B) Furthermore, the sample was reduced with dithiothreitol and investigated by SDS-PAGE, showing that each mAb was degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. [Figure 81]This report provides the results of mAb binding assays in Raji cells to test the effects of various mutations in CH2 of anti-CD38 isatuximab IgG1 and IgG2 mAbs with and without H429F modification. mAbs were titrated 3-fold sequentially, and binding was quantified by flow cytometry using goat anti-human IgG Fc-FITC secondary reagent. Binding profiles of Isa-IgG variants are shown: (A) IgG1-WT, IgG1-H429F, IgG1-LALA, and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F, and IgG1-H268E-A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F, and IgG2-Q274K-H429F. Note that in Panel B, the IgG1-WT and IgG1-H429F curves are similar to those in Panel A and are included for comparison. The level of nonspecific binding of the fluorescent conjugate to cells is shown (▲conjugate). MFI = median fluorescence intensity. Mean ± SEM, n=3. The P-value was determined by performing a two-way ANOVA for the main column effect and comparing it with IgG-WT using Dunnett's multiple comparison test. [Figure 82]This report presents the results of a competitive binding assay in Raji cells to test the effects of various mutations in the CH2 of anti-CD38 isatuximab IgG1 and IgG2 mAbs with and without H429F modification. The mAbs were titrated 3-fold sequentially, and the binding activity to competing biotinylated isatuximab IgG1-WT was determined by flow cytometry. The Isa-IgG competitors were (A) IgG1-WT, IgG1-H429F, IgG1-LALA, and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F, and IgG1-H268E-A327D-H429F; and (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F, and IgG2-Q274K-H429F. Note that in Panel B, the IgG1-WT and IgG1-H429F binding curves are similar to those in Panel A and are included for comparison. The P-value was determined by performing a two-way ANOVA for the main column effect and comparing it with IgG-WT using Dunnett's multiple comparison test. In all panels, the level of nonspecific binding of the fluorescent conjugate (streptavidin-FITC) to cells is shown (▲conjugate only), and the maximum binding of biotinylated Isa-IgG1-WT is shown (Isa-IgG1-WT-biotin). MFI = median fluorescence intensity. Mean ± SEM, n=2. [Figure 83]This report provides the results of a cytotoxicity assay testing the effects of various mutations in the CH2 of anti-CD38 Isa-IgG1 and IgG2 mAbs with and without H429F modification on the death of Raji cells in the presence of serum. The Isa-IgG variants were (A) IgG1-WT, IgG1-H429F, IgG1-LALA, and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F, and IgG1-H268E-A327D-H429F; and (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F, and IgG2-Q274K-H429F. Note that in Panel B, the same binding curves for IgG1-WT and IgG1-H429F as in Panel A are included for comparison. The p-value was determined by performing a two-way ANOVA for the main column effect and comparing it to IgG-WT using Dunnett's multiple comparison test. In all panels, the mutant mAb was compared to the relevant unmodified counterpart to determine the effect of the mutation, and the level of nonspecific target cell death (C' only) in the absence of the mAb is shown as a filled triangle (▲). Mean ± SEM, n=3. [Figure 84] The results demonstrate the death of Raji target cells in the presence of each mAb at the highest concentration tested (5 μg / mL) with and without human serum as a complement source (+C'), revealing that the observed CDC activity was dependent on the presence of complement. The Isa-IgG variants were (A) IgG1-WT, IgG1-H429F, IgG1-LALA, and IgG1-LALA-H429F, (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F, and IgG1-H268E-A327D-H429F, (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F, and IgG2-Q274K-H429F, with each experiment including an mAb-free control for nonspecific cytotoxicity. Note that Panel B includes the same data for IgG1-WT and IgG1-H429F as Panel A for comparison. Mean ± SEM, n=4. [Figure 85] This study provides results demonstrating the binding of purified C1q to target Ramos cells opsonized with anti-CD38 mAb. Ramos cells were opsonized with 5 μg / mL anti-CD38 mAb, followed by the addition of 10 μg / mL purified human C1q. C1q binding was detected using an anti-C1q rabbit polyclonal antibody, followed by a fluorescently labeled anti-rabbit-Fc probe. The Isa-IgG variants were (A) IgG1-WT, IgG1-H429F, IgG1-LALA, and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F, and IgG1-H268E-A327D-H429F; and (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F, and IgG2-Q274K-H429F. Note that in Panel B, the same data for IgG1-WT and IgG1-H429F as in Panel A are included for comparison. Gray bars indicate C1q binding to mAb-opsonized cells (●+C1q), and white bars indicate background fluorescence levels from mAb-opsonized cells in the absence of C1q (o-C1q). Background binding of C1q to mAb-free cells is shown compared to the "mAb-free" control. (Mean ± SD, n=2). MFI = Median fluorescence intensity. [Figure 86] This document provides size exclusion chromatography (SEC) elution profiles for S2P6 IgG1 mAbs. The mAbs were first purified from the culture supernatant by protein A affinity chromatography, followed by purification by SEC at pH 7.2. The SEC elution profiles for (A) S2P6-IgG1-WT and (B) S2P6-IgG1-H429F are shown, each consisting of a single, homogeneous IgG peak representing a monomer with minimal oligomeric species. [Figure 87]This report provides the results of SDS-PAGE analysis of SEC-purified mAbs, S2P6-IgG1-WT and S2P6-IgG1-H429F. Before reduction with dithiothreitol (DTT-), both mAbs migrated primarily with the expected mass of approximately 150 kDa of IgG(H2L2). After reduction with DTT (DTT+), each mAb was degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. [Figure 88] This document provides size exclusion chromatography (SEC) elution profiles for various S2P6 IgG2 mAbs generated. The mAbs were purified first by protein A affinity purification, followed by purification by SEC at pH 7.2, as shown. The SEC elution profiles for (A) S2P6-IgG2-WT, (B) S2P6-IgG2-H429F, (C) S2P6-IgG2-M1, (D) S2P6-IgG2-M1-H429F, (E) S2P6-IgG2-M2, (F) S2P6-IgG2-M2-H429F, (G) S2P6-IgG2-M3, and (H) S2P6-IgG2-M3-H429F are shown, each consisting of a single monomeric IgG peak with minimal oligomeric species. [Figure 89] (A) We provide the results of unreduced SDS-PAGE analysis of SEC-purified S2P6 IgG2 mAbs, showing that each mAb primarily migrated with the expected mass of approximately 150 kDa of IgG(H2L2). (B) We also show that the sample was reduced with dithiothreitol and investigated by SDS-PAGE analysis, showing that each mAb was degraded into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. [Figure 90]This paper provides results demonstrating antigen binding and cytotoxicity of rituximab-based mAb variants. (A) Anti-CD20 variants RIT-IgG1-WT and RIT-IgG1-H429F were titrated 3-fold consecutively from 10 μg / mL, and binding to Ramos-S cells was determined by flow cytometry using the FITC conjugate F(ab')2 fragment of goat anti-human IgG-Fc antiserum (n=4). Nonspecific binding was determined by staining cells with FITC goat anti-human IgG-Fc antiserum in the absence of mAbs (conjugate only). Ramos-S cells are Ramos cells expressing the SARS-CoV-2 (Wuhan) spike protein. (B) Anti-CD20 rituximab variants kill Ramos-S cell targets in the presence of serum. Purified mAbs were titrated 3-fold consecutively from 10 μg / mL, and CDC was measured by flow cytometry (n=3). Normal human serum diluted to one-third was used as the complement source. Concentration-dependent cytotoxic effects of RIT-IgG1-WT and RIT-IgG1-H429F are shown. Nonspecific target cell death (C' only) in the absence of mAbs is indicated by a filled star (★). [Figure 91] This report provides a graphical representation of the binding of SARS-CoV-2 spike-specific S2P6-based mAbs to Ramos-S cells. S2P6 mAbs, as shown, included unmodified IgG1, IgG2 H chains, or mutant H chain variants and were titrated sequentially 3-fold from 10 μg / ml. Their binding to Ramos-S cells was determined by flow cytometry using the FITC conjugate F(ab')2 fragment of goat anti-human IgG-Fc antiserum (n=4). Non-specific binding was determined by staining cells with FITC goat anti-human IgG-Fc antiserum in the absence of mAbs (conjugate only). All mAbs showed similar target cell binding. [Figure 92]This study provides results demonstrating that the S2P6 mAb variant kills Ramos-S cell targets in the presence of serum. Ramos-S cells are Ramos cells expressing the SARS-CoV-2 (Wuhan) spike protein. Purified mAbs were titrated 3-fold sequentially from 10 μg / mL (n=3), and CDC was measured by flow cytometry. Normal human serum diluted to 1 / 3 was used as a complement source. The concentration-dependent cytotoxic effects of S2P6 mAbs shown are (A) IgG1-WT, IgG1-H429F, IgG2-WT, and IgG2-H429F, (B) IgG2-M1-H429F, IgG2-M2-H429F, IgG2-M3-H429F, and (C) IgG2-M1, IgG2-M2, and IgG2-M3. For comparison, curves for IgG2-M1-H429F, IgG2-M2-H429F, and IgG2-M3-H429F from panel B are included. (D) Individual experiments from A-C were analyzed by setting a baseline using a complement-only control and calculating the area under the curve (AUC) as a plot of CDC death against log10 mAb concentration. Mean ± SD, n=3. P-values ​​were determined by one-way ANOVA and Tukey's multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001 [Figure 93] This provides CDC-based grouping of IgG and variant IgG compared to IgG2-WT. [Modes for carrying out the invention]

[0038] This disclosure relates to immunotherapy proteins comprising one or more immunoglobulin heavy chain polypeptides, each comprising one or more constant heavy chain domains, including one or more constant heavy chain domains, including one or more constant heavy chain domains, including one or more polypeptides comprising an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chains, and each of the one or more polypeptides additionally comprising at least one C1q binding modification.

[0039] The amino acid numbering used herein is based on the so-called Eu numbering system, which is related to the sequence numbering used to describe the human IgG1 myeloma protein called Eu (Edelman GM et al., Proc Natl Acad Sci USA 63(1):78-85, 1969). According to this system, for example, H429 of IgG1 (i.e., histidine at position 429) occurs at position 429 of the Eu sequence. Therefore, in any immunoglobulin molecule such as an antibody or a fragment thereof, or an antibody-like molecule, an amino acid at a given position number corresponds to or is related to the corresponding amino acid residue position number in the Eu sequence.

[0040] The heavy (H) chains of immunoglobulins such as IgG1 are modular, multifunctional monomeric polypeptides. (The immunoglobulin or antibody structure of a prototype with the H429 mutation can be thought to consist of a dimeric protein containing two monomeric body mass (H) chain polypeptides, each optionally associating with a light (L) chain, thus resulting in the H2L2 configuration (see Figure 2). However, other forms of immunoglobulin molecules can be formed, including when one heavy chain (H1) associates with one light (L1) chain in the H1L1 configuration, and when two heavy (H) chains dimerize in the absence of a light (L) chain to produce a heavy chain complex in the H2 configuration (Figure 2). When the light (L) and heavy (H) chains associate in the H1L1 configuration, the molecule can dimerize in this configuration to obtain a typical immunoglobulin structure designated as H2L2. This is exemplified by typical human IgG class immunoglobulins, where two heavy (H) chains are covalently bonded to each other and a light (L) chain is covalently bonded to each heavy (H) chain.)

[0041] The dimeric form of the H1L1 immunoglobulin molecule (i.e., the H2L2 protein) is the fundamental structural "unit" of all human Ig classes (i.e., IgG, IgE, IgD, IgA, and IgM), and indeed, it is also the basis for most mammalian immunoglobulin classes, with the exception of this form being known (for example, camelid immunoglobulins can form heavy chain dimers without a light chain (abbreviated as H2)). Thus, for example, the human immunoglobulin G (IgG) molecule exists physiologically as a single H2L2 unit in solution. However, other covalent higher-order oligomers of the basal H2L2 unit exist naturally, particularly common to IgM and IgA.

[0042] For example, IgM can form covalent pentamers or hexameric rings of the H2L2 form, where each H2L2 unit has a disulfide bond to an adjacent H2L2 unit, forming a pentamer oligomer (H2L2) 5 or a hexamer oligomer (H2L2) 6 (Eskeland T and TB Christensen, Scand J Immunol 4(3):217-228, 1975). Notably, the hexameric form of IgM is the most potent effector of the classical complement pathway (one of the two major effector systems of the innate immune system initiated by immunoglobulins), activated by avid binding of the soluble hexamer protein C1q (Eskeland T and TB Christensen, 1975, Randall TD et al., Proc Natl Acad Sci USA 89:962-966, 1992, Hughey CT). (et al., J Immunol 161:4091-4097, 1998, and Randall TD et al., Eur J Immunol 20:1971-1979, 1990). IgM is also well recognized as the most potent agglutinin due to its covalent oligomerization, which results in the presence of 10-12 specific and identical antigen-binding variable domains in each IgM pentamer or hexamer. Since individual antigen-recognition structures (e.g., Fab fragments) have a defined affinity for a target antigen site or epitope with strength defined by their monovalent binding to a single target structure, the presence of multiple antigen-recognition structures within a single molecule (e.g., 12 antigen-binding variable domains in each IgM hexamer) confers stronger or more avid binding of oligomeric immunoglobulins. Thus, oligovalent binding arises from the combined strength of the individual antigen-recognition interactions between the IgM oligomer and its target antigen epitope. In comparison, the avidity of IgG (which has two antigen-recognizing structures (Fab) per H2L2 unit) arises solely from the interaction of these two antigen-recognizing structures.

[0043] Immunoglobulins can also be considered modular, multifunctional proteins in which the target recognition structure is linked to the functionally activated structure by a flexible linker (i.e., the antigen recognition structure provided by the V domain of Fab is linked to the Fc region by a flexible hinge), and in the case of a typical immunoglobulin, each H chain and each L chain contains different domains that can be considered structural or functional modules (Figure 2). The H chain, in particular, contains a variable sequence antigen target recognition domain (V H The domain is followed by a series of constant domains that are specific to the Ig class heavy chain, such as IgG or IgA, and sequence analysis shows that these constant domains are related among immunoglobulin classes (see Figures 3 and 4). Therefore, V H The domain is followed by a first constant domain (CH1). This first constant domain is connected to a second constant domain (CH2) by a flexible polypeptide that acts as a linker known as a hinge region, followed by a third constant domain (CH3). In this way, a typical H chain consists of separate structural molecules from the amino terminus (i.e., the NH2 terminus V). H -CH1-hinge-CH2-CH3 (Figure 2), exemplified by the human IgG1 H chain sequence (SEQ ID NO: 3), is conserved across all human heavy chains (Figures 3, 4) exemplified by human IgG3 (SEQ ID NO: 4), IgG4 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgA1 (SEQ ID NO: 7), IgA2 (SEQ ID NO: 8), and mammalian immunoglobulins in general. Similarly, the light (L) chain has a variable antigen target recognition domain (V L The domains are followed by stationary domains specific to the light chain class.

[0044] It should be noted that polypeptides containing heavy chains of the IgG subclass contain a structurally specific segment called the hinge region. The hinge region of an IgG polypeptide is generally considered to consist of an upper hinge sequence, a core hinge sequence, and a lower hinge sequence. According to EU numbering, the upper hinge contains residues 216–225 (including 216 and 225), the core hinge contains residues 226–230 (including 226 and 230), and the lower hinge contains residues 231–237. It should be noted that the amino acid residues containing the structural lower hinge of an IgG polypeptide are encoded by the CH2 exon and may also be known in the literature as the proximal hinge region of CH2. In this specification, reference to CH2 includes reference to the lower hinge residues.

[0045] In some embodiments, the immunotherapy protein is a human immunotherapy protein. In some embodiments, the immunotherapy protein is a humanized or chimeric immunotherapy protein. In some embodiments, the immunotherapy protein is a non-human primate immunotherapy protein (e.g., monkey). In some embodiments, the immunotherapy protein is a rodent (e.g., mouse immunotherapy protein).

[0046] In one embodiment, the immunotherapy protein comprises one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and / or a hinge region. In one embodiment, the immunotherapy protein comprises the CH1 domain. In one embodiment, the immunotherapy protein comprises the CH2 domain. In one embodiment, the immunotherapy protein comprises the CH3 domain. In one embodiment, the immunotherapy protein comprises a hinge region. In one embodiment, the immunotherapy protein is derived from an IgG1 heavy chain polypeptide. In one embodiment, the immunotherapy protein is derived from an IgG2 heavy chain polypeptide. In one embodiment, the immunotherapy protein is derived from an IgG3 heavy chain polypeptide. In one embodiment, the immunotherapy protein is derived from an IgG4 heavy chain polypeptide.

[0047] Given their modularity, immunoglobulins offer a versatile platform for the creation of a diverse range of immunotherapeutic or diagnostic molecules, including bivalent antibodies, e.g., mAbs derived from classical hybridomas; heavy chain antibodies containing heavy chain dimers (e.g., camelid antibodies; Hamers-Casterman C et al., Nature 363:446, 1993); antibody-like (Ab-like) molecules (including several fusion proteins containing at least a CH3 domain (or at least a CH4 domain)); and other immunoglobulin derivatives known to those skilled in the art, as summarized in the "Periodic Table of Antibodies" (https: / / absoluteantibody.com / periodic-table-of-antibodies / #, the entire disclosure of which is incorporated herein by reference). Other specific examples include antibodies and antibody fragments, e.g., single-chain Fv(scFv) antibody fragments, asymmetric bispecific antibodies (WO2012 / 058768), asymmetric and bispecific antibody-like molecules with chain exchange domains (SEED or Seed bodies) (WO2007 / 110205), bivariable domain immunoglobulins (US7,612,181), knob-into-hole antibody forms (WO1998 / 050431), duobody molecules (WO2011 / 131746), IgG-like bispecific antibodies (Shen J et al., J Immunol Methods 318(1-2):65-74, 2007), and fusion proteins containing Fc or Fc region components, such as scFv fusions and double scFv fusions.

[0048] This specification uses numerous terms that are well known to those skilled in the art. Nevertheless, for the sake of clarity, many of these terms are defined below.

[0049] As used herein, the term "antibody" should be understood to include polyclonal antibodies (pAbs), monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, antibody mixtures (e.g., recombinant polyclonal antibodies), such as those generated by methods well known to those of skill in the art for producing multiple antibodies having different specificities from a single host cell line (e.g., Oligoclonics® technology: Merus BV, Utrecht, The Netherlands) or transgenic animals (unless otherwise specified). Further, it should be understood that an antibody can be of any immunoglobulin class (i.e., isotype) or allotype. Thus, for example, an antibody disclosed herein can be of an isotype selected from well-known immunoglobulin isotypes or can contain components from two or more isotypes (as can be the case for some chimeric antibody types). Also, an antibody can contain "mixed" chains, e.g., an antibody in which the H chain contains different H2 or H2L2 units (i.e., H x and H y (H x H y or L x H x H y L y )).

[0050] The term “antibody-like molecule” (Ab-like molecule) should be understood to refer to a protein that is not an antibody but contains minimally a target recognition structure (e.g., an antigen recognition sequence (e.g., a receptor or ligand such as the variable (V) domain or complementarity-determining region (CDR) of an immunoglobulin)) linked to at least the CH3 domain (or CH4 domain) of an immunoglobulin heavy (H) chain (providing a molecule having an “H-like” chain), including, for example, receptor fusion proteins containing at least the CH3 domain (or at least the CH4 domain) of the H chain. Examples of Ab-like molecules are those described above (Figure 2, Figure 53A, and / or Periodic Table of This includes those depicted in Antibodies. In some examples, an H-like chain containing at least a CH3 domain is thought to contain an Fc region component, which may be, for example, a complete Fc region, or simply the CH3 domain, or a CH2-CH3 or CH3-CH2 component. In other examples, such an H-like chain may further contain, for example, an immunoglobulin hinge sequence. The H-like chain may enable dimerization so that the antibody-like molecule may be provided in, for example, homodimeric or heterodimeric form. As depicted in Figure 53, in some embodiments, the Ab-like molecule may include an H2L2 form and may include a fusion protein containing the same or different target recognition structures (or enzymes and / or reporter molecules) such as "X1 X1 X1 X1", "X1 X1 X1 X2", "X1 X1 X2 X2", "X1 X1 X2 X3", or "X1 X2 X3 X4", where X1, X2, X3, and X4 represent different target recognition structures (or enzymes or reporter molecules).

[0051] The term “fusion protein” is well known to those skilled in the art and refers to a protein expressed from a DNA construct containing two or more open reading frames in a desired order, such that the protein may be considered a hybrid or chimeric protein. In some simple examples, a fusion protein comprises a protein (or fragment) of interest fused ("fused") to the amino-terminus (N-terminus) or carboxyl-terminus (C-terminus) of a partner polypeptide, such as a carrier protein (e.g., human serum albumin, HSA). As used herein, the term “fusion protein” will also be understood to refer to certain types of antibody-like molecules, such as fusion proteins containing a target recognition structure (e.g., an antigen recognition sequence (e.g., a receptor or ligand such as the variable (V) domain or complementarity-determining region (CDR) of an immunoglobulin)) linked to at least the CH3 domain (or at least the CH4 domain) of an immunoglobulin heavy (H) chain (see the example shown in Figure 2). Such fusion proteins are mixed in a manner similar to that described above for antibodies (e.g., bispecific antibodies) and antibody-like molecules, for example, such as when the fusion protein contains Fc fragments with different Fc regions (e.g., Fc1x and Fc2y). The Fc region may include a combined Fc region, each of which may optionally be linked to a different target recognition structure (e.g., ligand 1 and ligand 2). Those skilled in the art will understand that the target or antigen recognition structure may be linked to a polypeptide containing at least a CH3 domain (or an equivalent CH4 domain of IgE or IgM), which may include, for example, the entire H chain, or, for example, the complete Fc region, or simply the CH3 domain, or an H-like chain that may include CH2-CH3 or CH3-CH2 components. In other examples, such an H-like chain may further include, for example, an immunoglobulin hinge sequence and / or a CH1 domain. The CH1 domain may provide, for example, a suitable site for linking the target or antigen recognition structure.

[0052] The term “Fc fragment” (Fc or Fc portion), as used herein, refers to a dimer formed by covalent and / or non-covalent interactions between portions of an immunoglobulin heavy (H) chain (i.e., a dimer formed between two Fc regions of the H chain, each containing the CH2 and CH3 domains of the heavy chain and optionally a hinge sequence), which is largely involved in the activation of immunoeffector functions of immunoglobulins: in particular, the killing of target cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or trogocytosis. Cell-based effector responses initiated by antibodies and cell surface-located Fc receptors, which lead to the modulation and inhibition of the activity of innate and adaptive immune cells by inhibitory Fc receptors, effector responses of the innate immune system initiated by antibodies through activation of classical pathways of the complement system, cascades of proteins found in blood or biofluids that are important for the destruction of pathogens, and target death by direct lysis via complement-dependent cytotoxicity (CDC) and / or target phagocytosis (C'ADCP) via specific receptors of complement components. The Fc fragment also provides sites for inter-immunoglobulin association (self-assembly) that allow assembly (self-assembly) of immunoglobulin molecules into higher-order oligomers through covalent bonding between domains in the Fc region of one immunoglobulin's H chain with the Fc region of an adjacent immunoglobulin's H chain (for example, as seen in the pentameric and hexameric forms of IgM), and sites for non-covalent self-assembly, such as that seen in IgG when bound to an antigen, which leads to oligomerization, including hexamerization (Diebolder CA et al., Science 343(6176):1260-1263, 2014), and certain effector functions, among other properties.

[0053] As used herein, the term “Fc region component” should be understood to refer to a portion of the Fc region of an immunoglobulin heavy (H) chain, which includes at least a CH3 domain (or at least a CH4 domain), preferably a CH2 and CH3 domain, and optionally further includes an immunoglobulin hinge sequence (which may then include all or part of the lower hinge, core hinge, and upper hinge sequences) that can form an Fc fragment or Fc-like fragment (e.g., by dimerization). “Fc-like fragment” should be understood to refer to an Fc fragment-like structure, which includes a fragment or component of the Fc region, e.g., a CH3 domain (or CH4 domain) alone, or a CH3 domain in combination with a CH2 domain, and optionally further includes an immunoglobulin hinge sequence (which may include all or part of the lower hinge, core hinge, and upper hinge sequences).

[0054] As used herein, the term “to treat” includes the prevention and reduction of established symptoms of a disease or condition. Thus, the act of “treating” a disease or condition includes: (1) preventing or delaying the onset of clinical symptoms of a disease or condition in a subject who is afflicted with or prone to the disease or condition; (2) inhibiting the disease or condition (i.e., in the case of maintenance treatment, preventing, reducing, or delaying the onset or recurrence of the disease or condition, or at least one clinical or quasi-clinical symptom thereof); and (3) reducing or attenuating the disease or condition (i.e., causing regression of the disease or condition, or at least one clinical or quasi-clinical symptom thereof).

[0055] As used herein, the term “manufacturing of a pharmaceutical product” includes using one or more immunotherapy proteins as defined herein directly as a pharmaceutical product, or using them at any stage in the manufacture of a pharmaceutical product comprising one or more immunotherapy proteins as defined herein.

[0056] The term "effective dose" refers to an amount sufficient to achieve a beneficial or desired clinical outcome. An effective dose may be administered in one or more doses. Typically, an effective dose is sufficient to treat a disease or condition, or to alleviate, induce remission, stabilize, reverse, slow, or delay the progression of the disease or condition. As just one example, an effective dose of an immunotherapy protein, such as a mutant IgG1 antibody, may include, for example, about 0.1 to about 250 mg / kg body weight per day, more preferably about 0.1 to about 100 mg / kg body weight per day, and even more preferably about 0.1 to about 25 mg / kg body weight per day. However, notwithstanding the foregoing, it will be understood by those skilled in the art that the effective dose may vary and depend on various factors, including, for example, the age, weight, sex, and / or health of the subject being treated, the activity of the particular immunotherapy protein, the metabolic stability and duration of action of the particular immunotherapy protein, the route and timing of administration of the particular immunotherapy protein, the excretion rate of the particular immunotherapy protein, and the severity of the disease or condition being treated.

[0057] As shown in the examples provided below, the combination of hexamerization mutations and C1q binding modifications offers functional advantages. In one embodiment, the hexamerization mutation is a point mutation at the H429 position.

[0058] As illustrated in the examples provided below, mutant antibodies containing a point mutation at the H429 position have been found to confer significant functional changes. For example, IgG mutants with an H429F substitution exhibit enhanced ability to activate complement-dependent cytotoxicity (CDC) in complement assays (i.e., assays of complement function). Furthermore, fusion proteins containing an Fc region component with a point mutation (i.e., H429F) at position 429 fused to the angiotensin-converting enzyme 2 (ACE2) ectodomain (which can function as a "decoy" to block viral interactions and the entry of coronaviruses into host cells) may provide enhanced ability to deliver antiviral effects through CDC in infected cells, while similar fusion proteins with an H429Y substitution have been found to exhibit enhanced viral neutralization with little or no complementary activation (as immunotherapy proteins containing the H429Y-modified Fc component show inactivation of binding and activation with FcγR, particularly FcγRIIIa). While we do not wish to be constrained by theory, these effects may be due to the oligomerization of the antibody / fusion protein to an oligomer via self-assembly, either in solution or when binding to the relevant target molecule (e.g., the antigen to which the antibody variant is directed) via "target-on" oligomerization.

[0059] In this regard, it should be noted that while monomeric IgG1 and IgG3 can deliver CDC, the level of CDC can be considered low or "poor" compared to the levels achieved with natural pentameric or hexameric IgM. Furthermore, unlike IgM, IgG antibodies such as IgG1 and IgG3 are also relatively poor agglutinins due to their bivalent nature. Therefore, by enabling oligomerization (particularly after the immunoglobulin has bound to the antigen (i.e., "on-target" oligomerization) and possibly enhancing the known weak intrinsic self-association ability of the Fc of immunoglobulins such as IgG), immunotherapeutic proteins according to this disclosure may, by forming oligomers, provide a more optimal platform for complement system activation and other functions enhanced by self-association. This oligomerization may, in particular and / or optimally, involve the formation of a hexamer, such hexamerization has been visualized in the crystal structure of the anti-HIV antibody b12 (Saphire EO et al., Science 293:1155-1159, 2001), where the CH3 residue forms an interface between adjacent IgG:IgG molecules, thus forming a hexamer that optimally presents the binding sites of the six globular head domains of the C1q subunit that initiate the activation of the classical complement pathway.

[0060] Furthermore, many cellular molecules, including cell surface molecules, require substantial clustering by ligands (which may be soluble or other cell surface molecules) to induce signals that consequently induce cellular responses. Therefore, dimerization, typically achieved with antibodies, may not be sufficient to induce signals that lead to meaningful cellular responses. However, it is known that signal intensity can be increased by increasing the clustering of target molecules through approaches involving the addition of other entities that crosslink ligands (Chenoweth et al., Immunol Cell Biol 98:287-304, 2020). Thus, in the case of antibodies (such as mAbs) bound to their target molecules, such "super cross-clustering" or "hyper-clustering" of the target molecule can be achieved by using additional anti-immunoglobulin antibodies that crosslink the mAbs bound to the target molecule. While we do not wish to be bound by theory, it is thought that proteins such as mutant antibodies containing the H429F point mutation described above may similarly achieve "supercross-clustering or hyper-clustering" of target molecules through self-association upon binding to the target molecule. Depending on the nature of the target molecule, such clustering may induce an enhanced signaling response that could lead to, for example, cell proliferation (e.g., if the target molecule is, for example, CD3 or CD28), stimulation of inhibitory pathways to inhibit or reduce cellular responses (e.g., immune checkpoints; Chenoweth et al., reviewed in 2020 above), or stimulation of pathways that induce cell death, such as programmed cell death including apoptosis (see, for example, Ashkenazi A., Nat Rev Drug Discov 7(12):1001-1012, 2008). Stimulation of cellular pathways with mAbs for the development of therapeutic molecules is an approach used to treat a variety of diseases, including cancer, inflammatory and autoimmune diseases, infections, cardiovascular diseases, and others (Ashkenazi, 2008).

[0061] Accordingly, in one embodiment, the present disclosure provides an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component comprising at least a CH3 domain (or at least a CH4 domain), wherein the one or more polypeptides comprises an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0062] In some embodiments, the immunotherapy protein described herein comprises a dimeric immunotherapy protein comprising first and second immunoglobulin heavy chain polypeptides, each comprising an Fc region component containing CH2 and CH3 domains (so that the first and second polypeptides can form an Fc fragment or Fc-like fragment (e.g., by dimerization)), wherein at least one of the Fc region components of the first and second polypeptides comprises an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of the human IgG1 heavy chain. Therefore, immunotherapy proteins in such embodiments may be immunoglobulin molecules such as antibody-like molecules, including antibodies, dimeric polypeptides containing a pair of single-chain Fv polypeptides linked via an Fc fragment (i.e., scFv-Fc), or minibodies (i.e., proteins containing a pair of scFv polypeptides linked via a CH3 domain (see, for example, the discussion of polyvalent scFv-Fc and minibodies in Olafsen T et al., Generation of Antibody Fragments and Their Derivatives, Antibody Engineering Second edition, pp 69-84, 2010)).

[0063] In other embodiments, the immunotherapy proteins described herein comprise a partner polypeptide ligated to an Fc region component containing at least a CH3 domain (or at least a CH4 domain), the Fc region component comprising an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of the human IgG monohelic acid. Thus, immunotherapy proteins of such embodiments may be provided in the form of a fusion protein or a protein conjugate. As those skilled in the art will know, in a fusion protein, the partner polypeptide is covalently linked (i.e., "fused") to the Fc component (i.e., as a fusion partner) at the N-terminus or C-terminus (i.e., the Fc component) of the fusion partner via a peptide bond or linker sequence (e.g., a short peptide linker sequence such as an immunoglobulin hinge sequence or a well-known glycine-serine linker such as GGGGS), whereas in a protein conjugate, the partner polypeptide is chemically linked, for example, by a disulfide bond (e.g., through one or more cysteine ​​(C) residues), or by a crosslinking compound, for example, a homobifunctional crosslinking agent, for example, disuccinimidylsverate (DSS) (e.g., bis(sulfosuccinimidyl)sverate (BS)). 3It will be understood that the Fc component is covalently or non-covalently linked (i.e., as a conjugate partner) to the Fc component (i.e., as a conjugate partner) via disquimidyl tartrate (DST) for linking amine groups, or through heterobifunctional crosslinking agents such as m-maleimidobenzoyl-N-hydroxysuccinimide (MDS) and N-(ε-maleimidocaproloxy)succinimide (EMCS), or by other non-covalent bonds, such as hydrogen bonds. When the Fc component is a conjugate partner, the immunotherapy protein conjugate can be considered a crosslinking protein, and the Fc component may be conjugated to the partner polypeptide at the N-terminus or C-terminus, or otherwise at any other suitable site on the partner polypeptide. Alternatively, the Fc component (as a conjugate partner) may be conjugated to the partner polypeptide at its N-terminus or C-terminus, or at any other suitable site on the Fc component (e.g., within CH1 or the upper hinge sequence, if these are contained within the Fc region component).

[0064] The Fc region component of an immunotherapy protein (hereinafter also referred to as the "Fc component") may originate from one or more immunoglobulin types (e.g., IgG or IgA), for example, a heavy (H) chain polypeptide fragment corresponding to one produced by papain digestion (i.e., the polypeptide is cleaved within the upper hinge sequence to form constant heavy domain 2 (CH2; amino acids A231-K340 (Eu numbered) of human IgG1 heavy chain polypeptide), constant heavy domain 3 (CH3; amino acids G341-G446 or K447 (Eu numbered) of human IgG1 heavy chain polypeptide), and a lower hinge sequence (also known as the hinge proximal sequence of CH2; amino acids P232-P238 (Eu numbered)), and a core hinge sequence (amino acids C226-C 229), and may include full-length (i.e., "complete") Fc regions such as similar heavy chain polypeptide fragments that can be prepared by digestion of immunoglobulin heavy chain polypeptides with plasmin and human neutrophil elastase (NHE). Further examples of preferred Fc region components may include heavy chain polypeptide fragments containing all or part of the upper hinge sequence and constant heavy domain 1 (CH1), in addition to the CH2 and CH3 domains and lower and core hinge sequences. On the other hand, other preferred Fc region components may include heavy chain polypeptide fragments containing only the CH3 domain (e.g., amino acids G341-G446 or K447 (Eu numbered) of human IgG1 heavy chain polypeptide) or fragments thereof. In addition, preferred Fc region components include IgG1 This can include heterogeneous ("hybrid") CH3 domains, such as fragments derived from the CH3 domain and CH3 domains with chain exchange operation domains (SEEDs) containing other proteins such as IgA (Davies et al., Prot Eng Des Sel 23(4):195-202, 2009).

[0065] In some embodiments, the Fc region component is derived from a human immunoglobulin heavy chain polypeptide (e.g., as shown in Figures 3 and 4).

[0066] In other embodiments, the Fc region component is derived from an IgG heavy chain polypeptide, preferably IgG1 or IgG3 (e.g., human IgG1 or IgG3) heavy chain polypeptide, and more preferably IgG1. In some embodiments, the Fc region is glycosylated. In some embodiments, the Fc region does not contain the E430G substitution.

[0067] The Fc region component includes an amino acid substitution at the position corresponding to H429 (Eu numbering) in the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. If the Fc region component originates from a different immunoglobulin type or isotype (or from an immunoglobulin from a different species), those skilled in the art will understand that the position corresponding to H429 in human IgG1 heavy chain polypeptide IgG1 can be easily determined, for example, by routine sequence alignment (as shown, for example, in Figures 3 and 4). In some embodiments, the H429 amino acid substitution is an aromatic or cyclic amino acid. In some embodiments, the H429 amino acid substitution is an aromatic amino acid. In some embodiments, the H429 amino acid substitution is a cyclic amino acid. In some embodiments, the H429 amino acid substitution is a hydrophobic amino acid. In some embodiments, the H429 amino acid substitution is a highly hydrophobic amino acid (e.g., F or Y). In some embodiments, the H429 amino acid substitution has hydrophobicity of T or greater. In some embodiments, the H429 substitution is not a neutral amino acid (e.g., not glycine). In some embodiments, the H429 substitution is neither glycine nor leucine. In some embodiments, the H429 substitution is not glycine. In some embodiments, the H429 substitution is not leucine.

[0068] Suitable mutations at position 429 include the following: H → X, where X is selected from phenylalanine (H429F), glutamic acid (H429E), glutamine (H429Q), serine (H429S), alanine (H429A), threonine (H429T), tyrosine (H429Y), leucine (H429L), valine (H429V), glycine (H429G), tryptophan (H429W), arginine (H429R), and proline (H429P).

[0069] Suitable mutations at position 429 include the following: H → X, where X is selected from tyrosine (H429Y), phenylalanine (H429F), tryptophan (H429W), glutamic acid (H429E), aspartic acid (H429D), glutamine (H429Q), serine (H429S), asparagine (H429N), and threonine (H429T).

[0070] Some preferred amino acid substitutions at the position corresponding to H429 in the amino acid sequence of human IgG monohelic acid include H→Y (e.g., H429Y) substitutions and H→F (e.g., H429F) substitutions.

[0071] In some embodiments, H429 is substituted with phenylalanine (H429F). In some embodiments, H429 is substituted with glutamic acid (H429E). In some embodiments, H429 is substituted with glutamine (H429Q). In some embodiments, H429 is substituted with serine (H429S). In some embodiments, H429 is substituted with alanine (H429A). In some embodiments, H429 is substituted with threonine (H429T). In some embodiments, H429 is substituted with leucine (H429L). In some embodiments, H429 is substituted with valine (H429V). In some embodiments, H429 is substituted with glycine (H429G). In some embodiments, H429 is substituted with tryptophan (H429W). In some embodiments, H429 is substituted with arginine (H429R). In some embodiments, H429 is substituted with and proline (H429P).

[0072] As used herein, “C1q binding modification” refers to a modification that increases or decreases the C1q binding of an immunotherapy protein described herein. In some embodiments, this modification directly decreases / increases the binding of the immunotherapy protein to C1q compared to a control immunotherapy protein lacking C1q binding modification. In some embodiments, this modification indirectly decreases / increases the binding of the immunotherapy protein to C1q compared to a control immunotherapy protein lacking C1q binding modification. For clarity, the term “C1q binding modification” does not require, but is not excluded, that the modification itself binds to C1q.

[0073] In some embodiments, the C1q binding modification is located in the CH1 domain of the immunotherapy protein. In some embodiments, the C1q binding modification is located in the CH2 domain of the immunotherapy protein. In some embodiments, the C1q binding modification is located in the CH3 domain of the immunotherapy protein. In some embodiments, the C1q binding modification is located in the hinge region of the immunotherapy protein. In some embodiments, the mutation is located in the lower hinge. In some embodiments, the mutation is located in the upper hinge.

[0074] In some embodiments, the C1q bond modification is selected from modifications in C1q group 1, C1q group 2, C1q group 3, C1q group 4, and C1q group 5 as listed in Table 6. In some embodiments, the C1q bond modification is a modification in C1q group 1 as listed in Table 6. In some embodiments, the C1q bond modification is a modification in C1q group 2 as listed in Table 6. In some embodiments, the C1q bond modification is a modification in C1q group 3 as listed in Table 6. In some embodiments, the C1q bond modification is a modification in C1q group 4 as listed in Table 6. In some embodiments, the C1q bond modification is a modification in C1q group 5 as listed in Table 6.

[0075] In some embodiments, the C1q binding modification is selected from modifications in C1q group 1, C1q group 2, and C1q group 3, and this modification increases C1q binding compared to control IgG2 immunotherapy proteins and / or control IgG1 immunotherapy proteins lacking C1q binding modification.

[0076] In some embodiments, the C1q binding modification is selected from modifications in C1q group 4 and C1q group 5, and this modification reduces C1q binding compared to control IgG2 immunotherapy proteins and / or control IgG1 immunotherapy proteins lacking C1q binding modification.

[0077] In some embodiments, the polypeptide is selected from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the polypeptide is IgG1. In some embodiments, the polypeptide is IgG2. In some embodiments, the polypeptide is IgG3. In some embodiments, the polypeptide is IgG4.

[0078] In one embodiment, the polypeptide is IgG1, and the C1q binding modification is located in the CH1 domain, which is a C substitution at the position corresponding to S131 (Eu numbering) in the amino acid sequence of the human IgG1 heavy chain polypeptide.

[0079] In one embodiment, the polypeptide is IgG1, IgG2, IgG3, or IgG4, the modification is in the hinge region, and the modification is a substitution at positions 216-225 or 217-225 (Eu numbering) of the human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. In one embodiment, the substitution in the hinge region is selected from RKCCVE217-225 substituted with PKSCDKTHT, EPKSCDKTHT216-225 substituted with ERKCCVE, and EPKSCDKTHT216-225 substituted with ESKYGPP (Eu numbering). In one embodiment, the substitution in the hinge region is RKCCVE217-225 substituted with PKSCDKTHT. In one embodiment, the substitution in the hinge region is EPKSCDKTHT216-225 substituted with ERKCCVE. In one embodiment, the replacement in the hinge region is EPKSCDKTHT216~225 replaced with ESKYGPP.

[0080] In one embodiment, the polypeptide is IgG2, the modification is in the CH2 domain, and the modification is a substitution at a position corresponding to PVA233-236, L328, or S267 (Eu numbering) of human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. In one embodiment, the substitution in the CH2 domain is selected from PVA233-236 substituted with EFLG, PVA233-236 substituted with ELLG, and PVA233-236 substituted with EFEG. In one embodiment, the substitution in the CH2 domain is PVA233-236 substituted with EFLG (Eu numbering). In one embodiment, the substitution in the CH2 domain is PVA233-236 substituted with ELLG (Eu numbering).

[0081] In one embodiment, the polypeptides are IgG1, IgG3, and IgG4, the modification is in the CH2 domain, and the modification is an E substitution at the position corresponding to L235 (Eu numbering) of the human IgG1, IgG3, or IgG4 heavy chain polypeptide.

[0082] In one embodiment, the polypeptide is IgG1, IgG2, IgG3, or IgG4, the modification is in the CH2 domain, and the modification is a substitution at a position corresponding to QYN295-297, YNS296-298, ED269-270, L328, or S267 (Eu numbering) of human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. In one embodiment, the substitution in the CH2 domain is selected from QYN295-297 substituted with NST, YNS296-298 substituted with NSS, ED269-270 substituted with AA, L328 substituted with F, and S267 substituted with E (Eu numbering). In one embodiment, the substitution in the CH2 domain is QYN295-297 substituted with NST. In one embodiment, the substitution in the CH2 domain is YNS296-298 substituted with NSS. In one embodiment, the substitution in the CH2 domain is ED269-270 substituted with AA. In one embodiment, the substitution in the CH2 domain is L328 substituted with F and S267 substituted with E. In one embodiment, the substitution in the CH2 domain is L328 substituted with F. In one embodiment, the substitution in the CH2 domain is S267 substituted with E. In one embodiment, the polypeptide is IgG2, IgG3, or IgG4, the modification is in the CH2 domain, and the modification is a substitution at the position corresponding to Q274 (Eu numbering) of the human IgG2, IgG3, or IgG4 heavy chain polypeptide. In one embodiment, the substitution in the CH2 domain is Q274 substituted with K.

[0083] In one embodiment, the polypeptide is IgG1, the modification is in the CH2 domain, and the modification is a substitution at the position corresponding to K274 (Eu-numbered) of the human IgG1 heavy chain polypeptide. In one embodiment, the substitution in the CH2 domain is K274 substituted with Q. In one embodiment, the polypeptide is IgG1 or IgG3, the modification is in the lower hinge, and the modification is a substitution at the position corresponding to LL234-235. In one embodiment, the substitution in the lower hinge is LL234-235 substituted with AA.

[0084] In one embodiment, the polypeptide is IgG4, the modification is in the lower hinge, and the modification is a substitution at the position corresponding to FL234-235. In one embodiment, the substitution in the lower hinge is FL234-235 substituted with AA. In one embodiment, the polypeptide is IgG2, the modification is in the CH2 domain, and the modification is a substitution at the position corresponding to PVA234-236. In one embodiment, the substitution in the lower hinge is selected from PVA234-235 substituted with AAG.

[0085] In one embodiment, the polypeptide is IgG1, IgG2, IgG3, or IgG4, the modification is in the CH3 domain, and the modification is a substitution at a position corresponding to E430 or H435 (Eu numbering) of the human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. In one embodiment, the substitution in the CH3 domain is selected from E430 substituted with G and H435 substituted with A.

[0086] In one embodiment, the polypeptide is IgG1, IgG2, IgG3, or IgG4, and the modification is in the CH2 and CH3 domains. In one embodiment, the polypeptide is IgG1, and the modification is in the CH2 and CH3 domains. In one embodiment, the polypeptide is IgG2, and the modification is in the CH2 and CH3 domains. In one embodiment, the mutation in the CH2 domain is PVA233-236 substituted with EFLG, and the modification in the CH3 domain is E430G. In one embodiment, the mutation in the CH2 domain is PVA233-236 substituted with ELLG, and the modification in the CH3 domain is E430G. In one embodiment, the mutation in the CH2 domain is PVA233-236 substituted with EFEG, and the modification in the CH3 domain is E430G. In one embodiment, the polypeptide is IgG2, the mutation in the CH2 domain is PVA233-236 substituted with EFLG, and the modification in the CH3 domain is E430G. In one embodiment, the polypeptide is IgG2, the mutation in the CH2 domain is PVA233-236 substituted with ELLG, and the modification in the CH3 domain is E430G.

[0087] In one embodiment, the polypeptide is IgG3, the modification is in the CH3 domain, and the modification is a substitution at positions corresponding to N392, M397, and R435 (Eu-numbered) of the human IgG3 heavy chain polypeptide. In one embodiment, the substitutions in the CH3 domain include N392 substituted with K, M397 substituted with V, and R435 substituted with H (Eu-numbered).

[0088] In one embodiment, the polypeptide is IgG2, the C1q binding modification is located in the CH1 domain, and the modification is a substitution with S at the position corresponding to C131 (Eu numbering) in the amino acid sequence of human IgG1 heavy chain polypeptide.

[0089] In one embodiment, the polypeptide is IgG2, and the C1q binding modifications are PVA233-236 substituted with EFLG, C131 substituted with S, and C220 substituted with S.

[0090] In one embodiment, the polypeptide is IgG2, and the C1q binding modifications are PVA233-236 substituted with EFLG, C131 substituted with S, and C219 substituted with S.

[0091] In one embodiment, the polypeptide is IgG2, and the C1q binding modifications are PVA233-236 substituted with EFLG, C131 substituted with S, and ERKCCVE substituted with IgG1 216-225EPKSCDKTHT.

[0092] In one embodiment, the polypeptide is IgG1, and the C1q binding modification is PVA233-236 substituted with EAAGG.

[0093] In one embodiment, the polypeptide is IgG1, and the C1q binding modification is K274 substituted with Q.

[0094] In one embodiment, the polypeptide is IgG2, IgG3, or IgG4, and the C1q binding modification is K-substituted Q274.

[0095] In some embodiments, the C1q bond modification is not E430 substituted with G. In some embodiments, the C1q bond modification is neither L328 substituted with F nor S267 substituted with E. In some embodiments, the C1q bond modification is neither L328 substituted with F nor S267 substituted with E.

[0096] In one embodiment, the immunotherapy protein is an anti-CD20 antibody. In one embodiment, the immunotherapy protein is an anti-trinitrophenyl antibody (anti-TNP antibody). In one embodiment, the immunotherapy protein is selected from rituximab, ofatumumab, obinutuzumab, isatuximab, trastuzumab, and pertuzumab. In one embodiment, the immunotherapy protein is selected from rituximab, trastuzumab, and pertuzumab. In one embodiment, the immunotherapy protein is rituximab. In one embodiment, the immunotherapy protein is trastuzumab. In one embodiment, the immunotherapy protein is pertuzumab. In one embodiment, the immunotherapy protein is selected from rituximab (Rit), Rit-IgG2 FLGG HF, Rit-IgG2-FLGG EG, Rit-IgG2-LLGG HF, Isa-IgG2-M1-H429F, Isa-IgG2-M2-H429F, Isa-IgG2-M3-H429F, S2P6-IgG2-M1-H429F, S2P6-IgG2-M2-H429F, and S2P6-IgG2-M3-H429F. In one embodiment, the immunotherapy protein includes Rit-IgG2-FLGG HF. In one embodiment, the immunotherapy protein includes Rit-IgG2 FLGG EG. In one embodiment, the immunotherapy protein includes Rit-IgG2-LLGG HF. In one embodiment, the immunotherapy protein includes Isa-IgG2-M1-H429F. In one embodiment, the immunotherapy protein comprises Isa-IgG2-M2-H429F. In one embodiment, the immunotherapy protein comprises Isa-IgG2-M3-H429F. In one embodiment, the immunotherapy protein comprises S2P6-IgG2-M1-H429F. In one embodiment, the immunotherapy protein comprises S2P6-IgG2-M2-H429F. In one embodiment, the immunotherapy protein comprises S2P6-IgG2-M3-H429F.

[0097] In one embodiment, the immunotherapy protein comprises: a) H429 substituted with an aromatic amino acid or a cyclic amino acid, with C1q modification being PVA233-236 substituted with EFLG; b) H429 substituted with an aromatic amino acid or a cyclic amino acid, with C1q modification being PVA233-236 substituted with ELLG; c) H429 substituted with a hydrophobic amino acid, with C1q modification being PVA233-236 substituted with EFLG; and d) H429 substituted with a hydrophobic amino acid, with C1q modification being PVA233-236 substituted with ELLG.

[0098] In one embodiment, the immunotherapy protein is IgG2, which includes further mutations that create a disulfide between the light chain and the upper hinge region of the polypeptide. In one embodiment, the disulfide bond is formed between C214 and C219. In one embodiment, the disulfide bond is formed between C214 and C220. In one embodiment, further mutations are serine substitution of M1, C131 and serine substitution of C220. In one embodiment, further mutations are serine substation of M2, C131 and serine substitution of C219. In one embodiment, further mutations are serine substation of M3, C131 and substitution of ERKCCVE with EPKSCDKTHT in the IgG2 upper hinge.

[0099] In one embodiment, the immunotherapy protein includes modifications selected from the following: a) H429 substituted with F, wherein the C1q modification includes PVA233-236 substituted with EFLG; b) H429 substituted with F, wherein the C1q modification includes PVA233-236 substituted with ELLG; c) H429 substituted with Q, wherein the C1q modification includes PVA233-236 substituted with EFLG; d) H429 substituted with Q, wherein the C1q modification includes PVA233-236 substituted with ELLG H429 containing PVA233~236, e) H429 substituted with E, where the C1q modification is H429 containing PVA233~236 substituted with EFLG, f) H429 substituted with E, where the C1q modification is H429 containing PVA233~236 substituted with ELLG, g) H429 substituted with S, where the C1q modification is H429 containing PVA233~236 substituted with EFLG, h) H429 substituted with S, where the C1q modification is HVA233~236 substituted with ELLG H429, i) H429 substituted with A, including PVA233~236 with C1q modification substituted with EFLG, j) H429 substituted with A, including PVA233~236 with C1q modification substituted with ELLG, k) H429 substituted with Y, including PVA233~236 with C1q modification substituted with EFLG, l) H429 substituted with Y, including PVA233~236 with C1q modification substituted with ELLG, m) H429 with F H429 is a modified H429, where the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and C220 substituted with serine; H429, n)F-substituted H429, where the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and C219 substituted with serine; H429, o)F-substituted H429, where the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and IgG1H429 is ERKCCVE substituted with EPKSCDKTHT, H429 is substituted with p)F, and the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with serine, and C220 substituted with serine, H429 is substituted with q)F, and the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with serine, and C219 substituted with serine, as well as H429 is substituted with r)F, and the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with serine, and ERKCCVE in the IgG2 upper hinge substituted with EPKSCDKTHT.

[0100] In one embodiment, the immunotherapy protein includes H429 substituted with F, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with F, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein includes H429 substituted with Q, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with Q, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein includes H429 substituted with E, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with E, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein includes H429 substituted with S, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with S, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein includes H429 substituted with A, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with A, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein includes H429 substituted with Y, and the C1q modification includes PVA233-236 substituted with EFLG. In one embodiment, the immunotherapy protein includes H429 substituted with Y, and the C1q modification includes PVA233-236 substituted with ELLG. In one embodiment, the immunotherapy protein contains H429 substituted with F, the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and C220 is substituted with serine. In another embodiment, the immunotherapy protein contains H429 substituted with F, the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and C219 is substituted with serine.In one embodiment, the immunotherapy protein contains F-substituted H429, the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and ERKCCVE in the IgG2 upper hinge is substituted with IgG1 216-225EPKSCDKTHT. In another embodiment, the immunotherapy protein contains F-substituted H429, the C1q modification is PVA233-236 substituted with ELLG, C131 is substituted with serine, and C220 is substituted with serine. In yet another embodiment, the immunotherapy protein contains F-substituted H429, the C1q modification is PVA233-236 substituted with ELLG, C131 is substituted with serine, and C219 is substituted with serine. In one embodiment, the immunotherapy protein comprises H429 substituted with F, C1q modification is PVA233-236 substituted with ELLG, C131 is substituted with serine, and ERKCCVE in the IgG2 upper hinge is substituted with IgG1 216-225EPKSCDKTHT.

[0101] In one embodiment, C1q binding modification increases complement-based lysis compared to a control immunotherapy protein lacking C1q binding modification. In another embodiment, C1q binding modification decreases complement-based lysis compared to a control immunotherapy protein lacking C1q binding modification. In one embodiment, the control antibody is wild-type IgG2. In another embodiment, the control antibody is wild-type IgG1.

[0102] In one embodiment, the immunotherapy protein includes at least two C1q binding modifications. In another embodiment, the immunotherapy protein includes at least three C1q binding modifications.

[0103] In some embodiments, the immunotherapy proteins described herein may include one or more further mutations (e.g., amino acid substitutions). For example, the Fc component may include one or more sequence mutations known to those skilled in the art (see, for example, examples listed in Table 1 of Wang et al., Protein Cell 9(1):63-73, 2018, the entire disclosure of which is incorporated herein by reference), such as mutations that can modulate FcγR binding (e.g., S239D / I332E (Eu numbering) of IgG1 that increases FcγRIIIa binding); mutations to improve antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., S239D / I332E (Lazar GA et al., Proc Natl Acad Sci USA 103(11):4005-4010, 2006); opsonization (e.g., G236A / S239D / I332E; Richards JO et al., Mol Cancer Ther These may include mutations that improve complement activation (e.g., H268F / S324T; Moore GL et al., MAbs 2:181-189, 2010), or mutations that reduce effector function (e.g., IgG1:L234A / L235A; IgG4:F234A / L235A; Xu D et al., Cell Immunol 200(1):16-26, 2000), and mutations that confer enhanced binding to neonatal Fc receptors (FcRn), such as M252Y / S254T / T256E, to increase the in vivo half-life and thereby improve pharmacokinetics (PK) (Dall'Acqua WF et al., J Immunol 169(9):5171-5180, 2002).

[0104] Based on the observation of C1q binding by mAbs shown in Figures 57, 58, and 59, 71, 78, and 85, as well as the grouping in Table 6, and the sequence alignments in Figure 56 and Tables 7, 8, and 9, additional mutations can be incorporated into the IgG2 backbone, particularly mutations in cysteine ​​residues involved in disulfide bonds from the light chain to the heavy chain and / or between the heavy chains (position 131 in CH1, or in the upper hinge at positions 219 and 220 in IgG1, IgG2, IgG4, or IgG4 (Eu numbering)). Such mutations can be used alone or in combination with, for example, C1q binding modifications, e.g., C131S in CH1 of IgG2, or S131C in CH1 of IgG1, IgG3, or IgG4. In some embodiments, the C1q binding modification is used in combination with C219S and / or C220S. In some embodiments, the C1q binding modification is used in combination with C219S. In some embodiments, the C1q binding modification is used in combination with C220S. In some embodiments, the C1q binding modification is used in combination with C219S and / or C220S in IgG2. Other point mutations may also be useful. For example, the mutation K274Q at position 274 in IgG1 may result in a decrease in C1q binding (Figure 85), which is inferred from the reduced CDC activity of this mutant compared to its IgG1-WT counterpart (Figure 84). This point mutation may be used in combination with other mutations, such as those targeting the disulfide bond in CH1 or the upper hinge.

[0105] In one embodiment, further mutations are substitutions selected from substitution of K274 with Q, Q274 with K, C219 with S, S219 with C, and C220 with S.

[0106] In one embodiment, the polypeptide is IgG2, and the polypeptide includes further mutations that prevent the formation of an intra-heavy-chain disulfide bond in the upper hinge region of the polypeptide. In one embodiment, the intra-heavy-chain disulfide bond is between C131 and C219, or between C131 and C220. In one embodiment, the polypeptide includes a mutation at one or more positions C131, C219, or C220, the mutation being a substitution for an amino acid other than cysteine. In one example, the substitution is a substitution for serine. In one example, the mutation is at positions C131 and C219, or C131 and C220.

[0107] In one embodiment, the polypeptide is IgG2, and the polypeptide includes further mutations that form a disulfide bond between the light chain and the upper hinge region of the polypeptide. In one embodiment, the disulfide bond is formed between C214 of the light chain and C219 of the heavy chain, or between C214 of the light chain and C220 of the heavy chain. In one embodiment, further mutations are M1, substitution of C131 with S, and substitution of C220 with S. In one embodiment, further mutations are M2, substitution of C131 with S, and substitution of C219 with S. In one embodiment, further mutations are M3, a substation with S at C131, and substitution of ERKCCVE with EPKSCDKTHT in the IgG2 upper hinge.

[0108] In one embodiment, the immunotherapy protein is an anti-CD20 antibody. In another embodiment, the immunotherapy protein is an anti-trinitrophenyl antibody (anti-TNP antibody).

[0109] Other possible mutations in immunotherapy protein components include mutations to enhance complement activation, such as amino acid mutations at the position corresponding to K447 (Eu numbering) in the amino acid sequence of human IgG1 heavy chain polypeptide, in particular K447X, where X is selected from null (i.e., K447del; amino acid deletion or cleavage of the Fc component), K447X, and glutamic acid (i.e., K447E) (see van der Bremer ETJ et al., mAbs 7(4):672-680, 2015). Furthermore, immunotherapy protein components may also include mutations that modulate glycosylation to eliminate FcγR and complement C1 binding and / or activation (e.g., mutations at the position corresponding to Asn297 (Eu numbering) of the amino acid sequence of human IgG1 heavy chain polypeptides such as N297A, N297Q, or N297G (Wang et al., 2018 above) to provide a site with modified glycosylation (e.g., absence of glycan at position 297).

[0110] Alternatively, immunotherapy protein components may also be modified to achieve modifications that specifically enhance activity via glycosylation; FcγRIIIa, by producing immunotherapy proteins in the presence of kifunensin (a mannosidase inhibitor that interferes with the normal maturation of N-linked glycans, including core fucosylation of N-linked glycans). Furthermore, Fc components lacking core fucosylation of Asn297 glycan can also be achieved by culturing host cells expressing the immunotherapy protein using a fucosylation inhibitor (e.g., 2-fluoroperacetylated fucose, or similar), or by modifying these pathways through the expression of enzymes that modify the glycosylation pathway (e.g., GDP-6-deoxy-D-lyxo-4-hexose reductase; Neha M et al., J Biotech 5:100015, 2020), or by gene repression (e.g., siRNA silencing of the α-1,6-fucosyltransferase gene FUT8; Imai-Nishiya H et al., BMC Biotechnol 7:84, 2007), or knockout (Yamane-Ohnuki N et al., Biotechnol Bioeng 87:614-622, 2004).

[0111] The immunotherapy proteins described herein may be monomers, dimers, or oligomers in solution (for example, in physiological saline with a neutral pH such as a physiological pH of about 7.4).

[0112] For example, in some embodiments, the immunotherapy protein comprises monomers in saline, each monomer containing one copy of the Fc region component, while in other embodiments, the immunotherapy protein comprises dimers in saline, where the Fc region component self-associates (i.e., forms a dimer) by non-covalent bonds such as hydrogen bonds, or, in particular, by the formation of disulfide bonds through one or more cysteine ​​(C) residues, if located within hinge sequences, especially core hinge sequences (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003). Therefore, in some embodiments, the Fc domain component includes a core hinge sequence that allows the immunotherapy protein to self-assemble (i.e., form a dimer) by forming an interchain disulfide bond between one or more cysteine ​​(C) residues in the core hinge sequences of two Fc domain components, while in some other embodiments, the Fc domain component includes a CH3 domain that allows CH3:CH3 self-assembly by non-covalent interactions, or the Fc domain component includes CH3 and CH2 domains that allow self-assembly by CH2:CH2 and CH3:CH3 non-covalent interactions. Note that in the following examples and figures, the dimeric form of the immunotherapy protein is considered to be a single molecule (i.e., each contains two copies of the protein dimerized through the Fc domain component) and is referred to as monomer / monomeric.

[0113] In other embodiments, the immunotherapy protein comprises an oligomer in saline (at a neutral pH such as a physiological pH of approximately 7.4), and the immunotherapy protein comprises an amino acid substitution at the position corresponding to H429 in the amino acid sequence of the human IgG1 heavy chain polypeptide, thereby enabling the self-assembly of Fc or Fc-like fragments containing the mutated Fc component into a soluble oligomer form in saline (for example, in some specific embodiments, the oligomer form of the immunotherapy protein may comprise six dimeric proteins (i.e., a hexameric form) containing a total of 12 copies of the immunotherapy protein, for example, an oligomer form containing, for example, 3, 4, 5, 6, or 12 copies assembled from the dimeric form of the immunotherapy protein). In such embodiments, the amino acid substitution at the position corresponding to H429 in the amino acid sequence of the human IgG1 heavy chain polypeptide is H429X 1 It could be, X 1 It is selected from tyrosine (i.e., H429Y), methionine, isoleucine, leucine, tryptophan, and valine, Preferably, the immunotherapy protein contains an H→Y (i.e., H429Y) substitution.

[0114] In yet another embodiment, the immunotherapy protein may form an oligomer upon binding to the relevant target through “on-target” oligomerization. Such oligomerization may occur by the immunotherapy protein being a monomer or dimer in physiological saline (at a neutral pH such as about 7.4 physiological pH) and involves an amino acid substitution at the position corresponding to H429 in the amino acid sequence of the human IgG1 heavy chain polypeptide, enabling on-target oligomerization to oligomer forms (e.g., forms including 3, 4, 5, 6, or 12 copies of the immunotherapy protein (e.g., a hexamer in dimeric form)). In such embodiments, the amino acid substitution at the position corresponding to H429 in the amino acid sequence of the human IgG1 heavy chain polypeptide is H429X 2 This is possible, and in the formula, X 2It is selected from phenylalanine (i.e., H429F), glutamic acid, glutamine, and serine, Preferably, the immunotherapy protein contains an H→F (i.e., H429F) substitution.

[0115] Alternatively, the oligomeric forms of the immunotherapy proteins described herein may be produced by using other techniques well known to those skilled in the art, such as the use of Fc multimers (stradomers®) (Fitzpatrick EA et al., Front Immunol 11, article 496, 2020) containing a ligated multimerization domain (MD) sequence from the hinge region or isoleucine zipper (ILZ) of human IgG2 to the N-terminus or C-terminus of mouse IgG2a, as well as multimerization sequences from IgM (Melcheil et al., Sci Rep 1:124 doi:10.1038 / srep0012, 2011), or docking and dimerization sequences from unrelated proteins such as cyclic adenosine monophosphate-dependent protein kinases and A-kinase anchored proteins (Rossi EA et al., Bioconjug Chem 23(3):309-323, 2012).

[0116] It has been further found that the immunotherapy proteins described herein may exhibit enhanced binding to the neonatal Fc receptor (FcRn) through amino acid substitution at the H429 (Eu-numbered) position in the amino acid sequence of the human IgG1 heavy chain. Since FcRn is known to "recycle" antibodies to control how long antibodies persist in the body, this is expected to mean that the immunotherapy proteins will exhibit a longer in vivo half-life (i.e., compared to equivalent immunotherapy proteins without the mutation at the H429 position), thereby improving pharmacokinetics (PK) (Ward ES and RJ Orber, Trends Pharmacol Sci 39(10):892-904, 2018).

[0117] Dimeric immunotherapy proteins (e.g., immunoglobulin molecules) The immunotherapy proteins described herein may be dimeric immunotherapy proteins comprising an immunoglobulin molecule, the immunoglobulin molecule comprising first and second immunoglobulin heavy chain polypeptides, each comprising an Fc region component comprising at least a CH3 domain (or at least a CH4 domain). The Fc region components of the first and second immunoglobulin heavy chain polypeptides may self-associate (i.e., form a dimer) by non-covalent bonds such as hydrogen bonds, or, in particular, by the formation of disulfide bonds through one or more cysteine ​​(C) residues if located within a hinge sequence, particularly a core hinge sequence (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003), or otherwise be linked by crosslinking compounds such as those described above (e.g., by crosslinking the CH3 domain of the Fc region component). Therefore, immunotherapy proteins may be immunoglobulin molecules such as antibodies or antibody derivatives, for example, scFv-Fc (dimers of scFv polypeptides are formed through dimerization of the Fc region component), minibodies (dimers of scFv polypeptides are formed through linking of CH3 domains / CH4 domains), or any other suitable Fc-containing antibodies or derivatives known to those skilled in the art (for example, as summarized in the "Periodic Table of Antibodies" above).

[0118] Therefore, dimeric immunotherapy proteins must have at least one antigen-recognition structure, or in other words, an antigen-binding region (e.g., a variable domain V such as a Fab fragment). L and V HThe scFv may further include an antigen-binding region. The antigen-binding region may specifically bind to therapeutically important antigens or epitopes, such as cancer-related antigens such as cancer antigens present on the surface of cancerous cells (e.g., CD20, CD38, and CD52 antigens found on the surface of CLL cells, and cell surface antigens differentially expressed and / or present in cancer cells such as mucins (e.g., MUC-1) or carbohydrates (e.g., Lewis X) that are overexpressed in some breast and pancreatic cancers), autoantigens (e.g., autoantigens associated with SLE or multiple sclerosis (MS)), allergens (e.g., bee venom), antigens associated with other inflammatory diseases such as immune complex vasculitis, antigens from transplanted tissues or organs, or antigens of infectious agents such as bacteria, yeasts, parasites, or viral pathogens (e.g., antigens of SARS-CoV-2 virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Respiratory Syncytial Virus (RSV), or Dengue Virus).In other examples, the antigen-binding domain may specifically bind to cell surface molecules known to induce cell proliferation (i.e., the cell surface molecules may be, for example, CD3 or CD28) and / or to cell surface molecules known to stimulate inhibitory pathways for inhibiting or reducing cellular responses (i.e., the cell surface molecules may be, for example, immune checkpoint molecules, e.g., 4-1BB (CD137), differentiation antigens (CD40, CD154), OX40 receptors). TNFRSF4 (CD134), tumor necrosis factor receptor type II (TNFR2, CD120b), glucocorticoid-inducible TNFR-related proteins (GITR, TNFRSF18, CD357), differentiation antigen group 27 (CD27), T cell immunoglobulin and mucin domain-containing-3 (TIM-3), B and T lymphocyte attenuation factor (BTLA, CD272), lymphocyte activation gene-3 (LAG3, CD223), cytotoxic T lymphocyte-related protein 4 (CTLA4, C These may include D152), inducible T cell costimulatory receptor (ICOS, CD278), differentiation antigen group 28 (CD28), T cell immune receptor with Ig and ITIM domains (IGIT, Vstm3), programmed cell death ligand 1 (PDL-1, CD274), and programmed cell death protein 1 (PD-1, CD279). Oligomerization of immunotherapy proteins can result in enhanced crosslinking (e.g., superclustering), which in turn can result in enhanced signaling that induces, for example, enhanced cell proliferation or inhibited / reduced cellular responses (i.e., through enhanced stimulation of inhibitory pathways). In yet another example, antigen-binding domains may specifically bind to cell surface molecules known to induce cellular responses selected from stimulation of inhibitory pathways to inhibit or reduce responses. In yet another example, antigen-binding domains may specifically bind to cell surface molecules that induce cell death when engaged with other molecules such as ligands or agonist mAbs.Such cell surface molecules include, for example, those of the TNF receptor superfamily (also known as TNFRSF), which includes tumor necrosis factor receptor 1 (TNFR1, TNFRSF1A, CD120a), Fas (CD95, APO-1), death receptor 3 (DR3, TNFRSF25), death receptor 4 (DR4, CD261, TNFRSF10A, TRAILR1), death receptor 5 (DR5, CD262, TNFRSF10B, TRAILR2), and death receptor 6 (CD358, TNFRSF21). For example, under physiological conditions, the binding of a TNF-related apoptosis-inducing ligand (TRAIL) to its receptor, DR5 (Carneiro BA et al., Nat Rev Clin Oncol 17(7):395-417, 2020), or the binding of a FAS ligand to its cell surface receptor, FAS(CD95), induces oligomerization that initiates signaling leading to cell death (Leukocyte and Stromal Cell molecules: The CD Markers, by Zola H et al., page 195, John Wiley & Sons, 2007). For example, other molecules (not related to TNFRSF) that can induce apoptotic signals leading to cell death when crosslinked by mAbs or fragments thereof include CD38 (see Gambles MT et al., Molecules 26(15):4658, 2021), CD20 (see Shan D et al., Cancer Immunol Immunother 48:673-683, 2000, and Cardarelli PM et al., Cancer Immunol Immunother 51:15-24, 2002), and CD52 (see Rowan W et al., Immunology 95:427-436, 1998). These molecules can also be favorably targeted by dimeric immunotherapy proteins so that oligomerization can result in enhanced crosslinking and induction of apoptotic signals leading to cell death, as desired in target cells.

[0119] In some embodiments, immunoglobulin molecules according to this disclosure may include two antigen-binding regions, each of which specifically binds to a different antigen or epitope. As used herein, “antigen” is any substance that elicits an immune response (antibody) in the body. As used herein, “epitope,” also known as “antigenic determinant,” is a portion of an antigen recognized by an antigen-recognition structure.

[0120] In some embodiments, the immunoglobulin molecule according to this disclosure, which is an antibody, may be, for example, an IgD, IgE, or IgM isotype, but is preferably an IgA or IgG isotype, such as an antibody of any of the human IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 subtypes.

[0121] The first and second immunoglobulin heavy chain polypeptides of a dimerized immunotherapy protein containing an immunoglobulin molecule may be the same (i.e., homodimer) or different (i.e., heterodimer).

[0122] In some embodiments, the immunoglobulin molecule according to this disclosure, which is an antibody, may be an antibody that forms oligomers either in solution (e.g., in saline at a neutral pH such as about 7.4 physiological pH) or through self-association of the antibody to form an oligomer (e.g., a hexamer) when binding to a relevant target (e.g., an antigen to which the antibody variant is directed) via "target-on" oligomerization.

[0123] For example, without wishing to be constrained by theory, an immunoglobulin molecule that is an antibody containing an H429Y amino acid substitution in the Fc region component may form oligomers in solution (e.g., in physiological saline at physiological pH), which may lead to an overall increase in the strength of binding to target binding partners (e.g., cancer antigens, bacterial or viral pathogen antigens, or other soluble target molecules or molecular complexes) (e.g., enhanced avidity). Therefore, in relation to antibodies directed against cancer antigens present on the surface of cancerous cells, antibodies according to this disclosure (in the form of oligomers in solution) may exhibit an enhanced ability to bind to cancerous cells, leading to their destruction, for example, by complement-mediated CDC or by phagocytosis by macrophages and / or ADCCs. Similarly, in relation to antibodies directed against soluble target molecules or molecular complexes, antibodies according to this disclosure (in the form of oligomers in solution) may exhibit an enhanced ability to bind to soluble target molecules or molecular complexes, leading to their removal by phagocytosis by macrophages.

[0124] On the other hand, immunoglobulin molecules that are antibodies containing the H429F amino acid substitution in the Fc region component are thought to be able to form oligomers upon binding to relevant targets, such as the C1q complement protein complex, with the Fc component assembled "on the target" presenting a stabilized and optimal platform for enhanced avidity, thereby leading to complement activation and subsequent complement-dependent cytotoxicity (CDC). Therefore, in relation to antibodies directed against cancer antigens present on the surface of cancer cells, antibodies according to this disclosure may exhibit an enhanced ability to activate complement on cancer cells, leading to their destruction.

[0125] In some embodiments, an immunoglobulin molecule according to this disclosure may be provided or used as a first immunoglobulin molecule having a first antigen-binding domain directed to a first antigen, in combination with a second immunoglobulin molecule (according to this disclosure) having a second antigen-binding domain directed to a second antigen. As just one example, in such a combination, the first antigen may be a cancer antigen present on the surface of cancerous cells (e.g., CD38 found on the surface of CLL cells), and the second antigen may be a death receptor (e.g., DR5). Oligomerization of the first and second immunoglobulin molecules may lead to the formation of heterooligomers, such as heterohexamers, in which different cell surface targets are incorporated into a cluster, which may lead to enhanced induction of apoptotic signals resulting in cell death (e.g., when a death receptor is targeted), for example, through target cell specificity achieved via binding to a cancer antigen present on cancerous cells.

[0126] Fusion / Conjugate Immunotherapy Proteins The immunotherapy proteins described herein may include fusion proteins or protein conjugates comprising a partner polypeptide linked to an Fc region component containing at least a CH3 domain (or at least a CH4 domain).

[0127] Fusion proteins or protein conjugates according to this disclosure may be monomers, dimers, or oligomers. For example, in some embodiments, the fusion protein or protein conjugate comprises monomers, each monomer comprising one copy of the partner polypeptide (or a fragment thereof) and one copy of the Fc region component, while in other embodiments, the immunotherapy protein comprises dimers, where the Fc region component self-associates (i.e., forms a dimer) either by non-covalent bonds such as hydrogen bonds, or, in particular, by the formation of disulfide bonds through one or more cysteine ​​(C) residues, if located within a hinge sequence, particularly a core hinge sequence (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003). In such a dimer, the immunotherapy protein comprises two Fc region components (which associate with each other to form, for example, an Fc fragment or an Fc-like fragment) and two fusion / conjugate partner polypeptides (or their two fragments), and thus can be considered bivalent with respect to the partner polypeptide (or its fragments). The two fusion / conjugate partner polypeptides (or their two fragments) of the dimeric fusion protein or protein conjugate can be the same (i.e., homodimer) or different (i.e., heterodimer). In some embodiments of such a dimer, as depicted in Figure 53A, the fusion protein is antibody-like (Ab-like) and may have, for example, an H2 form or an H2L2 form (the monomeric fusion protein is produced using a light (L) chain and then, optionally, fused / conjugated with a partner polypeptide (such as a target recognition structure)). To make it easy to understand, each target recognition structure of an Ab-like molecule having the H2 or H2L2 form can be the same or different, such as "X1 X1 X1 X1", "X1 X1 X1 X2", "X1 X1 X2 X2", "X1 X1 X2 X3", or "X1 X2 X3 X4", where X1, X2, X3, and X4 each represent a different target recognition structure. Furthermore, one or more of the target recognition structures can be replaced by alternative partner polypeptide types, such as enzymes or reporter molecules.

[0128] Partner polypeptides may provide beneficial functions and / or characteristics to immunotherapy proteins.

[0129] In some embodiments, the partner polypeptide may be a cell surface receptor polypeptide (or a fragment thereof) or a co-receptor polypeptide (or a fragment thereof).

[0130] For example, a partner polypeptide can be a cell surface molecule such as a cell surface receptor polypeptide (or fragment thereof) that can bind to a viral structural protein so that the immunotherapy protein can function as a “decoy” to block viral interaction and the entry of the virus into host cells. Therefore, some examples of such cell surface receptor polypeptides (or fragment thereof) that may contain a partner polypeptide include, to name just a few, angiotensin-converting enzyme 2 (ACE2) ectodomain (ACE2 is the cell entry receptor for SARS-CoV-2), nucleolin (the cell entry receptor for RSV), dipeptidyl peptidase 4 (DPP4, CD26), Hsp70 (the cell entry receptor for Japanese encephalitis virus), hepatitis A virus cell receptor 1 (HAVCR1 / TIM-1; the cell entry receptor for hepatitis A virus and Ebola virus), and differentiation antigen group 155 (C Examples include D155 (a cell entry receptor for poliovirus), glucose transporter 1 (GLUT1; a cell entry receptor for human T-cell leukemia virus 1), proto-oncogene tyrosine protein kinase MER (MERTK; a host factor that promotes swine fever virus entry), TYRO3 protein tyrosine kinase (TYRO3; a cell surface protein associated with lymphocytic choriomeningitis (LMCV) infection), AXL (a cell surface protein associated with lymphocytic choriomeningitis (LMCV) infection), and cluster differentiation 4 receptor (CD4 receptor; a cell entry receptor for human immunodeficiency virus (HIV)). In embodiments where the partner polypeptide is a cell surface receptor polypeptide (or fragment thereof) capable of binding to a viral structural protein, a point mutation at the position of the Fc region component corresponding to H429 (Eu numbering) in the amino acid sequence of the human IgG1 heavy chain may, for example, provide the immunotherapy protein with an enhanced ability to provide antiviral effects through the CDC of infected cells (e.g., if the mutation is H429F) or confer enhanced viral neutralization to the immunotherapy protein (e.g., if the mutation is H429Y).In other examples, the partner polypeptide may be a ligand for a cell surface molecule known to induce cell proliferation (i.e., the cell surface molecule may be, for example, CD3 or CD28) and / or a ligand for a cell surface molecule known to stimulate inhibitory pathways to inhibit or reduce cellular responses (i.e., the cell surface molecule may be, for example, immune checkpoint molecules such as 4-1BB, CD40, OX40, TNFR2, GITR, CD27, TIM-3, BTLA, LAG3, CTLA4, ICOS, CD28, TIGIT, PDL-1, and PD-1, and the original immunotherapy protein Gomerization can result in enhanced cross-linking (e.g., supercross-clustering), which in turn can result in enhanced signaling that induces, for example, enhanced cell proliferation or inhibited / reduced cellular responses (i.e., through enhanced stimulation of inhibitory pathways). In yet another example, a partner polypeptide can be a ligand for cell surface molecules that induce cell death when engaged with other molecules such as ligands or agonist mAbs (e.g., the aforementioned cell surface molecules of TNFRSF (e.g., TNFR1, Fas, DR3, DR4, DR5, and DR6), as well as other molecules, e.g., CD38, CD20 (Shan). (as per et al., 2000 and Cardarelli et al., 2002) and CD52 (Rowan et al., 1998). Accordingly, in some embodiments, the immunotherapy proteins described herein may comprise a dimerized fusion protein or protein conjugate comprising, for example, TRAIL (i.e., a ligand for DR4 or DR5) as a partner polypeptide linked to an Fc region component comprising at least a CH3 domain including an H429F amino acid substitution. When the ligand binds to a cell surface molecule, oligomerization of the immunotherapy proteins of these embodiments may result in enhanced crosslinking and induction of apoptotic signals to induce cell death, as desired in target cells.

[0131] If the partner polypeptide is a coreceptor polypeptide (or a fragment thereof), the coreceptor polypeptide (or fragment thereof) may, among other things, be CXC chemokine receptor 4 (CXCR4), CC chemokine receptor 5 (CCR5) (a coreceptor of the CD4 receptor that binds to the HIV viral glycoprotein gp120 and allows HIV to fuse with the host cell membrane), tetraspanin and occludin (coreceptors required to enable infection by hepatitis C virus (HCV)), and Gas6 (a ligand for receptors (such as AXL and TYRO3) that bind together with phosphatidylserine shown by viruses including West Nile virus, Zika virus, and Ebola virus, and which facilitates the entry of such viruses into host cells).

[0132] In another example, a partner polypeptide can be a cell surface receptor polypeptide (or a fragment thereof) that can bind to a ligand for a cell surface receptor. Therefore, some examples of such polypeptides (or fragments thereof) include cytotoxic T lymphocyte-associated protein 4 (CTLA4) (or its soluble extracellular fragment). CTLA4 functions as an immune checkpoint, downregulating the immune response. Immunotherapy proteins containing fusion proteins or protein conjugates, including a CTLA4 partner polypeptide (e.g., a CTLA4-Fc component fusion protein), may exhibit enhanced binding to the CTLA4 ligand by forming dimers or oligomers, thereby potentially serving as decoys to provide various therapeutic effects for the treatment of tumors (e.g., melanoma and colorectal cancer) as well as various autoimmune diseases such as SLE and rheumatoid arthritis (RA). Further examples of suitable cell surface receptor polypeptides (or fragments thereof, such as ectodomains) capable of binding to ligands for cell surface receptors include other immune checkpoints (e.g., PD1) and other cytokine receptors, such as interleukin-1 receptor (IL-1R), interleukin-6 receptor (IL-6R), tumor necrosis factor receptor-2 (TNFR2, also known as CD120b), or receptors for cytokines of the TGF-β superfamily (see review in Czajkowsky DM et al., Mol Med 4(10):1015-1028, 2012). Immunotherapy proteins containing IL-1R (or fragments thereof) fusion proteins or protein conjugates could potentially be used, for example, in anti-IL-1 therapy for the treatment of type 2 diabetes, and immunotherapy proteins containing IL-6R (or fragments thereof) fusion proteins or protein conjugates could potentially be used, for example, in anti-IL-6 therapy for the treatment of tumors and RA. Immunotherapy proteins containing TNF-R2 or its ectodomain, or fusion proteins or protein conjugates thereof, may potentially be used in the treatment of rheumatoid arthritis (RA) or other inflammatory diseases or conditions.

[0133] While we do not wish to be bound by theory, it is conceivable that fusion proteins or protein conjugates (whether in monomeric or dimeric form) according to this disclosure, including an H429Y amino acid substitution in the Fc domain component, may form oligomers in solution (e.g., in saline at a neutral pH such as physiological pH of about 7.4), which could lead to an overall increase in the strength of binding to a target binding partner (e.g., a ligand for a viral structural protein or cell surface receptor, or other soluble target molecules or molecular complexes) (e.g., enhanced avidity). Thus, in relation to fusion or conjugate proteins containing cell surface receptor polypeptides or fragments thereof that can bind to viral structural proteins, this would result in enhanced avidity of binding to viruses and provide enhanced viral neutralization (perhaps conferred by crosslinking and / or agglutinating viral particles (virions)). However, in at least some embodiments, it is conceivable that such soluble oligomers may be substantially unable to bind to the Fc receptor, and therefore viral neutralization may be possible with little or no complement activation.

[0134] On the other hand, and again, without wanting to be constrained by theory, it is conceivable that fusion proteins or protein conjugates (whether in monomeric or dimeric form) containing the H429F amino acid substitution in the Fc region component may form oligomers upon binding to relevant targets (e.g., viral structural proteins or ligands for cell surface receptors), so that the Fc component assembled "on the target" presents a stabilized and optimal configuration for binding to the C1q complement protein complex (e.g., with enhanced avidity), thereby leading to complement activation, and subsequently enhanced complement-based effector functions such as complement-dependent cytotoxicity (CDC).

[0135] Immunotherapy proteins according to this disclosure can be produced according to any of the standard methodologies known to those skilled in the art. For example, those skilled in the art can easily prepare an immunotherapy fusion protein by generating a construct containing a polynucleotide sequence encoding a fusion protein using standard molecular biology techniques, introducing this construct into a suitable host cell for the expression of the fusion protein (e.g., human kidney (HEK) host cells or derivatives thereof, e.g., Expi293 cells (Thermo Fisher Scientific)), or a host animal (e.g., a pig, monkey, rabbit, or mouse), culturing the host cells according to a standard culture protocol, and recovering the expressed fusion protein from the culture supernatant using, for example, one of the known and suitable methodologies for purification (e.g., affinity chromatography (e.g., protein A), ion exchange chromatography (IEX), size exclusion chromatography (SEC), and a combination thereof). In some embodiments, the immunotherapy protein is produced by a transgenic animal (an animal modified to express the immunotherapy protein or nucleic acid described herein). Immunotherapy proteins, which are immunoglobulin molecules such as antibodies or Ab-like molecules, can be prepared using similar methodologies. That is, those skilled in the art can prepare a variable weight (V) of a suitable antibody (e.g., one containing an antigen-binding region that binds to the antigen of interest). H ) and light (V LBy generating a construct comprising a polynucleotide sequence encoding the ) region sequence and, for example, the constant heavy (CH) region from an IgG1 antibody, mutant antibodies (i.e., antibodies containing amino acid substitutions at the position corresponding to H429 (Eu numbering) in the amino acid sequence of the human IgG1 heavy chain) can be easily prepared and incorporated into the polynucleotide sequence encoding the CH3 region by standard molecular biology techniques such as site-directed mutagenesis and polynucleotide sequence alteration to encode mutations at H429 (e.g., H429F and H429Y). Similar to the preparation of immunotherapy fusion proteins, the construct can be introduced into suitable host cells (e.g., human kidney (HEK) host cells or derivatives thereof) cultured according to a standard culture protocol and expressed. The mutant antibodies can be purified from the culture supernatant using any known and suitable methodology for purification, such as affinity chromatography. Furthermore, when affinity chromatography (e.g., protein A) is used for purification, especially when the immunotherapy protein is in the form of an Ab-like molecule, the use of mild elution conditions, such as using an elution buffer containing a low concentration of arginine (e.g., less than 130 mM) and having a pH of 5 or less, has been found to be advantageous in suppressing aggregate formation. This can be achieved, for example, by a standard liquid chromatography system capable of delivering a buffer gradient to the column, in this case, for example, a linear gradient of up to 35% of 130 mM arginine (pH 4.0).

[0136] In some preferred embodiments, the recovery of the expressed immunotherapy protein according to this disclosure is as follows: (i) When it is desirable that the immunotherapy protein be provided in monomeric form, it is preferably carried out under conditions of a weakly acidic pH (e.g., pH 6.5, preferably pH 5.5, and more preferably pH 5.0, which is below a neutral pH), or (ii) If it is desirable that the immunotherapy protein be provided in oligomeric form (e.g., hexamer), it is preferably carried out under conditions of a substantially neutral pH (e.g., a pH in the range of 7.0 to 8.5, preferably 7.5 to 8.0, or more preferably a physiological pH of about 7.4), or (iii) When it is particularly desirable that the immunotherapy protein be provided as an antibody-like molecule, the process is preferably carried out using an affinity chromatography method that uses an elution buffer containing a low concentration of arginine (e.g., less than 130 mM) and a pH of 5.0 or less (preferably about 4.0).

[0137] In some specific embodiments, the recovery of the expressed immunotherapy protein according to this disclosure includes, for example, recovery by size exclusion chromatography (SEC) under weakly acidic pH conditions for the production of the monomeric form of the immunotherapy protein, or under substantially neutral pH conditions for the production of the immunotherapy protein in oligomeric form. SEC may optionally be followed by a recovery step including ion exchange chromatography (IEX).

[0138] The US FDA classified bsAbs into two main classes based on their mechanism of action: cell-bridging bsAbs and antigen-crosslinking bsAbs (non-cell-bridging molecules, Labrijn et al, 2019). Most cell-bridging bsAbs are designed for cancer treatment by linking immune cells to malignant cells. By sequential binding, i.e., by initially binding to cancer cells due to a higher affinity for tumor antigens, cell-bridging bsAbs can reduce nonspecific side effects, lower doses, and improve specificity and efficacy compared to mAbs. In contrast, antigen-crosslinking bsAbs target two antigens or two receptors simultaneously. Their primary MoA is either blocking cell growth / survival signals or activating immune cells (Engelman et al, 2007). Antigen-crosslinking bsAbs act essentially similarly to mAbs, except that they bind to two different targets.

[0139] In one embodiment, the Disclosure provides the use of immunotherapy proteins described herein for treating or preventing a disease or condition in a subject, the disease or condition may be selected from, for example, autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection, infectious diseases, and proliferative disorders.

[0140] In one embodiment, the present disclosure provides the use of the immunotherapy proteins described herein in the manufacture of a pharmaceutical product for treating or preventing a disease or condition, the disease or condition being selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection, infectious diseases, and proliferative diseases.

[0141] In one embodiment, the present disclosure provides a method for treating or preventing a disease or condition, the method comprising administering an effective amount of the immunotherapy protein described herein to a target, the disease or condition being selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection, infectious diseases, and proliferative diseases.

[0142] Diseases and conditions that can be treated or prevented by the methods described herein include autoimmune diseases and conditions such as SLE and MS, other inflammatory diseases (e.g., immune complex vasculitis), infectious and proliferative disorders (in particular solid tumors such as breast cancer), hematological malignancies, adenocarcinomas and lymphomas, including lymphoproliferative disorders (LPDs) such as leukemia (e.g., acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL)), and multiple myeloma (MM) and X-linked proliferative disorders.

[0143] The methods described herein are typically applied to the treatment of diseases or conditions in human subjects. However, subjects may also be selected from, for example, domestic animals (e.g., cattle, horses, pigs, sheep, and goats), companion animals (e.g., dogs and cats), and exotic animals (e.g., non-human primates, tigers, elephants, etc.).

[0144] In some embodiments, if the immunotherapy protein comprises a fusion protein comprising a partner polypeptide linked to an Fc region component containing at least a CH3 domain (or at least a constant heavy domain 4 (CH4) domain), and the partner polypeptide is a cell surface receptor polypeptide (or fragment thereof) capable of binding to a structural protein of the virus, thereby allowing the immunotherapy protein to function as a “decoy” to block viral interaction and the entry of the virus into a host cell, then the method described herein may further include administering an antibody directed against the virus (i.e., the target virus). For example, if the immunotherapy protein contains an ACE2 polypeptide (or a fragment thereof) that binds to the RBD of the CoV-2 spike protein, the antibody may be selected from, for example, broadly neutralizing coronavirus mAbs (i.e., bNmAbs that can neutralize multiple coronavirus types or strains), broadly reactive coronavirus mAbs (i.e., mAbs that cannot neutralize but can bind to multiple coronavirus types or strains), broadly neutralizing SARS-CoV-2 mAbs (i.e., bNmAbs that can neutralize multiple SARS-CoV-2 strains), and broadly reactive SARS-CoV-2 mAbs, broadly neutralizing coronavirus spike stem-specific mAbs, broadly reactive coronavirus spike stem-specific mAbs, broadly neutralizing SARS-CoV-2 spike stem-specific mAbs, and broadly reactive SARS-CoV-2 spike stem-specific mAbs. In some specific embodiments, the antibody may target an epitope of a SARS-CoV-2 structural protein other than the spike protein (S), such as an envelope protein (E), a membrane protein (M), or a nucleocapsid protein (N). In some other specific embodiments, the antibody may target an epitope on the spike protein (S), for example, but at a site different from the RBD.More generally, when the immunotherapy protein includes a cell surface receptor polypeptide other than an ACE2 polypeptide, a co-receptor polypeptide, or a fragment thereof, the antibody directed against the target virus may be selected from, for example, an antibody that broadly neutralizes a class / family of viruses (e.g., human immunodeficiency virus (HIV)), an antibody that is broadly reactive to a class / family of viruses, an antibody that broadly neutralizes strains of a specific virus type (e.g., a bNmAb capable of neutralizing multiple HIV-1 strains), and an mAb that is broadly reactive to a specific virus type. As shown below, the immunotherapy proteins and antibodies directed against the target virus of this disclosure have been found to work synergistically to enhance CDC death in cells (e.g., virus-infected cells).

[0145] Similarly, if the immunotherapy protein comprises a fusion protein comprising a partner polypeptide linked to an Fc region component comprising at least a CH3 domain (or at least a constant heavy domain 4 (CH4) domain), and the partner polypeptide is, for example, directed to a therapeutically important target (e.g., the partner polypeptide is a cell surface receptor polypeptide (or fragment thereof) that can bind to CTLA4, which functions as an immune checkpoint and downregulates the immune response), then the method herein may further comprise administering an antibody directed to that target (e.g., an antibody that binds to CTLA4) to provide an enhanced response, such as enhanced CDC death of cells (e.g., cancer cells).

[0146] If the method described herein involves administering an antibody in addition to the fusion protein (i.e., as described in the previous two paragraphs), preferably the antibody is, for example, H429X 2 The formula includes an Fc region component containing an amino acid substitution at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG monohelic acid polypeptide, where X 2This is selected from phenylalanine (H429F), glutamic acid (H429E), glutamine (H429Q), serine (H429S), alanine (H429A), threonine (H429T), tyrosine (H429Y), leucine (H429L), valine (H429V), glycine (H429G), tryptophan (H429W), arginine (H429R), and proline (H429P).

[0147] In one embodiment, the present disclosure provides a pharmaceutical composition or pharmaceutical comprising an immunotherapy protein as defined herein and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0148] Immunotherapy proteins may be administered in combination with one or more additional agents for the treatment of a particular disease or condition being treated. For example, in relation to the treatment of proliferative disorders, immunotherapy proteins may be used in combination with other agents for treating cancer (e.g., antitumor agents, taxoids including cisplatin, gemcitabine, cytosine arabinoside, doxorubicin, epirubicin, and taxol; topoisomerase inhibitors, such as etoposide; cell proliferation inhibitors, such as tamoxifen; aromatase inhibitors (e.g., anastrozole); and growth factor function inhibitors (e.g., antibodies such as the anti-erbB2 antibody trastuzumab (Herceptin®)). In some embodiments, immunotherapy proteins may be administered with one or more additional agents, which may also be immunotherapy proteins according to this disclosure. For example, a first immunotherapy protein according to this disclosure If the immunotherapy protein includes an ACE2 polypeptide (or a fragment thereof) that binds to the RBD of the CoV-2 spike protein, the second immunotherapy protein of this disclosure may be an antibody directed against the target CoV-2 virus, in particular an antibody targeted to an epitope of a different structural protein (e.g., envelope protein (E), membrane protein (M), or nucleocapsid protein (N)), or an antibody targeted to an epitope on the same structural protein (i.e., spike protein (S)) but at a site different from the RBD. As shown below, such combinations of immunotherapy proteins have been found to work synergistically to enhance CDC death in cells (e.g., virus-infected cells).

[0149] When used in combination with other drugs, immunotherapy proteins may be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. When administered in separate pharmaceutical compositions, the immunotherapy proteins and other drugs may be administered simultaneously or sequentially in any order (e.g., a few seconds, a few minutes, or even a few hours (e.g., 2 to 48 hours)).

[0150] Immunotherapy proteins can be formulated into pharmaceutical compositions having pharmaceutically acceptable carriers, diluents, and / or excipients. Examples of suitable carriers and diluents are well known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995. Examples of suitable excipients for the various different forms of pharmaceutical compositions described herein are found in Handbook of Pharmaceutical Excipients, 2, edited by A. Wade and P.J. Weller. nd This can be found in Edition, (1994). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol. Examples of suitable diluents include ethanol, glycerol, and water. The selection of carriers, diluents, and / or excipients may be made in relation to the intended route of administration and standard pharmaceutical practice.

[0151] Pharmaceutical compositions comprising immunotherapy proteins as defined herein may further include any suitable binders, lubricants, suspending agents, coating agents, and solubilizers. Examples of suitable binders include natural sugars such as starch, gelatin, and glucose; anhydrous lactose, free-flow lactose, beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, tragacanth, or sodium alginate; carboxymethylcellulose; and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, and even colorants may be provided in the pharmaceutical composition. Examples of preservatives include esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0152] Pharmaceutical compositions comprising immunotherapy proteins as defined herein are typically adapted for intravenous or subcutaneous administration. Therefore, pharmaceutical compositions may include solutions or emulsions prepared from sterile or bactericidal solutions that can be injected into a subject. Pharmaceutical compositions may be formulated in unit dosage forms (i.e., in the form of a unit dose, or in the form of separate parts comprising multiple or subunits of a unit dose).

[0153] The immunotherapy proteins, uses, and pharmaceutical compositions of this disclosure are further described below with reference to the following non-limiting examples. [Examples]

[0154] Example 1: ACE-2-Fc fusion protein containing the H429 mutation and activity analysis Methods and materials Constructs and fusion proteins The amino acid sequence of the human ACE2 polypeptide is available from the European Nucleotide Archive (ENA, European Molecular Biology Laboratory) under accession number BAB40370. The protein's ectodomain (amino acids 19-740; shown as SEQ ID NO: 1 in Table 1) contains a catalytic domain and a correctrin domain. In this example, different forms of ACE2 ectodomains were produced and studied; in particular, a cleaved (tr)ACE2 ectodomain containing amino acids 19-615 of the mature ACE2 polypeptide (referred to as trACE2, shown as SEQ ID NO: 2 in Table 1) excluding the correctrin domain, a full-length (fl)ACE2 ectodomain (flACE2; containing amino acids 19-740 of the mature ACE2 polypeptide; SEQ ID NO: 1 in Table 1), and an enhanced flACE2 ectodomain (EflACE2) containing a triple mutation within the ACE2 polypeptide that has been reported to improve its binding affinity to the S protein (Chan et al., 2020, above). These proteins were produced as fusion proteins containing an Fc region component derived from human IgG1, and trACE2-Fc, flACE2-Fc, and EflACE2-Fc were generated according to standard techniques (see Table 2).

[0155] For example, the construct encoding the trACE2 ectodomain in pcDNA3.4 (Thermo Fisher Scientific) was prepared by binding the polynucleotide sequence encoding the trACE2 ectodomain to a synthetic sequence encoding the linker and to IgG1 Fc having the amino acid sequence of accession number AXN93652.1 (Immunoglobulin gamma 1 constant region, partial [Homo sapiens]; National Center for Biotechnology Information (NCBI) database). To generate the flACE2-Fc expression construct, the trACE2 construct was subjected to KpnI digestion, followed by insertion of a codon-optimized polynucleotide sequence encoding the ACE2 collectrin domain (GeneArt, Thermo Fisher Scientific). To generate the EflACE2-Fc expression construct, a construct equivalent to that encoding flACE2-Fc but with three mutations (i.e., T27Y, L79T, and N330Y; Chan et al.) was prepared. We used a synthetic polynucleotide sequence having sACE2.v2.4) as described above by al., 2020. In addition, we produced variants of the fusion protein by incorporating H429F and H429Y mutations into the Fc component, which was introduced by cleavage at a specific AfeI site within the IgG Fc coding sequence and subsequent insertion of appropriate mutagenic oligonucleotides using NEBuilder (New England Biolabs, Ipswich, MA, United States of America) according to the manufacturer's instructions for use.

[0156] The H429 residue occupies a “filled” site within the IgG1 Fc structure (see Figure 1), occupied by a histidine (His / H) residue, which is also found at the corresponding positions of Fc fragments of all other human immunoglobulin classes (Figures 3, 4), and thus, for example, in human IgG1, IgG2, IgG3, and IgG4, as well as in the corresponding positions of primate IgG subclasses and some mouse IgG subclasses. For example, the structure of human IgG1-Fc published in 1981 (PDB:1Fc1; Deisenhofer, 1981 above) shows that H429 is not a surface-accessible residue (Figure 1), and in the space-filling representation of Fc, H429 is only “visible” when the overlapping residues are rendered in a non-space-filling manner, as shown in Figure 1C. Furthermore, analysis of residues adjacent to H429 indicates that H429 is located beneath the side chains of these residues, and calculation of the accessible surface area (ASA) for the Fc residue indicates that H429 is inaccessible to the solvent (0% ASA (Å) 2 (Figure 1C). H429 is also not a surface-accessible residue within other Fc structures, including Fc within the structure of the anti-HIV mAb (PDB:1HZH; Saphire et al., above) which forms a hexameric ring structure, but H429 does not form part of the interface. Similarly, analysis of the Fc structure of the anti-Lewis Y mAb (Ramsland et al., J Immunol 187(6):3208-3217, 2011) showed that H429 is also embedded in this antibody. [Table 1-1] [Table 1-2]

[0157] Expression of the fusion proteins was performed using transient transfection of Expi293 cells (Thermo Fisher Scientific). All expressed fusion proteins were first purified from the culture supernatant by ion exchange chromatography (IEX), followed by further purification by size exclusion chromatography (SEC). In particular, the supernatant of Expi293 cells transiently transfected for the expression of each ACE2-Fc fusion protein (the Fc region was due to wild-type (WT) hIgG1 Fc as described in the previous paragraph) was broadly dialyzed against 10 mM Tris-HCl pH 8.0 and applied to a High-Q column (BioRad Laboratories, Hercules, CA, United States of America). The bound proteins were eluted with a linear gradient into buffer A containing 0.4 M NaCl. The fractions were investigated by SDS-PAGE, and those fractions containing the flACE2-Fc WT fusion protein were pooled, concentrated using a 30 kDa cutoff filtration device (Pall Corporation, Port Washington, NY, United States of America), and separated by size exclusion chromatography (SEC) using a Superose 6 column (GE Life Sciences, Chicago, IL, United States of America).

[0158] SARS-CoV-2 RBD-Ig and RBD AviTag have been previously described. RBD AviTag was biotinylated in situ using Expi293BirA cells (Wines BD et al., J Immunol 197(4):1507-1516, 2016).

[0159] Lamelli native PAGE (N-PAGE), 150V, 2.5 hours, 4℃ was performed according to Wines BD et al., J Immunol 162(4):2146-2153, 1999).

[0160] Virus neutralization assay Antiviral titers were determined using the previously described microneutralization assay with SARS-CoV-2 (CoV / Australia / VIC01 / 2020) (Juno JA et al., Nat Med 26(9):1428-1434, 2020).

[0161] Biolayer Interferometry The affinity of the ACE2-Fc fusion protein for CoV-2S RBD was measured on an Octet RED96e (ForteBio, Fremont, CA, United States of America). All assays were performed at 25°C using an anti-human IgG Fc capture (AHC) biosensor chip (ForteBio) in kinetic buffer (phosphate-buffered saline (PBS) pH 7.4 supplemented with 0.1% (w / v) bovine serum albumin (BSA) and 0.05% (v / v) TWEEN-20). After a 60-second (60s) biosensor baseline step, the fusion protein (20 mg / mL) was loaded onto the anti-human IgG Fc capture (AHC) biosensor by immersing the sensor chip for 200 seconds and then washing it in kinetic buffer for 60 seconds. For most fusion proteins, association measurements were performed by immersion in a 2-fold dilution series of 16–250 or 500 nM SARS-CoV-2 RBD for 180 seconds, followed by measurement of dissociation in dynamic buffer for 180 seconds. For EflACE2-Fc WT, a 2-fold dilution series of 2–31 or 63 nM was used. The biosensor chips were regenerated five times using a 5-second cycle in 10 mM glycine pH 1.5 and a 5-second cycle in dynamic buffer. Baseline drift was corrected by subtracting the mean shift of fusion protein-loaded sensors not incubated with SARS-CoV-2 RBD and unloaded sensors incubated with SARS-CoV-2 RBD. DTo determine the values ​​and dynamic parameters, a curve fitting analysis was performed using Octet Data Analysis 10.0 software with a global-fit 1:1 model. Curves that could not be fitted were excluded from the analysis. The reported dynamic constants represented two independent experiments.

[0162] Flow cytometry analysis of ACE2-Fc fusion protein and recombinant dimer rsFcγR binding 5 × 10⁶ Ramos cells (Ramos-S cells; Lee WS et al., medRxiv doi:10.1101 / 2020.12.13.20248143,2020) expressing spike protein, transfected with ACE2-Fc fusion protein or rituximab (a chimeric mAb targeted to CD20) at 5 μg / ml or the indicated concentration, are mixed with 5 × 10⁶ Ramos cells (Ramos-S cells; Lee WS et al., medRxiv doi:10.1101 / 2020.12.13.20248143,2020) in 25 μl of PBS (PBS / BSA / G) containing 0.5% (w / v) BSA and 1 mM glucose. 6 Cells were incubated on ice for 30 minutes at a concentration of cells / ml, then washed twice with PBS / BSA / G, incubated with PE or FITC conjugate anti-human IgG-Fc for 30 minutes on ice, washed again, and resuspended in 25 μl of PBS / BSA / G.

[0163] The binding of dimeric recombinant soluble FcγR (rsFcγR) was evaluated as previously described (Wines et al., 2016, see above). ACE2-Fc opsonized Ramos-S cells or rituximab opsonized cells were resuspended in 0.5 μg / ml biotinylated dimer rsFcγRIIa (H131 allele morphology) or dimer rsFcγRIIIa (V158 allele morphology), or in BSA / PBS / G, incubated on ice for 30 minutes, and then incubated on ice for 20 minutes with 1 / 500 streptavidin-APC (or anti-hIgG-Fc FITC to confirm ACE2-Fc opsonization). The cells were washed, resuspended in PBS / BSA / G, and analyzed on a Canto® II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, United States of America).

[0164] Complement-dependent cytotoxicity (CDC) assay CDC was measured using Ramos-S cells opsonized with ACE2-Fc functional protein or a control mAb, or rituximab, and then incubated with human serum as a complement source. Therefore, Ramos-S cells were first incubated with the aforementioned fusion protein or rituximab (5 × 10⁶ minutes on ice in 25 μl of PBS / BSA / G) for 30 minutes. 6 Cells were then washed and resuspended in 1 / 3 diluted normal human serum at 37°C for 30 minutes. Cells were washed twice with PBS, dead cells were counted by staining with 1 / 500 Zombie Green (Zombie Green fixable viability kit, BioLegend, San Diego, CA, United States of America, according to the manufacturer's instructions for use), fixed with 2% paraformaldehyde in PBS, and analyzed on a Canto® II flow cytometer (Becton Dickinson).

[0165] Complement-fixed immunoassay for ACE2-Fc fusion protein 96-well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated overnight with 5 μg / ml avidin in PBS, blocked, and then incubated at room temperature for 1 hour with either a serial 2-fold dilution or a single concentration (2.5 μg / ml) of biotinylated RBD in 0.1% casein (Hartley et al., Science Immunology 5(54) doi:10.1126 / sciimmunol.abf8891, 2020). ACE2-Fc fusion protein was then added over the indicated concentration range. To measure C1q fixation, plates were incubated with 10 μg / ml purified human C1q (Merck Millipore, Burlington, MA, United States of America) at room temperature for 30 minutes, followed by incubation with a 1 / 2000 dilution of rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:45 2019) at room temperature for 1 hour. For measuring C5b-C9 fixation, plates were incubated with 10% fresh human serum at room temperature for 30 minutes, followed by incubation with a 1 / 2000 dilution of rabbit anti-C5b-C9 (Merck Millipore) at room temperature for 1 hour, washed, then incubated with goat anti-rabbit IgG (Merck Millipore) conjugated to HRP in a 1 / 2000 dilution at room temperature for 1 hour, followed by incubation with TMB substrate (Life Technologies Corporation, Carlsbad, CA, United States of America) at room temperature for 15-20 minutes. The reactivity was stopped using 1M sulfuric acid, and the absorbance was measured at OD450nm. Test samples and reagents were prepared in PBS supplemented with 0.1% (w / v) casein, and the plate was washed three times between each step with PBS containing 0.05% (v / v) TWEEN-20. Samples were tested in double decans, and background reactivity was corrected using negative control wells from which ACE2-Fc protein was omitted. Means and SEM results from independent experiments are shown.

[0166] RBD variant multiplex assay Custom multiplex arrays were produced using SARS-1 S1 subunits (ACROBiosystems, Newark, DE, United States of America), SARS-CoV-2 S1, HCoV NL63 S1 and S2 subunits (Sino Biological Inc., Beijing, China), NL63 S trimers (BPS Bioscience, San Diego, CA, United States of America), hexahistidine-tagged RBD WT protein (amino acids 19-613), and 21 variants identified from the GISAID RBD surveillance repository, expressed from pcDNA3 in Expi293 cells and purified by affinity chromatography. Bead coupling, washing steps, and data acquisition on a FlexMap3D® analyzer (Luminex Corporation, Austin, TX, United States of America) were as previously described (Lee et al., 2020, see above). In summary, direct binding of ACE2-Fc fusion proteins trACE2-Fc, flACE2-Fc, and EflACE2-Fc at 0.5–250 nM was detected using 25 μl of 1.3 μg / ml anti-human IgG R-phycoerythrin conjugate (Southern Biotech, Birmingham, AL, United States of America). 50 To determine the data, the 3-parameter agonist versus response curve (r 2The assay was performed using a ratio of >0.85. In the competitive assay, RBD or S1 coupling beads were incubated with 20 μl of 25 μg / ml biotinylated Avi-tagged ACE2 at room temperature for 2 hours in the presence of various non-biotinylated ACE2-Fc fusion protein "competitors" at 1–280 nM. Binding of biotinylated ACE2 (aa19–615) was first performed using 4 μg / ml streptavidin, R-phycoerythrin conjugate (SAPE) (Thermo Fisher Scientific) (1 hour), followed by 10 μg / ml R-phycoerythrin, biotin-XX conjugate (Thermo Fisher Scientific) (1 hour).

[0167] FcγRIIIa-NF-κB-RE Nanoluciferase Reporter Assay The FcγRIIIa-NF-κB-RE nanoluciferase reporter assay was performed using IIA1.6 / FcR-γ / FcγRIIIa V158 cells (NanoLuc, pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro], Promega Corporation, Madison, WI, United States of America) expressing NF-κB response element-driven nanoluciferase, essentially as previously described (Lee et al., 2020, above). In summary, Ramos cells expressing spike-IRES-orange 2 were used as target cells and incubated with the agonist and FcγRIIIa / NF-κB-RE reporter cells for 5 hours, after which induced nanoluciferase was measured using the Nano-Glo substrate (Promega Corporation).

[0168] Results and Discussion Construction and production of ACE2-Fc fusion proteins We analyzed the ability of these proteins to produce a series of ACE2-Fc fusion proteins (Table 2), neutralize SARS-CoV-2 infection, and mediate Fc-dependent effector function, which is typically attributed to the mechanism of action of antibodies. [Table 2]

[0169] In an attempt to enhance the avidity of binding to the SARS-CoV-2 spike protein, or to confer and improve Fc-dependent effector function, three versions of the ACE2 ectodomain were fused to the Fc region of IgG1 that was either unmodified or altered by mutation (i.e., substitution of histidine 429 with phenylalanine (H429F) or tyrosine (H429Y)) or modified glycosylation (i.e., lack of core fucose; trACE2-Fc-kif).

[0170] Purification of ACE2-Fc fusion protein The proteins were produced in Expi293 cells, purified by ion exchange (IEX), and then purified by size exclusion chromatography (SEC) at pH 7.4 (Figure 5). All fusion proteins showed an IEX purification profile similar to that of flACE2-Fc-WT (Figure 5A), containing a major elution peak (peak*, Figure 5A), and SDS-PAGE analysis of the collected fractions showed a single major species of approximately 270 kDa (Figure 5B). With the exception of those fusion proteins containing the H429Y mutation, SEC analysis and purification confirmed the presence of the major monomer species (Figure 5C) (Note: The monomer species is considered to be a single molecule (i.e., a monomer molecule) containing two (dimerized) copies of each ACE2-Fc fusion protein collected for further analysis), for example, for flACE2-Fc-WT, only trace amounts of higher molecular weight oligomers and other impurities were evident (Figure 5). Indeed, the presence of similar monomeric species was also evident after IEX purification of all fusion proteins using unmodified Fc-WT or Fc region components containing H429F modification (not shown). For fusion proteins containing the H429Y mutation, SEC analysis (Figure 5D) (i.e., the major IEX peak from the flACE2-Fc H429Y mutant, not shown), as well as the major IEX peaks from trACE2-Fc H429Y and EflACE2-Fc H429Y proteins (not shown)) showed a significant amount of oligomer (Y oli ) and monomer (Y mn The existence of these species was revealed, and their functional activity was subsequently evaluated separately.

[0171] ELISA for the binding of SARS-CoV-2S to the ACE2-Fc fusion protein RBD. The binding of trACE2-Fc WT, flACE2-Fc WT, and EflACE2-Fc WT fusion proteins to the SARS-CoV-2 receptor-binding domain (RBD) (Figure 6A-C) is generally similar (i.e., respectively, EC 50At 0.35 nM, 0.27 nM, and 0.25 nM (Figure 6D), the enhanced intrinsic affinity of EflACE2-Fc was not as evident in the binding of this bivalent form to RBD, as demonstrated in fusion with mouse IgG1 Fc (RBD-Ig). The binding activity of various fusion proteins with the mutated Fc component was also slightly lower in the monomer flACE2-Fc-H429Y(EC) 50 Except for the 0.48 nM variant (Figure 6D) and other H429Y Fc variants that similarly tended to have lower affinity levels, they were comparable (Figure 6).

[0172] The oligomerization of the ACE2-Fc H429Y fusion protein is pH-dependent. The oligomerization of fusion proteins containing the mutated H429Y Fc component was investigated by SEC isolation at pH 5.0 of flACE2-Fc-H429Y prepared by IEX. In contrast to SEC at pH 7.4 (Figures 7A and 7B), SEC at pH 5.0 (Figure 7B) showed a larger proportion of monomeric Y. mn This was clarified, and N-PAGE showed that it was homogeneously purified (see Figure 7C: Lane 1, Lane 2). Purified monomer flACE2-Fc-H429Y mn The sample (pH 5.0) was re-analyzed by dialysis at pH 7.4, followed by N-PAGE (Figure 7C) and SEC at pH 7.4 (Figure 7D). Re-exposure to pH 7.4 resulted in oligomer Y oli and monomer Y mn This yields a mixture of species (see Figure 7C; lane 2 pH 5.0, lane 5 pH 7.4), showing that some equilibrium between these forms occurs at a neutral pH. flACE2-Fc-H429Y prepared at pH 5.0 mn This variant showed binding to RBD-Ig comparable to other flACE2-Fc WT fusion proteins and Fc variants (Figure 7E). Furthermore, the high number of oligomers was not observed in fusion proteins containing the phenylalanine-substituted H429F Fc region component, clearly indicating a correlation with the tyrosine substitution of histidine 429.

[0173] Evaluation of virus neutralizing efficacy The antiviral activity of the ACE2-Fc fusion protein was determined by a microneutralization assay of SARS-CoV-2 infection in Vero cells (Figure 8), EC 50 The endpoint corresponds to the neutralization of approximately 99% of the inoculated virions (Khoury DS et al., Nat Rev Immunol 20(12):727-738, 2020).

[0174] The SARS-CoV-2 neutralization endpoint of the cleaved ectodomain, trACE2 (2.70 μM), was improved by approximately 10-fold by fusion to the wild-type Fc region of IgG1 (trACE2-Fc WT, 283 nM) (Figure 8). The improved potency was consistent with the improved avidity of binding to the SARS-CoV-2 spike RBD due to ACE2-Fc bivalentity resulting from the fusion of the ACE2 ectodomain to the IgG Fc region, and was similar to that of flACE2-Fc WT. EflACE2-Fc-WT showed a further improvement of approximately 20-fold (11 nM) compared to flACE2-Fc WT and trACE2-Fc WT fusion proteins, and approximately 200-fold improvement compared to unfused trACE2.

[0175] Analysis of Fc modifications revealed several interesting differences. Firstly, the H429Y mutation in trACE2-Fc and flACE2-Fc improved their virus-neutralizing efficacy (Figure 8), which was surprising because the neutralizing function of the ACE2-Fc fusion protein arises far enough away from the modification at the H429 position (i.e., in the fusion protein, the ACE2 polypeptide is expected to neutralize the virus, but the H429 residue is distal to ACE2, located in the CH3 domain of the Fc component). Secondly, the oligomeric form of trACE2-Fc-H429Y isolated by SEC at pH 7.4 oli The neutralizing activity (21.9 nM) was found to be 13-fold enhanced compared to trACE2-Fc WT. The Y oligomer of flACE2-Fc-H429Y, which contains two (dimerized) copies of the ACE2-Fc fusion protein, was found to be Y. oli , and monomer Y mnBoth forms (Figure 5B) (endpoint of 10.0 and 20.9 nM, respectively; Figure 8) showed greater potency than the flACE2-Fc WT (124 nM) fusion protein and were similar to virus neutralization achieved with EflACE2-Fc WT (10.6 nM), but EflACE2-Fc-H429Y oli appeared more potent (4 nM), representing >200-fold greater neutralizing activity than that of non-fused trACE2 and >600-fold greater in the case of EflACE2-Fc-H429Y (4 nM) than that of EflACE2-Fc WT (10.6 nM) (Figure 8). Thus, the Fc-H429Y mutation increased SARS-CoV-2 neutralization in trACE2-Fc and flACE2-Fc fusion protein formats and tended towards greater levels of potency when combined with triple mutations of ACE2 in the inherently higher affinity EflACE2-Fc.

[0176] Phenylalanine substitution of histidine 429 (H429F) in the ACE2-Fc fusion protein did not enhance neutralization.

[0177] Interaction of the ACE2-Fc fusion protein with FcγR The interaction of ACE2-Fc fusion proteins with FcγRIIa and FcγRIIIa was evaluated by flow cytometry using ACE2-Fc-opsonized Ramos-S cells and dimeric recombinant soluble FcγR (Wines et al., 2016, supra). All trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins bound to FcγRIIa and FcγRIIIa (Figures 9A, 9B), but the variant proteins containing the mutated H429Y Fc component mostly removed binding to both of these Fc receptor types.

[0178] Activation of FcγRIIIa by the ACE2-Fc fusion protein Antibody-dependent cytotoxicity (ADCC) and Fc-dependent clearance of viruses are important antiviral effector mechanisms that can play a protective role during SARS-CoV-2 infection (Li D et al., bioRxiv doi:10.1101 / 2020.12.31.424729,2021, and Shafer A et al., J Exp Med 218(3):e20201993,2021). Therefore, in this experiment, we evaluated the ability of trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins, as well as variant proteins, to activate FcγRIIIa. All Ramos-S cells opsonized with ACE2-Fc fusion proteins containing the wild-type (WT) Fc region were found to initiate FcγRIIIa activation (Figure 10). However, it was surprising that the flACE2-Fc fusion protein induced a higher level of activation than trACE2-Fc, indicating that including a collectrin domain in the ACE2 component of the fusion protein substantially and surprisingly improves the efficacy of FcγRIIIa activation by the ACE2-Fc fusion protein. Furthermore, it was found that the increased affinity of the flACE2-Fc fusion protein for the SARS-CoV-2 spike protein did not alter the level of FcγRIIIa activation and was equivalent to that of flACE2-Fc (Figure 10D).

[0179] The production of the trACE2-Fc fusion protein, trACE2-Fc-kif, in the presence of kifunensin (van Berkel PHC et al., Biotechnol Bioeng 105 (20:350-357, 2010), also improves the moderate level of FcγRIIIa activation exhibited by trACE2-Fc to a level equivalent to that of flACE2-Fc and EflACE2-Fc fusion proteins (Figure 10D), and CD20 +The therapeutic anti-CD20 mAb, rituximab, on Ramos-S cells approached levels (Figure 10A, 10D). The mannosidase inhibitor kifunensin prevents normal N-linked glycosylation, including core fucosylation, and in immunoglobulins, the absence of fucose on heavy chain glycans at Asn297 is known to improve FcγRIIIa binding and activation (Ferrara C et al., Proc Natl Acad Sci USA 108(31):12669-12674, 2011). Therefore, it was hypothesized that similar treatment of flACE2-Fc and EflACE2-Fc fusion proteins, or amino acid residue substitutions to increase affinity for FcγRIIIa (Wang et al., 2018 above), would likely further improve their FcγRIII activating potency.

[0180] Modification of trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins by including the H429F Fc component mutation did not affect FcγRIII activation by opsonized Ramos-S cells (Figure 10). In contrast, the inclusion of the mutated H429Y Fc component in all ACE2-Fc fusion proteins eliminated cellular FcγRIIIa activation, which was consistent with the aforementioned failure of binding to the Fc receptor, particularly FcγRIIIa (Figure 9B). Thus, while enhancing viral neutralization, the H429Y modified Fc component in trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins was inactive in FcγR binding (Figure 9) and in cellular activation via FcγRIIIa (Figure 10). The hierarchy of FcγRIIIa activation by fusion proteins is flACE2-Fc WT = EflACE2-Fc > trACE2-Fc, emphasizing that the presence of the correctrin domain is a crucial component in the optimal activation of FcγRIIIa by ACE2-Fc fusion proteins.

[0181] Complement fixation, activation, and lysis of SARS-CoV-2 spike cells by ACE2 Fc protein. The ACE2-Fc fusion protein containing a mutated Fc region component was investigated for its ability to fix complement components C1q and C5-C9 (Figures 11A-F) in ELISA-based analysis using avidin-immobilized RBD-biotin, and, importantly, for its ability to mediate complement-dependent death of cells expressing the SARS-CoV-2 spike protein (Figure 11G). When RBD was restricted (Figures 11A, 11B), the activity of the fusion protein containing the mutated H429F Fc component was remarkably enhanced compared to its wild-type Fc region counterpart. Additionally, binding of C5b-9 (Figures 11E, 11F), which form membrane attack complexes on cells, was comparable for the fusion protein containing the mutated H429F Fc region component. Furthermore, despite their oligomerization (Figures 6 and 7), a feature associated with superior CoV-2 neutralizing activity (Figure 8), both trACE2-Fc-H429Y and flACE2-Fc-H429Y fusion proteins showed little difference in C1q or C5-9 fixation compared to their wild-type Fc region-containing counterparts (Figures 11A-F). Moreover, the trACE2-Fc fusion protein was clearly more effective than flACE2-Fc in fixating complement C1q (see Figures 11C, 11E, as well as Figures 11D, 11F). This is likely because the presence of the correctrin dimerization domain in flACE2-Fc reduces the segmental flexibility of the fusion protein, thereby negatively impacting complement C1q binding. However, this difference is significantly reduced by including the H429F Fc mutation in the trACE2-Fc-H429F and flACE2-Fc-H429F fusion proteins, particularly evident in the fixation of the C5b-C9 complex (Figures 11E, 11F).

[0182] Surprisingly, the fusion protein containing the mutated H429F Fc region component was the only highly active ACE2-Fc fusion protein in serum complement-dependent cytotoxicity (CDC) of Ramos-S cells (Figure 11G). Despite their ability to fix C1q and C5b-9 in ELISA assays, trACE2-Fc WT, as well as trACE2-Fc-kif fusion proteins and flACE2-Fc WT, failed to induce complement-mediated cell death and, despite their higher affinity for the SARS-CoV-2 spike protein, failed to induce EflACE2-Fc WT. trACE2-Fc-H429Y mn The fusion protein was also weakly active in complement-mediated death.

[0183] Since it is well known that activation of the complement cascade can also lead to phagocytosis of cells, microorganisms, or particles opsonized with C1q (or other complement fragments such as C3b or C3bi; Ricklin D et al., Immunol Rev 274(1):33-58, 2016) that bind to specific cell surface receptors such as CR1 or CR3 on phagocytic cells (Vandendriessche S et al., Front Cell Dev Biol 9:624025, 2021), the observed enhancement of Fc-dependent complement lysis by ACE2-Fc fusion proteins containing the H429Y mutation is thought to reflect a similar enhancement in complement-dependent phagocytosis of ACE2-Fc coated targets (e.g., virions) via complement receptors.

[0184] conclusion Immunotherapy proteins in the form of fusion proteins containing angiotensin-converting enzyme 2 (ACE2) polypeptide (or a fragment thereof) fused to an Fc region component containing the H429 mutation have been found to offer considerable potential for the treatment or prevention of coronavirus infection. For example, the selection of the full length or cleavage (i.e., fragment) of ACE2, as well as various modifications of the Fc component, can also allow for considerable "modification" of antiviral agents to modify the action on which antiviral effects are achieved. For instance, by including the Fc component in an immunotherapy protein containing the H429Y substitution, oligomeric immunotherapy proteins exhibiting increased viral neutralization and inactivation of FcγR binding and activation can be produced. Conversely, by including the Fc component in an immunotherapy protein containing the H429F substitution, antiviral effects, including complement-dependent cytotoxicity (CDC) of spike protein-expressing cells (e.g., infected cells), can be achieved. Thus, these unique Fc mutations enable complementary approaches to modulate the function of the Fc region component, which can be useful in the development of therapeutically important ACE2-Fc fusion proteins and other fusion proteins by allowing for the selection of desired functional profiles.

[0185] Example 2: Chimeric anti-TNP IgG1 antibody containing the H429 mutation and activity analysis Methods and materials Construction of anti-TNP human IgG and mutant anti-TNP human IgG plasmid constructs Variable weight of mouse anti-trinitrophenyl (anti-TNP) antibody TIB142 (V H ) and light (V LChimeric anti-TNP human IgG antibody constructs consisting of a region sequence and a sequence from the constant weight (CH) region of a human IgG subclass have been previously described in detail as -hIgG1 (Patel D et al., J Immunol 184(11):6283-6292, 2010), hIgG2, and hIgG4 (Wines et al., 2016, see above). All chimeric antibody sequences were subcloned into pCR3 vectors. Using standard molecular biology techniques, mutations for H429F, H429Q, H429E, H429S, H433A, and H435A substitutions were generated in cDNA sequences encoding the Fc region component.

[0186] Expression and production of IgG antibodies by Expi293 cells IgG antibodies were produced in Expi293 human fetal kidney cells as previously described (Wines et al., 2016, see above). In summary, Expi293 cells were maintained in Expi293 Expression Medium (Gibco, Waltham, MA, United States of America) for both cell growth and protein production. The cells were simultaneously transfected with IgG heavy chain plasmid (15 ug) and light chain plasmid (15 μg) diluted in Opti-MEM I reduced serum medium (Gibco) using the Expifectamine transfection kit (Life Technologies), and then cultured for 4 days. The culture supernatant was clarified by centrifugation and filtered through a 0.2 μm filter. Subsequently, IgG was purified by affinity chromatography using a Hi-Trap HP protein A column (GE Healthcare Life Sciences, Marlborough, MA, United States of America), eluted with 0.1 M citrate, pH 3.5, followed by neutralization with 1 M Tris-HCl, pH 9.0, and dialyzed against PBS pH 7.5. Aggregates were removed by subsequent gel filtration on a Superose 6 10 / 300GL column (GE Healthcare Life Sciences), and the monomeric IgG peak fraction was collected. The antigen-binding activity of all antibody preparations was tested on BSA-TNP by ELISA as described (Wines et al, 2016, above).

[0187] Evaluation of antigen-binding activity by monoclonal anti-TNP mAbs Anti-TNP mAbs were evaluated by ELISA for their antigen-binding activity by binding to TNP-haptenylated BSA (TNP-BSA), as previously described in Wines et al., 2016.

[0188] ELISA immunoassay for detecting complement fixation by mAbs 96-well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated overnight at 4°C with 20 μg / ml TNP-BSA in PBS. The following morning, the plates were blocked at 37°C for 2 hours with 0.1% (w / v) casein in PBS, and then incubated at room temperature for 2 hours with anti-TNP mAb (concentration of 4–0.125 μg / ml). To measure C1q fixation, TNP:anti-TNP plates were incubated at room temperature for 30 minutes with 10 μg / ml purified human C1q (Merck Millipore), and then incubated at room temperature for 1 hour with a 1 / 2000 dilution of rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:45 2019). In experiments to measure C5b-C9 fixation, TNP:anti-TNP plates were incubated with 10% fresh human serum at room temperature for 30 minutes, followed by incubation with a 1 / 2000 dilution of rabbit anti-C5b-C9 (Merck Millipore) at room temperature for 1 hour, followed by washing. Then, they were incubated with goat anti-rabbit IgG (Merck Millipore) conjugated to HRP in a 1 / 2000 dilution at room temperature for 1 hour, followed by incubation with TMB substrate (Life Technologies) at room temperature for 15-20 minutes. Reactivity was stopped using 1M sulfuric acid, and absorbance was measured at OD450nm. Test samples and reagents were prepared in 0.1% (w / v) casein in PBS, and the plates were washed three times between each step using 0.05% (v / v) Tween20 in PBS. Samples were tested in double decans, and background reactivity was corrected using negative control wells with the antibody omitted.

[0189] Results and Discussion Anti-TNP antibody construct The TNP-WT mAb used in this example comprises one of two human heavy chain isotypes, firstly, a TNP-specific V of the mouse monoclonal antibody TIB142 fused to the CH1-hinge-CH2-CH3 domain of human IgG1, encoded by cDNA (SEQ ID NO: 33), in the order from N-terminus to C-terminus. HThe first is an IgG1 isotype polypeptide containing the domain (SEQ ID NO: 32) (Patel D et al., J Immunol 184:6283-6292, 2010); and the second is a TNP-specific V of mouse monoclonal antibody TIB142 fused to the CH1-hinge-CH2-CH3 domain of human IgG2, encoded by a codon-optimized cDNA having the sequence shown in SEQ ID NO: 35, in the order from N-terminus to C-terminus. H It was an IgG2 isotype polypeptide (SEQ ID NO: 34) containing the domain. The TNP-specific light chain polypeptide (SEQ ID NO: 36) was a TNP-specific V of mAb TIB142 fused to the human kappa chain constant domain, encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 37. L It included the domain.

[0190] IgG modification enhances complement fixation. When antibodies such as mAbs bind to their target antigens, the classical complement pathway is activated, potentially leading to physiological effects including target cell destruction via complement-dependent lysis (CDC) or complement-mediated antibody-dependent phagocytosis (C'ADCP) by specialized phagocytic cells via specific receptors for complement proteins and fragments. Activation of this major amplification effector system of innate immunity is critically dependent on and initiated by the binding of the hexameric complement protein C1q, which is part of the C1 complex. This binding of C1q by antibodies bound to its target antigen ultimately leads to the formation of membrane attack complexes (MACs) that include other complement components (C5b, C6, C7, C8, C9).

[0191] Since the H429F Fc region component mutation conferred an effective CDC to the ACE2-Fc fusion protein (see Example 1), the effects of these (Figure 12) and several other mutations (Figure 13) were evaluated in human IgG1 under various conditions of antigen density or input mAb concentration. The Fc component mutations were investigated by ELISA for complement fixation in relation to intact (chimeric) IgG1 antibodies having a mouse V domain that recognizes the TNP hapten and the human IgG1 constant domain. Surprisingly, the substitution of histidine at position 429 in the TNP-IgG1-H429F mAb resulted in enhanced C1q binding compared to the C1q binding of the unmodified TNP-IgG1-WT mAb (Figure 12). Next, TNP-specific mAbs were evaluated for the generation of membrane attack complex proteins (Figure 12). Again, the TNP-IgG1-H429F mAb exhibits enhanced complement activation, as seen by increased MAC protein (C5b, C6, C7, C9) formation, and importantly, this is perfectly consistent with its observed enhanced ability to bind to C1q conferred by the H429F mutation. Thus, modification at position 429 can unexpectedly alter the properties of an antibody. This enhanced function is surprising because amino acid 429 is embedded within the CH3 domain of IgG1, away from the complement C1q binding site within CH2 of the H chain and from the binding site to the leukocyte Fc receptor (Hogarth PM and Pietersz GA. Nature Reviews Drug Discovery 11:311-331, 2012; Chenoweth et al., 2020 above).

[0192] The H429F mutation in IgG1 antibody enhanced both C1q (Figure 12A) and C5b-C9 fixation (Figure 12B). In particular, in C1q fixation, the complement-enhancing activity of mutant IgG1 antibodies containing the H429F mutation was most evident when a lower density of TNP:BSA antigen was opsonized by the mutated IgG (Figure 12). Therefore, in relation to both ACE2-Fc fusion protein and intact IgG1 antibody, the H429F Fc component mutation enhances complement activation.

[0193] The antigen-binding activities of anti-TNP mAb, TNP-IgG1-WT, TNP-IgG2-WT, and the modified (variant) mAb, TNP-IgG1-H429F, were evaluated by ELISA for binding to TNP-haptenylated BSA (TNP-BSA) as described (Wines et al., 2016 supra). All of the mAbs showed substantially equivalent binding activity to TNP-BSA (Figure 12C), indicating that the enhanced complement activation by the Fc-modified anti-TNP antibodies is due to the Fc mutations and not due to differences in antigen-binding activity.

[0194] Conclusion The data obtained in this example demonstrated that the IgG-H429F variant enables enhanced C1q and C5b-C9 fixation to allow for increased complement activation and, thus, can be expected to induce useful complement-based effector functions.

[0195] Example 3 Production and Analysis of Modified Forms of Therapeutic Antibodies Containing the H429 Mutation In this example, the effect of modification at position H429 (i.e., by amino acid substitution) on antibody properties was evaluated using a number of irrelevant chimeric mAbs containing different antigen-binding variable domains that bind to irrelevant epitopes within the range of different target molecules on different cell types. These antibodies were "based on" various commercially used and, in some cases, clinically important mAbs. Specifically, the experiments aimed to determine the potential functional effects of amino acid substitution at position 429 in antibodies having the V domains of anti-HER2 mAb trastuzumab and pertuzumab, anti-CD20 mAb having the V domain of rituximab or 11B8, and anti-CD38 mAb daratumumab.

[0196] Methods and Materials Antibodies and Antibody Constructs Synthetic DNA corresponding to DNA sequences obtained by RT-PCR or immunoglobulin variable and constant sequences was assembled using standard molecular biology techniques, including ligation and Gibson assembly (NEBuilder, New England Biolabs), or as complete synthetic DNA encoding the entire immunoglobulin H and L chains. These sequences were used in expression vectors such as pcDNA3.1, pcDNA3.4 (Thermo Fisher Scientific), and pCIneo (Promega Corporation).

[0197] The unmodified mAb used in this example was formalized in human IgG heavy chain and produced using human kappa light chain. The unmodified mAb exhibited specific V of the indicated mAb. H and V L This includes the domain and is referred to as the wild-type (WT) form. For example, the "WT" trastuzumab antibody used in this example is the wild-type (WT) HER2-specific trastuzumab antibody as previously described. H and V L Includes the domain (https: / / go.drugbank.com / drugs / DB00072).

[0198] More specifically, the trastuzumab-WT mAb used in this example is trastuzumab fused to the CH1-hinge-CH2-CH3 domain of human IgG1 from N-terminus to C-terminus, and contains HER2-specific nucleotides. H The HER2-specific heavy chain polypeptide was encoded by codon-optimized DNA having the sequence described in https: / / go.drugbank.com / drugs / DB00072 (its amino acid sequence is provided as SEQ ID NO: 12) and shown as SEQ ID NO: 13, including the domain. Similarly, the polypeptide of the anti-HER2 mAb trastuzumab light chain was as described in https: / / go.drugbank.com / drugs / DB00072 (SEQ ID NO: 14), and was fused to the human kappa constant domain, trastuzumab's HER2-specific V LEncoded by codon-optimized DNA containing a domain and having the sequence shown as sequence number 15.

[0199] The 11B8-WT mAb used in this embodiment is a CD20-specific 11B8 fused to the CH1-hinge-CH2-CH3 domain of human IgG1, in the order from N-terminus to C-terminus. H The CD20-specific heavy chain polypeptide (US Patent No. 8,529,902) (SEQ ID NO: 16), which includes the domain, is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 17. Similarly, the polypeptide of the anti-CD20 mAb 11B8 light chain is as previously described (SEQ ID NO: 18), and contains the CD20-specific V of 11B8 fused to the human kappa constant domain. L It is encoded by codon-optimized DNA containing a domain and having a sequence shown as sequence number 19.

[0200] The daratumumab-WT mAb used in this example is a daratumumab mAb fused to the CH1-hinge-CH2-CH3 domain of human IgG1 from the N-terminus to the C-terminus, with CD38-specific V H The CD38-specific heavy chain polypeptide (https: / / go.drugbank.com / drugs / DB09331) (SEQ ID NO: 20), which includes the domain previously described, is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 21. Similarly, the polypeptide of the anti-CD38 mAb daratumumab light chain is the CD38-specific V of the daratumumab mAb fused to the human kappa constant domain. L The domain, as previously described at https: / / go.drugbank.com / drugs / DB09331 (SEQ ID NO: 22), is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 23.

[0201] The pertuzumab-WT mAb used in this example is HER2-specific IgG1 fused to the CH1-hinge-CH2-CH3 domain of human IgG1 in the order of N-terminus to C-terminus. HThe HER2-specific heavy chain polypeptide, including the domain, is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 25, as previously described at https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 24). Similarly, the polypeptide of the anti-HER2 mAb trastuzumab light chain is encoded by the HER2-specific V of pertuzumab mAB fused to the human kappa constant domain. L The domain, as previously described at https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 26), is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 27.

[0202] The rituximab-WT mAb used in this example is a CD20-specific vituximab fused to the CH1-hinge-CH2-CH3 domain of human IgG1 from the N-terminus to the C-terminus. H It is encoded by codon-optimized DNA containing a CD20-specific heavy chain polypeptide (described at https: / / go.drugbank.com / drugs / DB00073) (SEQ ID NO: 28) that includes the domain, and having the sequence shown as SEQ ID NO: 29. Similarly, the CD20-specific V of rituximab fused to the human kappa constant domain. L The polypeptide of the anti-CD20 mAb rituximab light chain, including the domain, as previously described at https: / / go.drugbank.com / drugs / DB000723 (SEQ ID NO: 30), and the codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0203] Synthesis of unmodified and mutated heavy chains Antibody expression vectors were generated by standard methods known to those skilled in the art. In summary, the antibody expression vectors consisted of synthetic polynucleotide sequences encoding antibody heavy or light chains, appropriately positioned within plasmids such as pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors for antibodies of different specificities were created using existing variable domains (V Hor V L It was produced by cleaving at the restriction site at the boundary of the ) domain. A new synthetic DNA encoding a new V domain and adjacent to a sequence (e.g., 25 nucleotides) homologous to the cleaved vector was then incorporated by reaction with NEBuilder (New England Biolabs) according to the manufacturer's instructions for use.

[0204] Fc variants were produced by synthesizing a synthetic polynucleotide sequence encoding the variant, by cleaving the Fc coding sequence of an antibody expression plasmid with an appropriate restriction enzyme, and by incorporating novel mutagenic synthetic DNA through a reaction using NEBuilder (New England Biolabs) according to the manufacturer's instructions for use.

[0205] Expression of antibody constructs Antibody expression was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific). Expi293F cells were cultured in EXPI expression medium (Life Technologies) and 24 hours before transfection of the cells, 2 × 10⁶ antibodies were expressed. 6 The solution was divided into concentrations of 10 cells / ml. On the day of transfection, 7.5 × 10⁶ cells were used. 710

[0206] Cell cultures were collected and centrifuged at 2500 rpm for 20-30 minutes. The supernatant was filtered through a 0.2 μm high-flow filter (Sartorius AG, Gottingen, Germany) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0207] Purification of mAbs with protein A affinity mAbs were purified from the supernatant of transfected Expi293F cells by protein A affinity chromatography. In summary, a Hi-trap® high-performance protein A column (GE Healthcare Life Sciences) was washed, equilibrated in binding buffer (20 mM NaH2PO4 pH 7.0), loaded with cell culture supernatant, washed with binding buffer to baseline OD 280 nm, eluted the bound antibody with 0.1 M sodium citrate tribasic dihydrate (pH 3.5), collected a 1 ml fraction, immediately neutralized with 1 M Tris-HCl pH 9.0, and pooled the antibody-containing fraction.

[0208] mAb size exclusion chromatography (SEC) After protein A affinity purification, the antibodies were further purified and characterized using size exclusion chromatography (SEC). The purified protein A antibodies were concentrated to OD280 nm at 6–8 using a 30 kDa molecular weight cutoff centrifuge concentrator device (Merck-Millipore). A Superose 6 10 / 300GL column (GE Healthcare Life Sciences) was equilibrated in PBS pH 7.2, then loaded with the concentrated protein A affinity purified antibody, separated in PBS at a flow rate of 0.5 ml / min, and a 0.5 ml fraction was collected. For some mAbs, since SEC was performed at pH 5.0, the concentrated protein A purified mAbs were dialyzed overnight in buffer (100 mM sodium citrate, 100 mM NaCl, pH 5.0) and then applied to a Superose 6 10 / 300 column pre-equilibrium in the same buffer. Dialysis-treated antibodies were applied to a Superose SEC column at a flow rate of 0.5 ml / min, and a 0.5 ml fraction was collected from the column.

[0209] Evaluation of antigen binding by monoclonal antibodies Purified antibodies were tested for antigen binding before functional analysis. mAbs that recognize cell surface antigens were tested for antigen recognition by flow cytometry as previously described in Trist et al., J Immunol 192(2):792-803, 2014. Anti-CD20 mAbs based on rituximab or 11B8 and anti-CD38 mAbs based on daratumumab were tested for binding to Ramos lymphoma cells expressing CD20 and CD38. Anti-HER2 mAbs based on trastuzumab and pertuzumab mAbs were tested in the HER2-expressing ovarian cancer cell line SK-OV-3. In summary, the mAbs were titrated by serial 2-fold dilution in 25 μl of FACS buffer (PBS with 0.5% (w / v) BSA). Then, 5 × 10⁻⁶ 625 microliters of target cells at a concentration of / ml were added to a titration mAb and incubated on ice for 30 minutes. The cells were then washed twice in FACS buffer, resuspended in 50 μL of anti-human IgG(Fab')2-Alexa 647 conjugate, incubated on ice for 30 minutes, washed twice in cold FACS buffer, and then resuspended in 200 μL of FACS buffer. The cells were analyzed by flow cytometry using a BD FACSCanto® II flow cytometer.

[0210] Flow cytometry detection of C1q binding to cells opsonized with monoclonal antibodies C1q binding to antibody-opsonized cells was evaluated by flow cytometry. Ramos lymphoma cells or SK-OV-3 adenocarcinoma cells (5 × 10⁶ in FACS buffer) 6 The cells ( / ml) were incubated on ice for 30 minutes with serial dilutions of either an anti-CD20 rituximab-based antibody or an anti-HER2 trastuzumab-based antibody. The cells were washed twice in FACS buffer, resuspended in normal human serum diluted 1 / 3 in FACS buffer, and incubated on ice for a further 30 minutes. The cells were then washed twice in cold FACS buffer, and bound C1q was detected by resuspending the cells in rabbit antiserum (1:500 dilution) for detecting human C1q, and incubated on ice for a further 30 minutes. The cells were then washed twice in cold FACS buffer, resuspended for 30 minutes in phycoerythrin conjugate donkey antiserum for detecting rabbit antibodies, washed twice more, and resuspended in 200 μL of ice-cold FACS buffer before analysis on a BD FACSCanto® II flow cytometer (Becton Dickinson).

[0211] Cellular complement-dependent lysis CDC was measured by flow cytometry using the Zombie Green Fixable Viability Kit (BioLegend) after opsonization of target cells with mAbs. The target mAb was successively diluted 2-fold in 25 μl of PBS / BSA / G (PBS containing 0.5% (w / v) BSA and 1 mM glucose) or Lebowitz-15 (L-15) medium (containing 0.5% (w / v) BSA and lacking phenol red) starting at the initial concentrations shown in the figure. Therefore, for each WT mAb or modified mAb (i.e., H429 mutant mAb), or combination thereof, 5 × 10⁶ mAbs in 25 μl of PBS / BSA / G was measured. 6 25 μl of target cells at a concentration of / ml were then added to a titration mAb and incubated on ice for 30 minutes. The cells were then washed twice in buffer, resuspended in 50 μl of complement (normal human serum (NHS) thawed immediately before use and diluted 1:3 in buffer), and incubated at 37°C for 30 minutes. After incubation, the cells were washed twice in cold buffer without BSA, then resuspended in 50 μl of Zombie Green (prepared in DMSO according to the manufacturer's instructions; BioLegend), diluted 1 / 500 in PBS, incubated on ice for a further 30 minutes, and protected from light. These cells were then washed once in buffer, resuspended in 50 μl of 2% paraformaldehyde / BSA / PBS / G, incubated on ice for 30 minutes, then washed once in PBS / BSA, and resuspended in 200 μl of PBS / BSA / G. Cells were analyzed by flow cytometry using a BD FACSCanto® II flow cytometer (Becton Dickinson).

[0212] MAb collaboration to enhance C1q binding and CDC 5 × 10 in 25 μl of PBS / BSA / G 5Ramos cells at a concentration of 1 / 1 cells / ml were incubated with rituximab-H429F, which was 2-fold diluted in the presence of 0.025 μg / ml daratumumab-WT, or 2-fold diluted in the presence of 0.025 μg / ml daratumumab-H429F mutant or 0.5 μg / ml 11B8-H429F mutant, in the presence of a 1 / 3 dilution of normal human serum at 37°C for 30 minutes, washed twice with PBS, incubated on ice for 30 minutes with 1 / 500 Zombie Green, washed again with PBS / BSA / G, and fixed with 2% paraformaldehyde. The cells were washed, resuspended in PBS / BSA / G, and analyzed on a BD FACSCanto® II flow cytometer (Becton Dickinson).

[0213] Results and Discussion Antigen binding by monoclonal antibodies The antigen-binding ability of the purified antibody used in this example was tested and confirmed prior to functional analysis.

[0214] A purified mAb for detecting cell surface antigens was used on antigen-positive Ramos cells (CD20). + CD38 + ) or SK-OV-3 cells (HER2 +Binding to any of the mAbs was also tested by flow cytometry, and all mAbs showed readily detectable levels of antigen binding (Figure 14). Furthermore, within each group, modified mAbs carrying mutations in the IgG heavy chain showed similar binding activity to the unmodified (WT) form (for example, the anti-CD20 mAb rituximab-WT, as well as the rituximab-H429F and rituximab-H429Y mutants, showed comparable homogeneous binding to CD20-expressing Ramos cells). Similarly, the anti-CD38 daratumumab-WT mAb and its CH3-modified mutant, daratumumab-H429F, and the anti-CD20 11B8-WT and its CH3-modified mutant all gave binding profiles comparable to their respective wild-type forms (Figure 14). Furthermore, the anti-HER2 mAb trastuzumab-WT and its modified CH3 mutant form, trastuzumab-H429F, and trastuzumab-H429Y showed readily detectable and equivalent binding to SK-OV-3 adenocarcinoma cells, similar to pertuzumab-WT and its modified CH3 mutant (Figure 14).

[0215] The effect of modification at position 429 on antibody properties was further investigated using mAbs that bind to cell surface molecules. In particular, it was found that substitution at H429 alters the physical characteristics of the antibody and selectively promotes antibody oligomerization. For example, using variants of rituximab antibodies with structurally similar introduced tyrosine and phenylalanine amino acids, namely rituximab-H429F mAb and rituximab-H429Y mAb, both rituximab-H429F mAb and rituximab-H429Y mAb eluted from the protein A affinity column as a single homogeneous peak with elution characteristics similar to unmodified rituximab-WT IgG (Figure 15A). However, purification by size exclusion chromatography (SEC) at pH 7.2 (Figure 16A) revealed that rituximab-H429Y mAb IgG exhibited a remarkably different SEC profile (i.e., compared to the profiles for rituximab-WT and rituximab-H429F antibodies). Specifically, the rituximab-H429Y mAb SEC profile showed two distinct peaks, rituximab-WT and rituximab-H429F One IgG peak (to the right of the vertical line in Figure 16A) was identified, which coincided with a single IgG peak in the SEC of the mAb, as well as a second peak (see Figure 16A, to the left of the vertical line) that is thought to contain the pre-formed oligomer (IgG(oli)) of rituximab-H429Y, indicating an equilibrium between the formation of oligomeric and non-oligomeric forms. The presence of both oligomeric and non-oligomeric IgG in rituximab-H429Y mAb was surprising, as it differed from rituximab-WT IgG, and even more surprising was that this differed from what was observed in rituximab-H429F mAb. Therefore, the different biophysical properties of rituximab-H429Y and rituximab-H429F mAbs are determined solely by the presence of hydroxyl in tyrosine at position 429 in rituximab-H429Y mAb.

[0216] Oligomers observed in SEC IgG of rituximab-H429Y mAb using SDS-PAGE (IgG)oli It was revealed that the oligomeric (IgG) and non-oligomeric (IgG) forms behave similarly to each other, as well as to rituximab-WT IgG and rituximab-H429F mAb IgG. That is, despite its oligomeric nature, rituximab-H429Y IgG oli Under non-reducing conditions, it migrated as a single, approximately 150 kDa IgG species identical to its non-oligomeric rituximab-H429Y IgG(H2L2) morphology, and importantly, it demonstrated that the oligomerization of the mAb promoted by the H429Y mutation was essentially non-covalent (Figure 17A). Furthermore, this indicates that rituximab-H429Y migrates as a pre-formed oligomer of IgG (IgG(IgG, H2L2)) resulting from the non-covalent association between the IgG heavy chains containing the H429Y mutation. oli It was clearly demonstrated that both ) can exist at pH 7.2. Further analysis under reducing conditions by reduction in dithiotheitol (DTT) showed that these non-covalent oligomeric and non-oligomeric rituximab-H429Y IgG were degraded into species of approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain, identical to the expected SDS-PAGE features of rituximab-WT and rituximab-H429F variants.

[0217] Therefore, the H429Y modification confers a new character to rituximab, and in solution at pH 7.2, the mAb exists as a pre-formed non-covalent oligomer of IgG, equilibrated with a single IgG molecule. In contrast, rituximab-H429F and rituximab-WT mAbs exist only as a single IgG species in solution. Thus, the selection of amino acids at position H429 has unpredictable effects on the physical properties of rituximab IgG.

[0218] Furthermore, these effects of mutations at position H429 were found to be independent of those in the V domain. That is, the equivalent evaluation of H429 substitution in trastuzumab, an mAb that detects a cell surface molecule (i.e., HER2) structurally different from CD20 and unrelated to rituximab, achieved similar results (see Figures 15B, 16B, and 17B). In particular, elution of trastuzumab-WT IgG from a protein A affinity matrix in citrate buffer, pH 3.0 yielded a homogeneous single peak (Figure 15B), similar to the elution of trastuzumab-H429F and trastuzumab-H429Y mutant mAbs (the single IgG peak coincided with the obtained peak for unmodified trastuzumab-WT IgG mAb). Next, size exclusion chromatography (SEC) at pH 7.2 (Figure 16B) revealed that unmodified trastuzumab-WT and trastuzumab-H429F mAb each contained the expected single major IgG species, while trastuzumab-H429Y mAb, in contrast, showed two distinct peaks (Figure 16B). The first trastuzumab-H429Y IgG peak was consistent with the single IgG peak observed for trastuzumab-WT mAb and trastuzumab-H429F mAb (Figure 16B, to the right of the vertical line), while the second trastuzumab-H429Y IgG peak was consistent with oligomeric IgG (IgG). oli ) contained (Figure 16B, left side of the vertical line). In addition, SDS-PAGE (Figure 17B) showed that under non-reducing conditions (i.e., no disulfide bond reduction), the IgG peaks of trastuzumab-WT and trastuzumab-H429F mAb shifted by the expected mass of 150 kDa, respectively. And, in the case of trastuzumab-H429Y mAb, SDS-PAGE (Figure 17B) showed that under non-reducing conditions, the oligomer trastuzumab-H429Y (IgG) oliThis showed that both the ) and non-oligomeric (IgG H2L2) species migrated similarly as a single 150kDa species. This clearly demonstrated that trastuzumab-H429Y can exist at pH 7.2 as both a single IgG and a pre-formed oligomer of IgG resulting from the association of non-covalent bonds between IgG heavy chains carrying the H429Y mutation.

[0219] Therefore, the amino acid substitution at position 429 in the trastuzumab heavy chain confers the same properties as those observed for equivalent substitutions in rituximab-based mAbs, and thus the impact of the H249Y mutation on the physical characteristics (particularly oligomerization) of the mutated mAb is significant, affecting antibody specificity, molecular targeting, epitope, and V H and V L It is independent of the domain.

[0220] Pre-formed oligomeric and non-oligomeric H429Y IgG antibodies exhibit enhanced CDC. While we do not wish to be constrained by theory, oligomerization, particularly hexamerization, of mAbs is thought to provide an optimal basis for C1q binding and activation of the complement cascade, leading to complement-dependent effector responses (e.g., phagocytosis or death of target cells by complement-dependent cytotoxicity (CDC)). Such oligomers / hexamers can be formed either in solution or on a target (i.e., "on-target" oligomerization or assembly).

[0221] The effect of H429Y modification of the CH3 domain of the IgG heavy chain on antibody effector function was evaluated by complement-dependent cytotoxicity assay (CDC) (see Figure 18). As an example, the effect of the H429 mutation on CDC efficacy was determined for both oligomeric and non-oligomeric forms of rituximab-H429Y, using CD20-positive Ramos lymphoma cells as target cells. Therefore, both oligomeric rituximab-H429Y (IgG(oli)) and non-oligomeric IgG(H2L2) forms (Figure 18A), separated by SEC at pH 7.2, were evaluated for CDC efficacy and compared with that of unmodified rituximab-WT IgG. Ramos cells were incubated in the presence of serial dilutions of rituximab-WT IgG, non-oligomeric rituximab-H429Y IgG(H2L2) (p1 fraction in Figure 18A), or oligomeric rituximab-H429Y IgG H2L2 (p2 fraction in Figure 18A). Normal human serum (diluted to 1 / 3) was then added as a complement source, and the percentage of Ramos cells killed by CDC at each mAb concentration was determined by flow cytometry (Figure 18B). Non-oligomeric rituximab-H429Y IgG(H2L2) (p1 fraction) showed a remarkable enhancement in CDC efficacy compared to unmodified rituximab-WT. Rituximab-H429Y(IgG) in non-covalent oligomeric IgG form. oli The (p2 fraction) also showed enhanced CDC compared to rituximab-WT. Interestingly, both the oligomeric p2 and non-oligomeric p1 forms exhibited similarly enhanced CDC efficacy, consistent with IgG(H2L2) which forms hexamers in solution at pH 7.2 before binding to target cells (H429Y oligomer), or, in the case of the non-oligomeric form, forms hexamers on the target cell surface after antigen binding, thereby providing an optimal Fc configuration for C1q binding and thus enhancing complement activation, as observed with the mAb-H429Y form of IgG.

[0222] Therefore, the substitution of H429 with tyrosine not only alters the physical properties of the mAb, enabling oligomerization in solution, but also enhances complement-dependent death of target cells, regardless of whether IgG is oligomerized in solution or on the target.

[0223] Oligomerization of mAbs containing the H429Y mutation is controlled by altering the pH. The non-covalent oligomerization properties of mAbs containing H429Y modification in the CH3 domain were further investigated by changing the pH of the buffer (environment) (Figure 18C). In particular, trastuzumab-H429Y mutant mAbs (purified by protein A affinity chromatography) were either dialyzed in buffer at pH 7.2 and then subjected to SEC at pH 7.2, or dialyzed in citrate buffer at pH 5.0 and then subjected to SEC at pH 5.0. At pH 7.2, both oligomeric and non-oligomeric IgG were present, but as the pH of the buffer was decreased, the formation of IgG hexamer oligomers was completely reversed, and only the IgG(H2L2) species was evident (Figure 18C). Therefore, the H429Y modification of the CH3 domain promotes pH sensitivity and non-covalent oligomerization of mAbs, thereby promoting oligomerization / hexamerization in solution or on targets that enhances complement effector potency. Furthermore, the ability to control oligomerization through pH changes may be useful in the production of immunoglobulins possessing this modification.

[0224] The H429F mutation enhances activation of the serum complement cascade. The extent to which modification of the CH3 domain at H chain position 429 affects C1q binding was investigated by flow cytometry on cells treated with unrelated mAbs that recognize CD20 or HER2 and have the H429F mutation in the CH3 of their heavy chains. The results are shown in Figures 19A-D. C1q binding was evaluated on CD20 Ramos cells treated with either rituximab-WT mAb or rituximab-H429F mAb (Figures 19A, 19B). Similarly, C1q binding was also measured on HER2-positive SK-OV-3 cells treated with trastuzumab-WT mAb or trastuzumab-H429F mAb (Figures 19C, 19D). C1q bound to Ramos cells treated with rituximab-WT mAb (Figure 19A) and SK-OV-3 cells treated with trastuzumab-WT (Figure 19C) in a detectable manner, exceeding the background control. Importantly, C1q binding to both rituximab-H429F-treated Ramos cells (Figure 19B) and trastuzumab-H429F-treated SK-OV-3 cells (Figure 19D) was enhanced compared to rituximab-WT and trastuzumab-WT. Therefore, the substitution of H429 in the CH3 domain of the heavy chain enhanced C1q binding in these unrelated anti-CD20 and anti-HER2 mAbs. Thus, it is also evident that this functional enhancement by H429 modification is independent of the antibody variable region, as well as the detected molecular target and epitope. Furthermore, the enhanced C1q binding is consistent with enhanced antibody-dependent activation of complement in the classical complement pathway, which leads to the development of MACs via CDC, resulting in cell lysis.

[0225] Therefore, since the binding of the C1q component of the C1 complex initiates the classical complement cascade leading to cell lysis, the effect of enhanced C1q binding on CDC killing efficacy was also determined using rituximab-H429F mAb and compared to the killing efficacy of unmodified rituximab-WT (Figure 19E). In particular, the percentage of Ramos cells killed by CDC was assessed by flow cytometry at each of the indicated mAb concentrations (Figure 19E). The remarkable enhancement of C1q binding conferred by H429F modification of the CH3 domain in rituximab-H429F mAb (Figure 19A compared to Figure 19B) as well as the CDC killing efficacy (EC 50 ) was also dramatically improved (>10-fold) compared to CDC with unmodified rituximab-WT (Figure 19E). This remarkable improvement in C1q binding and CDC killing efficacy by the H429F mutation is consistent with oligomerization (particularly hexamerization) of the antibody on the target surface, which appears to provide an optimal configuration of mAb Fc for binding to C1q, which is itself a hexamer, as exemplified by the analysis of rituximab-H429F mAb.

[0226] Enhanced complement activation by mAbs with the H429 mutation is epitope-independent. As discussed above, H429 substitution in anti-CD20 rituximab mAbs was found to strongly increase their CDC activity. To evaluate whether the enhancement of antibody function could be applied to different epitopes within a single molecular target, complement activation was investigated using a second anti-CD20 mAb. The results are shown in Figures 20-22.

[0227] The type II anti-CD20 mAb, 11B8, exhibits inherently poor CDC activity, but importantly, it also detects an epitope distinct from that of the type I anti-CD20 mAb rituximab (Meyer S et al., Br J Haematol 180(6):808-820, 2018). 11B8-WT mAbs containing the H429F substitution and their variants were produced as described above and purified separately by protein A affinity chromatography (Figure 20). A single homogeneous IgG peak was obtained for both 11B8-WT and 11B8-H429F mAbs (Figure 20), indicating that the modification did not alter the purification characteristics of the modified mAb compared to the unmodified 11B8-WT mAb. Furthermore, size exclusion chromatography demonstrated that 11B8-WT mAbs, like 11B8-H429F mAbs, contained a single non-oligomeric IgG (H2L2) species (Figure 21A). This was confirmed by SDS-PAGE analysis (Figure 21B) that revealed the expected 150 kDa IgG species before reduction of the disulfide bond in wild-type and mutant mAbs (Figure 21B) that were degraded into heavy chain species of approximately 50 kDa and light chain species of 25 kDa after reduction in DTT.

[0228] The CDC efficacy of 11B8 WT was also compared with that of the H429-modified antibody 11B8-H429F (Figure 22). 11B8-WT and 11B8-H429F were titrated in the presence of normal human serum as a complement source, and the percentage of Ramos cell death was evaluated for each mAb concentration. The 11B8-WT mAb failed to induce significant CDC, while, in contrast, the Fc-modified 11B8-H429F mAb was found to mediate potent CDC (Figure 22). Therefore, CDC efficacy was enhanced in two distinct and unrelated mAbs, rituximab-H429F and 11B8-H429F (compare Figures 19E and 22), indicating that CDC improvement by H429-position modification can be achieved in different mAbs targeting distinct epitopes within the same target molecule. This also indicates that the enhanced CDC achieved by CH3 modification is independent of the variable domain of the modified antibody.

[0229] The enhancing effect of H429F is independent of the antibody, molecular target, and cell type. The enhanced complement activation efficacy conferred by H429 substitution to other mAbs was evaluated in daratumumab, an antibody that recognizes a fourth unrelated molecular target, namely CD38, a cell surface molecule structurally unrelated to CD20 or HER2 (de Weers M et al., J Immunol 2011;186:1840-1848, 2011; Overdijk mB et al., MAbs 7:311-321, 2015). Daratumumab-WT mAbs were formalized as human IgG1 and kappa light chain mAbs as described above. Mutations of this antibody were manipulated, and daratumumab-H429F mAbs were constructed by substituting the H429 residue with phenylalanine. In each case, the daratumumab-based IgG mAbs exhibited comparable features and eluted from the protein A affinity column as a single homogeneous peak (Figure). Further purification by SEC yielded a single IgG(H2L2) peak for the daratumumab-WT mAb, and a similar peak for the daratumumab-H429F mutant mAb (Figure 24A). Furthermore, SDS-PAGE confirmed that the SEC-purified mAb contained the expected 150 kDa IgG species (before disulfide bond reduction), as well as approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in DTT (Figure 24B).

[0230] The CDC efficacy of daratumumab-H429F mAb was compared with that of daratumumab-WT mAb (Figure 25). Each mAb was individually titrated by serial 2-fold dilution in the presence of normal human serum as a complement source. The percentage of CD38-expressing Ramos cells killed was evaluated for each mAb concentration and is shown in Figure 25. Both daratumumab-WT and daratumumab-H429F mAb achieved approximately 80% death of Ramos lymphoma cells, but daratumumab-H429F mAb exhibited greater CDC efficacy and an approximately 15-fold higher EC50. Next, the improved CDC efficacy of daratumumab-H429F mAb was investigated using CDC-resistant CD38-positive KMS-12-PE myeloma cells (Figure 26A). Daratumumab-WT mAb, even at a concentration (5 μg / ml) five times higher than the concentration required to maximally kill Ramos lymphoma cells (1 μg / ml), showed barely detectable CDC in KMS-12-PE cells (approximately 80% lysis; see Figure 25), exceeding background control lysis (approximately 20%) in the presence of complement (C' only, no mAb). In contrast, potent CDC in KMS-12-PE cells was mediated by daratumumab-H429F mAb (Figure 26A). Further experiments evaluated CDC potency on CD38-expressing SUP-15 acute lymphoblastic leukemia (ALL) cells. As can be seen from Figure 26B, SUP-15 cells also resisted CDC killing by daratumumab-WT mAb but were readily killed by the daratumumab-H429F mutant mAb. Therefore, substitution at position 429 of the CH3 domain not only enhances CDC against certain targets but can also rescue CDC that is effective against lysis-resistant targets. Furthermore, the enhanced CDC efficacy against the CD38 target (structurally distinct from CD20 detected by rituximab and 11B8, and HER2 detected by trastuzumab) also demonstrates that the improved efficacy is independent of the target and detected epitope, and therefore independent of the V domain.

[0231] H429 substitution promotes functional synergies between mAbs directed to different epitopes. We investigated the functional synergies between mAbs and those mediated by H429 modification by determining the degree of C1q binding in a mixture of mAbs (trastuzumab and pertuzumab) targeting separate epitopes in HER2.

[0232] The purification characteristics of pertuzumab-WT mAb and a variant in which H429 was replaced with phenylalanine (i.e., pertuzumab-H429F mAb) are shown in Figures 27 and 28A. For each mAb, a single equivalent IgG peak was obtained from protein A affinity chromatography (Figure 27), and a single homogeneous non-oligomeric IgG species was observed upon further purification by size exclusion chromatography (Figure 28A, to the right of the vertical line). SDS-PAGE analysis (Figure 28B) confirmed that these peaks contained a 150 kDa IgG(H2L2) species that degrades to a heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa after reduction (Figure 28B).

[0233] The synergy between unrelated anti-HER2 mAbs detecting different epitopes in HER2, trastuzumab-WT, and pertuzumab-WT was determined by evaluating the degree of C1q binding in a mixture of mAbs quantified by flow cytometry (Figure 29A). Ovarian cancer cells SK-OV-3 treated with a mixture of HER2 mAbs at equal concentrations, trastuzumab-H429F containing H429F modification, or pertuzumab-H429F showed enhanced C1q binding (MFI=10,877) compared to that observed when the mAbs were used individually at the same concentration (Figure 29A), compared to pertuzumab-H429F (MFI=798) or trastuzumab-H429F (MFI=1739).

[0234] The synergistic enhancement of C1q binding was further evaluated by titrating individual anti-HER2 mAbs or a pair of mAbs, for each mAb alone, or in a 1:1 ratio of mAbs starting at 2.5 μg / ml:2.5 μg / ml (Figure 29B), with 2-fold serial dilutions from a starting concentration of 5 μg / ml (Figure 29B). Trastuzumab-H429F and pertuzumab-H429F acted synergistically to further enhance complement activation, particularly when the mAb concentration was restricted (e.g., at 1.25 μg / ml; indicated by arrows in Figure 29B). The C1q binding (MFI=6327) of a mixture of 1.25 μg / ml trastuzumab-H429F and 1.25 μg / ml pertuzumab-H429F is greater than that of the individual mAbs used alone at the same or twice the concentration (i.e., trastuzumab-H429F MFI=603 at 1.25 μg / ml, or 1101 at 2.5 μg / ml, and pertuzumab-H429F MFI=457 at 1.25 μg / ml, or 658 at 2.5 μg / ml), and greater than that of a mixture of WT mAbs at the same 1:1 concentration (e.g., trastuzumab-WT at 1.25 μg / ml and pertuzumab-WT at 1.25 μg / ml). MFI=522), and even greater than that of wild-type mAbs used alone (i.e., trastuzumab-WT MFI=258 at 1.25 μg / ml, or 405 at 2.5 μg / ml) and pertuzumab-WT MFI=424 at 1.25 μg / ml, or 483 at 2.5 μg / ml). This improved complement-activating potency further suggests that greater functional potency can be achieved through synergistic effects in mixtures of CH3 domain-modified mAbs that recognize different epitopes.

[0235] Modification of the CH3 domain allows mAbs with different specificities to act synergistically in the CDC independently of their target. The synergistic effects of mAbs leading to greater functional potency, as reflected in enhanced C1q binding (Figure 29), were investigated for enhanced CDC lysis of targets using a mixture of mAbs detecting either two different molecular structures (i.e., CD20 and CD38) or two different epitopes within the same molecular structure, CD20. The results are shown in Figure 30.

[0236] The cooperation in CDC between antibodies detecting different molecular structures and different epitopes was determined using a mixture of rituximab-WT or rituximab-H429F mAb targeting CD20, in combination with an unrelated surface molecule, CD38-targeting daratumumab-WT or daratumumab-H429F mAb. When rituximab-WT mAb was titrated in the presence of 0.25 μg / ml daratumumab-WT mAb (Figure 30A), the cooperation in CDC was not evident; that is, the CDC observed with any concentration of rituximab-WT did not exceed the baseline CDC observed with 0.25 μg / ml daratumumab-WT alone (0 μg / ml rituximab, Figure 30A) or with any concentration of rituximab-WT alone (i.e., titrated in the absence of daratumumab-WT). However, in contrast to unmodified wild-type mAbs, rituximab-H429F and daratumumab-H429F mAbs acted to enhance CDC cell death (Figure 30B). In particular, synergy between mAbs in CDC mediation was readily observed in the 0.5 μg / ml to 0.125 μg / ml rituximab-H429F concentration range when used with 0.025 μg / ml daratumumab-H429F mAb (Note: Enhancement is indicated by the upward arrow in Figure 30B). CDC mediated by rituximab-H429F mAb with daratumumab-H429F mAb was considerably greater than that mediated by either H429F mAb alone (rituximab-H429F or 0.025 μg / ml daratumumab; enhancement is indicated by the upward arrow in Figure 30B).

[0237] We investigated the functional cooperation between mAbs within the same target molecule, while detecting different epitopes, in a mixture of different CD20 mAbs. The results are shown in Figure 30C. Titration of rituximab-H429F mAb in the presence (0.5 μg / ml) or absence of 11B8-H429F mAb revealed that the mAbs acted synergistically. The cooperation between the mAbs was readily detectable when the concentrations of both mAbs were limited (rituximab-H429F at 0.25 μg / ml to 0.031 μg / ml and 11B8-H429F at 0.05 μg / ml), and the CDC was greater than that of either mAb alone (enhancement is indicated by the upward arrow in Figure 30C).

[0238] Therefore, the cooperative and synergistic effects of mAbs due to modification of the antibody H chain at position 429, particularly H429F, are extensive in their effect. It promotes greater functional potency through cooperative and functional synergistic effects in a mixture of mAbs, regardless of the epitopes detected by individual mAbs (i.e., whether the epitopes are located on the same or different molecular targets).

[0239] Example 4: B lymphocyte death by anti-CD20 therapeutic antibody with H429 mutation. Monoclonal antibodies are used to treat inflammatory diseases such as autoimmune diseases by targeting normal (i.e., non-malignant) cells. For example, the anti-CD20 mAb rituximab is used to treat inflammatory diseases by targeting normal B lymphocytes known to express CD20 (Lee DSW et al., Nat Rev Drug Discov 20:179-199, 2021).

[0240] Methods and materials Isolation of leukocytes from human peripheral blood Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated venous blood (Vacutainer ACD-A Becton Dickinson) by centrifugation using a Ficol gradient. Purified cells from the plasma / Ficol interface were washed in flow cytometry buffer (L-15 medium lacking phenol red and containing 0.5% BSA (L15-BSA)) and 5 × 10⁻⁶ cells were obtained. 6 The solution was resuspended in L15-BSA to a concentration of / ml.

[0241] Cellular complement-dependent lysis CDC death of normal peripheral blood B lymphocytes in PBMCs, mediated by WT and mutated anti-CD20 mAbs, was measured by flow cytometry using the Zombie Green Fixable Viability Kit (BioLegend) as described above, after opsonization of cells with the mAb.

[0242] CDC was performed in a 96-well plate. Cells (25 μl, 5 × 10⁴) 6( / ml) was reacted with an equal volume of mAb in L15 - BSA for 30 minutes on ice, then washed twice in L15 - BSA (diluted in 100 - 200 μl of buffer and centrifuged (200 x g for 5 minutes at 4 °C)). Then, IgG - opsonized cells were resuspended in 50 μl of human serum diluted 1 / 3 in L15 - BSA as a source of complement and incubated at 37 °C for 30 minutes. Then, the treated cells were washed once in L15 - BSA, resuspended in 50 μl of anti - CD19 - APC antibody (BioLegend) in L15 - BSA, and further incubated on ice for 30 minutes. Then, after washing twice in L - 15 lacking BSA, the cells were resuspended in 50 μl of Zombie Green (diluted 1 / 500 in protein - free L - 15 medium or PBS according to the manufacturer's instructions) and incubated on ice for 30 minutes. The cells were washed once in L15 - BSA, fixed on ice by resuspending the cells in 2% paraformaldehyde in buffer for 30 minutes, washed once more, and then resuspended in 200 μl of L15 - BSA for flow cytometry analysis. B lymphocytes were identified by staining for CD19, and the percentage (%) of dead B lymphocytes specifically killed by CDC was determined as CD19 in PBMC samples treated with the negative control mAb trastuzumab - WT or trastuzumab - H429F antibody + Zombie green + CD19 compared to the background control of the cells + Zombie green + Enumerated as a percentage of the cells.

[0243] Results and Discussion CDC death of normal B lymphocytes by rituximab-H429F was examined by flow cytometry and compared with that of unmodified rituximab-WT (Figure 31). Rituximab-H429F showed greater efficacy, killing over 85% of B cells compared to the much lower mortality achieved by unmodified rituximab-WT, which killed only 30% of peripheral blood B lymphocytes (Figure 31A) (Figure 31B). Because HER2 is not expressed on B lymphocytes, there was no CDC of B lymphocytes with the negative control anti-HER2 mAb, trastuzumab-WT, or trastuzumab-H429F (Figure 31C, D).

[0244] Example 5: H429F modification improves the function of other types of immunoglobulins. Histidine 429 is conserved in equivalent positions across all human Ig classes and subclasses (Figures 3 and 4), namely in the CH3 domain of all IgG and IgA subclasses (Figure 3), IgD (Figure 4), and also in IgE and IgM, while the CH4 domain is equivalent to the CH3 domain of IgG (Figure 4). The effects of H429 modification on the function of other immunoglobulins were evaluated using human IgG3 and human IgG4 as examples.

[0245] Methods and materials Antibodies and antibody constructs The mAbs used in this example contained heavy chains of the IgG3 or IgG4 subclass shown in Table 5. The IgG3 heavy chains used herein contain three amino acid substitutions, N392K, M397V, and R435H, introduced to avoid aggregation of the purified antibody (Saito S et al., Prot Sci 28(5):doi:10.1002 / pro,2019).

[0246] The rituximab-IgG3 CD20-specific heavy chain polypeptide (SEQ ID NO: 38) is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 39, and includes the CD20-specific VH domain of rituximab (https: / / go.drugbank.com / drugs / DB00073) fused to the CH1-hinge-CH2-CH3 domain ...

Claims

1. An immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, each comprising one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3), and hinge region, wherein the one or more polypeptides comprises an amino acid substitution at a position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain, and the one or more polypeptides comprises at least one C1q linkage modification.

2. The immunotherapy protein according to claim 1, wherein the amino acid substitution at the position corresponding to H429 in the amino acid sequence of the human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide is an amino acid substitution from H to an aromatic amino acid or a cyclic amino acid (Eu numbering).

3. The immunotherapy protein according to claim 1 or 2, wherein the substitution at H429 is selected from H429 substituted with F, H429 substituted with Q, H429 substituted with E, H429 substituted with S, H429 substituted with A, H429 substituted with Y, H429 substituted with L, H429 substituted with V, H429 substituted with G, H429 W substituted with R, and H429 substituted with P (Eu numbering).

4. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with F (Eu numbering).

5. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with Q (Eu numbering).

6. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with E (Eu numbering).

7. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is S-substituted H429 (Eu numbering).

8. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with A (Eu numbering).

9. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with Y (Eu numbering).

10. The immunotherapy protein according to any one of claims 1 to 3, wherein the substitution at H429 is H429 substituted with V (Eu numbering).

11. The immunotherapy protein according to any one of claims 1 to 10, wherein the immunotherapy protein is an antibody that forms an oligomer through self-association in a solution at physiological pH.

12. The immunotherapy protein according to claim 11, wherein the immunotherapy protein is a hexamer.

13. The immunotherapy protein according to any one of claims 1 to 10, wherein the immunotherapy protein is an antibody that forms an oligomer when it binds to a relevant target through "target-on" oligomerization.

14. The immunotherapy protein according to claim 13, wherein the immunotherapy protein forms a hexamer when it binds to a relevant target through "target-on" oligomerization.

15. The immunotherapy protein according to any one of claims 1 to 14, wherein the C1q binding modification is selected from modifications in C1q group 1, C1q group 2, C1q group 3, C1q group 4, and C1q group 5.

16. The immunotherapy protein according to claim 15, wherein the C1q binding modification is selected from modifications in C1q group 1, C1q group 2, and C1q group 3, and the modification increases C1q binding compared to a control IgG2 immunotherapy protein and / or control IgG1 immunotherapy protein lacking the C1q binding modification.

17. The immunotherapy protein according to any one of claims 1 to 16, wherein the C1q binding modification increases complement-based lysis compared to a control immunotherapy protein lacking the C1q binding modification.

18. The immunotherapy protein according to claim 15, wherein the C1q binding modification is selected from modifications in C1q group 4 and C1q group 5, and the modification reduces C1q binding compared to a control IgG2 immunotherapy protein and / or a control IgG1 immunotherapy protein lacking the C1q binding modification.

19. The immunotherapy protein according to any one of claims 1 to 15 and 18, wherein the C1q binding modification reduces complement-based lysis compared to a control immunotherapy protein lacking the C1q binding modification.

20. The immunotherapy protein according to any one of claims 1 to 19, wherein the polypeptide is selected from IgG1, IgG2, IgG3, or IgG4.

21. The immunotherapy protein according to any one of claims 1 to 17 and 20, wherein the polypeptide is IgG1, the C1q binding modification is located in the CH1 domain, and the modification is a C substitution at a position corresponding to S131 (Eu numbering) of the amino acid sequence of human IgG1 heavy chain polypeptide.

22. The immunotherapy protein according to any one of claims 1 to 20, wherein the polypeptide is IgG1, IgG2, IgG3, or IgG4, the modification is in the hinge region, and the modification is a substitution at a position corresponding to 216-225 or 217-225 (Eu numbering) of a human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide.

23. The immunotherapy protein according to claim 22, wherein the substitution in the hinge region is selected from RKCCVE217-225 substituted with PKSCCDKTHT, EPKSCCDKTHT216-225 substituted with ERKCCVE, and EPKSCCDKTHT216-225 substituted with ESKYGPP.

24. The immunotherapy protein according to any one of claims 1 to 20, wherein the polypeptide is IgG2, the modification is in the CH2 domain, and the modification is a substitution at a position corresponding to PVA233-236, L328, or S267 (Eu numbering) of a human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide.

25. The immunotherapy protein according to claim 24, wherein the substitution in the CH2 domain is selected from PVA233-236 substituted with EFLG, PVA233-236 substituted with ELLLG, and PVA233-236 substituted with EFEG.

26. The immunotherapy protein according to any one of claims 1 to 15 or 18 to 20, wherein the polypeptide is IgG1, IgG3, and IgG4, the modification is located in the CH2 domain, and the modification is an E substitution at a position corresponding to L235 (Eu numbering) of the human IgG1, IgG3, or IgG4 heavy chain polypeptide.

27. The immunotherapy protein according to any one of claims 1 to 20, wherein the polypeptide is IgG1, IgG2, IgG3, or IgG3, the modification is in the CH2 domain, and the modification is a substitution at a position corresponding to QYN295-297, YNS296-298, ED269-270, L328, or S267 (Eu numbering) of a human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide.

28. The immunotherapy protein according to claim 27, wherein the substitution in the CH2 domain is selected from QYN295-297 substituted with NST, YNS296-298 substituted with NSS, ED269-270 substituted with AA, L328 substituted with F, and S267 substituted with E (Eu numbering).

29. The immunotherapy protein according to claim 24 or 25, wherein the substitution in the CH2 domain is PVA233-236 substituted with EFLG (Eu numbered).

30. The immunotherapy protein according to claim 24 or 25, wherein the substitution in the CH2 domain is PVA233-236 substituted with ELLLG (Eu numbered).

31. The substitution in the CH2 domain is i) S267 replaced with E and L328 replaced with F (Eu numbering), ii) S267 (Eu numbering) replaced with E, and iii) The immunotherapy protein according to claim 27 or 28, selected from L328 (Eu numbering) substituted with L328F.

32. The immunotherapy protein according to any one of claims 1 to 17 or 20, wherein the polypeptide is IgG1, IgG2, IgG3, or IgG4, the modification is in the CH3 domain, and the modification is a substitution at a position corresponding to E430 or H435 (Eu numbering) of a human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide.

33. The immunotherapy protein according to claim 32, wherein the substitution in the CH3 domain is selected from E430 substituted with G and H435 substituted with A.

34. The immunotherapy protein according to any one of claims 1 to 20, wherein the polypeptide is IgG3, the modification is in the CH3 domain, and the modification is a substitution at positions corresponding to N392, M397, and R435 (Eu numbering) of the IgG3 heavy chain polypeptide.

35. The immunotherapy protein according to claim 34, wherein the substitutions in the CH3 domain include N392 substituted with K, M397 substituted with V, and R435 substituted with H (Eu numbering).

36. The immunotherapy protein according to any one of claims 1 to 17 and 20, wherein the polypeptide is IgG2, the C1q binding modification is located in the CH1 domain, and the modification is a substitution with S at a position corresponding to C131 (Eu numbering) of the amino acid sequence of human IgG1 heavy chain polypeptide.

37. The immunotherapy protein according to any one of claims 1 to 17 and 20, wherein the polypeptide is IgG2, and the C1q binding modification is PVA233 to 236 substituted with EFLG, C131 substituted with S, and C220 substituted with S.

38. The immunotherapy protein according to any one of claims 1 to 17 and 20, wherein the polypeptide is IgG2, and the C1q binding modification is PVA233 to 236 substituted with EFLG, C131 substituted with S, and C219 substituted with S.

39. The immunotherapy protein according to any one of claims 1 to 17 and 20, wherein the polypeptide is IgG2, and the C1q binding modification is PVA233 to 236 substituted with EFLG, C131 substituted with S, and ERKCCVE substituted with IgG1 216 to 225EPKSCDKTHT.

40. The immunotherapy protein according to any one of claims 1 to 15, 19, and 20, wherein the polypeptide is IgG1 and the C1q binding modification is PVA233 to 236 substituted with EAAGG.

41. The immunotherapy protein according to any one of claims 1 to 15, 19, and 20, wherein the polypeptide is IgG1 and the C1q binding modification is K274 substituted with Q.

42. The immunotherapy protein according to any one of claims 1 to 15, 19, and 20, wherein the polypeptide is IgG2, IgG3, or IgG4, and the C1q binding modification is a K-substituted Q274.

43. The immunotherapy protein according to any one of claims 1 to 42, wherein the immunotherapy protein is an anti-CD20 antibody.

44. The immunotherapy protein according to any one of claims 1 to 43, wherein the immunotherapy protein is selected from rituximab, ofatumumab, obinutuzumab, isatuximab, trastuzumab, and pertuzumab.

45. The immunotherapy protein according to any one of claims 1 to 44, wherein the C1q binding modification is located in the CH1 domain, hinge region, CH2 domain, or CH3 domain, or a combination thereof.

46. The immunotherapy protein according to any one of claims 1 to 45, wherein the immunotherapy protein is selected from rituximab (Rit)-IgG2 FLGG HF, Rit-IgG2 FLGG EG, Rit-IgG2 LLGG HF, Isa-IgG2-M1-H429F, Isa-IgG2-M2-H429F, Isa-IgG2-M3-H429F, S2P6-IgG2-M1-H429F, S2P6-IgG2-M2-H429F, and S2P6-IgG2-M3-H429F.

47. The immunotherapy protein according to any one of claims 1 to 46, wherein the immunotherapy protein optionally comprises a constant heavy domain 3 (CH3) and a constant heavy domain 2 (CH2) of an immunoglobulin heavy chain in combination with a lower, core, and / or upper hinge sequence.

48. The immunotherapy protein according to any one of claims 1 to 47, wherein the immunotherapy protein is a bispecific immunotherapy protein.

49. The immunotherapy protein according to any one of claims 1 to 48, wherein the immunotherapy protein comprises at least two C1q binding modifications.

50. The immunotherapy protein according to any one of claims 1 to 49, wherein the immunotherapy protein comprises at least three C1q binding modifications.

51. The polypeptide described above, i) Amino acid substitution at the position corresponding to 274 (Eu numbering) in the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain, ii) Amino acid substitutions at the position corresponding to 219 (Eu numbering) in the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain, iii) Amino acid substitutions at the position corresponding to 220 (Eu numbering) in the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain, and iv) The immunotherapy protein according to any one of claims 1 to 50, comprising a further mutation selected from one or more amino acid substitutions at positions corresponding to 219 and 220 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

52. The immunotherapy protein according to claim 51, wherein the further mutation is a substitution at a position corresponding to C219 (Eu numbering) or C220 (Eu numbering) in the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

53. The immunotherapy protein according to claim 51, wherein the further mutation is a substitution selected from substitutions by Q at K274, substitution by K at Q274, substitution by S at C219, substitution by C at S219, and substitution by S at C220.

54. The immunotherapy protein according to any one of claims 1 to 20, 22 to 25, 27 to 32, 36 to 39, and 43 to 53, wherein the polypeptide is IgG2, and the polypeptide comprises a further mutation that prevents the formation of an intrachain disulfide bond in the upper hinge region of the polypeptide.

55. The immunotherapy agent according to claim 54, wherein the heavy chain disulfide bond is located between C131 and C219, or between C131 and C220.

56. The immunotherapy protein according to any one of claims 1 to 20, 22 to 25, 27 to 32, 36 to 39, and 43 and 53, wherein the polypeptide is IgG2, and the polypeptide comprises a further mutation that forms a disulfide bond between the light chain and the upper hinge region of the polypeptide.

57. The method according to claim 56, wherein the disulfide bond is formed between C214 of the light chain and C219 of the heavy chain, or between C214 of the light chain and C220 of the heavy chain.

58. The immunotherapy protein according to any one of claims 54 to 57, wherein the further mutations are substitutions of M1, C131 with S, and C220 with S.

59. The immunotherapy protein according to any one of claims 54 to 57, wherein the further mutations are substitutions of M2, C131 with S, and C219 with S.

60. The immunotherapy protein according to claim 54 or 57, wherein the further mutations are substations of M3, C131 by S, and substitution of ERKCCVE with EPKSCDKTHT in the IgG2 upper hinge.

61. The aforementioned immunotherapy protein a) containing H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, b) The H429 is substituted with F, and the C1q modification is PVA233-236 substituted with ELLG. c) Includes H429 substituted with Q, wherein the C1q modification is PVA233-236 substituted with EFLG. d) Includes H429 substituted with Q, wherein the C1q modification is PVA233-236 substituted with ELLG. e) containing H429 substituted with E, wherein the C1q modification is PVA233-236 substituted with EFLG. f) Includes H429 substituted with E, and the C1q modification is PVA233-236 substituted with ELLG. g) containing H429 substituted with S, wherein the C1q modification is PVA233-236 substituted with EFLG, h) containing H429 substituted with S, wherein the C1q modification is PVA233-236 substituted with ELLG. i) containing H429 substituted with A, wherein the C1q modification is PVA233-236 substituted with EFLG, j) Includes H429 substituted with A, wherein the C1q modification is PVA233-236 substituted with ELLG. k) Contains H429 substituted with Y, and the C1q modification is PVA233-236 substituted with EFLG. l) Contains H429 substituted with Y, and the C1q modification is PVA233-236 substituted with ELLG. m) Contains H429 substituted with F, the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with S, and C220 is substituted with S. n) containing H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with S, and C219 is substituted with S. o) Includes H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with S, and ERKCCVE in the upper hinge of IgG2 substituted with EPKSCDKTHT. p) Contains H429 substituted with F, and the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with S, and C220 substituted with S. q) Contains H429 substituted with F, and the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with S, and C219 substituted with S, and The immunotherapy protein according to any one of claims 1 to 60, comprising H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLLG, C131 substituted with S, and ERKCCVE in the IgG2 upper hinge substituted with IgG1 EPKSCDKTHT.

62. The immunotherapy protein according to claim 17 or 19, wherein the control immunotherapy protein is IgG1.

63. The immunotherapy protein according to claim 17 or 19, wherein the control immunotherapy protein is human IgG2.

64. An oligomer comprising the immunotherapy protein described in any one of claims 1 to 63.

65. A nucleic acid encoding an immunotherapy protein according to any one of claims 1 to 63.

66. The nucleic acid according to claim 65, wherein the nucleic acid is selected from RNA, DNA, or a combination thereof.

67. Use of an immunotherapy protein, oligomer, or nucleic acid according to any one of claims 1 to 66 for the treatment or prevention of a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative disorders.

68. Use of an immunotherapy protein, oligomer, or nucleic acid according to any one of claims 1 to 66 in the manufacture of a pharmaceutical product for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative disorders.

69. A method for treating or preventing a disease or condition, comprising administering an effective amount of an immunotherapy protein, oligomer, or nucleic acid described in any one of claims 1 to 66, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplant conditions or rejection reactions, infectious diseases, and proliferative diseases.

70. The use according to claim 67 or 68, or the method according to claim 69, wherein the immunotherapy protein or oligomer removes soluble molecules and / or immune complexes from circulation.

71. The use according to claim 67 or 68, or the method according to claim 69, wherein the immunotherapy protein enhances the death receptor signaling response.

72. The use according to claim 67 or 68, or the method according to claim 69, wherein the immunotherapy protein enhances complement-based lysis when administered to a subject.

73. A pharmaceutical composition or pharmaceutical product comprising an immunotherapy protein, oligomer, or nucleic acid according to any one of claims 1 to 66, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

74. A kit comprising at least one immunotherapy protein, oligomer, or nucleic acid according to any one of claims 1 to 66.

75. A method for producing an immunotherapy protein according to any one of claims 1 to 66, comprising culturing host cells containing a construct encoding the protein under conditions suitable for the expression of the protein, (i) A weakly acidic pH for recovering immunotherapy proteins in monomeric form, (ii) A method comprising recovering the immunotherapy protein in oligomeric form from the culture supernatant under substantially neutral pH conditions.

76. A method for producing an immunotherapy protein according to any one of claims 1 to 66, comprising: culturing host cells containing a construct encoding the protein under conditions suitable for the expression of the protein; and recovering the protein from the culture supernatant using an affinity chromatography method that includes arginine at a concentration of less than 130 mM and an elution buffer at pH 5.0 or lower.

77. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprises one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions.

78. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions, and the one or more polypeptides comprise an amino acid substitution with an aromatic, cyclic, or hydrophobic amino acid at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain.

79. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid, wherein the one or more polypeptides comprises one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions, and the one or more polypeptides comprises at least one C1q linkage modification.

80. A method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with an aromatic, cyclic, or hydrophobic amino acid; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the polypeptide comprises one or more of the constant heavy chain domain 5 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions.

81. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions.

82. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the one or more polypeptides comprise one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions, and the one or more polypeptides comprise amino acid substitutions by F, Q, E, S, A, Y, L, V, G, W, R, or P at the position corresponding to H429 (Eu numbering) of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain.

83. A method for producing / modifying an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P, wherein the one or more polypeptides comprises one or more of the constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions, and the one or more polypeptides comprises at least one C1q linkage modification.

84. A method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising: i) substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P; and ii) introducing a C1q modification as described in Table 5 into the immunotherapy protein, wherein the polypeptide comprises one or more of the constant heavy chain domain 5 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions.

85. The method according to any one of claims 77 to 84, wherein the C1q bond modification is selected from modifications in C1q group 1, C1q group 2, C1q group 3, C1q group 4, and C1q group 5.

86. An immunotherapy protein comprising a modified C1q linkage, comprising one or more immunoglobulin heavy chain polypeptides including one or more of the following: constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (CH2), constant heavy chain domain 3 (CH3) domains, and hinge regions, wherein the one or more polypeptides comprises amino acid substitutions by F, Q, E, S, A, Y, L, V, G, W, R, or P at the position corresponding to H429 (Eu numbering) of the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4 heavy chain.

87. A method for producing / modifying a C1q bond in an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P.

88. A method for enhancing complement-based lysis mediated by an immunotherapy protein comprising one or more immunoglobulin heavy chain polypeptides, comprising substituting an amino acid at the position corresponding to H429 (Eu numbering) in the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain with F, Q, E, S, A, Y, L, V, G, W, R, or P.

89. The method according to any one of claims 77, 79, 80, 81, 83, 84, 85, 87, and 88, wherein the substitution in H429 is selected from H429 substituted with F, H429 substituted with Q, H429 substituted with E, H429 substituted with S, H429 substituted with A, H429 substituted with Y, H429 substituted with L, H429 substituted with V, H429 substituted with G, H429 substituted with H429W, H429 substituted with R, and H429 substituted with P (Eu numbering).

90. The method according to claim 89, wherein the substitution in H429 is H429 substituted with F (Eu numbering).

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