Immunotherapeutic proteins

Abstract

A bispecific immunotherapeutic protein and methods of use and production thereof are disclosed wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy domain 4 (CH4) domain), wherein the one or more polypeptides comprise an amino acid substitution (Eu numbering) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain.

Description

Technical Field

[0001] This disclosure relates to a bispecific immunotherapy protein and methods of using and producing the same, wherein the immunotherapy protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)), wherein the one or more polypeptides comprises an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain. Background Technology

[0002] Monoclonal antibodies (mAbs) have become one of the most important and successful types of treatment, revolutionizing the treatment of cancer and inflammatory diseases such as autoimmune diseases. Many mAbs engineered on the backbone of IgG antibodies specifically utilize the powerful effector functions of the immune system by binding to both the target antigen through their variable Fab domain and the Fcγ receptor (FcγR) through their heavy chain, including the constant Fc portion (Fc or Fc fragment). This leads to the activation of inflammatory or killer cells. Additionally, the Fc of the antibody can activate blood proteins called complement (involving the aggregation of antibodies in a process known as “self-association” or “target assembly”) to achieve complement-dependent effector functions, such as complement-dependent cytotoxicity (CDC). These different effects work individually but in a complementary manner, enabling natural antibodies to destroy “targets” such as cancer cells and virus-infected cells (or the virus itself), as well as bacteria, parasites, and other pathogens. However, they can also be advantageously utilized through the action of engineered mAbs (i.e., therapeutic mAbs) designed for various medical treatments, such as cancer therapies and the treatment of infections and inflammatory diseases and conditions.

[0003] Given the above, it is perhaps not surprising that a great deal of research has been applied to elucidate the details of Fc interactions (e.g., with FcγR and complement proteins such as C1q and membrane attack complex (MAC) proteins C4-C9) and to identify possible modifications (e.g., mutations) to Fc that could potentially enhance the efficacy of therapeutic mAbs. However, to date, few therapeutic mAbs have included such Fc mutations; the most well-known are antibodies in which the heavy chain glycan has been engineered to lack fucose, thereby resulting in enhanced FcγRIII interactions, such as obinutuzumab and margetuximab (which contain a six-point mutation in the heavy chain of the IgG1 antibody), and other antibodies in clinical trials but not yet approved, such as various HexaBody™ mAbs (GenMab BV; Copenhagen, Denmark), which contain a triple mutation in the Fc fragment of IgG1, namely, amino acid substitutions at positions 345 (i.e., E345R), 430 (i.e., E430G), and 440 (i.e., S440Y) (referred to as "IgG-RGY" (see International Patent Publication No. WO 2014 / 006217)), or a single mutation at position 430 (i.e., E430G) of the IgG1 Fc (de Jong RN et al., *PLoSBiol* 14(1):e1002344, 2016. This E430G mutation has been shown to enhance the ability of IgG1 antibodies to form hexamers on target molecules, and IgG1 mutants including the E430G mutation have been reported to exhibit strongly enhanced CDC conditioned on antigen binding at the target cell site (de Jong et al., 2016, ibid.). Therefore, it will be apparent to those skilled in the art that there is a need in the art for modifications that enhance the therapeutic efficacy of immunotherapeutic proteins (e.g., mAbs). Summary of the Invention

[0004] In the work leading to this disclosure, the inventors generated a series of mutant antibodies (particularly mutant immunoglobulin G (IgG) molecules) comprising point mutations at various locations, for example, at and around the interface between the CH2 and CH3 domains of a heavy chain polypeptide. Some molecules, including the mutation at position 429, were found to be at “buried” or inaccessible sites within the IgG1 structure (see [link to previous work]). Figure 1The Fc region is occupied by histidine (His / H) residues to indicate, for example, enhanced activation of the complement system via the classical complement pathway, which includes C1q binding, membrane attack complex formation, and complement-dependent cytotoxicity in complement function assays. Additionally, evidence obtained by the inventors indicates that some molecules containing an Fc region component with a mutation at position 429 are capable of forming oligomers in solution or upon binding to an associated target. This disclosure relates to bispecific immunotherapeutic proteins and their potential therapeutic use, wherein the bispecific immunotherapeutic protein comprises or is modified to comprise an Fc region component containing a mutation at position 429 (or a corresponding position) of the Fc region component. This disclosure also relates to microantibodies and their potential therapeutic use, wherein the microantibody comprises or is modified to comprise an Fc region component containing a mutation at position 429 (or a corresponding position) of the Fc region component.

[0005] Therefore, in one aspect, the present invention provides a bispecific immunotherapy protein comprising two antigen recognition structures targeting two different epitopes, and comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)), wherein the one or more polypeptides comprises an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain.

[0006] On one hand, the present invention provides an oligomer comprising a bispecific immunotherapeutic protein as described herein.

[0007] On the one hand, the present invention provides a nucleic acid that encodes a bispecific immunotherapy protein as described herein.

[0008] On one hand, the present invention provides the use of a bispecific immunotherapy protein, oligomer, or nucleic acid as described herein for treating or preventing 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, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0009] On one hand, the present invention provides the use of a bispecific immunotherapy protein, oligomer, or nucleic acid as described herein, wherein the bispecific immunotherapy protein, oligomer, or nucleic acid is used to prepare a medicament 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, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0010] On one hand, the present invention provides a method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of a bispecific immunotherapeutic protein, oligomer, or nucleic acid as described herein, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0011] On one hand, the present invention provides a pharmaceutical composition or drug comprising a bispecific immunotherapeutic protein, oligomer or nucleic acid as described herein, and pharmaceutically acceptable carriers, diluents and / or excipients.

[0012] On one hand, the present invention provides a kit comprising at least one bispecific immunotherapeutic protein, oligomer or nucleic acid as described herein.

[0013] On one hand, the present invention provides a method for generating a bispecific immunotherapeutic protein as described herein, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant under the following conditions:

[0014] (i) A weakly acidic pH for recovering bispecific immunotherapeutic proteins in monomeric form; or

[0015] (ii) A substantially neutral pH for the recovery of bispecific immunotherapeutic proteins in oligomeric form.

[0016] On one hand, the present invention provides a method for generating a bispecific immunotherapeutic protein as described herein, the method comprising culturing a host cell containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant, for example, using a method comprising affinity chromatography, the method may comprise using an elution buffer containing arginine at a concentration of less than 130 mM and a pH less than or equal to 5.0.

[0017] On one hand, the present invention provides a method for generating / modifying a bispecific immunotherapy protein, the method comprising substituting an amino acid (Eu number) located at a position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain, wherein the immunotherapy protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)).

[0018] On one hand, the present invention provides a microantibody comprising one or more immunoglobulin heavy chain polypeptides containing an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)), wherein the one or more polypeptides comprises an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain.

[0019] On one hand, the present invention provides an oligomer comprising microantibodies as described herein.

[0020] On the one hand, the present invention provides a nucleic acid that encodes a microantibody as described herein.

[0021] On one hand, the present invention provides a method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of a microantibody, oligomer, or nucleic acid as described herein, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0022] On one hand, the present invention provides a pharmaceutical composition or drug comprising microantibodies, oligomers or nucleic acids as described herein, and pharmaceutically acceptable carriers, diluents and / or excipients.

[0023] On one hand, the present invention provides a kit comprising at least one microantibody, oligomer or nucleic acid as described herein.

[0024] On one hand, the present invention provides a method for producing microantibodies as described herein, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant under the following conditions:

[0025] (i) A weakly acidic pH for recovering bispecific immunotherapeutic proteins in monomeric form; or

[0026] (ii) A substantially neutral pH for the recovery of bispecific immunotherapeutic proteins in oligomeric form.

[0027] On one hand, the present invention provides a method for generating microantibodies as described herein, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for expressing the protein, and recovering the protein from the culture supernatant, for example, using a method comprising affinity chromatography, the method using an elution buffer containing arginine at a concentration of less than 130 mM and a pH less than or equal to 5.0.

[0028] On one hand, the present invention provides a method for generating / modifying microantibodies, the method comprising substituting an amino acid (Eu number) located at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain, wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptides containing an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)). Attached Figure Description

[0029] Figure 1Illustrations of the structure of the Fc fragment of human IgG1 are provided, showing the inaccessible site of the H429 residue within the Fc fragment. These illustrations are reproduced from Deisenhofer J., Biochemistry 20:2361-2370, 1981, human IgG1-Fc (PDB: 1Fc1), rendered with space padding. (A) Side view of the Fc, where the A chain is shown in black, the B chain in light gray, and the N-linked glycan in dark gray. The boxed area of ​​chain A indicates the enlarged area in insets B and C; (B) the enlarged boxed area in inset A, thus indicating in gray the solvent-accessible amino acids of chain B labeled according to the Eu number of human IgG1: methionine (M428), glutamic acid 430 (E430), alanine 431 (A431), leucine 432 (L432), and histidine H435 at position 428, which is above and obscures histidine 429 (H429) buried in Fc; (C) a view of inset B after the solvent-accessible residues shown in inset B have not been rendered (i.e. removed from the view). Until now, the buried H429 shown in gray CPK has become clearly visible below these covered adjacent solvent-accessible residues shown in inset B. The residue-accessible surface area (ASA) calculated using PISA (i.e., “Protein Interfaces, Surfaces and Assembly” service PISA; http: / / www.ebi.ac.uk / pdbe / prot_int / pistart.htm, and Krissinel E and K Henrick. Journal of Biochemistry (J Mol Biol) 372:774-797, 2007) indicated that H429 was unreachable, with a recorded value of 0.0;

[0030] Figure 2 Illustrations of immunoglobulins (antibodies) and antibody-like molecules are provided, thus illustrating the modular nature of antibodies:

[0031] Small left image:A representation of the prototype immunoglobulin structure is provided—immunoglobulins comprise chains of variable (V) and constant (C) domains arranged in a self-assembled heavy (H) chain and optionally a light (L) chain. Domains are further identified by the chain (e.g., CH3 is the third constant domain of the heavy chain). The heavy chain (H) typically further includes different linker or hinge sequences between Fab and Fc fragments. As shown in the inset (residue H429 is indicated in the CH3 domain), some H chains contain three constant H domains, and others contain four constant H domains (e.g., IgM and IgE), where CH4 is the equivalent domain of 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 typically contain the antibody. On the other hand, in antibodies including a light (L) chain, the two antibody H chains typically dimerize, and the L chain is associated with each H chain (i.e., H2L2). Some antibodies lacking strong H-chain interactions (e.g., human IgG4) may sometimes be antibody “half-molecules” consisting of a monomeric H chain and an 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 of the heavy chain, a CH2 domain, and a CH3 domain).

[0032] Small image on the right:This provides a representation that illustrates the modular nature of immunoglobulins, allowing for flexibility in the production of Ab-like molecules and fusion proteins (including immunoglobulin moieties). The molecules shown all comprise an "H-like" chain containing at least one CH3 domain. Specifically, each of the described Ab-like molecules consists of a V domain for target antigen recognition linked by a linker sequence to a CH3 domain from, for example, the Fc region of IgG (in variants of the shown molecules, the V domain may be an equivalent CH4 domain from IgE or IgM). The CH3 domain may carry mutations such as the mutation at position 429 (Eu number) as described herein. The V and CH3 domains can be linked by sequences that may include heterolinking sequences, synthetic linking sequences, and / or other homologous or heterologous sequences or domains linking the V and CH3 domains (e.g., as depicted in the example shown, the V and CH3 domains can be linked via CH1-CH2 or CH2 with or without additional short polypeptide sequences such as heterolinking sequences or immunoglobulin hinge sequences), and the CH1, CH2, and hinge sequence modules can be provided in different combinations and orders, not limited to the example shown. The molecules shown are each in monomeric form and can dimerize to form homodimers or heterodimers. Similarly, for the fusion proteins shown, each fusion protein is composed of a target recognition portion (depicted as "X") linked by a linking sequence to at least one CH3 domain (or an equivalent CH4 domain of IgE or IgM) from the Fc region of, for example, IgG. Similarly, the CH3 domain may include mutations such as the mutation at position 429 (Eu number), and the target recognition portion and the H chain module (e.g., the CH3 domain, CH2-CH3, or CH3-CH2 component) are linked by sequences that may include heterologous or synthetic linker sequences and / or other homologous or heterologous sequences or domains that link the V and CH3 domains (e.g., CH1-CH2 or CH2, with or without additional short polypeptide sequences such as heterologous or synthetic linker sequences or immunoglobulin hinge sequences).

[0033] Figure 3The conservation of domain structure and sequence homology in immunoglobulin G (IgG) and immunoglobulin A (IgA) molecules is demonstrated: sequence comparisons of the hinge and constant domains of the human immunoglobulin heavy (H) chain sequence are provided, thus depicting the domain-based structure of the H chain (i.e., CH1-hinge-CH2-CH3 defined by IgG1; the Eu numbering of the first amino acid of each IgG1 domain and hinge is shown below (inverted triangle): CH1 domain, amino acids 118 to 215; hinge amino acids 216 to 230; CH2 domain amino acids 231 to 340; and CH3 domain amino acids 341 to 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 of human IgG1 across other IgG and IgA subclasses is also shown (indicated by arrows). The amino acid sequences were derived from open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). The accession numbers for the H chains 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. Clustal was used for alignment, except for manually aligned hinges.

[0034] Figure 4Histidine at position 429 in IgG1 is shown to be conserved across all immunoglobulin classes. A comparison of the amino acid sequence of the IgG1 CH3 domain with 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, and in the equivalent domains (CH4) of IgA subclasses IgA1 and IgA2, IgD, and IgE and IgM. The IgG1 CH3 domain is defined and numbered according to Eu designations, and the first amino acid in the CH3 domain is indicated by an inverted triangle above glycine. The amino acid sequence shown is derived from an open reading frame translation of a sequence from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). The accession numbers for the H chain sequence 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.

[0035] Figure 5 The results of purification of the ACE2-Fc H429Y fusion protein according to this disclosure are shown: (A) anion exchange (IEX) chromatography of flACE2-Fc-WT (flow through (ft), eluted fraction and wash), wherein the peaks containing ACE2-Fc are highlighted with *; (B) SDS-PAGE of the flow through (ft) and IEX peak fractions, wherein flACE2-Fc H429Y migrates above 250 kDa and low molecular weight impurities are marked with †; (C) size exclusion chromatography (SEC) of the IEX fraction containing flACE2-Fc-WT (comparative) using a Superose 6 column, wherein oligomers (oli), monomers (mn) and low molecular weight impurities (†) are indicated; and (D) SEC of the IEX fraction containing flACE2-Fc H429Y, thus showing a high proportion of oligomeric species. Monomer (mn) species are considered to be single molecules (i.e. monomer molecules) containing two copies of the corresponding ACE2-Fc fusion polypeptide that self-associate through the Fc region components.

[0036] Figure 6Graphical results illustrating the SARS-CoV-2 RBD binding activity of the ACE2-Fc fusion protein according to this disclosure are provided. Binding of the ACE2-Fc fusion protein to immobilized RBD-Ig was determined by ELISA: (A) trACE2-Fc-WT, (B) flACE2-Fc-WT, and (C) EflACE2-Fc-WT and its variants:

[0037] F includes the ACE2-Fc protein containing the mutated H429F Fc region component.

[0038] Y oli SEC-purified ACE2-Fc protein, including the mutated H429Y Fc region component (oligomeric protein).

[0039] Ymn comprises SEC-purified ACE2-Fc protein containing a mutated H429Y Fc region component (monomer protein; a fusion protein containing two copies of ACE2-Fc protein dimerized by the Fc region component).

[0040] The trACE2-Fc-WT protein produced by kif in the presence of the mannosidase inhibitor kifunensine. (D) EC50 of trACE2-Fc-WT, flACE2-Fc-WT, and variant proteins. 50 The overview of the binding constants demonstrates that the apparent RBD binding affinity of flACE2-Fc-WT is higher than that of trACE2-Fc-WT, and the binding affinity of the flACE2-Fc-H429Y monomer is weaker (Ymn; i.e., the flACE2-Fc-H429Y fusion protein containing two copies dimerized through the Fc region). Welch's unpaired t-test showed p = 0.0332 (*), < 0.0001 (****).

[0041] Figure 7Results illustrating the formation of pH-dependent oligomers of the flACE2-Fc H429Y fusion protein according to this disclosure are provided: 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 produced a larger proportion of monomeric (mn) fusion protein separated than at pH 7.4. (C) Native PAGE of ACE2-Fc H429Y (1 µg). 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 redialyzed against PBS pH 7.4 and purified by SEC, and the mn fraction collected as shown in inset D. (D) SEC chromatogram of oligomers analyzed in lane 1 of small figure C at pH 5.0 after redialysis with PBS at pH 7.4 and SEC performed in PBS at pH 7.4. (E) flACE2-Fc-H429Y monomer (Y) prepared at pH 5. mn The improved RBD-Ig binding activity. A monomer is considered a single molecule (i.e., a monomer molecule) containing two copies of the corresponding ACE2-Fc fusion protein dimerized through the Fc region component;

[0042] Figure 8 Results are provided demonstrating the SARS-CoV-2 neutralizing efficacy of the ACE2-Fc fusion protein according to this disclosure affected by ACE2 scaffolding (i.e., truncated or full-length) and Fc mutations. The neutralizing efficacy of the ACE2 peptide and three groups of ACE2-Fc-WT fusion proteins and variant proteins was determined by titration to the cytopathic effect (CPE) endpoint in a micro-neutralization assay. The fusion proteins are trACE2-Fc, flACE2-Fc, and EflACE2-Fc WT, as well as the Fc variants H429F, F; and the H429Y oligomer on the SEC, Y. oli ; and the H429Y monomer on SEC, Y mn(Contains two copies of the fusion protein dimerized via the Fc region). The trACE2-Fc fusion protein includes the glycan-modified trACE2-Fc-kif. Neutralization endpoints were compared with ACE2 and ACE2-Fc WT using Dunnett's multiple comparisons test, mean ± SEM, ANOVA. p = 0.1234 (not significant), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****);

[0043] Figure 9 Results of assays assessing the interaction between the ACE2-Fc fusion protein and FcγR according to this disclosure are provided. Ramos-S cells were conditionally charged with the ACE2-Fc WT fusion protein and its variant (5 µg / ml). Biotinylated (A)-dimer rsFcγRIIa or (B)-dimer rsFcγRIIIa probes, followed by streptavidin-APC, bound to conditionated cells, and the binding was described as median fluorescence intensity (median FI) determined by flow cytometry. FcR binding activity of the ACE2-Fc fusion protein was readily detectable except for the ACE2-Fc H429Y fusion protein, which showed significantly reduced FcR binding activity (3 replicas, mean ± SEM).

[0044] Figure 10 Results of assays assessing the ability of the ACE2-Fc fusion protein according to this disclosure to mediate cell activation via FcγRIIIa are provided. The results indicate that flACE2-Fc protein is a potent activator of FcγRIIIa, except that the Fc H429Y mutant in any ACE2 format failed to stimulate it. Defucosylated trACE2-Fc-kif is also a potent activator of FcγRIIIa. Ramos-S target cells were conditioned with the following: (A) trACE2-Fc, (B) flACE2-Fc and (C) EflACE2-Fc, WT and variant proteins (including H429F, F); H429Y, oligomers, Y oli H429Y monomer, Y mn(A fusion protein containing two copies of a component dimerized via the Fc region); or a trACE2-Fc kif produced by trACE2-Fc WT in Expi293 cells in the presence of the mannosidase inhibitor chifine. In some experiments, Ramos-S target cells were conditioned with anti-CD20 mAb rituximab RIT. These conditioned targets were incubated with FcγRIIIa-NF-κB-RE nanoluciferase reporter cells, and FcγRIIIa activation was measured by induction of nanoluciferase (RLU). Data were fitted to agonist response curves to estimate EC50. 50 (D) shows the EC from the curve fitting. 50 (nM) values. Mean ± SEM of ANOVA using Dunnett's multiple comparison test compared to trACE2-Fc WT, n ≥ 4. p = 0.0021 (**), 0.0002 (***), < 0.0001 (****);

[0045] Figure 11 Results demonstrating the strong immobilization of complement in Ramos-S target cells and guidance of complement-dependent cytotoxic killing by an ACE2-Fc fusion protein comprising Fc region components with H429F and H429Y mutations according to this disclosure are provided; as determined by ELISA analysis of complement binding activity of trACE2-Fc (A, C, E) or flACE2-Fc (B, D, F) bound to SARS-CoV-2 RBD-biotin by avidin-captured protein bound to a plate: binding of C1q to ACE2-Fc fusion protein variants (2.0 μg / ml) on a plate at different concentrations of avidin-captured RBD (A, B); titration of C1q (C, D) or C5b-9 (E, F) binding to serially diluted ACE2-Fc fusion protein variants bound to avidin-captured RBD-biotin (2.5 μg / ml) (mean ± SEM); two independent ELISA assays. (G) Flow cytometry analysis of complement-dependent cytotoxicity (CDC) (killing %) of conditioned Ramos-S cells in the presence of 1 / 3 diluted normal human serum as a complement source (ECGs from curve fitting are shown). 50 (nM) value);

[0046] Figure 12Results are provided showing that IgG1 antibodies containing the Fc region component with the H429F mutation having a CH3 domain strongly immobilize complement and show the same antigen binding assay. (A, B) Different concentrations (20 μg / ml - 0.625 μg / ml) of TNP-BSA antigen were adsorbed into the wells of an ELISA plate and reacted with a single concentration (2 μg / ml) of chimeric anti-TNP human IgG1 and IgG2 mAb containing unmodified wild-type (WT) heavy chains or with 2 μg / ml of anti-TNP mAb containing IgG1 heavy chains including the Fc component mutation H429F (TNP-IgG1-H429F). Immobilization with (A) purified human C1q and C1q detected with anti-C1q rabbit polyclonal antibody and (B) treatment of antibody-conditioned TNP-BSA-coated wells with human serum as a complement source, and detection of membrane attack complex (C5b-C9) formation with anti-C5b-C9 rabbit polyclonal antibody. (C) provides the results of an ELISA showing the antigen-binding activity of the chimeric anti-TNP mAb used in insets A and B, including unmodified mAbs (TNP-IgG1-WT and TNP-IgG2-WT mAbs) and an H429-modified mAb, namely TNP-IgG1-H429F. ELISA plates were coated with TNP-BSA, then titrated with anti-TNP mAb, and antibody binding was detected using HRP-conjugated anti-human IgG. All mAbs showed similar antigen-binding activity;

[0047] Figure 13 Results of C1q fixation and MAC (C5b-9) formation, as shown by mAbs containing IgG1 heavy chains modified with glutamate, glutamine, or serine at position 429 of the heavy chain, are provided. TNP-BSA (20 μg / ml) was adsorbed into the wells and reacted with chimeric human anti-TNP mAbs, titrated across a concentration range of 4 μg / ml – 0.125 μg / ml. The tested mAbs contained unmodified wild-type (WT) heavy chains of IgG1 (TNP-IgG1-WT) (inset AD) or IgG2 (TNP-IgG2-WT) (inset AC), or IgG1 H chains containing mutant TNP-IgG1-H429Q, TNP-IgG1-H429E (A, C), or TNP-IgG1-H429S (B, D). The wells coated with antibody-conditioned TNP-BSA were treated with human serum as a complement source. The fixation of C1q (A, B) was detected using anti-C1q rabbit polyclonal antibody, and the formation of membrane attack complex (C5b-C9) (C, D) was detected using rabbit anti-C5b-C9 polyclonal antibody.

[0048] Figure 14Flow cytometry results were provided for the quantification of SEC-purified mAbs by binding to target cell surface antigens. Unmodified (WT) rituximab, daratumumab-WT, 11B8-WT mAbs, and their CH3-modified mutants carrying mutations H429F or H429Y were evaluated by flow cytometry on Ramos lymphoma cells expressing CD20 and CD38, showing peak fractions of p1 and IgG (IgG H2L2). oli The binding activity of peak fraction p2 was evaluated. The binding activity of unmodified trastuzumab-WT, pertuzumab-WT, and its CH3 heavy chain mutant carrying the H429F mutation was assessed on HER2-expressing SK-OV-3 cells.

[0049] Figure 15 Elution chromatograms are provided for the protein A affinity purification characteristics of rituximab-WT, trastuzumab-WT, and their mutants carrying modifications in the CH3 region of the heavy chain. IgG was recovered from the column after elution with sodium citrate buffer, pH 3.0: (A) Elution curve based on anti-CD20 rituximab mAb. Rituximab-WT IgG and each mutant IgG eluted as a single homogeneous peak; (B) Elution curve based on anti-HER2 trastuzumab mAb. Trastuzumab-WT IgG and each mutant IgG eluted as a single homogeneous peak;

[0050] Figure 16 Size exclusion chromatography (SEC) curves revealing how the H429 mutation can alter the physical properties of IgG are provided. Following protein A affinity purification, mAbs were further purified by SEC at pH 7.2: (A) The SEC curves of unmodified rituximab-WT (WT) and rituximab-H429F (H429F) mutants consisted of a single homogeneous IgG peak (fraction to the right of the vertical dashed line) and a minimal number of oligomeric species (fraction to the left of the vertical dashed line). Thus, for example, the SEC curve of unmodified rituximab-WT showed a single dominant species corresponding to IgG (H2L2) as expected, as confirmed by SDS-PAGE analysis. Figure 17(A) , in which it migrates as expected with an unreduced IgG mass of approximately 150 kDa, and upon reduction in DTT, it breaks down into its approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species. On the other hand, rituximab-H429Y mAb contains both non-oligomeric IgG and oligomeric IgG; (B) SEC curves of protein A purified anti-HER2 trastuzumab wild-type (WT) (i.e., the unmodified form of trastuzumab) and its mutants including amino acid substitutions of H429F or H429Y. For trastuzumab-WT and H429F mutant mAbs, a single peak of non-oligomeric IgG is observed to the right of the vertical dashed line, but trastuzumab-H429Y mutant IgG (H429Y) contains both non-oligomeric IgG and oligomeric IgG. The non-oligomeric IgG peak of trastuzumab-H429Y is consistent with the non-oligomeric IgG peak of unmodified trastuzumab-WT or its H429F variant and the equivalent rituximab-based mAb in inset (A). The SEC curve of oligomeric IgG in trastuzumab-H429Y is consistent with that of oligomeric IgG in rituximab-H429Y in inset (A).

[0051] Figure 17 Images obtained from SDS-PAGE of mAbs purified from SEC are provided. mAbs purified by SEC were analyzed by SDS-PAGE in 5–15% gradient gels with or without disulfide bond reduction: (A) Under non-reduction 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 disintegrated as expected into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains. Prior to reduction, rituximab-H429Y mAb in both non-oligomeric IgG (HY IgG (H2L2)) and oligomeric (HY IgG (oli)) forms (see [link to DTT]). Figure 16(A) Both migrated as a single 150 kDa IgG, i.e., the H2L2 species. After reduction, both forms migrated as approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains; WT = wild type, HF = H429F, HY = H429Y; M = molecular weight marker, whose mass (kD) is shown on the left; (B) Under non-reducing conditions, the IgG peaks of trastuzumab-WT (WT IgG) and trastuzumab-H429F (HF IgG) migrated as a single species of IgG, i.e., the expected molecular size of approximately 150 kDa H2L2. After reduction with dithiothreitol (DTT), the 150 kDa IgG disintegrated as expected into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains. Before reduction, trastuzumab-H429Y mAb in both non-oligomeric IgG (HY IgG(H2L2)) and oligomeric (HY IgG(oli)) forms migrated identically as a single 150 kDa IgG species under both non-reducing and reducing conditions, as expected, with approximately 50 kDa of heavy (H) chain and approximately 25 kDa of light (L) chain; WT = wild type, HF = H429F, HY = H429Y; M = molecular weight marker, whose mass (kD) is shown on the left.

[0052] Figure 18 Experimental results are provided demonstrating that the oligomeric and non-oligomeric forms of the H429Y-modified IgG antibody exhibit equivalent CDC potency, and their formation is pH-sensitive: (A) SEC of rituximab-H429Y at pH 7.2 shows the presence of two major IgG forms (see also...). Figure 16(A) Non-oligomeric IgG (H2L2) (to the right of the vertical dashed line, indicated as p1) and oligomeric IgG (IgG(oli)) (to the left of the vertical dashed line, indicated as p2) were collected separately to further evaluate complement-dependent cytotoxicity (CDC) potency; (B) CDC potency was determined by flow cytometry using Ramos lymphoma cells and diluted 1 / 3 normal human serum as complement sources, based on plot A, and non-oligomeric (p2) and non-oligomeric (p1) IgG forms and SEC-purified unmodified rituximab-WT IgG. The killing effect on Ramos cells conditionated with non-oligomeric rituximab H429Y p1 (hollow square), oligomeric rituximab-H429Y p2 (triangle), or unmodified rituximab-WT (solid circle) was determined. The control background CDC in the absence of mAb (C' without mAb) is shown as a filled rhombus; and (C) IgG oligomer formation is pH sensitive. Trastuzumab-H429Y purified from protein A was analyzed by size exclusion chromatography (SEC) at pH 7.2 (left inset) or pH 5.0 (right inset). At pH 7.2, both oligomeric (IgG(oli)) and non-oligomeric IgG were present; however, at pH 5.0, only a single peak corresponding to non-oligomeric IgG was present.

[0053] Figure 19Results show that the H429F mutation in the CH3 domain of the IgG H chain effectively promotes C1q binding and complement-dependent cell-mediated cytotoxicity. Flow cytometry analysis depicts C1q binding (fluorescence intensity) in Ramos lymphoma cells conditioned with unmodified rituximab-WT (A) or modified rituximab-H429F mAb (B), or SK-OV-3 ovarian cancer cells conditioned with trastuzumab-WT (C) or modified trastuzumab-H429F mAb (D). Cells conditioned with mAb were treated with normal human serum as the complement source, and C1q binding was detected by staining with an anti-C1q rabbit polyclonal antibody. C1q binding to mAb-conditioned cells is shown in a histogram without shaded areas, and C1q background binding controls (i.e., cells treated with serum complement in the absence of mAb) are shown in a histogram with gray shaded areas. The median fluorescence intensity (MFI) for each histogram is indicated in parentheses. (E) Complement-dependent cytotoxicity (CDC; i.e., kill % as detected by Zombie Green) of Ramos lymphoma cells conditioned with rituximab WT (solid circle) or its mutated variant mAb rituximab-H429F (solid square) was determined by flow cytometry using normal human serum as the complement source. The mutation at position 429 in rituximab-H429F significantly enhanced CDC compared to rituximab-WT mAb.

[0054] Figure 20 The results of protein A affinity chromatography for type II anti-CD20 11B8-WT mAb and mutant mAb including the H429 substitution are shown. 11B8-WT mAb (WT) was generated from wild-type human IgG1 heavy chain. 11B8-H429F (H429F) was generated from a modified IgG1 heavy chain in which the histidine at position 429 in the CH3 domain of the Ig heavy chain was replaced by phenylalanine.

[0055] Figure 21Results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of 11B8-WT mAb and 11B8-H429F (H429F) mutants are shown. (A) Chromatogram of mAb purified by SEC at pH 7.2; monomeric non-oligomeric IgG is shown to the right of the vertical dashed line, and (B) SDS-PAGE analysis (5-15% gradient gel) of SEC-purified monomeric IgG mAb from inset A. Before reduction (non-reduction), all SEC-purified antibodies migrated at the expected molecular size of approximately 150 kDa for IgG, i.e., H2L2. After reduction (reduction) with DTT, all antibodies were dissociated as expected into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains. WT = wild type, HF = H429F, M = molecular weight marker, whose mass (kD) is shown on the left;

[0056] Figure 22 Results are provided demonstrating the CDC potency of type II anti-CD20 mAb conferred by the H429 modification of the CH3 domain. CDC is generated by 11B8-WT mAb (solid circles) or the CH3-mutant 11B8-H429F mAb (solid squares). Complement-dependent cleavage was determined using Ramos lymphoma cells conditioned with the indicated concentration of mAb and diluted 1 / 3 normal human serum as complement sources. CDC (kill %) was determined by flow cytometry using Zombie Green. Control CDC in the absence of mAb (C' without mAb) is shown as filled rhombuses. 11B8-WT mAb failed to mediate CDC, while the 11B8-H429F mutant mAb mediated effective complement-dependent cleavage in lymphoma cells (WT = wild type).

[0057] Figure 23 The results of protein A affinity chromatography for anti-CD38 daratumumab-WT mAb and daratumumab-H429F mAb are shown. The elution chromatograms show that each antibody eluted as a single, homogeneous peak;

[0058] Figure 24The results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of daratumumab-WT mAb and the mutant mAb daratumumab-H429F are shown. (A) Chromatogram of SEC-purified mAb. In each case, the mAb contained a single non-oligomeric IgG peak (fraction to the right of the vertical dashed line) and no oligomeric species (fraction to the left of the vertical dashed line); (B) SDS-PAGE analysis (5-15% gradient gel) of SEC-purified IgG from small figure A. Before reduction (non-reduction), all antibodies migrated at the expected molecular size of approximately 150 kDa IgG (i.e., H2L2). After reduction (reduction) with DTT, all antibodies were dissociated as expected into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains. WT = wild type, HF = H429F, M = molecular weight marker, whose mass (kD) is shown on the left;

[0059] Figure 25 Results showing that H429F substitution significantly enhanced complement-dependent cleavage of lymphoma cells by daratumumab-WT are presented. The figure shows the CDC levels (kill %) of daratumumab-WT mAb (solid circles) and daratumumab-H429F (solid squares). Background cleavage (solid triangles) was determined in the absence of mAb but in the presence of complement. CDC was determined by flow cytometry using Zombie Green. Daratumumab-H429F mAb-mediated complement-dependent cleavage of lymphoma cells was more efficient than that mediated by unmodified daratumumab-WT.

[0060] Figure 26 Results of complement-dependent cleavage of myeloma and leukemia cells resistant to CD38 mAb lysis by H429F substitution are presented. CDC generated by complement-dependent cleavage was determined using: (A) KMS-12-PE myeloma cells conditioned with the indicated concentration of daratumumab-WT mAb (solid circles) or daratumumab-H429F mutant mAb (solid squares). Background cleavage (solid triangles) was determined in the absence of mAb but in the presence of complement; and (B) SUP-15 acute lymphoblastic leukemia (ALL) cells conditioned with the indicated concentration of daratumumab-WT mAb (solid circles) or daratumumab-H429F mutant mAb (solid squares). Background cleavage (solid triangles) was determined in the absence of mAb but in the presence of complement. CDC (kill %) was determined by flow cytometry using Zombie Green.

[0061] Figure 27Results of protein A affinity chromatography for anti-HER2 mAb pertuzumab-WT and CH3 variants are shown. The elution chromatograms show that each antibody eluted as a single, homogeneous peak;

[0062] Figure 28 Results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of the anti-HER2 mAb pertuzumab-WT and CH3 H429F variants are shown. (A) Chromatogram of SEC-purified mAbs. The mAbs were further purified by size exclusion chromatography (SEC) at pH 7.2 after protein A affinity chromatography. Chromatograms of unmodified pertuzumab-WT and the Fc-modified variant pertuzumab-H429F (H429F) are shown. In each case, the protein A-purified mAb contained a single non-oligomeric IgG peak (fraction to the right of the vertical dashed line) and no oligomeric species (fraction to the left of the vertical dashed line). (B) SDS-PAGE analysis (5-15% gradient gel) of SEC-purified IgG from inset A. All SEC-purified antibodies migrated at the expected molecular size of approximately 150 kDa for IgG (i.e., H2L2) prior to reduction (non-reduction). After reduction with DTT, all antibodies were degraded as expected into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains. WT = wild type, HF = H429F, M = molecular weight marker, expressed in kilodaltons (kD);

[0063] Figure 29Results are provided illustrating the synergistic and functional effects in a mixture of mAbs including those with amino acid substitutions at position H429; specifically, results show that H429F-modified mAbs can be combined to enhance C1q binding to target cells: (A) Flow cytometry histograms (unfilled histograms) of C1q binding to HER2-expressing SK-OV-3 cells conditioned with trastuzumab-H429F alone, pertuzumab-H429F alone, or a 1:1 mixture of trastuzumab-H429F and pertuzumab-H429F. Filled histograms show background binding of C1q to unconditionated cells. Median fluorescence intensity (MFI) values ​​for each histogram are also shown. C1q binding was detected using an anti-C1q-specific polyclonal rabbit antibody; (B) Titration of enhanced functional synergistic effects. SK-OV-3 cells were conditioned with titrated individual anti-HER2 mAbs, trastuzumab-WT, pertuzumab-WT, trastuzumab-H429F, pertuzumab-H429F, or a 1:1 mixture of trastuzumab-WT and pertuzumab-WT, or trastuzumab-H429F and pertuzumab-H429F. Double-headed vertical arrows illustrate the broad synergy in the mixture of trastuzumab-H429F and pertuzumab-H429F, where antibody concentration is limiting compared to the binding of C1q to any single mAb.

[0064] Figure 30Graphical results are provided illustrating the synergistic and functional effects of H429-modified mAbs in enhancing complement-dependent killing (CDC) of target cells. The indicated rituximab-based mAbs were titrated individually on Ramos cells (light gray column) or in the presence of fixed concentrations of the indicated anti-CD38 or anti-CD20 mAb (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, (C) Rituximab-HF titrated in the presence of 0.5 μg / ml 11B8-H429F. CDC (kill %) was determined by flow cytometry using Zombie Green. The horizontal dashed lines in (B, C) indicate the percentage of CDC kills obtained in the absence of (0 µg / ml) rituximab-based mAbs but in the presence of only (B) 0.025 µg / ml daratumumab-H429F or (C) 0.5 µg / ml 11B8-H429F. The black arrows in (B, C) indicate the enhanced CDC of the mAb mixture compared to the enhanced CDC of the individual mAbs (i.e., CDC of rituximab-H429F alone at the indicated concentrations (light column) or 0.025 µg / ml daratumumab-H429F or 0.5 µg / ml 11B8-H429F (dark column), 0 µg / ml rituximab).

[0065] Figure 31 Rituximab antibody containing the H429F mutation provides a target for CD19 in normal peripheral blood. +Flow cytometry analysis of the CDC efficacy of B lymphocytes. Results indicated that rituximab-H429F mAb exhibited more effective CDC killing against normal peripheral blood B cells compared to unmodified rituximab-WT. Peripheral blood mononuclear cells were treated with the indicated mAb and human complement, and the proportion of live or dead B cells was determined by identifying B cells with anti-CD19 staining and by identifying dead cells with Zombie Green (ZG) staining. Cell histograms show the Zombie Green median fluorescence intensity (ZG MFI) (CD19 median fluorescence intensity (CD19 MFI)) of CD19-gated B cells. In each cell histogram, the percentage of dead cells (ZG positive) is shown in the upper segment Q1 of each cell histogram 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); the percentage of live B cells is shown in the lower segment Q2.

[0066] Figure 32 Results are provided demonstrating that the effect of H429 modification on antibody function is not limited to IgG1, but extends to other immunoglobulin types. Here, the CDC potency of rituximab antibodies formatted with (A) IgG3 or (B) IgG4 subclass heavy chains modified with wild-type (WT) or H429F (HF) against Ramos lymphoma cells was evaluated.

[0067] Figure 33 Results demonstrating effective CDC (cytotoxicity percentage) in target cells mediated by mAb are provided, depending on the presence of both the monoclonal antibody and serum complement. CDC (cytotoxicity percentage) of rituximab-H429F, daratumumab-H429F, or 11B8-H429F on Ramos cells or daratumumab-H429F on SUP-15 cells are shown. A = Percentage of kill in the presence of the indicated mAb and human serum as a complement source; B = Ligation in the presence of only the mAb at the same concentration as in A; C = Ligation in the presence of only complement.

[0068] Figure 34Results of complement-dependent cleavage induced by H429F substitution in leukemia cells resistant to unmodified anti-CD38 mAb isatuximab are presented. CDC generated by complement-dependent cleavage was determined using SUP-15 acute lymphoblastic leukemia (ALL) cells conditioned with indicated concentrations of isatuximab-WT mAb (dashed lines with filled circles) and isatuximab-H429F mutant mAb (solid lines with filled squares). Background cleavage (unfilled circles) was determined in the absence of mAb but in the presence of only complement (C' only, no mAb). Background cleavage induced by 5 μg / ml mAb in the absence of complement was determined for isatuximab-WT mAb (inverted unfilled triangles) and isatuximab-H429F mutant mAb (hollow unfilled squares). CDC (kill %) was determined by flow cytometry using Zombie Green.

[0069] Figure 35 Flow cytometry results were provided for the binding of purified DR5-specific mAbs containing unmodified wild-type (WT) H chains of human IgG1 or IgG2 subclasses, or containing human IgG1 or human IgG2 subclass heavy chains modified by replacing histidine 429 with phenylalanine (H429F), to Colo205 colorectal cells. The inset shows: (A) binding of BDR5-1WT containing the wild-type H chain of the human IgG1 subclass or binding of BDR5-1HF mAb containing the IgG1 heavy chain modified with H429F; (B) binding of BDR5-2WT containing the unmodified heavy chain of the human IgG2 subclass or binding of BDR5-2HF mAb containing the IgG2 heavy chain modified with H429F; (C) binding of TDR5-1WT mAb containing the unmodified heavy chain of the human IgG1 subclass or binding of TDR5-1HF mAb containing the IgG1 heavy chain modified with H429F; and (D) binding of TDR5-2WT containing the unmodified heavy chain of the human IgG2 subclass or binding of TDR5-2HF mAb containing the IgG2 heavy chain modified with H429F. The mAb was titrated sequentially twofold, and its binding activity on Colo205 cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. All insets show the non-specific binding level of the fluorescent conjugate to cells (▲ conjugate); MFI = median fluorescence intensity;

[0070] Figure 36Flow cytometry results were provided for binding of purified DR5-specific mAbs containing unmodified wild-type (WT) H chains of human IgG1 or human IgG2 subclasses, or containing human IgG1 or human IgG2 heavy chains modified by replacing histidine 429 with phenylalanine (H429F), to target Ramos lymphoma cells. The inset shows: (A) binding of BDR5-1WT containing an unmodified heavy chain of human IgG1 subclass or binding of BDR5-1HF mAb containing an IgG1 heavy chain modified with H429F; (B) binding of BDR5-2WT containing an unmodified heavy chain of human IgG2 subclass or binding of BDR5-2HF mAb containing an IgG2 heavy chain modified with H429F; (C) binding of TDR5-1WT containing an unmodified heavy chain of human IgG1 subclass or binding of TDR5-1HF mAb containing an IgG1 heavy chain modified with H429F; and (D) binding of TDR5-2WT containing an unmodified heavy chain of human IgG2 subclass or binding of TDR5-2HF mAb containing an IgG2 heavy chain modified with H429F. The mAb was titrated sequentially twofold, and its binding to Ramos cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. In all small plots, the level of nonspecific binding to cells was determined using an irrelevant IgG antibody (negative IgG), and background binding of the fluorescent anti-IgG conjugate to cells is shown (▲ conjugate); MFI = median fluorescence intensity;

[0071] Figure 37 Flow cytometry results were provided for the binding of purified DR5-specific mAbs to target KMS-12-PE myeloma cells to unmodified wild-type (WT) H chains containing human IgG1 or human IgG2 subclasses, or to human IgG1 or human IgG2 heavy chains modified by replacing histidine 429 with phenylalanine (H429F). The binding activities of DR5-specific mAbs, BDR5, and TDR5 are shown, including unmodified wild-type H chains containing human IgG1 subclasses (BDR5-1WT, TDR5-1WT) or human IgG2 subclasses (BDR5-2WT, TDR5-2WT), or Fc-mutated H chains containing H249F mutations in IgG1 subclasses (BDR5-1HF, TDR5-1HF) or IgG2 subclasses (BDR5-2HF, TDR5-2HF). KMS12-PE cells were incubated with 5 μg / ml of mAb. Binding activity was quantified by flow cytometry using an anti-IgG secondary reagent labeled with FITC goat anti-hIgG Fc. Background control (conjugate) binding only, along with background fluorescence of cells only, is shown.

[0072] Figure 38 Results demonstrating Colo205 colorectal cell viability are provided in the presence of purified DR5-specific mAbs containing either an unmodified wild-type H chain of human IgG1 or human IgG2 subclasses or an H chain containing an Fc mutation of human IgG1 or human IgG2 subclasses with the H429F mutation. In the presence of each of the mAbs below 20 μg / ml, 10,000 cells were cultured in each well of a 96-well plate: TDR5-1WT mAb containing an unmodified wild-type H chain of human IgG1 subclasses, or TDR5-1HF mAb containing an H chain of human IgG1 subclasses with the H429F mutation, or TDR5-2WT mAb containing an unmodified wild-type H chain of human IgG2 subclasses, or TDR5-2HF mAb containing an H chain of human IgG2 subclasses with the H429F mutation. Additionally, Colo205 cells were cultured alone in the presence of a mixture of 10 μg / ml TDR5-2HF and 10 μg / ml BDR5-2HF mAb (TDR5-2HF + BDR5-2HF), both containing the Fc mutation of human IgG2 H chain carrying the H429F mutation. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (ab-free). Viability is expressed as: [experimentally determined mAb or control treatment Abs450 nm - background Abs450 of cell culture medium], and the mean Abs450 and four replicate values ​​are shown.

[0073] Figure 39Results demonstrating Ramos lymphoma cell survival are provided in the presence of purified DR5-specific mAbs containing unmodified wild-type or Fc-mutated H chains of human IgG1 or IgG2 subclasses with the H429F mutation. In the presence of each of the mAbs below 20 μg / ml, 10,000 cells were cultured in each well of a 96-well plate: TDR5-1WT mAb containing unmodified wild-type H chains of human IgG1 subclasses, or TDR5-1HF mAb containing H chains of human IgG1 subclasses with the H429F mutation, TDR5-2WT mAb containing unmodified wild-type H chains of human IgG2 subclasses, or TDR5-2HF mAb containing H chains of human IgG2 subclasses with the H429F mutation. Additionally, Ramos cells were cultured alone in the presence of a mixture of 10 μg / ml TDR5-2HF and 10 μg / ml BDR5-2HF mAb (TDR5-2HF + BDR5-2HF), both containing the Fc mutation of human IgG2 H chain carrying the H429F mutation. Cell viability was quantified using a CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (ab). Viability is expressed as: [experimentally determined mAb or control treatment Abs450 nm - background Abs450 of cell culture medium], and the mean Abs450 and four replicate values ​​are shown.

[0074] Figure 40Results demonstrating the survival of Colo205 colorectal cells in the presence of a mixture of purified DR5-specific mAbs, wherein the mAbs contain wild-type or Fc-modified H chains of the same IgG subclass. BDR5 and TDR5 mAbs contain unmodified wild-type H chains of human IgG1 or human IgG2 subclasses, or heavy chains containing H429F-modified IgG1 or IgG2 subclasses. Colo205 cells (30,000 cells / well in 96-well plates) were cultured in serial 2-fold dilutions of a 1:1 mixture of mAbs containing the same H chains. The starting concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. Therefore, the mixtures used are: BDR5-1WT mAb containing unmodified wild-type IgG1 H chain mAb and TDR5-1WT containing unmodified wild-type IgG1 H chain (BDR5-1WT + TDR5-1WT); BDR5-2WT mAb containing unmodified wild-type IgG2 H chain mAb and TDR5-2WT containing unmodified wild-type IgG2 H chain (BDR5-2WT + TDR5-2WT); BDR5-1HF mAb containing H429F-modified IgG1 H chain and TDR5-1HF mAb containing H429F-modified IgG1 H chain (BDR5-1HF + TDR5-1HF); and BDR5-2HF mAb containing H429F-modified IgG2 H chain and TDR5-2HF mAb containing 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 (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (ab), and maximum cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [experimentally determined mAb or control Abs450 nm - background Abs450 of the cell culture medium];

[0075] Figure 41Results demonstrating the survival of Colo205 colorectal cells in the presence of different pairs of purified DR5-specific mAbs, wherein the mAbs comprise wild-type or Fc-mutant H chains of different IgG subclasses. The mAbs comprise H chains of human IgG1 or human IgG2 subclasses comprising wild-type heavy chains or heavy chains modified with H429F. Colo205 cells (10,000 cells / well in 96-well tissue culture plates) were cultured in a 1:1 mixture of mAbs serially diluted 2-fold from 1 μg / ml. The initial concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. The mixtures of wild-type mAbs used were: a mixture of BDR5-2WT containing wild-type human IgG2 H chain and TDR5-1WT containing unmodified wild-type human IgG1 H chain (BDR5-2WT + TDR5-1WT); a mixture of BDR5-1WT containing wild-type human IgG1 H chain and TDR5-2WT containing unmodified wild-type human IgG2 H chain (BDR5-1WT + TDR5-2WT); and a control mixture of BDR5-2WT containing unmodified wild-type human IgG2 H chain and TDR5-2WT containing unmodified wild-type human IgG2 H chain (BDR5-2WT + TDR5-2WT). Figure 40 The mixtures of DR5 mAbs containing the H chain of the H429F Fc mutation observed are: a mixture of BDR5-2HF containing the H chain of human IgG2 containing the H429F Fc mutation and TDR5-1HF containing the H chain of human IgG1 containing the H429F Fc mutation (BDR5-2HF + TDR5-1HF); a mixture of BDR5-1HF containing the H chain of human IgG1 containing the H429F Fc mutation and TDR5-2HF containing the H chain of human IgG2 containing the H429F Fc mutation (BDR5-2HF + TDR5-2HF); and a positive killing control mixture of BDR5-2HF containing the H chain of human IgG2 containing the H429F Fc mutation and TDR5-2HF containing the H chain of human IgG2 containing the H429F Fc mutation (BDR5-2HF + TDR5-2HF), as shown in [reference to previous text]. Figure 38 and Figure 40 The results were observed in [the study]. Cell viability was quantified using the CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (ab), and maximum cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [experimentally determined mAb or control Abs450 nm - background Abs450 of cell culture medium];

[0076] Figure 42 Results showing the survival rates of Colo205 cells in the presence of paired mixtures of BDR5 and TDR5 mAbs at different ratios are provided. Ten thousand Colo205 cells were cultured for 48 hours in the presence of a single mAb BDR5-1WT or TDR5-1WT containing a wild-type IgG1 H chain, or a single mAb BDR5-1HF or TDR5-1HF containing an H chain modified with H429F, or a mixture of BDR5-1HF and TDR5-1HF mAbs, at the following ratios: 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixtures shown in the figure, BDR5-1HF mAb is designated as B-1HF, and TDR5-1HF is designated as T-1HF. Controls include the maximum cell survival rate in the absence of mAbs (ab-free) or the maximum cell death control 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 (Abs450 nm) is a measure of cell viability and viability. Viability is expressed as: [experimentally determined mAb or control Abs450 nm - background Abs450 of the cell culture medium];

[0077] Figure 43 Results showing the survival of Ramos lymphoma cells in the presence of paired mixtures of BDR5 and TDR5 mAbs at different ratios are provided. 10,000 Ramos cells were cultured for 48 hours in the presence of individual mAbs BDR5-1WT or TDR5-1WT containing wild-type H chains, or individual mAbs BDR5-1HF or TDR5-1HF containing H chains modified with H429F, or mixtures of BDR5-1HF and TDR5-1HF mAbs. In the mixtures shown in the figure, BDR5-1HF mAb is designated B-1HF, and TDR5-1HF is designated T-1HF. Cell viability was quantified using a CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (Abs 450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (ab), and maximum cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [experimentally determined mAb or control Abs450 nm - background Abs450 of cell culture medium];

[0078] Figure 44Results are provided for the quantitative detection of the binding of DR5-specific mAbs to Colo205 cells containing different epitopes and wild-type or Fc-mutated heavy chains of human IgA2 subclasses. The mAbs used as tissue culture supernatants from appropriately transfected Expi293 cells contained unmodified wild-type IgA2 heavy chains (BDR5-A2WT) and (TDR5-A2WT) or Fc-mutated IgA2 H chains carrying the H429F mutation of the human IgA2 subclass (BDR5-A2HF) and (TDR5-A2HF). Background fluorescence (without ab), determined solely by measuring the binding of the fluorescent conjugates, is shown together with non-specific fluorescence (cell-only).

[0079] Figure 45 Results demonstrating Colo205 cell viability are provided in the presence of two different DR5-specific mAbs containing heavy chains of human IgA2 subclasses. Colo205 cells (10,000 cells / well in a 96-well plate) were cultured for 48 hours in serially 2-fold dilutions of tissue culture supernatant from Expi293 cells to produce… Figure 44 The DR5 IgA 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 carrying H429F modification; and TDR5-A2HF containing heavy chains of human IgA2 subclasses carrying H429F modification. Cell viability was quantified using a CCK8 colorimetric cell viability assay, where the absorbance of the cell culture supernatant (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (abs-free), and maximum cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [Experimentally determined Abs450 nm - background Abs450 of cell culture medium];

[0080] Figure 46Results demonstrating enhanced killing of Ramos lymphoma cells by mixtures of H429F-modified mAbs targeting different molecular targets are provided. Ramos cells were cultured alone at 10,000 cells / well, or co-cultured with their Fc-modified counterparts, in the presence of either exatuximab (Isa-WT) containing the H chain of IgG1 WT, or BDR5-1 (BDR5-1WT) containing the H chain of IgG1 WT, or a 1:1 mixture of the two mAbs (Isa-WT + BDR5-1WT). All mAbs were present in the culture at 10 μg / ml, and for mixtures, each mAb was 10 µg / ml, with a total mAb concentration of 20 µg / ml. Cell viability was quantified using a CCK8 colorimetric cell viability assay after 48 hours of culture, where the absorbance (Abs450 nm) of the CCK8-treated cell supernatant was a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (Ab-free), and maximum cell death was determined by incubating cells with SDS prior to CCK8 treatment. Viability is expressed as: [Experimentally determined Abs450 nm - Background Abs450 of cell culture medium], and the mean Abs450 and four replicate values ​​are shown.

[0081] Figure 47 Results are provided illustrating complement-dependent cleavage (CDC) in leukemia cells resistant to cleavage of unmodified anti-CD38 mAb mezagitamab induced by H429F substitution. CDC generated by complement-dependent cleavage was determined using Ramos lymphoma cells specifically conditioned with either mezagitamab-WT mAb or mezagitamab-H429F mutant mAb at the indicated concentrations. Background cleavage was determined in the absence of mAb but with only complement (C' only). CDC (kill %) was determined by flow cytometry using Zombie Green.

[0082] Figure 48It is shown that additional mutations in the K439E or S440K in the Fc region fraction can suppress the enhanced CDC of H429F-modified rituximab, and the combination of rituximab-H429F / K439E and rituximab-H429F / S440K restores the effective CDC killing effect on Ramos cells. (A) Ramos lymphoma cells were conditioned with rituximab-WT (WT) or rituximab-H429F (H429F) mAb alone, or with rituximab-H429F containing the additional H chain mutation K439E (H429F / K439E), or with rituximab-H429F containing the additional H chain mutation S440K (H429F / S440K), or with a paired mixture of rituximab-H429F / S440K and rituximab-H429F / K439E (H429F / K439E + H429F / S440K). Background lysis was determined in the absence of mAb but with only complement present (C' without mAb). CDC (kill %) of mAb was measured by flow cytometry using Zombie Green in the presence of 1 / 3 diluted human serum. Enhanced CDC mediated by rituximab H429F modification was inhibited by suppressing Fc:Fc interactions of K439E or S440K mutants, but was found to be completely reversible in mixtures of the two IgG mutants. (B) For binding assays, proteins were titrated sequentially twice, and binding activity was quantified by flow cytometry using FITC-conjugated anti-hIgG-Fc secondary reagents (on Ramos cells). The level of nonspecific background binding of the fluorescent conjugate to cells is shown (conjugate only); MFI = median fluorescence intensity. All mAbs showed nearly identical binding to CD20;

[0083] Figure 49 Results of flow cytometry analysis of the binding (A, C) and CDC (B, D) of purified flACE2-Fc fusion proteins (A, B) and SARS-CoV spike-specific mAbs (C, D) using Ramos-S cells are provided. For the binding assay, the proteins were sequentially titrated twice, and binding activity (on Ramos-S cells) was quantified by flow cytometry using FITC-conjugated anti-hIgG-Fc secondary reagents. The level of nonspecific background binding of the fluorescent conjugates to the cells is shown (conjugate); MFI = median fluorescence intensity. For the CDC assay, the proteins were sequentially titrated twice, and human serum was used as the complement source. CDC lysis was quantified using Zombie Green. Background lysis by complement in the absence of antibodies (C' only without mAb) was identified and shown; and

[0084] Figure 50Graphical results from flow cytometry analysis are provided, showing the synergistic and functional effects of the H429F-modified flACE2-Fc fusion protein and the H429F-modified anti-SARS-CoV-2 mAb in further enhancing complement-dependent killing (CDC). (A) Titration of mAb S2P6-H429F (S2P6-HF) or S2P6-WT alone, or in the presence of a fixed concentration of flACE2-Fc-H429 fusion protein (1 μg / ml final concentration) (S2P6-HF + flACE2-Fc-HF), mediates 23.4% CDC killing when used alone (indicated by hollow diamonds), or titration in the presence of a fixed concentration of flACE2-Fc-WT (final 1 μg / ml) (S2P6-WT + flACE2-Fc-WT), mediates 5.0% killing % when used alone (filled diamonds). Background cleavage via complement in the absence of mAb or Fc fusion protein is shown (C' only); arrows indicate instances of maximum synergy; (B) Evaluation of CDC killing efficacy of H429F-modified flACE2-Fc on Ramos-S cells. mAb CC40.8-H429F (CC40.8-HF) and CV3-25-H429F (CV3-25-HF) were used alone (2.5 μg / ml final concentration) 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 is 2.5 μg / ml and the final concentration of flACE2-Fc-H429F is 1 μg / ml). Killing efficacy was evaluated in flow cytometry assays using Zombie Green. Also shown are flACE-2-Fc-WT-mediated CDC kill % and background cleavage via complement in the absence of mAb or Fc fusion protein (C' only). Four replicate values ​​and SEM are shown. Mean kill % values ​​are shown above each column;

[0085] Figure 51The results presented show three formats of ACE2-Fc fusion proteins containing Fc region components with wild-type sequences or H429F mutations that bind equivalently to Ramos-S target cells: trACE2-Fc, flACE2-Fc, and EflACE2-Fc; 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 insets, the non-specific binding level of the fluorescent conjugate to cells is shown (conjugate only); MFI = median fluorescence intensity. EC50 (nM) values ​​from curve fitting are shown.

[0086] Figure 52 The results presented show ACE2-Fc fusion proteins in three formats (trACE2-Fc, flACE2-Fc, and EflACE2-Fc) containing Fc region components with wild-type sequences or H429F mutations, strongly immobilizing complement and direct complement-dependent cytotoxicity (CDC) in Ramos-S target cells; as determined by flow cytometry analysis of CDCs from conditioned 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 subplots, only the non-specific complement killing level (C' only) is shown in the absence of ACE2-Fc fusion proteins. EC50 (nM) values ​​for curve fitting from the H429F protein are shown. The fit of the (nd)WT protein could not be determined;

[0087] Figure 53 Illustrations of immunoglobulins (antibodies) and immunoglobulin (antibody)-like molecules are provided, illustrating the modular nature of antibodies:

[0088] (A) Left inset: Definitions of immunoglobulin molecular chains and components are as follows Figure 2 The left-hand side diagram indicates and is also defined within it.

[0089] The inset image in the middle provides an example of an Ab-like fusion protein, demonstrating how the modular nature of immunoglobulins allows for flexibility in the production of Ab-like molecules (as shown in...). Figure 2(As provided in the text). In the specifically described 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 connected to both the H chain and the L chain constant domain at the CH1 domain, respectively, so that it can be assembled into the H2L2 Ab-like fusion protein).

[0090] The right inset illustrates possible Ab-like fusion proteins that contain fusions with different target recognition structures (or enzymes and / or reporter molecules) in any specific combination (e.g., “X1 X1 X1 X1”, “X1 X1 X1 X2”, “X1 X1 X2 X2”, “X1X1 X2 X3”, “X1 X2 X3 X4”; where “X1”, “X2”, “X3”, and “X4” represent different target recognition structures (or enzymes or reporter molecules)).

[0091] (B) SDS-PAGE analysis of the EflACE2-Ab-like-WT fusion protein eluted from the protein A affinity matrix with 0.4 M arginine (pH 4) revealed a fully disulfide-linked molecule (lane 1) conforming to the H2L2 Ab-like configuration. This molecule contains an EflACE2 polypeptide fused to the immunoglobulin constant heavy chain (EflACE2-CH), which is self-assembled with an equivalent EflACE2 polypeptide fused to the immunoglobulin light chain constant domain (EflACE2-CL). Upon reduction with dithiothreitol, both chains (i.e., EflACE2-CH (ACE2-CH) and EflACE2-CL (ACE2-CL); lane 2) were individually degraded.

[0092] The results provided in (C, D) indicate that the EflACE2-Ab-like-H429F fusion protein (H429F) strongly directs CDC in Ramos-S target cells, while the corresponding fusion protein with the wild-type sequence (WT) of the H chain is ineffective. CDC in opsonized Ramos-S cells was determined using normal human serum in the presence of a 1 / 3 dilution as a complement source. This effective CDC mediated by the EflACE2-Ab-like-H429F fusion protein, compared to the EflACE2-Ab-like-WT fusion protein, cannot be attributed to different levels of opsonization, as the binding of these two proteins to Ramos cells expressing the SARS-CoV-2 spike is comparable. Binding was determined by flow cytometry analysis using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. MFI = median fluorescence intensity; background binding of the anti-Ig fluorescent conjugate is shown as (conjugate only).

[0093] Figure 54The results of purification of the EflACE2-Ab-like-Fc-H429F fusion protein using protein A and elution with 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 combined and concentrated protein A fractions containing EflACE2-Ab-like-Fc-H429F using a Superose 6Increase 10 / 300 column with the indicated oligomeric material (HMW); and (C) SDS-PAGE analysis of the combined protein A eluent and the combined SEC monomer fractions under non-reducing (DTT-free, dithiothreitol-free) and reducing (DTT-containing) conditions.

[0094] Figure 55 Results demonstrating Colo205 colorectal cell viability are provided in the presence of purified DR5-specific mAbs containing either a single H429F mutation (HF) or L234A, L235A, and H429F mutations (LA / LA / HF). The mAbs were titrated individually or in combination (BDR5-1LA / LA / HF + TDR5-1LA / LA / HF), and compared to a mixture of BDR5-1HF + TDR5-1HF, where both mAbs in the mixture contain only H429F-modified IgG1 H chains; Colo205 cells (10,000 cells / well in a 96-well plate) were cultured in sequential 2-fold dilutions of the indicated mAbs. Individual mAb titrations were started at 1 μg / ml. For mixture titrations, the initial concentration of 1 µg / ml comprised 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 (Abs450 nm) is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibodies (Abs-free), and maximum cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [experimentally determined mAb at Abs450 nm or control treatment Abs450 nm - background Abs450 of the cell culture medium];

[0095] Figure 56The combination of H429-substituted enhanced IgG-like fusion protein and bNAb (A) broadly neutralizes mAb in coronaviruses and (B) Ab-like decoys with H429-substituted enhanced Fc interactions can be combined to form (C) a mixed hexamer. (D) bNAb CV3-25 and Fc-ACE2 fusions, when both are incorporated into the H429F modification, synergistically induce complement-mediated SARS-CoV-2 spike cell killing more effectively than either agent alone (n = 4).

[0096] Figure 57 Images of the supernatant from transfected Expi293 cells expressing H429-modified microantibodies are provided by SDS-PAGE. Analysis of the supernatant by SDS-PAGE in 5–15% gradient gels showed the presence or absence of disulfide bond reduction: In lanes 1–4, under non-reducing conditions, the H429-modified microantibodies BDR5-Fab-CH3-H429F (lane 1) and TDR5-Fab-CH3-H429F (lane 2) migrated as a single species at the expected molecular size of approximately 125 kDa, containing (VH-CH1-linker-CH3)2L2. Upon DTT reduction (lanes 5 and 6), these microantibodies, as expected, disintegrated into approximately 34 kDa VH-CH1-linker-CH3 chains and approximately 25 kDa light (L) chains. In lane 3, under non-reducing conditions, the H429-modified microantibody TR2-3-HF (lane 3) migrated as a single species with a expected molecular size of approximately 105 kDa (scFv-linker-CH2-CH3)2. After DTT reduction (lane 7), these microantibodies disintegrated as expected into a single scFv-linker-CH2-CH3 chain of approximately 51.5 Da. In lane 4, under non-reducing conditions, the H429-modified antibody b12 (lane 3), serving as a comparison of the microantibody, migrated as a single species with a expected molecular size of approximately 150 kDa IgG (i.e., H2L2). After reduction in DTT (lane 8), this mAb disintegrated as expected into a heavy (H) chain of approximately 50 kDa and a light (L) chain of approximately 25 kDa. In lanes 5-7, heavy chain species are marked with a single asterisk (*) and in lane 8, b12 heavy chain species are marked with a double asterisk (**).

[0097] Figure 58Characterization of bispecific bsCD37 V10 mAb containing the H chain with wild-type histidine at position 429 (V10 WT), or a histidine 429 phenylalanine mutation (V10 H429F), or a histidine 429 phenylalanine mutation and a serine 354 cysteine ​​mutation (V10CC H429F) is provided. Size exclusion chromatography (SEC) of bsmAbs is shown as a thick line and molecular weight standards as dashed lines, with their mass in kDa and retention times indicated by vertical arrows. SDS-PAGE of each bsmAb under reducing (R) or non-reducing (NR) conditions is shown. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0098] Figure 59 The binding and CDC potency of CD37 bsmAb on Daudi cells are shown. (A) Binding of CD37xCD37-V10-WT, CD37xCD37-V10-H429F, and CD37xCD37-V10CC-H429F bsmAbs was assessed by flow cytometry after successive 3-fold titrations of bsmAb. (B) CDC potency was assessed on Daudi cells after successive 2-fold titrations of bsmAb.

[0099] Figure 60 The binding and CDC potency of CD37 bsmAb on Ramos cells are shown. (A) Binding of CD37xCD37-V10-WT, CD37xCD37-V10-H429F, and CD37xCD37-V10CC-H429F mAbs were assessed by flow cytometry after 3-fold sequential titrations of bsmAb. (B) CDC potency was assessed on Ramos cells after 2-fold sequential titrations of bsmAb; inflection points are indicated by vertical arrows, with the CDC indicator concentration less than 1.1 µg / mL in stage 1 (left arrow) and the CDC indicator concentration greater than 1.1 µg / mL in stage 2 (right arrow).

[0100] Figure 61 The thermal stability of CD37 bsmAb is shown. The thermal degeneration of (A) CD37xCD37-V10-WT, (B) CD37xCD37-V10-H429F and (C) CD37xCD37-V10CC-H429F mAb was measured using SYRO-Orange, and the observed transition temperature is shown in (D) from the dF / dT curve.

[0101] Figure 62The properties of BDR5xTDR5 V10-WT, H429F bsmAb, and BDR5xTDR5 V11 bsmAb containing the H chain with a wild-type histidine (WT) or histidine 429 phenylalanine mutation (H429F) at position 429 are shown. Size exclusion chromatography (SEC) of bsmAbs is shown as a thick line and molecular weight standards as dashed lines. Their mass is in kDa, and retention time is indicated by vertical arrows. SDS-PAGE of each bsmAb under reducing (R) or non-reducing (NR) conditions is shown. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0102] Figure 63 The efficacy of H429F modification was demonstrated to be enhanced by bispecific DR5 mAb. COLO205 cells were treated with BDR5xTDR5 bsmAb in the format V10-WT, V10-H429F, or V11-H429F. Surviving cells were enumerated after 2 days and normalized relative to untreated cells. 'V-10 WT' refers to a bsmAb containing a bispecifically paired V10 mutation, while 'V10-H429F' or 'V11-H429F' indicates additional inclusions modified with H429F. Mixed-effects analysis of the main column effect was compared with Tukey's multiple comparison analysis, n = 8, except for BDR5xTDR5 V11-H429F, n = 9;

[0103] Figure 64 Results of treatment of COLO205 cells with the indicated concentration of BDR5xTDR5-V10-H429F bsmAb are shown, with and without 5 μM pan-cysteine ​​inhibitor z-VAD. Surviving cells are listed after 1 day and normalized relative to untreated cells. Comparisons were performed using two-way ANOVA and Tuki multiple comparison test, n = 3.

[0104] Figure 65 Characterization of CD3xCD28-V10 bsmAbs containing the H chain with a wild-type histidine (WT) or histidine 429 phenylalanine mutation (H429F) at position 429 is provided. Size exclusion chromatography (SEC) of the bsmAbs is shown as a thick line and molecular weight standards as dashed lines. Mass is expressed in kDa, and retention time is indicated by vertical arrows. SDS-PAGE of each bsmAb was performed under reducing (R) or non-reducing (NR) conditions. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0105] Figure 66 The activation of T cells by the CD3xCD28 bispecific mAb is shown. Cells stimulated with the IL-2 promoter-luciferase Jurkat reporter gene were subjected to bispecific mAbs, anti-CD3, anti-CD28, or anti-HEL human IgG1. Cell activation was measured by luciferase expression as mean relative light units (RLU) ± SEM of three replicates.

[0106] Figure 67 The properties of PD-L1x4-1BB-V10 bsmAbs containing the H chain with a wild-type histidine (WT) or histidine 429 phenylalanine mutation (H429F) at position 429 are provided. Size exclusion chromatography (SEC) of the bsmAbs is shown as a thick line and molecular weight standards as dashed lines. Mass is expressed in kDa, and retention time is indicated by vertical arrows. SDS-PAGE of each bsmAb was performed under reducing (R) or non-reducing (NR) conditions. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0107] Figure 68 Cell activation is shown after co-culturing GS-J7 / 4-1BB / PD-1 / NFAT-NFκB-luciferase reporter T cells with BS-C3 / PD-L1 cells in the presence of indicated concentrations of bsmAb PD-L1x4-1BB V10-WT or bsmAb PD-L1x4-1BB V10-H429F or control IgG1. Cell activation was measured by luciferase expression. Data are presented as mean relative optical units (RLU) ± SEM of three replicates.

[0108] Figure 69 The characteristics of HER2x4-1BB-V10 bsmAbs containing the H chain with a wild-type histidine (WT) or histidine 429 phenylalanine mutation (H429F) at position 429 are provided. Size exclusion chromatography (SEC) of the bsmAbs is shown as a thick line and molecular weight standards as dashed lines. Mass is expressed in kDa, and retention time is indicated by vertical arrows. SDS-PAGE of each bsmAb was performed under reducing (R) or non-reducing (NR) conditions. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0109] Figure 70Cell activation was demonstrated after co-culturing GS-H3 / 4-1BB clone 16 / NFAT-NFκB-luciferase reporter T cells with SK-BR-3 cells in the presence of indicated concentrations of bsmAb HER2x4-1BB V10-WT or bsmAb HER2x4-1BBV10-H429F or control IgG1. Cell activation was measured by luciferase expression. Data are expressed as mean relative optical units (RLU) ± SEM of three replicates.

[0110] Figure 71 Characterization of CD20xCD3-V10 bsmAbs containing the H chain with a wild-type histidine (WT) or histidine 429 phenylalanine mutation (H429F) at position 429 is provided. Size exclusion chromatography (SEC) of the bsmAbs is shown as a thick line and molecular weight standards as dashed lines. Mass is expressed in kDa, and retention time is indicated by vertical arrows. SDS-PAGE of each bsmAb was performed under reducing (R) or non-reducing (NR) conditions. Arrows indicate the position of intact IgG or heavy (H) or light (L) chains; molecular weight markers (M1) and mass in kDa are shown.

[0111] Figure 72 Cell activation as measured by luciferase is shown. GS-J2C clone 16 / NFAT-luciferase reporter T cells were cultured with (A) or without (B) Raji cells in the presence of indicated concentrations of bsmAb CD20xCD3V10-WT or bsmAb CD20xCD3 V10-H429F or control IgG1. Cell activation was measured by luciferase expression. Data are expressed as mean relative optical units (RLU) ± SEM of two replicates.

[0112] Figure 73 Cell viability is shown. COLO205 cells were treated with an equimolar mixture of IgG1-based BDR5 and TDR5 mAbs or also IgG1-based (A) a mixture of BDR5-WT and TDR5-WT or BDR5xTDR5 V10WT bsmAb or (B) V10 bsmAb of BDR5-H429F and TDR5-H429F or BDR5xTDR5-V10-H429F mbsMab. Viable cells were listed after 1 day of culture and normalized relative to untreated cells; mAb concentrations were the total concentrations of mAbs in the mixture (µg / ml).

[0113] Figure 74Cell viability is shown. COLO205 cells were treated with an equimolar mixture of BDR5 and TDR5 mAbs, formatted as (A) IgG2-WT, (B) IgG2-H429F, or (C) IgG1-WT, in the absence or presence of 5 µg / mL dimer rsFcγRIIa-H131 or dimer rsFcγRIIb. Viable cells were listed after 1 day of culture and normalized relative to untreated cells. mAb concentration is the total concentration (µg / ml) of mAbs in the mixture. Detailed Implementation

[0114] This disclosure relates to immunotherapeutic proteins comprising one or more polypeptides containing Fc region components, wherein the one or more polypeptides comprise an amino acid substitution (mutation) (Eu number) at a position corresponding to H429 (within the CH3 domain) of the amino acid sequence of the heavy (H) chain of human immunoglobulin G1 (IgG1).

[0115] This disclosure relates to a bispecific immunotherapy protein comprising an Fc region component, wherein the one or more polypeptides comprise an amino acid substitution (mutation) (Eu number) at a position corresponding to H429 (within the CH3 domain) of the amino acid sequence of the heavy (H) chain of human IgG1, IgG2, IgG3, or IgG4.

[0116] This disclosure relates to a microantibody comprising an Fc region component, wherein the one or more polypeptides comprise an amino acid substitution (mutation) (Eu number) at a position corresponding to H429 (within the CH3 domain) of the amino acid sequence of the heavy (H) chain of human IgG1, IgG2, IgG3, or IgG4.

[0117] The amino acid numbering used in this paper is based on the so-called Eu numbering system, which relates to the sequence numbering used in describing the human IgG1 myeloma protein known as 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) appears at position 429 of the Eu sequence. Therefore, in any immunoglobulin molecule such as an antibody or its fragment or antibody-like molecule, the amino acid at a given position number relates to or corresponds to the position number of the corresponding amino acid residue in the Eu sequence.

[0118] The heavy (H) chains of immunoglobulins such as IgG1 are modular, multifunctional, but monomeric polypeptides. The prototype immunoglobulin or antibody structure (with the H429 mutation) can be considered as a dimer protein comprising two monomeric heavy (H) chain polypeptides, each optionally associated with the light (L) chain, and thus exists in the H2L2 format (see [link to relevant documentation]). Figure 2 However, other forms of immunoglobulin molecules can be formed, including one heavy chain (H1) and one light chain (L1) associated in an H1L1 format, and other forms in which the two heavy (H) chains can form a dimer in the absence of the light (L) chain to produce a heavy chain complex in an H2 format. Figure 2 When the light (L) chain and heavy (H) chain associate in the form of H1L1, the molecule can dimerize in this configuration to produce the typical immunoglobulin structure designated as H2L2. This is exemplified by the typical human IgG class of immunoglobulins, in which the two heavy (H) chains are covalently bound to each other, and the light (L) chain is covalently bound to each heavy (H) chain.

[0119] The dimer form of the H1L1 immunoglobulin molecule (i.e., the H2L2 protein) is the basic structural "unit" of all human Ig classes (i.e., IgG, IgE, IgD, IgA, and IgM), and in fact, the basis of most mammalian immunoglobulin classes, noting that exceptions to this format are known (e.g., camel immunoglobulins can form heavy-chain dimers without light chains (abbreviated as H2)). Thus, for example, the human immunoglobulin G (IgG) molecule physiologically exists in solution as a single H2L2 unit. However, other covalently bound higher-order oligomers of basic H2L2 units do exist in nature and are common, specifically for IgM and IgA.

[0120] For example, IgM can form covalent pentameric or hexameric rings in the form of H2L2, where each H2L2 unit has a disulfide bond with an adjacent H2L2 unit to form pentameric oligomers (H2L2)5 or hexameric oligomers (H2L2)6 (Eskeland T and TB Christensen, Scandinavia Journal of Immunology 4(3):217-228, 1975), and it is noteworthy that 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 affinity binding to the soluble hexameric protein C1q (Eskeland T and TB Christensen, 1975 ibid.; Randall TD et al., Proceedings of the National Academy of Sciences 89:962-966, 1992; Hughey CT et al., *Journal of Immunology* 161:4091-4097, 1998; and Randall TD et al., *Eur J Immunology* 20:1971-1979, 1990. IgM is also recognized as the most potent lectin due to its covalent oligomerization, which results in 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 defined affinities for target antigen sites or epitopes, the strength of these antigen recognition structures is defined by their monovalent binding to a target structure. Therefore, the presence of multiple antigen recognition structures in a molecule (e.g., the 12 antigen-binding variable domains in each IgM hexamer) confers stronger or more affinity binding to oligomeric immunoglobulins. Thus, oligovalent binding arises from the combined strength of individual antigen recognition interactions between the IgM oligomer and its target antigen epitope. In contrast, the affinity of IgG (which has two antigen recognition structures (Fab) / H2L2 units) comes solely from the interaction between these two antigen recognition structures.

[0121] Immunoglobulins can also be viewed as modular, multifunctional proteins, where target recognition structures are connected to functional activation structures via flexible linkers (i.e., the antigen recognition structure provided by the V domain of the Fab is connected to the Fc region via a flexible hinge), and where, in the case of typical immunoglobulins, each H chain and each L chain contains different domains, which can be considered as structural or functional modules. Figure 2 The H chain specifically contains a variable sequence antigen target recognition domain (V). HFirst, there is a series of heavy chain-specific constant domains, followed by a series of Ig class-specific constant domains (e.g., IgG or IgA), and sequence analysis showed that these constant domains are correlated among immunoglobulin classes (see [link to relevant documentation]). Figure 3 and 4 Therefore, V H Following the first constant domain is the first constant domain (CH1). This first constant domain is connected to the second constant domain (CH2) via a flexible polypeptide that acts as a linker known as the hinge region. Following the second constant domain is the third constant domain (CH3), and thus a typical H-chain consists of discrete molecular structures starting from the amino terminus (i.e., NH2-terminal V). H -CH1-hinge-CH2-CH3 ( Figure 2 ), and is illustrated by the human IgG1 H chain sequence (SEQ ID NO: 3), and is conserved across all human heavy chains (). Figure 3 , 4 This is 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), IgE (SEQ ID NO: 9), IgM (SEQ ID NO: 10), and IgD (SEQ ID NO: 11), as well as general mammalian immunoglobulins. Similarly, the light (L) chain contains a variable antigen target recognition domain (V... L First, there is the structural domain, followed by the constant structural domain specific to the light chain class.

[0122] Given their modular nature, immunoglobulins provide a universal platform for creating a variety of immunotherapeutic or diagnostic molecules, including bivalent antibodies such as classic hybridoma-derived mAbs, 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 some fusion proteins containing at least one CH3 domain (or at least one CH4 domain)), and other immunoglobulin derivatives known to those skilled in the art as outlined 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 such as single-chain Fv antibody fragments, asymmetric bispecific antibodies (WO 2012 / 058768), chain exchange engineered domains (SEED or Seed bodies), which are asymmetric and bispecific antibody-like molecules (WO 2007110205); dual variable domain immunoglobulins (US 7,612,181), mortar antibody formats (WO 1998 / 050431), duobody molecules (WO 2011 / 131746), IgG-like bispecific antibodies (Shen J et al., Journal of Immunol Methods 318(1-2):65-74, 2007), and fusion proteins containing Fc or Fc region components, such as scFv fusion bodies and double scFv fusion bodies.

[0123] This specification uses many terms that are well known to those skilled in the art. Nevertheless, for clarity, many of these terms are defined below.

[0124] As used herein, the term “antibody” should be understood to include (unless specifically indicated otherwise) polyclonal antibodies (pAb), monoclonal antibodies (mAb), chimeric antibodies, humanized antibodies, and antibody mixtures (e.g., recombinant polyclonal antibodies), such as antibodies produced by methods well known to those skilled in the art for producing multiple antibodies with different specificities from a single host cell line (e.g., Oligocolonics® technology: Merus BV, Utrecht, The Netherlands) or transgenic animals. Furthermore, it should be understood that antibodies can be any class of immunoglobulins (i.e., isotypes) or allotypes. Thus, for example, antibodies disclosed herein can be isotypes selected from well-known immunoglobulin isotypes, or contain components from more than one isotype (e.g., this may be the case for some types of chimeric antibodies). Similarly, antibodies can contain “mixed” chains; for example, antibodies containing H2 or H2L2 units where the H chains are different (i.e., H… x and H y (H) x H y or L x H x H y L y )).

[0125] The term "antibody-like molecule" (Ab-like molecule) should be understood to refer to a protein that is not an antibody and minimally contains a target recognition structure (e.g., a receptor or ligand such as an antigen recognition sequence, or a variable (V) domain or complementarity-determining region (CDR) of an immunoglobulin) linked to at least one CH3 domain (or CH4 domain) of the immunoglobulin heavy (H) chain (providing an "H-like" chain for the molecule), and includes, for example, receptor fusion proteins containing at least one CH3 domain (or at least one CH4 domain) of the H chain. Examples of Ab-like molecules include the Ab-like molecules mentioned above (such as...). Figure 2 , Figure 53 A and / or Ab-like molecules as described in the antibody periodic table). In some instances, H-like chains containing at least one CH3 domain are considered to contain an Fc region component and can be, for example, the entire Fc region, or simply the CH3 domain, or a CH2-CH3 or CH3-CH2 component. In other instances, such H-like chains may further contain, for example, an immunoglobulin hinge sequence. H-like chains can enable dimerization, allowing antibody-like molecules to be provided, for example, in the form of homodimers or heterodimers. Figure 53As depicted herein, in some embodiments, the Ab-like molecule may comprise the H2L2 format and may comprise, for example, 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).

[0126] The term "fusion protein" is well known to those skilled in the art and refers to a protein expressed in a desired order by a DNA construct comprising two or more open reading frames, such that the protein can be considered a hybrid or chimeric protein. In some simple examples, a fusion protein comprises a protein (or fragment) of interest linked ("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 comprising a target recognition structure (e.g., a receptor or ligand such as an antigen recognition sequence, or a variable (V) domain or complementarity-determining region (CDR) of an immunoglobulin), said target recognition structure being linked to at least one CH3 domain (or at least one CH4 domain) of the immunoglobulin heavy (H) chain (see [link to relevant documentation]). Figure 2 (Examples shown in the text). Such fusion proteins may include, for example, mixed Fc regions (e.g., in a manner similar to that described above for antibodies (e.g., bispecific antibodies) and antibody-like molecules), such that the fusion protein includes, for example, Fc fragments, wherein the Fc regions (chains) are different (e.g., Fc1x and Fc2y), and wherein each of the Fc regions 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 one CH3 domain (or an equivalent CH4 domain of IgE or IgM), which may, for example, contain an intact H-chain or an H-like chain, which may contain, for example, an intact Fc region or only a CH3 domain or a CH2-CH3 or CH3-CH2 component. In other examples, such H-like chains may further include, for example, an immunoglobulin hinge sequence and / or a CH1 domain. The CH1 domain may, for example, provide a suitable site for linking the target or antigen recognition structure.

[0127] As used herein, the term "Fc fragment" (Fc or Fc moiety) refers to a dimer formed by covalent and / or non-covalent interactions between portions of the 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), and said dimer is responsible for the extensive activation of immunoglobulin's immune effector functions: particularly cell-based effector responses initiated by antibodies and Fc receptors located on the cell surface, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibodies. Complement-dependent cell-mediated phagocytosis (ADCP) or cytotoxicity kills target cells, and this also leads to the regulation and inhibition of the activity of innate and adaptive immune cells by inhibitory Fc receptors; and effector responses of the innate immune system initiated by antibodies activating the complement system via the classical pathway of the complement system, which is a protein cascade found in blood or biological fluids that is important for the destruction of pathogens and involves killing targets by direct cleavage of complement-dependent cytotoxicity (CDC) and / or killing targets by phagocytosis of targets via specific receptors of complement components (C'ADCP). The Fc fragment also provides association sites between immunoglobulin molecules, thereby allowing immunoglobulin molecules to assemble (self-associate) into higher-order oligomers (e.g., through covalent binding between a domain in the Fc region of the H chain of one immunoglobulin and a domain in the Fc region of the H chain of an adjacent immunoglobulin (as seen in the pentamer and hexamer forms of IgM) and non-covalent self-association such as seen in IgG, which, when bound to an antigen, leads to oligomerization, among other properties, including hexamer formation (Diebolder CA et al., Science 343(6176):1260-1263, 2014) and certain effector functions).

[0128] As used herein, the term "Fc region component" should be understood to refer to a portion of the Fc region of the immunoglobulin heavy (H) chain in which H429 is located, said Fc region component comprising at least one CH3 domain (or at least one CH4 domain), but preferably comprising both CH2 and CH3 domains, and optionally further comprising an immunoglobulin hinge sequence (which may comprise all or part of a lower hinge, core hinge, and upper hinge sequence in sequence), capable of forming (e.g., by dimerization) an Fc fragment or an Fc-like fragment. "Fc-like fragment" should be understood to refer to an Fc fragment-like structure, but said Fc-like fragment comprises a fragment or component of the Fc region, for example, a single CH3 domain (or CH4 domain) or a combination of a CH2 and CH3 domain, and optionally further comprises an immunoglobulin hinge sequence (which may comprise all or part of a lower hinge, core hinge, and upper hinge sequence in sequence).

[0129] It should be noted that polypeptides containing the heavy chain of the IgG subclass contain structurally unique segments known as hinge regions. 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 designations, the upper hinge contains residues 216-225 (including the terminal values), the core hinge contains residues 226-230 (including the terminal values), and the lower hinge contains residues 231-237. It should be noted that the amino acid residues of the lower hinge of IgG polypeptides are encoded by the CH2 exon and are also referred to in the literature as the CH2 hinge proximal region. In this application, references to CH2 include references to the lower hinge residues.

[0130] 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).

[0131] As used in this article, a “bispecific immunotherapy protein” is an immunotherapy protein that contains two antigen recognition structures targeting two different epitopes. Those skilled in the art will understand that numerous platform technologies are available for generating bispecific immunotherapeutic proteins, and that bispecific immunotherapeutic proteins as described herein can be generated using bispecific immunotherapeutic protein generation platforms known in the art, such as those described in the following literature: Ma J et al., Frontiers in Immunology 12:626616, 2021; Wang Q et al., Antibodies 8(3):43, 2019; Wang S et al., EMBO Molecular Medicine 3(9):e14291, 2021; Suurs FV et al., Pharamcology & Therapeutics 201:103-119, 2019; or Brinkmann U and Kontermann RE, MAb 9(2): 182-212, 2017; Dillion M et al., MAb 9(2): 213-230, 2017; Wu L et al., Cancer 14: 2518, 2022. Note that throughout the instructions, the bispecific antibody is referred to as "bsmAb".

[0132] Bispecific antibodies can be generated in different formats (Carter P et al., *Journal of Immunological Methods* 248:7-15, 2001). Typically, bsmAbs can contain two heavy chains or portions thereof, and two light chains, in the classic H2L2 format of immunoglobulins produced using methods such as the knock-in-hole method (Dillon M et al. 2017; Merchant et al., *Nature Biotechnology*, 16, 677-81 1998) or the Duobody method (Labrijin AF et al., *Proceedings of the National Academy of Sciences*, 110:5145-50, 2013). Each H chain contains at least one variable domain and, in many cases, one or more constant domains, including the CH domain of CH3, which is indicated in this paper as suitable for incorporation into H429F modification. In prototypical IgG-like bispecific antibodies, a first combination of heavy and light chains (HL), sometimes referred to (in this paper) as arm-A, can detect epitopes different from those detected by a second combination of heavy and light chains, sometimes referred to (in this paper) as arm-B. In this prototypical IgG-like (H2L2) bsmAb, arm-A contains light and heavy chains that can be represented as LAHA, and arm-B can similarly be represented as LBHB, and a bsmAb containing a proper assembly of arms-A and arm-B is represented as LAHAHBLB. In some cases, the bispecific antibody may contain only the heavy chain (H2), each H chain containing... Figure 2 The VH domains, which are different antigen-specific combinations of one or more constant domains, are described in the literature.

[0133] Furthermore, in other instances, bsmAbs can be generated by methods that incorporate an antigen recognition structure for detecting one antigen into an H2L2 or H2 mAb that specifically recognizes a second antigen, thereby creating a bispecific entity. For example, the fusion of a single-chain Fv (scFv) or domain antibody with an H2L2 mAb (Gu, C. et al., *Acta Pharmacol Sinica*, 43, 672-680, 2022), or in another instance, the fusion of two divalent but monospecific H2L2 or H2 mAbs to produce a polyvalent, such as a tetravalent (H4L4 or H4) bsmAb.

[0134] In some embodiments, the bispecific immunotherapy protein is generated using a platform selected from: DEKK, ART-Ig, CrossMab, DuoBody, Ortho-Fab, chain exchange engineered domain (SEED), mortis, DuetMab, κλ body, YBODY, antibody-based bispecific binding to T cell receptors (BEAT), cross-variable domain (CODV), dual variable domain (DVD)-Ig, Adaptir, dual-action antigen-binding fragment (Fab) (DAF), Wuxibody, FIT-Ig, TsBsIgG, Triomab, and XmAb. In one embodiment, the bispecific immunotherapy protein is generated using the DEKK platform. In one embodiment, the bispecific immunotherapy protein is generated using the ART-Ig platform. In one embodiment, the bispecific immunotherapy protein is generated using the CrossMab platform. In one embodiment, the bispecific immunotherapy protein is generated using the DuoBody platform. In one embodiment, the bispecific immunotherapy protein is generated using the Ortho-Fab platform. In one embodiment, the bispecific immunotherapy protein is generated using the SEED platform. In one embodiment, the bispecific immunotherapy protein is generated using a mortar and pestle platform. In one embodiment, the bispecific immunotherapy protein is generated using a DuetMab platform. In one embodiment, the bispecific immunotherapy protein is generated using a YBODY platform. In one embodiment, the bispecific immunotherapy protein is generated using a BEAT platform. In one embodiment, the bispecific immunotherapy protein is generated using a COVD platform. In one embodiment, the bispecific immunotherapy protein is generated using a DVD-Ig platform. In one embodiment, the bispecific immunotherapy protein is generated using a DAF platform. In one embodiment, the bispecific immunotherapy protein is generated using a Wuxibody platform. In one embodiment, the bispecific immunotherapy protein is generated using a FIT-IG platform. In one embodiment, the bispecific immunotherapy protein is generated using a TsBsIgG platform. In one embodiment, the bispecific immunotherapy protein is generated using a Triomab platform. In one embodiment, the bispecific immunotherapy protein is generated using an XmAb platform. In some embodiments, the bispecific immunotherapy protein is a cell-bridged bispecific immunotherapy protein. In some embodiments, the bispecific immunotherapy protein is an antigen-crosslinked bispecific immunotherapy protein.

[0135] In some embodiments, a bispecific immunotherapeutic protein is generated using a platform bsmAb format containing an Fc region, the format being selected from formats having a common light chain and a heterologous H chain, HL chain half-molecule exchange, or heterologous chains (H chain and / or L chain) and tandem and fusion domains for directed or forced assembly.

[0136] In some embodiments, the bispecific immunotherapeutic protein is generated using a platform bsmAb format containing an Fc region selected from the following: CrossMab, scFv-Fab IgG, DART-Fc, CODV-Fab-TL, HLE-BiTE, LP-DART, F(ab)3 CrossMab, IgG-(scFv)2, Bs4Ab, DVD-Ig, mAb2, F(ab)4 CrossMab, tetravalent DART-Fc, (scFv)4-Fc, CODV-Ig, “two-in-one” and tandem VHH-Fc, for example as described in Labrijn et al., Nature Reviews Drug Discovery, 18, 585-608, 2019.

[0137] In some embodiments, the epitope is located on a different cell. In some embodiments, the binding of a bispecific immunotherapy protein to an epitope can promote interactions between cells such as immune cells and cancer or virus-infected cells or microorganisms. This may lead to an immune response.

[0138] In some embodiments, the two epitopes are located on the same molecule (bi-complementary sites). In some embodiments, the two epitopes are located on the same molecule, and the bispecific immunotherapy binds to a single molecule (binding to both epitopes on the same molecule). In some embodiments, the two epitopes are located on the same molecule, and the bispecific immunotherapy binds to two molecules (binding to two epitopes on two different molecules, such as binding to two CD37 molecules on the same cell or two CD37 molecules on different cells).

[0139] In some embodiments, the epitope is contained in two different molecules. In some epitopes, the molecule is located on the same cell. In some embodiments, the molecule is located on different cells.

[0140] In some embodiments, one or two epitopes are located on a cell surface molecule. In some embodiments, one or two epitopes are located on a soluble molecule. In some embodiments, two epitopes are located on a cell surface molecule. In some embodiments, one epitope is located on a cell surface molecule. In some embodiments, one epitope is located on a soluble molecule. In some embodiments, two epitopes are located on a soluble molecule. In some embodiments, one epitope is located on a cell surface molecule, and one epitope is located on a soluble molecule.

[0141] In some embodiments, one epitope is located on a cancer cell and another epitope is located on an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In one embodiment, the immune cell is a natural killer cell. In one embodiment, the immune cell is a myeloid cell. In one embodiment, the immune cell is a phagocyte. In some embodiments, the epitope is located on the same cell. In some embodiments, one epitope is located on a cell and another epitope is located on a microorganism. In some embodiments, the microorganism is selected from eukaryotes, bacteria, and viruses. In some embodiments, the microorganism is SARS-CoV-2.

[0142] In some embodiments, the bispecific immunotherapy protein is a T-cell-stimulated immunotherapy protein. In some embodiments, the bispecific immunotherapy protein is a T-cell-redirected killer immunotherapy protein. In some embodiments, the bispecific immunotherapy protein is a natural killer cell-redirected killer immunotherapy protein. In some embodiments, the bispecific immunotherapy protein is a phagocyte-redirected killer immunotherapy protein. In some embodiments, the bispecific immunotherapy protein is a complement-redirected killer immunotherapy protein.

[0143] In some embodiments, one or both of the epitopes are located on: cancer-associated antigens, autoantigens, allergens, antigens associated with inflammatory diseases, antigens from transplanted tissues or organs, or antigens of infectious agents such as eukaryotes, soluble molecules, bacterial or viral pathogens.

[0144] In some embodiments, the bispecific immunotherapy protein is selected from the bispecific antibodies described in the following literature: Ma J et al., ibid., 2021; Wang Q et al., ibid., 2019; Wang S et al., ibid., 2021; SuursFV et al., ibid., 2019; or Brinkmann U and Kontermann, RE, ibid., 2017. In some embodiments, the bispecific immunotherapy protein is bicomplementary.

[0145] In some embodiments, one or both of the epitopes are located on a protein selected from: differentiation cluster 3 (CD3), B cell maturation antigen (BCMA), FcγRIII, FcγRIIb, FcγRIIa, FcαRI, FcγRI, native cytotoxicity triggering receptor 1 (NKp46), cytotoxic cell lectin-like receptor K1 (NKG2D), native cytotoxicity triggering receptor (NKp30), death receptor 5 (DR5), TNP conjugate (2,4,6-trinitrophenyl conjugate), biotin conjugate, hapten conjugate, interleukin-2 (IL-2), interleukin-4 ( IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-17 (IL-17), Tumor Necrosis Factor (TNF), Tumor Necrosis Factor α (TNFα), Human Epidermal Growth Factor Receptor-2 (HER2), Differentiation Cluster 19 (CD19), Differentiation Cluster 20 (CD20), Differentiation Cluster 22 (CD22), Differentiation Cluster 27 (CD27), Differentiation Cluster 28 (CD28), Differentiation Cluster 30 (CD30), Differentiation Cluster 33 (CD35) 3) Differentiation clusters 37 (CD37), 38 (CD38), 39 (CD39), 47 (CD47), 52 (CD52), 79 (CD79), 123 (CD123), C-type lectin-like molecule-1 (CLL-1), C-type lectin domain family 12 member A (CLEC12A), δ-like 4 (DLL4), differentiation antigen cluster 135 (FLT-3), Fc receptor-like protein 5 (FcRH5), G protein-coupled receptor class C5 member D (GPRC5D), carcinoembryonic antigen (CEA), epithelial cells Adhesion molecule (EpCAM), prostate-specific membrane antigen (PSMA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte activation gene-3 (LAG-3), epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (cMet), glycoprotein 100 (GP100), angiopoietin-2 (Ang-2), vascular endothelial growth factor A (VEGF-A), tumor-associated glycoprotein 72 (Tag72), and sealing protein 18 (CLDN18).2) Differentiation cluster 66 (CD66), CD300-like family member F (CD300f), phosphatidylinositol proteoglycan 2 (GPC2), FMS-associated receptor tyrosine kinase 3 (FLT3), CD276 molecule (B7-H3), cell adhesion molecule (CEA), delta-like canonical notch ligand 3 (DLL3), disialotyl ganglioside (GD2), glycoprotein A33 (gpA33), phosphatidylinositol proteoglycan 3 (GPC3), guanylate cyclase 2C (GUCY2C), major histocompatibility complex, class IA (HLA-A), MAGE family members Member A4 (MAGE-A4), mucin 1 cell surface associated (MUC1), mesothelin (MSLN), mucin 16 cell surface associated (MUC16), mucin 17 cell surface associated (MUC17), New York esophageal squamous cell carcinoma 1 (NY-ESO1), PRAME nuclear receptor transcription factor (PRAME), prostate stem cell antigen (PSCA), somatostatin receptor 2 (SSTR2), STEAP family member 1 (STEAP1), differentiation cluster 127 (4-1BB), glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), and surviving SARS-CoV-2. The following proteins are involved: S protein, HER2 (receptor tyrosine kinase ERBB2, also known as ERBB2), complement 5 (C5), insulin-like growth factor 1 receptor (IGF1R), hepatocyte growth factor receptor (HGFR), folate receptor α, interleukin-31 (IL31), hepatitis A virus cell receptor 2 (HAVCR2), V-domain-containing T cell activation inhibitor 1 (VTCN1), tumor-associated calcium signaling transducer 2 (TACSTD2), fibroblast growth factor receptor 2 (FGFR2), and glutamate carboxypeptidase (FOLH1).

[0146] In some embodiments, one or both of the epitopes are located on a protein selected from: CD37, HER2 C5, IGF1R, HGFR, folate receptor α, IL31, HAVCR2, VTCN1, TACSTD2, FGFR2, FOLH1, and PD1.

[0147] In some embodiments, the two epitopes are located on proteins selected from: CD37, HER2 C5, IGF1R, HGFR, folate receptor α, IL31, HAVCR2, VTCN1, TACSTD2, FGFR2, FOLH1, and PD1.

[0148] In one embodiment, one or both of the epitopes are CD37 epitopes. In one embodiment, one or both of the epitopes are BDR5 epitopes. In one embodiment, one or both of the epitopes are DR5 epitopes. In one embodiment, one or both of the epitopes are CD3 epitopes. In one embodiment, one or both of the epitopes are CD20 epitopes. In one embodiment, one or both of the epitopes are CD19 epitopes. In one embodiment, one or both of the epitopes are CD28 epitopes. In one embodiment, one or both of the epitopes are PD-L1 epitopes. In one embodiment, one or both of the epitopes are 4-1BB. In one embodiment, one or both of the epitopes are HER2 epitopes. In one embodiment, one or both of the epitopes are BCMA epitopes. In one embodiment, one or both of the epitopes are FcγRIIb epitopes. In one embodiment, one or both of the epitopes are FcαR1 epitopes. In one embodiment, one or both of the epitopes are FcγR1 epitopes. In another embodiment, one or both of the epitopes are TNP epitopes.

[0149] In some embodiments, one epitope is CD3, and another epitope is located on a protein selected from: BCMA, CD20, CD19, CD38, PSMA, Tag72, HER2, EGFR, MAGE antigen (e.g., MAGEA4), PD-L1, CLDN18.2, CD300f, ROR1, EpCAM, GPC2, FLT3, OKT3, UTCH1, L2K, TR66, ZWB67, and guanylate cyclase.

[0150] In some embodiments, the epitopes are each located on one of the following protein pairs: HER2 and FcγRIII; BCMA and FcγRIII; PDL-1 and 4-1BB; HER2 and 4-1BB; CD3 and BCMA; CD3 and CD20; CD3 and CD19; FcγRIIb and TNP-BSA; CD3 and CD28; CD3 and CD123; CD3 and CD33; CD3 and CD37; CD3 and CD38; CD3 and CLEC12A; CD3 and FLT-3; CD3 and survival protein; CD20 and CD47; CTLA-4 and PD-1; LAG-3 and PD-1; PD-1 and PD-L1; CD30 and FcγRIII; CD3 and GP100; CD20 and CD47; CTLA-4 and PD-1; LAG-3 and PD-1; PD-1 and PD-L1; CD20 and EpCam; EGFR and cMet; Ang-2 and VEGF-A; and CD3 and BCMA. In some embodiments, the epitope is located on one of the following protein pairs: HER2 and FcγRIII; BCMA and FcγRIII; CD3 and BCMA; CD3 and CD20; CD3 and CD19; FcγRIIb and TNP-BSA; CD3 and CD28; CD3 and CD123; CD3 and CD33; CD3 and CD37; CD3 and CD38; CD3 and CLEC12A; CD3 and FLT-3; CD3 and surviving protein; CD20 and CD47; CTLA-4 and PD-1; LAG-3 and PD-1; PD-1 and PD-L1; and CD30 and FcγRIII CD3. In some embodiments, the epitope is located on the proteins HER2 and FcγRIII. In some embodiments, the epitope is located on the proteins BCMA and FcγRIII. In some embodiments, the epitope is located on PDL-1 and 4-1BB. In some embodiments, the epitope is located on HER2 and 4-1BB. In some embodiments, the epitope is located on proteins CD3 and CD20. In some embodiments, the epitope is located on proteins CD3 and BCMA. In some embodiments, the epitope is located on proteins FcγRIIb and TNP-BSA. In some embodiments, the epitope is located on proteins CD3 and CD28. In some embodiments, the epitope is located on proteins CD3 and CD123. In some embodiments, the epitope is located on proteins CD3 and CD33. In some embodiments, the epitope is located on proteins CD3 and CD37. In some embodiments, the epitope is located on proteins CD3 and CD38. In some embodiments, the epitope is located on proteins CD3 and CLEC12A. In some embodiments, the epitope is located on proteins CD3 and FLT-3. In some embodiments, the epitope is located on proteins CD3 and viable white matter.In some embodiments, the epitope is located on proteins CD20 and CD47. In some embodiments, the epitope is located on proteins LAG-3 and PD-1. In some embodiments, the epitope is located on proteins PD-1 and PD-L1. In some embodiments, the epitope is located on proteins CD30 and FcγRIII. In some embodiments, the epitope is located on proteins CD20 and EpCam. In some embodiments, the epitope is located on proteins CD3 and CD19. In some embodiments, the epitope is located on proteins FIXa and FX. In some embodiments, the epitope is located on proteins EGFT and cMet. In some embodiments, the epitope is located on proteins CD3 and GP100. In some embodiments, the epitope is located on proteins Ang-2 and VEGF-A. In some embodiments, the epitope is located on proteins PD-1 and CTLA-4. In some embodiments, one of the epitopes is located on CD3. In some embodiments, CD3 is a T cell. In some embodiments, one of the epitopes is located on BCMA. In some embodiments, one of the epitopes is located on FcγRIII. In some embodiments, one of the epitopes is located on FcγRIIb. In some embodiments, one of the epitopes is located on TNP-BSA. In some embodiments, one of the table bits is located on CD20. In some embodiments, one of the table bits is located on CD19. In some embodiments, one of the table bits is located on CD28.

[0151] In some embodiments, the cell surface molecules are cell surface molecules on T cells. In some embodiments, the cell surface molecules are cell surface molecules on B cells. In some embodiments, the cell surface molecules are cell surface molecules on natural killer cells. In some embodiments, the cell surface molecules are cell surface molecules on bone marrow cells. In some embodiments, the cell surface molecules are cell surface molecules on phagocytes.

[0152] In some embodiments, the cell surface molecules are selected from: CD3, CD19, CD20, CD28, DR5, HER2, FcγRIII, and FcγRIIb. In some embodiments, the soluble molecules are selected from: IL-6, IL-8, IL-10, IL-12, and SARS-CoV-2 S protein. In some embodiments, the soluble molecules are synthetic molecules (e.g., TNP-BSA, biotin).

[0153] In some embodiments, the soluble molecules are selected from the TNF superfamily (TNFSF), including tumor necrosis factor (TNF), lymphotoxin α (LT), TNFSF12 (TWEAK), TNFSF15 (TL1), Fas ligand (FASL), TNFSF14 (Tα1β2) (lymphotoxin β [LTβ] in combination with lymphotoxin α, TNFSF14), TNFSF14 (LIGHT), OX40 ligand (OX40L), CD70, TNFSF8 (CD30L), 4-1BB ligand (4-1BBL), GITR ligand (GITRL), CD40 ligand (CD40L), TNFSF20 (BAFF), THFSF13 (APRIL), RANK ligand (RANKL), neurotrophic factors, myelin-associated inhibitory factors, β-amyloid precursor protein fragments, and prion peptides.

[0154] In some embodiments, the soluble molecules are selected from cytokines, chemokines, adipokines, peptide hormones, and secretases.

[0155] In some embodiments, the cytokines are selected from one or more of the following: interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), and interleukin-12. Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL-14), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-17 (IL-17), Interleukin-18 (IL-18), Interleukin-19 (IL-19), Interleukin-20 (IL-20), Interleukin-21 (IL-21), Interleukin-22 (IL-22), Interleukin-23 (IL-23) Interleukin-24 (IL-24), Interleukin-25 (IL-25), Interleukin-26 (IL-26), Interleukin-27 (IL-27), Interleukin-28 (IL-28), Interleukin-29 (IL-29), Interleukin-30 (IL-30), Interleukin-31 (IL-31), Interleukin-32 (IL-32), Interleukin-33 (IL-33), Interleukin-34 (IL-34), Interleukin-35 (IL-35), Interleukin-36 (IL-36), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), Interferon-α (IFNα), Interferon-β (IFNβ), Interferon-γ (IFNγ), Tumor necrosis factor (TNF), Transforming growth factor β (TGFβ), Bone morphogenetic protein 5 (BMP-5), and Bone morphogenetic protein 7 (BMP-7).

[0156] In some embodiments, the chemokines are selected from one or more of the following: CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10 (MIP-1γ), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.

[0157] In some embodiments, the adipokines are selected from one or more of the following: leptin, adiponectin, apelin, chemerin, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1 / CCL2), plasminogen activator inhibitor-1 (PAI-1), retinol-binding protein 4 (RBP4), tumor necrosis factor, endothelin, retinin, serine protease inhibitor (SERPINA12), granulin precursor, C1q. The associated proteins include tumor necrosis factor-associated protein 4 (CTRP-4), leptin, adiponectin, apalamin peptide, chemokines, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1 / CCL2), plasminogen activator inhibitor-1 (PAI-1), retinol-binding protein 4 (RBP4), tumor necrosis factor, endothelin, retinin, serine protease inhibitor (SERPINA12), granulin precursor, C1q, and tumor necrosis factor-associated protein 4 (CTRP-4).

[0158] In some embodiments, the peptide hormone is selected from one or more of the following: adrenocorticotropic hormone (ACTH), aadropin, amylin, angiotensin, atrial natriuretic peptide (ANP), calcitonin, cholecystokinin (CCK), gastrin, ghrelin, glucagon, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), growth hormone, follicle-stimulating hormone (FSH), insulin, leptin, luteinizing hormone (LH), melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone (PTH), prolactin, renin, somatostatin, and thyroid-stimulating hormone (TSH). Thyroid-stimulating hormone-releasing hormone (TRH), vasopressin (also known as arginine vasopressin (AVP) or antidiuretic hormone (ADH), vasoactive intestinal peptide (VIP), growth hormone (GH1), gonadotropin-releasing hormone 1 (GNRH1), gonadotropin-releasing hormone 2 (GNRH2), growth hormone-releasing hormone (GHRH), parathyroid hormone-like hormone (PTHLH), adrenocorticotropic hormone-releasing hormone (CRH), anti-Müllerian hormone (AMH), human chorionic growth hormone 1 (CSH1), human chorionic growth hormone 2 (CSH2), promelanin-concentrating hormone (PMCH), and resistin (RETN).

[0159] In some embodiments, the secreted enzyme is selected from one or more of the following: interstitial collagenase (MMP1); gelatinase-A, 72 kDa gelatinase (MMP2); lysosome 1 (MMP3); matrix lysosome, PUMP 1 (MMP7); neutrophil collagenase (MMP8); gelatinase-B, 92 kDa gelatinase (MMP9); lysosome 2 (MMP10); lysosome 3 (MMP11); macrophage metalloelastase (MMP12); collagenase 3 (MMP13); ameliolytics (MMP20); X-MMP (MMP21); and epidermal growth factor (Epilysin) (MMP28).

[0160] In some embodiments, the cell surface molecules are selected from the following cell surface molecules on natural killer cells: NKp46, NKG2D, NKp30, and FcγRIII.

[0161] In some embodiments, the bispecific immunotherapy protein is selected from: blinatumomab, emicizumab, amivantamab-vmjw, tebentafusp-tebn, faricimab-svoa, cadonilimab, mosunetuzumab, teclistamab, catumaxomab, and odron. extamab, ozoralizumab, acapatamab, acasunlimab, anuctamab, epantomab, bafisontamab, bavunalimab, carbotamipr, cartunidumab, cefostamab, cibisatamab, ciduvectamig, cinrebafusp alfa, danvilostomig, denecimig, dilpacimab, duvortuxizumab, efdamrofusp alfa, elranatamab, emerfetamab, emirodatamab, englumafusp alfa, enristomig, epcoritamab, erfonrilimab, ertumaxomab, Fanastomig, faliximab, fazpilodemab, fidasimtamab, forimtamig, givastomig, glofitamab, gremubamab, gresonitamab, imvotamab, inezetamab, istirasumab, ivonescimab, izalontamab.Izalontamab brengitecan, izuralimab, linvoseltamab, lomvastomig, lorigerlimab, lutikizumab, mosunitozumab, navicixizumab, nezastomig, nivatrotamab, obteramig, obrindatam (ab), Onitumab, Pamvatamig, Pavurutamab, Petosemtamab, Pramotamab, Ragistomig, Remtolumab, Reozalimab, Rezetamig, Rilvegostomig, Rovelizumab, Runimotamab Sabestomig, simridarlimab, talquetamab, tarlatamab, tebobolimab, tebotelimab, tecaginlimab, teratolimab, tepoditamab, tibulizumab, tidutamab, tobemstomig, trontinemab, ubamata (e.g., mab), vanucizumab, vepsitamab, vibecotamab, volrustomig, vonsetamig, voxalatamab, vudalimab, xaluritamig, xirestomig, zenocutuzumab, zeripatamig, zifibancimig, or their derivatives.)

[0162] In some embodiments, the bispecific immunotherapy protein is selected from: bonnetumab; emecizumab; ervantumab-vmjw; tebfos-tebn; faliximab-svoa; cantulimumab; mosunituzumab; teritumumab; caputuzumab; onituzumab and orizumab or derivatives thereof.

[0163] In some embodiments, the bispecific immunotherapy protein is selected from OKT3, UTCH1, L2K, TR66, and ZWB67 or derivatives thereof.

[0164] There are many bispecific antibodies available on different platforms / formats and with varying specificities, containing Fc sequences that can be modified by H429F, including but not limited to: icarperatumab, icarsulimab, anucalutumab, epantuzumab, baphetumab, panalimab, carbotamiprid, cantulimab, caputuzumab, cisvirstatazumab, cebituxumab, stovectatrid, sibivop alpha, dannilopomib, denosimib, depaxicumab, dutuximab, emoxivip Alpha, Enatuzumab, Enatuzumab, Emicizumab, Enatuzumab, Engrumafap Alpha, Ensumi, Ecraletumab, Irelilimumab, Ertoxumab, Famami, Faliximab, Favipirabumab, Fidastatumab, Voritumib, Gastromoxine, Gastrostatumab, Ggrababumab, Imvoltamarab, Inellizumab, Estatolimab, Evoximarab, Elongatumab, Rencon Elongatumab, Izuralimab, Rivoxel Monoclonal antibodies, Losomi, Loregolizumab, Rugizumab, Moshunituzumab, Nasecoxizumab, Neostigmet, Enytozumab, Obertamig, Obrinda, Onituzumab, Pamvatamituz, Pavlutatamituz, Pysentozumab, Paramotuzumab, Lagistrom, Rentolu, Rizzolizumab, Rezetuzumab, Regsomi, Rochizumab, Lovezumab, Ranimozumab, Sabestrom, Sreylizumab, Taquetozumab, Talatozumab Tibenfos, terpolizumab, ticalizumab, teratozumab, taprizumab, tibulizumab, taprizumab, tosumi, teronizumab, ubatuzumab, vanusxizumab, vesutuzumab, vexutuzumab, vortami, vorciletusab, vodalizumab, caroliterizumab, ciretomi, zetuzumab, zetipazimi, zefcitami, and engineered bispecific mAbs with other specificities in these and other platforms / formats.

[0165] In some embodiments, the immunotherapeutic agent is a bispecific antibody selected from the following: zanidatamab, JSKN-003, gefurulimab, IBI-311, davutamig, IMGN-151, ATTO-1310, BP-1210, ATTO-003, BH-3737, BpAb-1, BH-3737, CB-108, CB-201, ivicentamab, and MEDI-4276. In some embodiments, the bispecific antibody is zanidatamab. In some embodiments, the bispecific antibody is JSKN-003. In some embodiments, gefurulimab is used. In some embodiments, the bispecific antibody is IBI-311. In some embodiments, the bispecific antibody is davutamig.

[0166] In some embodiments, the bicomponent antibody is IMGN-151. In some embodiments, the bicomponent antibody is ATTO-1310. In some embodiments, the bicomponent antibody is BH-3737. In some embodiments, the bicomponent antibody is BpAb-1. In some embodiments, the bicomponent antibody is BH-3737. In some embodiments, the bicomponent antibody is BpAb-1. In some embodiments, the bicomponent antibody is BH-3737. In some embodiments, the bicomponent antibody is CB-108. In some embodiments, the bicomponent antibody is CB-201. In some embodiments, the bicomponent antibody is evertuzumab. In some embodiments, the bicomponent antibody is MEDI-4276.

[0167] The mechanisms of action of therapeutic mAbs can include complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), antibody-dependent cytokinesis (ADCT), and induction of receptor signaling to induce cellular responses such as apoptosis. Bispecific mAbs may retain these activities, or they may be eliminated or enhanced by specific mutations in the Fc region.

[0168] Redirecting effector cells to kill cells, particularly malignant or virus-infected cells, is an important approach in the development of therapeutic bsmAbs. Bispecific mAbs bridge target cancer cells, enabling cancer cell antigens to bind to effector cells, bind to molecules on the effector cell surface, and activate those molecules. Typically, for T effector cells, target molecules can be components of T cell receptors such as CD3, or co-stimulatory molecules on T cells such as 4-1BB, OX40, CD30, GITR, or CD27 or CD28. In this way, the cytotoxicity of effector cells is triggered and directed to cancer cell targets outside of normal immune recognition mechanisms.

[0169] In some embodiments, the immunotherapeutic protein is a mini-body. As used herein, "mini-body" or "mini-bodies" refers to a class of bispecific immunotherapeutic proteins comprising two ScFvs fused to CH3. In one embodiment, the molecular weight of the mini-body is less than 130 kDa. In one embodiment, the molecular weight of the mini-body is less than 120 kDa. In one embodiment, the molecular weight of the mini-body is less than 110 kDa. In one embodiment, the molecular weight of the mini-body is less than 100 kDa. In one embodiment, the molecular weight of the mini-body is less than 90 kDa. In one embodiment, the molecular weight of the mini-body is less than 85 kDa. In one embodiment, the mini-body is selected from: 124I-A11, Df-IAB22M2C, 6B11-OCIK, and ALXN1720. In some embodiments, the mini-body is a bispecific immunotherapeutic protein.

[0170] As used herein, the term “treatment” includes textiles and the relief of identified symptoms of a disease or condition. Thus, acts of “treating” a disease or condition include: (1) preventing or delaying the onset of clinical symptoms of a developing disease or condition in a subject who has or is susceptible to said disease or condition; (2) suppressing the disease or condition (i.e.,, in the case of maintenance treatment, preventing, reducing or delaying the development of the disease or condition or its recurrence, or preventing, reducing or delaying at least one of its clinical or subclinical symptoms); and (3) alleviating or reducing the disease or condition (i.e., causing the disappearance of at least one of the disease or condition or its clinical or subclinical symptoms).

[0171] As used herein, the phrase “drug preparation” includes the use of one or more immunotherapeutic proteins as defined herein, either directly as a drug or in any stage of drug preparation comprising one or more immunotherapeutic proteins as defined herein.

[0172] The term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. An effective amount may be administered once or multiple times. Generally, an effective amount is sufficient to treat a disease or condition or otherwise alleviate, improve, stabilize, reverse, slow, or delay the development of a disease or condition. For example only, an effective amount of an immunotherapeutic protein, such as a mutant IgG1 antibody, may be contained between about 0.1 mg / kg body weight / day and about 250 mg / kg body weight / day, more preferably between about 0.1 mg / kg body weight / day and about 100 mg / kg body weight / day, and still more preferably between about 0.1 mg / kg body weight / day and about 25 mg / kg body weight / day. However, those skilled in the art will understand that the effective amount can vary and depends on a variety of factors, including the age, weight, sex, and / or health status of the subject receiving treatment, the activity of the specific immunotherapeutic protein, the metabolic stability and duration of action of the specific immunotherapeutic protein, the route and timing of administration of the specific immunotherapeutic protein, the excretion rate of the specific immunotherapeutic protein, and, for example, the severity of the disease or condition being treated.

[0173] As illustrated in the examples provided below, mutant antibodies containing a point mutation at position H429 have been found to confer significant functional changes. For example, IgG mutants with an H429F substitution have shown an enhanced ability to activate complement-dependent cytotoxicity (CDC) in complement assays (i.e., assays of complement function). Similarly, fusion proteins containing an Fc region component with a point mutation at position 429 (i.e., H429F) fused to the extracellular domain of angiotensin-converting enzyme 2 (ACE2) have been found to provide an enhanced ability to deliver antiviral activity through CDC in infected cells, while similar fusion proteins with an H429Y substitution have shown enhanced virus neutralization with little or no complement activation (because immunotherapeutic proteins containing an H429Y-modified Fc component have shown eliminated FcγR binding and activation, particularly with FcγRIIIa). While not wishing to be bound by theory, it is thought that these effects may arise from the oligomerization of proteins into oligomers when they bind to relevant target molecules (e.g., the antigen targeted by the antibody mutant) through self-association of the antibody / fusion protein in solution or through "on-target" oligomerization.

[0174] Furthermore, it should be noted that although monomeric IgG1 and IgG3 can deliver CDC, the level of CDC can be considered low or “poor” compared to the levels achieved by natural pentamer or hexamer IgM. In addition, unlike IgM, IgG antibodies such as IgG1 and IgG3 are also relatively poor lectins due to their divalent nature. Therefore, by achieving oligomerization (by potentially enhancing the known weak inherent self-association ability of the Fc of immunoglobulins such as IgG, especially after the immunoglobulin has already bound to the antigen (i.e., “on-target” oligomerization), the immunotherapeutic proteins according to this disclosure can provide a more optimized platform for complement system activation and other functions enhanced by self-association through the formation of oligomers. This oligomerization can particularly and / or optimally involve the formation of hexamers; and such hexamerization has been visualized in the crystal structure of the anti-HIV antibody b12 (Saphire EO et al., Science 293:1155-1159, 2001), in which CH3 residues form 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, which initiates activation of the classical complement pathway.

[0175] Furthermore, many cellular molecules, including cell surface molecules, require extensive clustering via ligands (which can be soluble or another cell surface molecule) to induce signaling, thereby inducing cellular responses. Therefore, dimerization typically achieved with antibodies may not be sufficient to induce signals leading to meaningful cellular responses. However, it is known that signal strength can be increased by enhancing the clustering of target molecules, for example, through the addition of other entities involving the addition of cross-linked ligands (Chenoweth AC et al., *Immunol Cell Biol* 98:287-304, 2020). Therefore, such “super-clustering” or “hyper-clustering” of target molecules can be achieved by using additional anti-immunoglobulin antibodies that cross-link the mAb bound to the target molecule, when an antibody (such as a mAb) binds to its target molecule. While not wishing to be bound by theory, it is thought that proteins, including mutant antibodies with the H429F point mutation mentioned above, can similarly achieve super-clustering or hyper-clustering of target molecules through self-association upon binding to the target molecule. Depending on the nature of the target molecules, such clustering may induce enhanced signaling responses that could lead to, for example, cell proliferation (e.g., when the target molecules are, for example, CD3 or CD28), stimulation of inhibitory pathways to suppress or reduce cellular responses (e.g., immune checkpoints; reviewed in Chenoweth et al., ibid. 2020), or stimulation of pathways that induce cell death, such as programmed cell death including apoptosis (see, e.g., Ashkenazi A., Nature Reviews Drug Discovery 7(12):1001-1012, 2008). Stimulating cellular pathways via mAbs to develop therapeutic molecules is one approach for treating a range of diseases, including cancer, inflammatory and autoimmune diseases, infections, cardiovascular diseases, and others (Ashkenazi, ibid. 2008).

[0176] Therefore, in one aspect, this disclosure provides an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptides containing an Fc region component, the Fc region component containing at least one CH3 domain (or at least one CH4 domain), wherein the one or more polypeptides include an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain.

[0177] In some embodiments, the immunotherapeutic protein as described herein comprises a dimer immunotherapeutic protein comprising a first immunoglobulin heavy chain polypeptide and a second immunoglobulin heavy chain polypeptide, the first and second immunoglobulin heavy chain polypeptides comprising an Fc region component including a CH2 domain and a CH3 domain (such that the first and second polypeptides can form (e.g., by dimerization) an Fc fragment or an Fc-like fragment), wherein the Fc region component of at least one of the first and second polypeptides comprises an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain. Therefore, the immunotherapeutic protein of such embodiments can be an immunoglobulin molecule, such as an antibody or antibody-like molecule, such as a dimer polypeptide containing a pair of single-chain Fv polypeptides linked by an Fc fragment (i.e., scFv-Fc) or a microantibody (i.e., a protein containing a pair of scFv polypeptides linked by a CH3 domain (see, for example, the discussion of multivalent scFv-Fc and microantibodies in Olafsen T et al., Generation of Antibody Fragments and Their Derivatives, Antibody Engineering, 2nd Edition, pp. 69-84, 2010)).

[0178] In other embodiments, the immunotherapeutic protein as described herein comprises a partner polypeptide linked to an Fc region component, the Fc region component comprising at least one CH3 domain (or at least one CH4 domain), wherein the Fc region component comprises an amino acid substitution (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain. Therefore, the immunotherapeutic protein of such embodiments can be provided as a fusion protein or a protein conjugate. Those skilled in the art will understand that in fusion proteins, the mate peptide is covalently linked (i.e., "fused") to the Fc component (i.e., as the fusion mate) 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) at the N-terminus or C-terminus of the fusion mate (i.e., the Fc component), while in protein conjugates, the mate peptide is linked via a chemical bond such as a disulfide bond (e.g., via one or more cysteine ​​(C) residues) or a cross-linking compound such as a bifunctional cross-linking agent such as disuccinimide octanoate (DSS) (e.g., bis(sulfosuccinimide)octanoate (BS)). 3Thermo Fisher Scientific, Waltham, MA, United States of America, connects the Fc region component (i.e., as a conjugate conjugate) covalently or nonvalently to the conjugate component, or disuccinimide tartrate (DST) to an amino or heterobifunctional crosslinking agent, such as m-maleimide benzoyl-N-hydroxysuccinimide (MDS) and N-(ε-maleimide propionyloxy)succinimide (EMCS), or via other nonvalent binding such as hydrogen bonding. When the Fc component is a conjugate conjugate, the immunotherapy protein conjugate can be considered a crosslinked protein, and the Fc component can be conjugated to the conjugate peptide at the N-terminus or C-terminus, but can also be conjugated in other ways at any other suitable site on the conjugate peptide. Alternatively, the Fc component (as a conjugate partner) may be conjugated to the partner polypeptide in other ways at the N-terminus or C-terminus of the Fc component or at any other suitable site on the Fc component (e.g., within CH1 or the upper hinge sequence, if these are included in the Fc region component).

[0179] The Fc region component of an immunotherapy protein (hereinafter also referred to as the "Fc component") may be derived from one or more immunoglobulin types (e.g., IgG or IgA) and may contain a full-length (i.e., "intact") Fc region, such as a heavy (H) chain polypeptide fragment corresponding to a heavy chain polypeptide fragment produced by papain digestion (i.e., wherein the polypeptide is cleaved within an upper hinge sequence to produce an Fc region comprising constant heavy domain 2 (CH2; amino acids A231 to K340 (Eu number) of the human IgG1 heavy chain polypeptide), constant heavy domain 3 (CH3; amino acids G341 to G446 or K447 (Eu number) of the human IgG1 heavy chain polypeptide), a lower hinge sequence (also referred to as the hinge proximal sequence of CH2; amino acids P232 to P238 (Eu number)), and a core hinge sequence (amino acids P232 to P238 (Eu number)). The Fc region component may contain fragments of heavy chain polypeptides containing only the CH2 and CH3 domains, as well as similar heavy chain polypeptide fragments that can be prepared by digesting immunoglobulin heavy chain polypeptides with plasmin and human neutrophil elastase (NHE). Other examples of suitable Fc region components may include fragments of heavy chain polypeptides containing, in addition to the CH2 and CH3 domains and the lower and core hinge sequences, all or part of the upper hinge sequence and constant heavy domain 1 (CH1). Alternatively, other suitable Fc region components may include fragments of heavy chain polypeptides containing only the CH3 domain (e.g., amino acids G341 to G446 or K447 (Eu number) of the human IgG1 heavy chain polypeptide) or fragments thereof. Additionally, suitable Fc region components may include heterologous (“hybrid”) CH3 domains, such as those derived from IgG1, such as IgA. The CH3 domain and other protein fragments in the form of chain-exchange engineered (SEED) domains of the CH3 domain (Davies et al., Protein Engineering Design and Selection, 23(4):195-202, 2009).

[0180] In some embodiments, the Fc region component is derived from human immunoglobulin heavy chain polypeptides (e.g., as in...). Figure 3 and 4 The human immunoglobulin heavy chain polypeptide shown in the image).

[0181] In other embodiments, the Fc region component is derived from an IgG heavy chain polypeptide, preferably an 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 E430G substitution.

[0182] The Fc region component contains an amino acid substitution (Eu number) at the position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3, or IgG4 heavy chain polypeptide. When the Fc region component originates from another immunoglobulin type or isotype (or from an immunoglobulin from another species), it should be understood that those skilled in the art can readily determine the position corresponding to H429 of the human IgG1 heavy chain polypeptide IgG1 by, for example, routine sequence alignment (e.g., as shown in...). Figure 3 and 4 (As shown). 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 a 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 not glycine or leucine. In some embodiments, the H429 substitution is not glycine. In some embodiments, the H429 substitution is not leucine.

[0183] Suitable mutations at position 429 include:

[0184] H→X, where X is selected from phenylalanine (H429F), glutamic acid (H429E), glutamine (H429Q), serine (H429S), alanine (H429A), tyrosine (H429Y), threonine (H429Y), leucine (H429L), valine (H429), glycine (H429G), tryptophan (H429W), arginine (H429R), and proline (H429P).

[0185] Suitable mutations at position 429 include:

[0186] 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).

[0187] Preferred amino acid substitutions at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide include H→Y (e.g., H429Y) substitution and H→F (e.g., H429F) substitution.

[0188] 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 (Y). In some embodiments, H429 is substituted with leucine (H429L). In some embodiments, H429 is substituted with valine (H429). 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 proline (H429P).

[0189] The amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide may be the only mutation in the Fc component. However, in some embodiments, the Fc component may contain one or more additional mutations (e.g., amino acid substitutions). For example, the Fc component may contain one or more sequence mutations known to those skilled in the art (see, for example, examples listed in Table 1 of Wang X et al., Protein Cell 9(1):63-73, 2018; the full disclosure of the literature is incorporated herein by reference), such as: mutations that may regulate FcγR binding (e.g., S239D / I332E (Eu number) that increases FcγRIIIa binding to IgG1); mutations that improve antibody-dependent cytotoxicity (ADCC), such as S239D / I332E (Lazar GA et al., Proceedings of the National Academy of Sciences 103(11):4005-4010, 2006); and mutations that modulate phagocytosis (e.g., G236A / S239D / I332E; Richards JO et al., Molecular Cancer Therapy 7:2517-2527). 2008) or complement activation (e.g., H268F / S324T; Moore GL et al., MAb 2:181-189, 2010), or reduced effector function (e.g. IgG1:L234A / L235A; IgG4:F234A / L235A; Xu D et al., Cell Immunol 200(1):16-26, 2000); and mutations conferring enhanced binding to neonatal Fc receptor (FcRn), such as M252Y / S254T / T256E (Dall'Acqua WF et al., Journal of Immunology 169(9):5171-5180, 2002), to increase in vivo half-life and thereby improve pharmacokinetics (PK).

[0190] Other mutations that may be included in the Fc region component include mutations for enhancing complement activation, such as amino acid mutations (Eu numbers) located at the position corresponding to K447 of the amino acid sequence of the human IgG1 heavy chain polypeptide; specifically K447X, where X is selected from null (i.e., K447del; amino acid deletion or truncation of the Fc component) and glutamate (i.e., K447E) (see van der Bremer ETJ et al., mAb 7(4):672-680, 2015). Furthermore, the Fc region component may also include mutations regulating glycosylation (e.g., mutations (Eu numbers) located at the position corresponding to Asn297 of the amino acid sequence of the human IgG1 heavy chain polypeptide, such as N297A, N297Q, or N297G (Wang et al., 2018 ibid.)) to provide a site with modified glycosylation (e.g., lack of glycan at position 297) to eliminate FcγR and complement C1 binding and / or activation.

[0191] Alternatively, the Fc region component can be treated to achieve modified glycosylation by generating an immunotherapeutic protein in the presence of a chiffon base (a mannosidase inhibitor that prevents the normal maturation of N-linked glycans, including the core fucosylation of N-linked glycans); this is a modification that specifically enhances activity via FcγRIIIa. Furthermore, the Fc component lacking the core fucosylation of Asn297 glycan can also be achieved by culturing host cells expressing immunotherapeutic proteins with fucosylation inhibitors (e.g., 2-fluoro-peracetylated fucose or analogues), by expressing enzymes that modify glycosylation pathways (e.g., GDP-6-deoxy-D-lythreo-4-hexylose reductase; Neha M et al., J Biotech 5:100015, 2020), or by gene repression of these pathways (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).

[0192] The immunotherapeutic proteins described herein can be monomeric, dimeric, or oligomeric in solution (e.g., in physiological saline at a neutral pH, such as about 7.4).

[0193] 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, wherein the Fc region component self-associates (i.e., forms a dimer) through non-covalent binding such as hydrogen bonding or through disulfide self-association via one or more cysteine ​​(C) residues, particularly within the hinge sequence, especially within the core hinge sequence (if present) (Yoo EM et al., Journal of Immunology 170:3134-3138, 2003). Therefore, in some embodiments, the Fc region component includes a core hinge sequence such that the immunotherapy protein can self-associate (i.e., form a dimer) by forming interchain disulfide bonds between one or more cysteine ​​(C) residues in the core hinge sequences of the two Fc region components. In other embodiments, the Fc region component includes a CH3 domain such that CH3:CH3 can self-associate via non-covalent interactions, or the Fc region component includes both a CH3 and a CH2 domain such that it can self-associate via non-covalent interactions of CH2:CH2 and CH3:CH3. It should be noted that in the examples and figures below, the dimer form of the immunotherapy protein is considered as a monomolecule (i.e., each molecule contains two copies of the protein dimerized by the Fc region component) and is referred to as a monomer.

[0194] In other embodiments, the immunotherapeutic protein comprises an oligomer in physiological saline (at a neutral pH, such as physiological pH of about 7.4), wherein the immunotherapeutic protein comprises an amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide, which enables an Fc or Fc-like fragment comprising a mutated Fc component to self-associate in physiological saline into a soluble oligomeric form (e.g., comprising 3, 4, 5, 6, or 12 copies of an oligomer assembled, for example, from a dimer form of the immunotherapeutic protein, such that in some specific embodiments, the oligomeric form of the immunotherapeutic protein may comprise six dimer proteins (i.e., a hexamer form), comprising a total of 12 copies of the immunotherapeutic protein). In such embodiments, the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide may be:

[0195] H429X 1 , where X 1 It is selected from tyrosine (i.e., H429Y), methionine, isoleucine, leucine, tryptophan and valine;

[0196] However, preferably, the immunotherapy protein contains an H→Y (i.e., H429Y) substitution.

[0197] In other embodiments, the immunotherapeutic protein can form oligomers upon binding to a relevant target via "on-target" oligomerization. Such oligomerization can occur with the immunotherapeutic protein, which is a monomer or dimer in saline (at neutral pH, such as physiological pH 7.4), and includes an amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide. This allows for oligomerization on the target into an oligomeric form (e.g., containing 3, 4, 5, 6, or 12 copies of the immunotherapeutic protein, e.g., a hexamer in a dimeric form). In such embodiments, the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide can be:

[0198] H429X 2 , where X 2 Selected from phenylalanine (i.e., H429F), glutamic acid, glutamine, and serine;

[0199] However, preferably, the immunotherapy protein contains H→F (i.e., H429F) substitution.

[0200] Alternatively, the oligomeric form of the immunotherapeutic protein described herein can be produced using other techniques well known to those skilled in the art, such as using Fc multimeric forms (stradomers) TM ), which contains a multimerizing domain (MD) sequence that links from the hinge region of human IgG2 or isoleucine zipper (ILZ) to the N-terminus or C-terminus of mouse IgG2a (Fitzpatrick EA et al., Front Immunol 11, Chapter 496, 2020), as well as using multimerizing sequences from IgM (Melcheil et al., Sci Rep 1:124 doi:10.1038 / srep0012, 2011), or docking and dimerizing sequences from irrelevant proteins such as cyclic adenosine monophosphate-dependent protein kinase and A-kinase anchoring protein (Rossi EA et al., Bioconjug Chem 23(3):309-323, 2012).

[0201] Further findings revealed that the immunotherapeutic protein described herein can exhibit enhanced neonatal Fc receptor (FcRn) binding via an amino acid substitution (Eu number) at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain. This is expected to mean that the immunotherapeutic protein will exhibit a longer in vivo half-life (i.e., relative to an equivalent immunotherapeutic protein without the mutation at position H429), and thereby improve pharmacokinetics (PK), since FcRn is known to “recycle” antibodies to control how long it persists in vivo (Ward ES and RJ Orber, Trends PharmacolSci 39(10):892-904, 2018).

[0202] Dimeric immunotherapeutic proteins (e.g., immunoglobulin molecules).

[0203] The immunotherapeutic proteins described herein can be dimer immunotherapeutic proteins comprising immunoglobulin molecules, wherein the immunoglobulin molecules comprise a first immunoglobulin heavy chain polypeptide and a second immunoglobulin heavy chain polypeptide, each comprising an Fc region component including at least one CH3 domain (or at least one CH4 domain). The Fc region components of the first and second immunoglobulin heavy chain polypeptides can be non-covalently linked, such as by hydrogen bonding, or by self-association (i.e., dimerization) through disulfide bonds formed by one or more cysteine ​​(C) residues, particularly within the hinge sequence, especially the core hinge sequence (if present) (Yoo EM et al., *Journal of Immunology* 170:3134-3138, 2003), or otherwise linked by cross-linking compounds, such as those mentioned above (e.g., by cross-linking the CH3 domain of the Fc region component). Therefore, the immunotherapy protein can be an immunoglobulin molecule, such as an antibody or antibody derivative, such as scFv-Fc (where a dimer of the scFv polypeptide is formed by dimers of Fc region components), a microantibody (where a dimer of the scFv polypeptide is formed by connecting CH3 domain / CH4 domains), or any other suitable Fc-containing antibody or derivative known to those skilled in the art (e.g., as outlined in the “Antibody Periodic Table” mentioned above).

[0204] Therefore, the dimer immunotherapy protein may further include at least one antigen recognition structure, or in other words, an antigen-binding region (e.g., scFv or containing a variable domain V). L and V HAntigen-binding regions, such as Fab fragments, can bind specifically to antigens or epitopes that have therapeutic significance; for example, cancer-related antigens such as cancer antigens present on the surface of cancer cells (e.g., differentially expressed and / or present cell surface antigens in cancer cells, such as CD20, CD38, and CD52 antigens found on the surface of CLL cells, and mucins (e.g., MUC-1) or carbohydrates (e.g., Lewis X) 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 bacterial, yeast, parasitic, or viral pathogens (e.g., antigens of SARS-CoV-2, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), respiratory syncytial virus (RSV), or dengue virus). In other instances, antigen-binding domains can specifically bind to cell surface molecules known to induce cell proliferation (i.e., cell surface molecules can be, for example, CD3 or CD28) and / or stimulate inhibitory pathways to suppress or reduce cellular responses (i.e., cell surface molecules can be, for example, immune checkpoint molecules such as 4-1BB (CD137), differentiation clusters (CD40, CD154), OX40 receptors (TNFRSF4, CD134), tumor necrosis factor receptor type II (TNFR2, CD120b), glucocorticoid-induced TNFR-related proteins (GITR, TNFRSF18, CD357), differentiation cluster 2, etc.). 7 (CD27), 3 containing T cell immunoglobulin and mucin domains (TIM-3), B and T lymphocyte attenuators (BTLA, CD272), lymphocyte activation gene-3 (LAG3, CD223), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), inducible T cell co-stimulatory (ICOS, CD278), differentiation cluster 28 (CD28), TT cell immune receptors with Ig and ITIM domains (IGIT, Vstm3), programmed death ligand 1 (PDL-1, CD274), and programmed cell death protein 1 (PD-1, CD279);Furthermore, oligomerization of the immunotherapeutic protein can produce enhanced cross-linking (e.g., superclustering), and consequently lead to enhanced signaling to induce, for example, enhanced cell proliferation or inhibited / reduced cellular responses (i.e., enhanced stimulation via inhibitory pathways). In yet another example, the antigen-binding region can specifically bind to cell surface molecules known for inducing cellular responses selected from stimulatory pathways to inhibit or reduce responses. In still yet another example, the antigen-binding region can specifically bind to cell surface molecules that, when bound by ligands such as agonist mAbs or other molecules, induce cell death. These cell surface molecules include, for example, cell surface molecules of the TNF-receptor superfamily (also known as TNFRSF), which include: tumor necrosis factor receptor 1, TNFRSF1A (CD120a); tumor necrosis factor receptor 2, TNFRSF1B (CD120b); lymphotoxin β receptor, TNFRSF3 (CD18); OX40, TNFRSF4 (CD134); CD40, TNFRSF5 (Bp50); Fas receptor, TNFRSF6 (Apo-1, CD95); and other related molecules. Bait receptor 3, TNFRSF6B (TR6, M68); CD27, TNFRSF7 (S152, Tp55); CD30, TNFRSF8 (Ki-1, TNR8); 4-1BB, TNFRSF9 (CD137); Death receptor 4, TNFRSF10A (TRAILR1, Apo-2, CD261); Death receptor 5, TNFRSF10B (TRAILR2, CD262); Decoy receptor 1, TNFRSF10C (TRAILR3, LIT, TRID, C) D263); decoy receptor 2, TNFRSF10D (TRAILR4, TRUNDD, CD264); RANK, TNFRSF11A (CD265); osteoprotein, TNFRSF11B (OCIF, TR1); TWEAK receptor, TNFRSF12A (Fn14, CD266); TACI, TNFRSF13B (IGAD2, CD267); BAFF receptor (BAFF-R), TNFRSF13C (CD268); herpesvirus entry mediator, TNFR SF14 (ATAR, TR2, CD270); Nerve growth factor receptor, TNFRSF16 (p75NTR, CD271); B cell maturation antigen, TNFRSF17 (TNFRSF13A, CD269, BCMA); Glucocorticoid-induced TNFR-related, TNFRSF18 (AITR, CD357); TROY, TNFRSF19 (TAJ, TRADE); RELT, TNFRSF19L (19L); Death receptor 6, TNFRSF21 (CD358);Death receptor 3, TNFRSF25 (Apo-3, TRAMP, LARD, WS-1); exoprotein A2 receptor, TNFRSF27 (XEDAR) (Wallach, Cold Spring Harb Perspect Biol 10:a028431, 2018; Ware, Immunol Rev 244:5-8, 2011; Locksley et al. Cell 104 (4) 487-501, 2001; Vanamee and Faustman Sci Signal, 2018). For example, under physiological conditions, the binding of TNF-related apoptosis-inducing ligand (TRAIL) to its receptor DR5 (Carneiro BA et al., Nature Review Clinical Oncology 17:395-417, 2020) or the binding of FAS ligand to its receptor FAS (CD95) on the cell surface induces oligomerization of signaling that leads to cell death (Leukocyte and Stromal Cell molecules: The CD Markers, from Zola H et al., p. 195, John Wiley & Sons, 2007). Other molecules (unrelated to TNFRSF) that can induce apoptotic signals leading to cell death when cross-linked, for example, via 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 advantageously targeted by dimeric immunotherapeutic proteins, such that oligomerization may lead to enhanced cross-linking and the induction of apoptotic signals, thereby resulting in potentially desired cell death in the target cells.

[0205] In some embodiments, the immunoglobulin molecule according to this disclosure may comprise two antigen-binding regions, each of which specifically binds to a different antigen or epitope. As used herein, an "antigen" is any substance that causes the body to produce an immune response (antibody) against it. An "epitaph," also referred to herein as an "antigenic determinant," is part of an antigen recognized by an antigen recognition structure.

[0206] In some embodiments, the immunoglobulin molecule according to this disclosure is an antibody, which may be, for example, an IgD, IgE or IgM isotype, but preferably an IgA or IgG isotype, such as an antibody of any of the human IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 subtypes.

[0207] The first and second immunoglobulin heavy chain polypeptides of the dimer immunotherapy protein containing immunoglobulin molecules can be the same (i.e., homodimers) or different (i.e., heterodimers).

[0208] In some embodiments, the immunoglobulin molecule according to this disclosure is an antibody, which may be an antibody that forms an oligomer by self-association in solution (e.g., in physiological saline at a neutral pH, such as about 7.4) through oligomerization into an oligomer (e.g., a hexamer), or an antibody that forms an oligomer by binding to an associated target (e.g., an antigen targeted by an antibody mutant) through “on-target” oligomerization.

[0209] For example, and although not wishing to be bound by theory, it is thought that immunoglobulin molecules comprising antibodies with H429Y amino acid substitutions in the Fc region can form oligomers in solution (e.g., in physiological saline at physiological pH), which may result in an overall increase in binding strength (e.g., enhanced affinity) to target-binding partners (e.g., cancer antigens, antigens of bacterial or viral pathogens, or other soluble target molecules or molecular complexes). Therefore, in the context of antibodies against cancer antigens present on the surface of cancer cells, antibodies according to this disclosure (in the form of oligomers in solution) may exhibit an enhanced ability to bind to cancer cells, thereby causing said cancer cells to be destroyed by, for example, CDC via complement or by phagocytosis of macrophages and / or ADCC; and in the context of antibodies 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, thereby causing said soluble target molecules or molecular complexes to be removed by phagocytosis of macrophages.

[0210] On the other hand, it is believed that immunoglobulin molecules containing H429F amino acid substitutions in the Fc region fraction can form oligomers upon binding to the relevant target, resulting in an "on-target" assembled Fc fraction exhibiting a stable optimal binding platform, whereby, for example, affinity for the C1q complement protein complex is enhanced, thereby leading to complement activation and consequently complement-dependent cytotoxicity (CDC). Therefore, in the context of antibodies against cancer antigens present on the surface of cancer cells, antibodies according to this disclosure can exhibit an enhanced ability to activate complement on cancer cells, thereby leading to the destruction of said cancer cells.

[0211] In some embodiments, the immunoglobulin molecule according to this disclosure may be provided or used as a first immunoglobulin molecule having a first antigen-binding region targeting a second antigen, in combination with a second immunoglobulin molecule having a first antigen-binding region targeting a first antigen. For example only, in such combinations, the first antigen may be a cancer antigen present on the surface of cancer 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 result in the formation of heterooligomers such as heterohexamers, in which different cell surface targets are integrated into the cluster, which may lead to cell death, for example, the induction of enhanced apoptotic signaling (e.g., where a death receptor has been targeted), where target cell specificity is achieved by binding to, for example, cancer antigens present on cancer cells.

[0212] Fusion / Conjugate Immunotherapy Proteins

[0213] Immunotherapy proteins as described herein may comprise fusion proteins or protein conjugates including a partner polypeptide linked to an Fc region component, wherein the Fc region component contains at least one CH3 domain (or at least one CH4 domain).

[0214] The fusion protein or protein conjugate according to this disclosure can be monomeric, dimeric, or oligomeric. For example, in some embodiments, the fusion protein or protein conjugate comprises monomers, each of which contains one copy of a partner polypeptide (or a fragment thereof) and one copy of an Fc region component, while in other embodiments, the immunotherapy protein comprises a dimer, wherein the Fc region component is self-associated (i.e., forms a dimer) by non-covalent binding such as hydrogen bonding or by forming disulfide bonds through one or more cysteine ​​(C) residues, particularly within the hinge sequence, especially within the core hinge sequence (if present) (Yoo EM et al., *Journal of Immunology* 170:3134-3138, 2003). In such dimers, the immunotherapy protein comprises two Fc region components (associated with each other to form, for example, an Fc fragment or an Fc-like fragment) and two fusion / conjugated partner polypeptides (or two fragments thereof), and can therefore be considered divalent relative to the partner polypeptide (or a fragment thereof). The two fusion / conjugation polypeptides (or two fragments thereof) of a dimer fusion protein or protein conjugate can be identical (i.e., homodimers) or different (i.e., heterodimers). In some embodiments of such dimers, such as in Figure 53 As depicted in A, the fusion protein is antibody-like (Ab-like) and can have, for example, an H2 or H2L2 format (where the monomeric fusion protein is generated from a light (L) chain, which can then optionally be fused / conjugated to a partner polypeptide (such as a target recognition structure). It will be readily understood that each target recognition structure of an Ab-like molecule having an H2 or H2L2 format 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. Similarly, one or more target recognition structures in the target recognition structure can be replaced by alternative partner polypeptide types such as enzymes or reporter molecules.

[0215] Couple body peptides can provide beneficial functions and / or properties for immunotherapy proteins.

[0216] In some embodiments, the mate polypeptide may be a cell surface receptor polypeptide (or a fragment thereof) or a co-receptor polypeptide (or a fragment thereof).

[0217] For example, the mate peptide can be a cell surface molecule such as a cell surface receptor peptide (or fragment thereof) capable of binding to the structural proteins of a virus, allowing the immunotherapeutic protein to act as a "decoy" to block viral interactions and viral entry into host cells. Therefore, some examples of such cell surface receptor peptides (or fragments thereof) that may include mate peptides include the extracellular domain of angiotensin-converting enzyme 2 (ACE2) (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 cluster 155 (CD155; poliovirus). Examples of viral entry receptors include, but are not limited to, the cell entry receptor for the virus (CIL), glucose transporter 1 (GLUT1; cell entry receptor for human T-cell leukemia virus 1), proto-oncogene tyrosine protein kinase MER (MERTK; a host factor that promotes the entry of classical swine fever virus), TYRO3 protein tyrosine kinase (TYRO3; a cell surface protein associated with lymphocytic choroid plexus meningitis (LMCV) infection), AXL (a cell surface protein associated with lymphocytic choroid plexus meningitis (LMCV) infection), and differentiation cluster 4 receptor (CD4 receptor; cell entry receptor for human immunodeficiency virus (HIV)). In embodiments where the mate polypeptide is a cell surface receptor polypeptide (or a fragment thereof) capable of binding to viral structural proteins, a point mutation (Eu number) located at the position of the Fc region component corresponding to H429 of the human IgG1 heavy chain amino acid sequence can, for example, provide the immunotherapeutic protein with an enhanced ability to provide antiviral activity via the CDC of infected cells (e.g., the mutation is H429F) or confer enhanced virus neutralization on the immunotherapeutic protein (e.g., the mutation is H429Y). In other instances, the mate peptide may be a ligand of a cell surface molecule known for inducing cell proliferation (i.e., the cell surface molecule may be, for example, CD3 or CD28) and / or stimulating inhibitory pathways to suppress 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 wherein oligomerization of the immunotherapy protein may result in enhanced cross-linking (e.g., supercrossing), and consequently enhanced signaling to induce, for example, enhanced cell proliferation or suppressed / reduced cellular responses (i.e., through enhanced stimulation of inhibitory pathways).In other instances, the partner polypeptide can be a ligand of a cell surface molecule that, when bound by a ligand or other molecule such as an agonist mAb (e.g., cell surface molecules of TNFRSF mentioned above (e.g., TNFR1, Fas, DR3, DR4, DR5, and DR6) and other molecules such as CD38, CD20 (Shan et al., ibid. 2000; and Cardarelli et al., ibid. 2002) and CD52 (Rowan et al., ibid. 1998)), induces cell death. Therefore, in some embodiments, the immunotherapeutic protein as described herein can comprise a dimer fusion protein or protein conjugate containing, for example, TRAIL (i.e., a ligand of DR4 or DR5) as a partner polypeptide linked to an Fc region component comprising at least one CH3 domain substituted with an H429F amino acid. When ligands bind to cell surface molecules, oligomerization of the immunotherapeutic proteins in these embodiments may lead to enhanced cross-linking and induce apoptosis signaling, resulting in potentially desired cell death in target cells.

[0218] When the mate polypeptide is a co-receptor polypeptide (or fragment thereof), the co-receptor polypeptide (or fragment thereof) may be, for example, CXC chemokine receptor type 4 (CXCR4), CC chemokine receptor type 5 (CCR5) (a co-receptor of the CD4 receptor that binds to HIV viral glycoprotein gp120 and enables HIV to fuse with the host cell membrane), tetraspanin or occludin (which is a co-receptor required for hepatitis C virus (HCV) infection), and Gas6 (which is a ligand for receptors (such as AXL and TYRO3)) that bind together with phosphatidylserine displayed on the virus, including West Nile virus, Zika virus, and Ebola virus, and promote the entry of such viruses into host cells, etc.

[0219] In another instance, the mate polypeptide can be a cell surface receptor polypeptide (or a fragment thereof) capable of binding to a cell surface receptor ligand. Thus, 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 and downregulates the immune response. Immunotherapy proteins containing fusion proteins or protein conjugates including CTLA4 mate polypeptides (e.g., CTLA4-Fc fraction fusion proteins) can exhibit enhanced binding to CTLA4 ligands by forming dimers or oligomers, thereby acting as decoys to produce various therapeutic effects for the potential treatment of tumors (e.g., melanoma and colorectal cancer) and various autoimmune diseases such as SLE and rheumatoid arthritis (RA). Other examples of suitable cell surface receptor peptides (or fragments thereof, such as extracellular domains) capable of binding to ligands of cell surface receptors include other immune checkpoints (e.g., PD1) and other cytokine receptors, such as receptors for interleukin-1 (IL-1R), interleukin-6 (IL-6R), tumor necrosis factor receptor-2 (TNFR2, also known as CD120b), or receptors for TGF-β superfamily cytokines (see review in Czajkowsky DM et al., *Molecular Medicine* 4(10):1015-1028, 2012). Immunotherapy proteins containing fusion proteins or protein conjugates including IL-1R (or fragments thereof) could potentially be used for anti-IL-1 therapy to treat, for example, type 2 diabetes, and immunotherapy proteins containing fusion proteins or protein conjugates including IL-6R (or fragments thereof) could potentially be used for anti-IL-6 therapy to treat, for example, cancer and RA. Immunotherapy proteins containing fusion proteins or protein conjugates of TNF-R2 or its extracellular domain may be used to treat RA or other inflammatory diseases or conditions.

[0220] While not wishing to be bound by theory, it is believed that fusion proteins or protein conjugates (whether in monomeric or dimer form) according to this disclosure, including H429Y amino acid substitutions in the Fc region component, can form oligomers in solution (e.g., in physiological saline at neutral pH, such as physiological pH of about 7.4), which may result in an overall increase in binding strength (e.g., enhanced affinity) to target-binding conjugates (e.g., ligands of viral structural proteins or cell surface receptors, or other soluble target molecules or molecular complexes). Thus, in the context of fusion or conjugate proteins comprising cell surface receptor peptides or fragments thereof capable of binding to viral structural proteins, this results in enhanced affinity for the virus to provide enhanced viral neutralization (possibly conferred by cross-linking and / or aggregation of viral particles (virions)). However, it is believed that, in at least some embodiments, such soluble oligomers are substantially unable to bind to Fc receptors and are therefore capable of viral neutralization with little or no complement activation.

[0221] On the other hand, and similarly not wanting to be bound by theory, it is believed that fusion proteins or protein conjugates (whether in monomeric or dimer form) that include H429F amino acid substitutions in the Fc region components can form oligomers when binding to relevant targets (e.g., ligands of viral structural proteins or cell surface receptors), resulting in a stable and optimal arrangement of the "target-assembled" Fc components for binding to the C1q complement protein complex (e.g., with enhanced affinity), thereby leading to complement activation and further enhancing complement-based effector functions such as complement-dependent cytotoxicity (CDC).

[0222] The immunotherapeutic proteins according to this disclosure can be produced according to any standard method known to those skilled in the art. For example, those skilled in the art can readily prepare immunotherapeutic fusion proteins by: generating a construct containing a polynucleotide sequence encoding a fusion protein using standard molecular biology techniques; introducing the construct into suitable host cells (e.g., human kidney (HEK) host cells or derivatives thereof, such as Expi293 cells (Thermo Fisher Scientific)) to express the fusion protein or a host animal (e.g., 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 any of the following methods: affinity chromatography (e.g., protein A), ion exchange chromatography (IEX), size exclusion chromatography (SEC), and combinations thereof). In some embodiments, the immunotherapeutic protein is produced by a transgenic animal (the animal being modified to express the immunotherapeutic protein or nucleic acid as described herein). Similar methods can be used to prepare immunotherapeutic proteins that are immunoglobulin molecules such as antibodies or Ab-like molecules. In other words, those skilled in the art can readily prepare mutant antibodies (i.e., antibodies containing an amino acid substitution (Eu number) at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain) by generating constructs, said constructs comprising a variable weight (V) encoding a suitable antibody. H ) and light (V) L The multinucleotide sequence of the region (e.g., including the multinucleotide sequence of the antigen-binding region that binds to the antigen of interest) and the constant weight (CH) region from, for example, an IgG1 antibody, are incorporated into the multinucleotide sequence encoding the CH3 region by standard molecular biology techniques such as site-directed mutagenesis, or by multinucleotide sequence alterations to encode mutations located at H429 (e.g., H429F and H429Y). Similar to the preparation of immunotherapeutic fusion proteins, the construct can be introduced into suitable host cells (e.g., human kidney (HEK) host cells or derivatives thereof), cultured according to standard culture protocols, and the expressed mutant antibody purified from the culture supernatant using any of the known suitable purification methods, such as affinity chromatography. Furthermore, it has been found advantageous to use mild elution conditions, such as using an elution buffer containing a low concentration of arginine (e.g., less than 130 mM) and a pH less than or equal to 5, when using affinity chromatography for purification (e.g., protein A), especially when the immunotherapeutic protein is in an Ab-like molecular form, to inhibit aggregate formation. This can be achieved, for example, by a standard liquid chromatography system that can deliver a buffer gradient to the column, in this case, a linear gradient to, for example, 35% of 130 mM arginine (pH 4.0).

[0223] In some preferred embodiments, the recovery of the immunotherapeutic protein expressed according to this disclosure is as follows:

[0224] (i) Preferably carried out under weakly acidic pH conditions (e.g., pH less than neutral pH, such as pH 6.5, preferably pH 5.5, and more preferably pH 5.0), wherein it is desirable to provide the immunotherapeutic protein in monomeric form; or

[0225] (ii) When it is desired that the immunotherapy protein be provided in an oligomeric form (e.g., as a hexamer), it is preferably carried out under substantially neutral pH conditions (e.g., 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

[0226] (iii) Preferably, the method is carried out using affinity chromatography, which uses an elution buffer containing a low concentration of arginine (e.g., less than 130 mM) and a pH less than or equal to 5.0 (preferably about pH 4.0), especially when it is desired that the immunotherapeutic protein is provided as an antibody-like molecule.

[0227] In some specific embodiments, the recovery of the immunotherapeutic protein expressed according to this disclosure comprises recovery by size exclusion chromatography (SEC), for example under weakly acidic pH conditions for producing the monomeric form of the immunotherapeutic protein, or under substantially neutral pH conditions for producing the immunotherapeutic protein in oligomeric form. If desired, SEC may be performed after a recovery stage comprising ion exchange chromatography (IEX).

[0228] In one embodiment, the bispecific immunotherapy protein is a mini-body. As used herein, a “mini-body” is a class of bispecific immunotherapy proteins consisting of two ScFvs fused to CH3. The ScFvs contain antigen recognition structures targeting different epitopes. In one embodiment, the molecular weight of the mini-body is less than 90 kDa. In another embodiment, the molecular weight of the mini-body is less than 85 kDa.

[0229] The US FDA classifies bsAbs into two main categories based on their mechanism of action: cell-bridging bsAbs and antigen-crosslinked bsAbs (non-cell-bridging molecules; Labrijn et al., 2019). Most cell-bridging bsAbs are designed for cancer therapy by linking immune cells to malignant cells. Through sequential binding—that is, binding first to cancer cells due to higher affinity for tumor antigens—cell-bridging bsAbs can improve specificity and efficacy, with fewer non-specific side effects and lower doses compared to mAbs. In contrast, antigen-crosslinked bsAbs target two antigens or two receptors simultaneously. Their primary MoA is blocking cell growth / survival signals or activating immune cells (Engelman et al., 2007). The function of antigen-crosslinked bsAbs is essentially similar to that of mAbs, except that they bind to two different targets.

[0230] On the one hand, this disclosure provides the use of an immunotherapeutic protein as described herein for treating or preventing a disease or condition in a subject, wherein the disease or condition may be selected from, for example, autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0231] On the one hand, this disclosure provides the use of an immunotherapeutic protein as described herein, which is used to prepare a medicament 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, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0232] On one hand, this disclosure provides a method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of an immunotherapeutic protein as described herein, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

[0233] Among diseases and conditions that can be treated or prevented by the methods described herein are autoimmune diseases and conditions such as SLE and MS, other inflammatory diseases (e.g., immune complex vasculitis), infectious diseases and proliferative diseases (especially solid tumors such as breast cancer), blood cancers such as lymphoproliferative disorders (LPD), including leukemias (e.g., acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL)), adenocarcinomas and lymphomas, as well as multiple myeloma (MM) and X-linked proliferative disorders.

[0234] The methods described herein will typically be applied to treat 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 wild animals (e.g., non-human primates, tigers, elephants, etc.).

[0235] In some embodiments, the immunotherapy protein comprises a fusion protein including a partner polypeptide linked to an Fc region component, the Fc region component including at least one CH3 domain (or at least one constant recombinant domain 4 (CH4) domain), and the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof) capable of binding to viral structural proteins, such that the immunotherapy protein can act as a "decoy" to block viral interactions and viral entry into host cells, and the method described herein may further comprise the administration of an antibody against the virus (i.e., the target virus). For example, when the immunotherapeutic 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, the following: broadly neutralizing coronavirus mAbs (i.e., bNmAbs that can neutralize multiple coronavirus types or strains), broadly reactive coronavirus mAbs (i.e., mAbs that can bind to multiple coronavirus types and strains but not neutralize them), 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, for example, target epitopes of SARS-CoV-2 structural proteins other than the spike protein (S), such as envelope proteins (E), membrane proteins (M), or nucleocapsid proteins (N). In some other specific embodiments, the antibody may target, for example, an epitope on the spike protein (S) but at a site different from the RBD. More generally, when the immunotherapeutic protein comprises a cell surface receptor polypeptide or co-receptor polypeptide or fragment thereof other than the ACE2 polypeptide, the antibody against the target virus may be selected, for example, antibodies with broad neutralization against a class of viruses / virus families (e.g., human immunodeficiency virus (HIV)), or antibodies with broad reactivity against a class of viruses or virus families, antibodies with broad neutralization against strains of a specific virus type (e.g., bNmAbs that can neutralize multiple HIV-1 strains), and mAbs with broad reactivity against a specific virus type. As shown below, it has been found that the immunotherapeutic proteins of this disclosure and antibodies against target viruses can synergistically enhance CDC killing of cells (e.g., virus-infected cells).

[0236] Similarly, when an immunotherapy protein comprises a fusion protein, the fusion protein comprises a partner polypeptide linked to an Fc region component containing at least one CH3 domain (or at least one constant recombinant domain 4 (CH4) domain), and the partner polypeptide targets, for example, a therapeutically significant target (e.g., a cell surface receptor polypeptide (or a fragment thereof) capable of binding to CTLA4, which functions as an immune checkpoint and downregulates the immune response). The method described herein may further comprise the administration of an antibody against the target (e.g., an antibody that binds to CTLA4) to provide, for example, an enhanced response, such as enhanced CDC killing of cells (e.g., cancer cells).

[0237] In cases where the method described herein includes, in addition to administering the fusion protein, administering an antibody (i.e., as described in the first two paragraphs), preferably, the antibody comprises an Fc region component containing an amino acid substitution (EU number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide, for example, H429X. 2 Amino acid substitution, where X 2 It is selected from phenylalanine (H429F), glutamic acid (H429E), glutamine (H429Q), serine (H429S), alanine (H429A), tyrosine (H429Y), threonine (H429T), leucine (H429L), valine (H429V), glycine (H429G), tryptophan (H429W), arginine (H429R) and proline (H429P).

[0238] On one hand, this disclosure provides a pharmaceutical composition or medicament comprising an immunotherapeutic protein as defined herein, and pharmaceutically acceptable carriers, diluents, and / or excipients.

[0239] Immunotherapy proteins can be administered in combination with one or more additional agents for treating a specific disease or condition being treated. For example, in the context of treating proliferative diseases, immunotherapy proteins can be used in combination with other drugs used to treat cancer, including, for example, antitumor drugs such as cis-platin, gemcitabine, cytosine arbinoside, doxorubicin, and epirubicin; taxoids including taxol; topoisomerase inhibitors such as etoposide; cell inhibitors such as tamoxifen; aromatase inhibitors (e.g., as anastrozole); and growth factor function inhibitors (e.g., antibodies such as anti-erbB2 antibody trastuzumab). In some embodiments, immunotherapy proteins can be administered with one or more additional agents, which may also be immunotherapy proteins according to this disclosure. For example, when the first immunotherapeutic protein according to this disclosure comprises an ACE2 polypeptide (or a fragment thereof) that binds to the RBD of the CoV-2 spike protein, the second immunotherapeutic protein of this disclosure may be an antibody against the target CoV-2 virus, particularly an antibody targeting a different structural protein (e.g., an envelope protein (E), a membrane protein (M), or a nucleocapsid protein (N)) or an epitope on the same structural protein (i.e., the spike protein (S)) that is different from the RBD site. As shown below, combinations of such immunotherapeutic proteins have been found to synergistically enhance CDC killing of cells (e.g., virus-infected cells).

[0240] When used in combination with other pharmaceutical agents, the immunotherapy protein can be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. If administered in separate pharmaceutical compositions, the immunotherapy protein and other pharmaceutical agents can be administered simultaneously or in any order (e.g., within seconds or minutes or even hours (e.g., 2 hours to 48 hours)).

[0241] Immunotherapy proteins can be formulated into pharmaceutical compositions with 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 forms of pharmaceutical compositions described herein can be found in *Handbook of Pharmaceutical Excipients*, 2nd edition, (1994), edited by A Wade and PJ Weller. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water. The selection of carriers, diluents, and / or excipients can be based on the intended route of administration and standard pharmaceutical practice.

[0242] Pharmaceutical compositions containing immunotherapeutic proteins as defined herein may further include any suitable binders, lubricants, suspending agents, coating agents, and solubilizers. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, and even dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and parabens. Antioxidants and suspending agents may also be used.

[0243] Pharmaceutical compositions comprising immunotherapeutic proteins as defined herein are generally suitable for intravenous or subcutaneous administration. Therefore, pharmaceutical compositions may comprise solutions or emulsions that can be injected into a subject and are prepared from sterile or sterilizable solutions. Pharmaceutical compositions may be formulated in unit dosage forms (i.e., in the form of discrete portions containing a unit dose or multiples of a unit dose or subunits).

[0244] The immunotherapeutic proteins, uses, and pharmaceutical compositions of this disclosure are further described below with reference to the following non-limiting examples.

[0245] Example

[0246] Example 1: ACE-2-Fc fusion protein containing the H429 mutation and its activity analysis

[0247] Methods and Materials

[0248] Constructs and fusion proteins

[0249] The amino acid sequence of the human ACE2 polypeptide was obtained from the European Nucleotide Archive (ENA, European Molecular Biology Laboratory) with accession number BAB40370. The extracellular domains of the protein (amino acids 19 to 740; shown as SEQ ID NO: 1 in Table 1) contain a catalytic domain and a collectrin domain. In this example, different forms of the ACE2 extracellular domain were generated and studied; specifically, a truncated (tr)ACE2 extracellular domain (containing amino acids 19 to 615 of the mature ACE2 polypeptide (named trACE2, shown as SEQ ID NO: 2 in Table 1) and excluding the collectrin domain), a full-length (fl)ACE2 extracellular domain (flACE2; containing amino acids 19 to 740 of the mature ACE2 polypeptide; SEQ ID NO: 1 in Table 1), and an enhanced flACE2 extracellular domain (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, ibid.). These proteins were generated as fusion proteins according to standard techniques, together with an Fc region component derived from human IgG1 to produce trACE2-Fc, flACE2-Fc, and EflACE2-Fc (see Table 2).

[0250] For example, a construct encoding the extracellular domain of trACE2 was prepared by linking a polynucleotide sequence encoding the trACE2 extracellular domain with a synthetic sequence encoding the adapter and a sequence encoding human IgG1 Fc (specifically, IgG1 Fc with accession number AXN93652.1, the amino acid sequence of which is the immunoglobulin γ1 constant region, part [Homo sapiens]; National Center for Biotechnology Information (NCBI) database). To generate the flACE2-Fc expression construct, the trACE2 construct was KpnI digested, followed by insertion of a codon-optimized polynucleotide sequence (GeneArt, Thermo Fisher Scientific) encoding the ACE2 collectrin domain. To generate the EflACE2-Fc expression construct, a synthetic polynucleotide sequence encoding flACE2-Fc was used, but with three mutations (i.e., T27Y, L79T, and N330Y; Chan et al., 2020). The synthetic polynucleotide sequence of sACE2.v2.4 described above. Additionally, according to the manufacturer's instructions, H429F and H429Y mutations incorporated into the Fc component are introduced by cleaving at a unique AfeI site within the IgG Fc coding sequence, and subsequently, a variant of the fusion protein is generated by inserting appropriate mutagenic oligonucleotides using NEBuilder (New England Biolabs, Ipswich, MA, United States of America).

[0251] The H429 residue occupies a "buried" site within the IgG1 Fc structure (see [link]). Figure 1 ), and is occupied by histidine (His / H) residues, which are also found at corresponding positions in the Fc fragments of all other human immunoglobulin classes ( Figure 3 , Figure 4 And thus found in corresponding positions in, for example, human IgG1, IgG2, IgG3, and IgG4, as well as primate IgG subclasses and some mouse IgG subclasses. For example, the structure of human IgG1-Fc published in 1981 (PDB:1Fc1; Deisenhofer, 1981 ibid.) indicates that H429 is not a surface-accessible residue. Figure 1 Furthermore, in the space-filling representation of Fc, H429 is only "visible" when the overlapping residues are presented in a non-space-filling manner, such as... Figure 1As shown in C. Similarly, analysis of residues adjacent to H429 indicates that H429 is located below the side chain of these residues, and calculation of the accessible surface area (ASA) of the Fc residues indicates that H429 is solvent inaccessible (0% ASA (Å)). 2 ), Figure 1 C). H429 is not a surface-accessible residue in other Fc structures, including those within the anti-HIV mAb structure that forms a hexameric ring (PDB: 1HZH; Saphire et al., ibid.), but H429 does not form part of the interface. Similarly, analysis of the Fc structure of anti-Lewis Y mAb (Ramsland et al., Journal of Immunology 187(6):3208-3217, 2011) indicates that H429 is also embedded in this antibody.

[0252] Table 1

[0253]

[0254] Fusion protein expression 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). Specifically, the supernatant of Expi293 cells transiently transfected to express the corresponding ACE2-Fc fusion protein (where the Fc region was based on the wild-type (WT) hIgG1 Fc mentioned in the preceding paragraph) was extensively dialyzed against 10 mM Tris-HCl at 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 to buffer A containing 0.4 M NaCl. Fractions were examined by SDS-PAGE, and fractions containing flACE2-Fc WT fusion protein were combined and concentrated using a 30 kDa cutoff filter (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).

[0255] 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., Journal of Immunology 197(4):1507-1516, 2016).

[0256] Lamelli natural PAGE (N-PAGE), 150 V, 2.5 h, 4 °C, according to Wines BD et al., Journal of Immunology 162(4):2146-2153, 1999.

[0257] Virus neutralization test

[0258] As previously described, antiviral titers were determined in a microneutralization assay using SARS-CoV-2 (CoV / Australia / VIC01 / 2020) (Juno JA et al., Nature Medicine 26(9):1428-1434, 2020).

[0259] Biological layer interferometry

[0260] The affinity of the ACE2-Fc fusion protein for CoV-2 S RBD was measured on an Octet RED96e (FortéBio, Fremont, CA, United States of America). All assays were performed at 25 °C using an anti-human IgG Fc capture (AHC) biosensor tip (FortéBio) 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 (60 s) biosensor baseline step, the fusion protein (20 mg / mL) was loaded onto the anti-human IgG Fc capture (AHC) biosensor by immersing the sensor tip for 200 seconds and then washing for 60 seconds in kinetic buffer. For most fusion proteins, correlation measurements were performed by immersing them in a 2-fold dilution series of SARS-CoV-2 RBD (16–250 nM or 500 nM) for 180 seconds followed by measuring dissociation in kinetic buffer for 180 seconds. For EflACE2-Fc WT, a 2-fold dilution series of 2–31 nM or 63 nM was used. The biosensor tip was regenerated five times using a cycle of 5 seconds in 10 mM glycine at pH 1.5 followed by 5 seconds in kinetic buffer, and baseline drift was corrected by subtracting the average offset of the fusion protein-loaded sensor not incubated with the SARS-CoV-2 RBD and the unloaded sensor incubated with the SARS-CoV-2 RBD. Curve fitting analysis was performed using Octet Data Analysis 10.0 software with a global fit 1:1 model to determine K. D Values ​​and kinetic parameters. Curves that cannot be fitted are excluded from the analysis. The reported kinetic constants represent two independent experiments.

[0261] The binding of the ACE2-Fc fusion protein and the recombinant dimer rsFcγR was determined by flow cytometry.

[0262] 5 µg / ml or the indicated concentration of ACE2-Fc fusion protein or rituximab (a chimeric mAb targeting CD20) was mixed with Ramos cells expressing the transfected spike protein (Ramos-S cells; Lee WS et al., medRxiv doi:10.1101 / 2020.12.13. 20248143, 2020) in 25 µl of PBS containing 0.5% (w / v) BSA and 1 mM glucose (PBS / BSA / G) at a concentration of 5 x 10⁻⁶. 6 Cells / ml were incubated on ice for 30 minutes, then washed twice with PBS / BSA / G, and incubated on ice for 30 minutes with PE or FITC-conjugated anti-human IgG-Fc. The cells were then washed again and resuspended in 25 μl PBS / BSA / G.

[0263] Binding to the dimeric recombinant soluble FcγR (rsFcγR) was assessed as previously described (Wines et al., 2016, ibid.). ACE2-Fc-conditioned Ramos-S cells or rituximab-conditioned cells were resuspended in 0.5 µg / ml biotinylated dimeric rsFcγRIIa (H131 allele form) or dimeric rsFcγRIIIa (V158 allele form) or BSA / PBS / G and incubated on ice for 30 min, followed by incubation on ice for 20 min with 1 / 500 streptavidin-APC (or anti-hIgG-Fc FITC for confirming ACE2-Fc conditioning). 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).

[0264] Complement-dependent cytotoxicity (CDC) assay

[0265] Ramos-S cells conditioned with ACE2-Fc functional protein or the control mAb rituximab were used to measure CDC, followed by incubation with human serum as a complement source. Therefore, Ramos-S cells were first incubated with the fusion protein or rituximab as described above (5 x 10⁻⁶ cells / mL). 6Cells / ml, in 25 µl PBS / BSA / G on ice for 30 min, then washed at 37°C before resuspending in 1 / 3 diluted normal human serum for 30 min. Cells were washed twice with PBS and then enumerated by staining with 1 / 500 Zombie Green (from the Zombie Green Fixable Viability Kit, BioLegend, San Diego, CA, United States of America, according to the manufacturer's instructions), fixed with PBS containing 2% paraformaldehyde, and analyzed on a Canto™ II flow cytometer (Beddy Medical).

[0266] Complement fixation immunoassay for ACE2-Fc fusion protein

[0267] Ninety-six-well MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated overnight with PBS containing 5 μg / ml avidin, blocked, and then incubated at room temperature for 1 hour with either serially diluted or single-concentration (2.5 μg / ml) 0.1% casein containing biotinylated RBD (Hartley et al., Science Immunology 5(54) doi:10.1126 / sciimmunol.abf8891, 2020). ACE2-Fc fusion protein was then added within the indicated concentration range. To measure C1q fixation, the plate was 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 1 / 2000 diluted rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:45 2019) at room temperature for 1 hour. In the assay for C5b-C9 fixation, the plate was 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, and then incubated with a 1 / 2000 dilution of HRP-conjugated goat anti-rabbit IgG (Merck Millipore) 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. Reactivity was stopped with 1 M sulfuric acid, and absorbance was measured at OD 450 nm. 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 duplicate, and background reactivity was corrected using a negative control well that omitted the ACE2-Fc protein. Mean values ​​and SEM results from independent experiments are shown.

[0268] RBD variant multiple assays

[0269] Customized multiplex arrays were generated using SARS-1 S1 subunit (ACROBiosystems, Newark, DE, United States of America), SARS-CoV-2 S1 and HCoV NL63 S1 and S2 subunits (Sino Biological Inc., Beijing, China), NL63 S trimer (BPSBioscience, San Diego, CA, United States of America), and hexahistine-labeled RBD WT protein (amino acids 19-613), along with 21 variants identified from the GISAID RBD monitoring library, expressed by pcDNA3 in Expi293 cells and purified by affinity chromatography. Bead conjugation, washing procedures, and data acquisition on a FlexMap3D™ analyzer (Luminex Corporation, Austin, TX, United States of America) were as previously described (Lee et al., 2020, ibid.). Briefly, direct binding of ACE2-Fc fusion proteins trACE2-Fc, flACE2-Fc, and EflACE2-Fc to 250 nM was detected using 25 µl of 1.3 µg / ml anti-human IgG R-phycoerythrin conjugate (SouthernBiotech, Birmingham, AL, United States of America) from 0.5 nM to 250 nM. The data were fitted to a 3-parameter agonist-response curve (r... 2 > 0.85), to determine each EC 50 In the competitive assay, in the presence of various unbiotinylated ACE2-Fc fusion protein “competitors,” RBD-coupled beads or S1-coupled beads were simultaneously incubated with 20 µl of 25 µg / ml biotinylated, AviTagged ACE2 at room temperature from 1 nM to 280 nM for 2 hours. Binding of biotinylated ACE2 (aa 19 to 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).

[0270] FcγRIIIa-NF-κB-RE Nanoluciferase Reporter Gene Assay

[0271] FcγRIIIa-NF-κB-RE nanoluciferase reporter gene assays were performed using IIA1.6 / FcR-γ / FcγRIIIa V158 cells expressing NF-κB response element-driven nanoluciferase (NanoLuc, pNL3.2.NF-κB-RE[NlucP / NF-κB-RE / Hygro], Promega Corporation, Madison, WI, United States of America) and were performed essentially as previously described (Lee et al., 2020 ibid.). In short, Ramos cells expressing spike-IRES-orange2 were used as target cells and incubated with the agonist and FcγRIIIa / NF-κB-RE reporter gene for 5 h, after which the induced nanoluciferase was measured using Nano-Glo substrate (Promega Corporation).

[0272] Results and discussion

[0273] Construction and generation of ACE2-Fc fusion protein

[0274] A series of ACE2-Fc fusion proteins were generated (Table 2) and their ability to neutralize SARS-CoV-2 infection and mediate Fc-dependent effector functions, which are typically attributed to the antibody's mechanism of action, was analyzed.

[0275] Table 2

[0276]

[0277] In an attempt to enhance affinity for the SARS-CoV-2 spike protein or confer and improve Fc-dependent effector function, the extracellular domains of three versions of ACE2 were fused with the Fc region of IgG1 that was either unmodified or altered by mutation (i.e., replacing histidine 429 with phenylalanine (H429F) or tyrosine (H429Y)) or by modified glycosylation (i.e., lacking core fucose; trACE2-Fc-kif).

[0278] Purification of ACE2-Fc fusion protein

[0279] Proteins were produced in Expi293 cells and purified by (an)ion exchange (IEX), followed by size exclusion chromatography (SEC) at pH 7.4. Figure 5 All fusion proteins showed IEX purification curves similar to those of flACE2-Fc-WT. Figure 5 A), which contains the main elution peak (peak). * ; Figure 5 A), and SDS-PAGE analysis of the collected fractions showed a single dominant species of approximately 270 kDa ( Figure 5 B). Apart from fusion proteins containing the H429Y mutation, SEC analysis and purification confirmed the presence of the major monomeric species ( Figure 5 C (Note: Monomeric species are considered to be single molecules (i.e., monomeric molecules) containing two (dimerized) copies of the corresponding ACE2-Fc fusion protein. These monomeric species are collected for further analysis, and only trace amounts of high molecular weight oligomers and other impurities are noticeable, such as flACE2-Fc-WT. Figure 5 In fact, the presence of similar monomeric species was also evident (not shown) after IEX purification of all fusion proteins using unmodified Fc-WT or Fc region components including H429F-modified Fc regions. For fusion proteins containing the H429Y mutation, SEC analysis ( Figure 5 D) (i.e., the major IEX peak from the flACE2-Fc H429Y mutant, not shown), and the major IEX peaks from the trACE2-Fc H429Y and EflACE2-Fc H429Y proteins (not shown), reveal the presence of a large number of oligomeric (Yoli) and monomeric (Ymn) species, the functional activities of which were subsequently assessed separately.

[0280] ELISA was used to bind the SARS-CoV-2 S ACE2-Fc fusion protein RBD.

[0281] 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 6 AC) are generally similar (i.e., EC) 50 The values ​​were 0.35 nM, 0.27 nM, and 0.25 nM, respectively. Figure 6 (D) The enhanced intrinsic affinity of EflACE2-Fc for binding to this bivalent form of RBD is not very pronounced, which appears upon fusion with mouse IgG1 Fc (RBD-Ig). The binding activity of various fusion proteins with the mutated Fc component is also equivalent, except for slightly lower affinity (EC). 50 The monomer flACE2-Fc-H429Y (with a concentration of 0.48 nM) Figure 6 D) and other H429Y Fc variants that also tend towards lower affinity levels ( Figure 6 ).

[0282] Oligopolymerization of the ACE2-Fc H429Y fusion protein is pH-dependent.

[0283] Oligopolymerization of the fusion protein, including the mutated H429Y Fc component, was examined by SEC separation at pH 5.0 using flACE2-Fc-H429Y prepared by IEX. This was compared with SEC separation performed at pH 7.4. Figure 5 D and Figure 7 Compared to A), SEC (conducted at pH 5.0) Figure 7 B) revealed a larger proportion of monomeric Y mn N-PAGE showed that it was purified to homogeneity ( Figure 7 C: Lane 1 compared to lane 2). Dialysis was performed at pH 7.4, followed by N-PAGE (…). Figure 7 C) and at pH 7.4 via SEC ( Figure 7 D) Both have effects on the purified monomer flACE2-Fc-H429Y mn Reanalysis was performed at pH 5.0. Re-exposure to pH 7.4 produced oligomer Y. oli and monomer Y mn Mixture of species ( Figure 7 C; Lane 2 pH 5.0 compared to Lane 5 pH 7.4), thus indicating some equilibrium among these forms at neutral pH. flACE2-Fc-H429Y prepared at pH 5.0 mn This demonstrates equivalent binding to RBD-Ig to other flACE2-Fc WT fusion proteins and Fc variants. Figure 7 E). It is also evident that the prevalence of oligomers is related to the tyrosine substitution of histidine 429, as this was not observed in fusion proteins with H429F Fc region components having phenylalanine substitution.

[0284] Assessment of virus neutralizing efficacy

[0285] The antiviral activity of the ACE2-Fc fusion protein was determined in a microneutralization assay of SARS-CoV-2 infection in Vero cells. Figure 8 ), of which EC 50 The endpoint corresponds to the neutralization of approximately 99% of the viral particles in the inoculum (Khoury DS et al., Nature Review Immunol 20(12):727-738, 2020).

[0286] The SARS-CoV-2 neutralizing endpoint of the truncated extracellular domain trACE2 (2.70 µM) was improved by approximately 10-fold through fusion with the wild-type Fc region of IgG1 (trACE2-Fc WT, 283 nM). Figure 8The improved efficacy is consistent with the improved affinity for binding to the SARS-CoV-2 spike RBD, as the ACE2-Fc bivalent resulting from the fusion of the ACE2 extracellular domain with the IgG Fc region is similar to that of flACE2-Fc WT. EflACE2-Fc-WT showed an approximately 20-fold (11 nM) improvement over flACE2-Fc WT and trACE2-Fc WT fusion proteins, and an approximately 200-fold improvement over the unfused trACE2.

[0287] Analysis of Fc modifications revealed several interesting differences. First, the H429Y mutation in trACE2-Fc and flACE2-Fc improved their viral neutralizing efficacy ( Figure 8 This is surprising because the neutralizing function of the ACE2-Fc fusion protein occurs at a modification far from the H429 site (i.e., in the fusion protein, the ACE2 peptide is expected to neutralize the virus, but the H429 residue is located distal to ACE2 in the CH3 domain of the Fc component). Secondly, trACE2-Fc-H429Y isolated by SEC at pH 7.4 was found... oli The neutralizing activity of the oligomeric form (21.9 nM) was 13 times greater than that of trACE2-Fc-WT. The oligomer Y of flACE2-Fc-H429Y... oli and monomer Y mn Both forms contain two (dimeric) copies of the ACE2-Fc fusion protein. Figure 5 B) (The endpoints were 10.0 nM and 20.9 nM, respectively; Figure 8 The fusion protein showed greater potency than flACE2-Fc WT (124 nM) and similar virus neutralization to that achieved with EflACE2-Fc WT (10.6 nM), but EflACE2-Fc-H429Y oli It appears to be more effective (4 nM), meaning that the neutralizing activity of EflACE2-Fc WT (10.6 nM) is more than 200 times that of unfused trACE2, or more than 600 times that of EflACE2-Fc-H429Y (4 nM). Figure 8 Therefore, the Fc-H429Y mutation increases SARS-CoV-2 neutralization in the trACE2-Fc and flACE2-Fc fusion protein formats, and also tends to have higher levels of potency when combined with the triple mutation of ACE2 with the inherently higher affinity of EflACE2-Fc.

[0288] The phenylalanine substitution (H429F) at histidine 429 of the ACE2-Fc fusion protein did not enhance neutralization.

[0289] ACE2-Fc Interaction between fusion protein and FcγR

[0290] Ramos-S cells conditioned with ACE2-Fc and dimeric recombinant soluble FcγR (Wines et al., 2016, ibid.) were used to assess the interaction of FcγRIIa and FcγRIIIa with ACE2-Fc fusion proteins by flow cytometry. The trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins all bound to FcγRIIa and FcγRIIIa. Figure 9 (A, 9B), however, variant proteins containing the mutated H429Y Fc component largely eliminate binding to both Fc receptor types.

[0291] Activation of FcγRIIIa by ACE2-Fc fusion protein

[0292] Antibody-dependent cytotoxicity (ADCC) and Fc-dependent viral clearance 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., Journal of Experimental Medicine 218(3):e20201993, 2021). Therefore, in this experiment, the ability of trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins and variant proteins to activate FcγRIIIa was evaluated. It was found that Ramos-S cells conditioned with ACE2-Fc fusion proteins containing the wild-type (WT) Fc region all initiated FcγRIIIa activation ( Figure 10 Surprisingly, the flACE2-Fc fusion protein induced a higher level of activation than trACE2-Fc, indicating that the collectrin domain included in the ACE2 component of the fusion protein substantially and surprisingly improved the efficacy of the ACE2-Fc fusion protein in activating FcγRIIIa. Furthermore, the increased affinity of the flACE2-Fc fusion protein for the SARS-CoV-2 spike protein was found not to alter the level of FcγRIIIa activation, which was equivalent to that of flACE2-Fc. Figure 10 D).

[0293] The presence of chifrine produced the trACE2-Fc fusion protein (van Berkel PHC et al., Biotechnology and Bioengineering 105(20:350-357, 2010), namely trACE2-Fc-kif, which also improved the moderate activation level of FcγRIIIa shown by trACE2-Fc, bringing it to a level equivalent to flACE2-Fc and EflACE2-Fc fusion proteins. Figure 10 D), and close to CD20 + Levels of therapeutic anti-CD20 mAb rituximab on Ramos-S cells ( Figure 10 A, 10D). Chifu base is a mannosidase inhibitor that prevents normal N-linked glycosylation, including core fucosylation, and in immunoglobulins, the lack of fucose on the heavy chain glycan at Asn297 is known to improve FcγRIIIa binding and activation (Ferrara C et al., Proceedings of the National Academy of Sciences 108(31):12669-12674, 2011). Therefore, it is hypothesized that similar treatments or amino acid residue substitutions to increase affinity for FcγRIIIa in flACE2-Fc and EflACE2-Fc fusion proteins (Wang et al., 2018 ibid.) may further improve their FcγRIII activation efficacy.

[0294] Modification of the trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins by mutations including the H429F Fc component does not affect the activation of FcγRIII in opsonized Ramos-S cells. Figure 10 In contrast, all ACE2-Fc fusion proteins, including the mutated H429Y Fc component, eliminated FcγRIIIa activation in cells, consistent with the aforementioned inability to bind to Fc receptors, particularly FcγRIIIa. Figure 9 B). Therefore, while enhancing virus neutralization, the H429Y-modified Fc component in the trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins has no activity for FcγR binding (B). Figure 9 ), and activates cells via FcγRIIIa ( Figure 10 The hierarchical structure for fusion protein activation of FcγRIIIa is flACE2-FcWT = EflACE2-Fc > trACE2-Fc, thus highlighting that the presence of the collectrin domain is a key component for optimal activation of FcγRIIIa by the ACE2-Fc fusion protein.

[0295] ACE2 Fc protein's role in complement fixation, activation, and lysis of SARS-CoV-2 spike cells

[0296] In an ELISA-based analysis using avidin-fixed RBD biotin, the ability of the ACE2-Fc fusion protein containing the mutated Fc region component to fix complement components C1q and C5-C9 was examined. Figure 11 AF) and, importantly, its ability to mediate complement-dependent killing of cells expressing the SARS-CoV-2 spike protein ( Figure 11 G). When RBD is restrictive ( Figure 11 When A, 11B), the fusion protein containing the mutated H429F Fc component showed surprisingly enhanced activity compared to the counterpart containing the wild-type Fc region. Additionally, C5b-9 (which forms a membrane attack complex on cells) Figure 11 The binding of E, 11F) is equivalent to a fusion protein containing a mutated H429F Fc region component. Furthermore, although oligomerization ( Figure 6 and 7 (characteristics associated with its superior CoV-2 neutralizing activity) Figure 8 However, compared to their wild-type Fc counterparts, both trACE2-Fc-H429Y and flACE2-Fc-H429Y fusion proteins showed little difference in C1q or C5-9 fixation. Figure 11 AF). Furthermore, it is clear that the trACE2-Fc fusion protein is more effective than flACE2-Fc in fixing complement C1q (compared to...). Figure 11 C, 11E and 11D, 11F); this may be due to the presence of the collectrin dimerization domain in flACE2-Fc reducing the segmental flexibility of the fusion protein and thus negatively affecting complement C1q binding. However, it is noteworthy that this difference is significantly reduced by the H429F Fc mutation in both trACE2-Fc-H429F and flACE2-Fc-H429F fusion proteins; this is particularly evident in the fixation of the C5b-C9 complex (C, 11E and 11D, 11F). Figure 11 E, 11F).

[0297] Surprisingly, the fusion protein containing the mutated H429F Fc region component is the only highly active ACE2-Fc fusion protein in Ramos-S cells exhibiting serum complement-dependent cytotoxicity (CDC). Figure 11 G). Although trACE2-Fc WT, trACE2-Fc-kif fusion protein, and flACE2-Fc WT have the ability to immobilize C1q and C5b-9 in ELISA assays ( Figure 11(A, 11C, 11E), but despite having a higher affinity for the SARS-CoV-2 spike protein, it still cannot induce complement-mediated cell death, nor can EflACE2-Fc WT. trACE2-Fc-H429Y mn The fusion protein also has activity in complement-mediated killing, albeit weakly.

[0298] Since it is well known that activation of the complement cascade can also lead to phagocytosis of cells, microbes, or particles, such as those modulated by C1q (or other complement fragments, such as C3b or C3bi; Ricklin D et al., Immunology Review 274(1):33-58, 2016) that bind to specific cell surface receptors on phagocytes, such as CR1 or CR3 (Vandendriessche S et al., Frontiers in Cell and Developmental Biology 9:624025, 2021); and the observed enhancement of Fc-dependent complement cleavage by the ACE2-Fc fusion protein with the H429Y mutation, it is believed that this enhancement will also be reflected in a similar enhancement of complement-dependent phagocytosis of ACE2-Fc-coated targets (e.g., viral particles) via complement receptors.

[0299] in conclusion

[0300] Immunotherapy proteins in fusion protein form have been found to offer considerable potential for treating or preventing coronavirus infection, comprising an angiotensin-converting enzyme 2 (ACE2) polypeptide (or a fragment thereof) fused to an Fc region component containing an H429 mutation. For example, selection of full-length or truncated ACE2 (i.e., fragments) and various modifications to the Fc component can also enable considerable “modulation” of antiviral agents to achieve antiviral effects. For instance, by including the Fc component in an immunotherapy protein containing an H429Y substitution, oligomeric immunotherapy proteins can be produced, exhibiting increased virus neutralization and elimination of FcγR binding and activation. On the other hand, by including the Fc component in an immunotherapy protein containing an H429F substitution, antiviral effects, including complement-dependent cytotoxicity (CDC) of cells expressing spike proteins (e.g., infected cells), can be achieved. Therefore, these unique Fc mutations enable complementary approaches to modulate the function of the Fc region component, which may facilitate the development of ACE2-Fc fusion proteins and other therapeutically significant fusion proteins by allowing selection of desired functional profiles.

[0301] Example 2: Chimeric anti-TNP IgG1 antibody containing the H429 mutation and its activity analysis

[0302] Methods and Materials

[0303] Production of anti-TNP human IgG and mutant anti-TNP human IgG plasmid constructs

[0304] The variable weight (V) of mouse anti-trinitrophenyl (anti-TNP) antibody TIB142 H ) and light (V) L Chimeric anti-TNP human IgG antibody constructs, consisting of sequences from the Fc region and the constant weight (CH) region from human IgG subclasses, have been previously described in detail. hIgG1 (Patel D et al., *Journal of Immunology* 184(11):6283-6292, 2010), hIgG2, and hIgG4 (Wines et al., 2016, ibid.). All chimeric antibody sequences were subcloned into the pCR3 vector. Mutations involving the substitutions of H429F, H429Q, H429E, H429S, H433A, and H435A were introduced into the cDNA sequences encoding the Fc region components using standard molecular biology techniques.

[0305] IgG antibodies were expressed and generated using Expi293 cells.

[0306] As previously described, IgG antibodies were generated in Expi293 human embryonic kidney cells (Wines et al., 2016, ibid.). In short, for both cell growth and protein production, Expi293 cells were maintained in Expi293 expression medium (Gibco, Waltham, MA, United States of America). Cells were simultaneously transfected using the Expifectamine transfection kit (Lifetechnologies) with IgG heavy chain plasmid (15 μg) and light chain plasmid (15 μg) diluted in Opti-MEM I reduced serum medium (Gibco), and then cultured for four days. The culture supernatant was clarified by centrifugation and filtered through a 0.2 μm filter. IgG was then purified by affinity chromatography using a Hi-Trap HP Protein A column (GE Healthcare LifeSciences, Marlborough, MA, United States of America). Elution was performed with 0.1 M citric acid at pH 3.5, followed by neutralization with 1 M Tris-HCl at pH 9.0 and dialyzing against PBS at pH 7.5. Aggregates were subsequently removed by gel filtration on a Superose 6 10 / 300GL column (GE Healthcare LifeSciences), and monomeric IgG peak fractions were collected. The antigen-binding activity of all antibody formulations was tested by ELISA on BSA-TNP as described (Wines et al., 2016, ibid.).

[0307] Antigen binding activity was assessed using monoclonal antibody TNP mAb.

[0308] Antigen-binding activity of anti-TNP mAb can be assessed by ELISA through binding to TNP haptenized BSA (TNP-BSA), as previously described in Winees et al., 2016, ibid.

[0309] ELISA Immunoassay for Complement Fixation Using mAb

[0310] Ninety-six-well MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated overnight at 4°C with PBS containing 20 μg / ml TNP-BSA. The next morning, the plates were blocked at 37°C with PBS containing 0.1% (w / v) casein for 2 hours, and then incubated at room temperature for 2 hours with anti-TNP mAb (concentrations between 4 μg / ml and 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), followed by incubation at room temperature for 1 hour with a 1 / 2000 dilution of rabbit anti-C1q IgG (Kurtovic L et al., BMC Medicine 17:45 2019). In the assay for C5b-C9 fixation, the TNP: anti-TNP plate was 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, then washed, and then incubated with a 1 / 2000 dilution of HRP-conjugated goat anti-rabbit IgG (Merck Millipore) at room temperature for 1 hour, followed by incubation with TMB substrate (Lifetech) at room temperature for 15–20 minutes. Reactivity was stopped with 1 M sulfuric acid, and absorbance was measured at OD450 nm. Test samples and reagents were prepared in PBS containing 0.1% (w / v) casein, and the plate was washed three times with PBS containing 0.05% (v / v) Tween 20 between each step. Samples were tested in duplicate, and background reactivity was corrected using negative control wells (with antibodies omitted).

[0311] Results and discussion

[0312] Anti-TNP antibody construct

[0313] The TNP-WT mAb used in this example contains either of two human heavy chain isotypes; firstly, an IgG1 isotype polypeptide (SEQ ID NO: 32) containing, in N-terminal to C-terminal order, the TNP-specific V of the mouse monoclonal antibody TIB142 fused with the CH1-hinge-CH2-CH3 domain of human IgG1 encoded by cDNA (SEQ ID NO: 33). H The domain (Patel D et al., *Journal of Immunology* 184: 6283-6292, 2010); and secondly, the IgG2 isotype polypeptide (SEQ ID NO: 34) contains, in the order from N-terminus to C-terminus, the TNP-specific V of the mouse monoclonal antibody TIB142 fused with the CH1-hinge-CH2-CH3 domain of human IgG2 encoded by codon-optimized cDNA having the sequence shown as SEQ ID NO: 35. H Domain. The TNP-specific light chain polypeptide (SEQ ID NO: 36) comprises a TNP-specific V domain of mAbTIB142 fused with a human κ chain constant domain encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 37. L Structural domain.

[0314] IgG modification enhances complement fixation

[0315] When antibodies such as mAbs bind to their target antigens, the classical complement pathway may be activated, leading to physiological effects including the destruction of target cells via complement-dependent cleavage (CDC) or complement-mediated antibody-dependent phagocytosis (C'ADCP) by specific phagocytes via specific receptors for complement proteins and fragments. Activation of this major amplification effector subsystem of innate immunity depends primarily on and is initiated by the binding of the hexameric complement protein C1q, which is part of the C1 complex. This binding of C1q to antibodies that bind to their target antigens ultimately results in the formation of the membrane attack complex (MAC), which includes other complement components (C5b, C6, C7, C8, C9).

[0316] Because the H429F Fc region component mutation conferred effective CDC in the ACE2-Fc fusion protein (see Example 1), these were evaluated in human IgG1 under different conditions of antigen density or infused mAb concentration. Figure 12 ) and several other mutations ( Figure 13The effects of this study were examined in ELISA for complement fixation with Fc component mutations in the context of a complete (chimeric) IgG1 antibody recognizing both the mouse V domain and the human IgG1 constant domain of the TNP hapten. Surprisingly, substitution of histidine at position 429 in the TNP-IgG1-H429F mAb resulted in enhanced C1q binding compared to unmodified TNP-IgG1-WT mAb. Figure 12 Next, the generation of the membrane attack complex protein of TNP-specific mAbs was evaluated. Figure 12 Similarly, TNP-IgG1-H429F mAb exhibits enhanced complement activation, as seen in increased formation of MAC proteins (C5b, C6, C7, C9), and importantly, this is entirely consistent with the enhanced C1q binding ability conferred by the observed H429F mutation. Thus, modification at position 429 can unexpectedly alter the properties of the antibody. This enhanced function is surprising because amino acid 429 is embedded within the CH3 domain of IgG1 and is located away from the complement C1q binding surface and the leukocyte Fc receptor binding site in the CH2 region of the H chain (Hogarth PM and Pietersz GA. *Nature Reviews Drug Discovery* 11:311-331, 2012; Chenoweth et al., 2020, ibid.).

[0317] The H429F mutation in IgG1 antibodies enhances C1q ( Figure 12 A) and C5b-C9 fixed ( Figure 12 B) Both. When lower-density TNP:BSA antigen is opsonized by mutated IgG, the complement-enhancing activity of mutant IgG1 antibodies containing the H429F mutation is most pronounced, particularly in C1q fixation. Figure 12 Therefore, in the context of the ACE2-Fc fusion protein and the intact IgG1 antibody, the H429F Fc component mutation enhances complement activation.

[0318] As described, the antigen-binding activity of anti-TNP mAb, TNP-IgG1-WT, TNP-IgG2-WT, and modified (mutant) mAb, TNP-IgG1-H429F, to TNP haptenized BSA (TNP-BSA) was assessed by ELISA (Wines et al., 2016, ibid.). All mAbs showed substantially equivalent binding activity to TNP-BSA. Figure 12 C), thus indicating that the enhanced complement activation of Fc-modified anti-TNP antibodies is due to Fc mutations, rather than differences in antigen-binding activity.

[0319] in conclusion

[0320] The data obtained in this example indicate that the IgG-H429F mutant can enhance C1q and C5b-C9 fixation, thereby increasing complement activation, and thus can be expected to induce useful complement-based effector functions.

[0321] Example 3: Generation and analysis of therapeutic antibodies in modified form containing the H429 mutation.

[0322] In this example, a number of irrelevant chimeric mAbs containing different antigen-binding variable domains were used to evaluate the effect of the modification site H429 (i.e., by amino acid substitution) on antibody properties, where the antigen-binding variable domain binds to irrelevant epitopes in a range of different target molecules on different cell types. These antibodies are “based” on various commercially available mAbs that are clinically significant in some cases. Specifically, the experiments aimed to determine the possible functional effects of the amino acid substitution at position 429 in antibodies carrying the V domains of anti-HER2 mAbs trastuzumab and pertuzumab; anti-CD20 mAb carrying the V domain of rituximab or 11B8; and anti-CD38 mAb daratumumab.

[0323] Methods and Materials

[0324] Antibodies and antibody constructs

[0325] DNA sequences obtained via RT-PCR, or synthetic DNA corresponding to the variable and constant sequences of immunoglobulins, are assembled using standard molecular biology techniques, including ligation and Gibson assembly (NEBuilder, New England Biolabs), or used as complete synthetic DNA to encode the complete H and L chains of immunoglobulins. These sequences are used in expression vectors such as pcDNA3.1, pcDNA3.4 (Thermo Fisher Scientific), and pCIneo (Promega).

[0326] The unmodified mAb used in this example is formatted on the human IgG heavy chain and generated using the human κ light chain. The unmodified mAb includes the specific V of the indicated mAb. H and V L The domain is referred to as the wild-type (WT) form; for example, as previously described, the “WT” trastuzumab antibody used in this example contains the wild-type (WT) HER2-specific V domain of trastuzumab. H and V L Structural domain (https: / / go.drugbank.com / drugs / DB00072).

[0327] More specifically, the trastuzumab WT mAb used in this example comprises the HER2-specific heavy chain polypeptide described at https: / / go.drugbank.com / drugs / DB00072 (whose amino acid sequence is provided as SEQ ID NO: 12), which contains, in N-terminal to C-terminal order, the HER2-specific V of trastuzumab fused with the CH1-hinge-CH2-CH3 domain of human IgG1. H The domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 13. Similarly, the peptide of the anti-HER2 mAb trastuzumab light chain is as described in https: / / go.drugbank.com / drugs / DB00072 (SEQ ID NO: 14) and comprises a HER2-specific V of trastuzumab fused with a human κ constant domain. L The structural domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 15.

[0328] The 11B8-WT mAb used in this example comprises a previously described CD20-specific heavy chain polypeptide (US Patent No. 8,529,902) (SEQ ID NO: 16), which contains, in N-terminal to C-terminal order, a CD20-specific V of 11B8 fused with the CH1-hinge-CH2-CH3 domain of human IgG1. H The domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 17. Similarly, the polypeptide against the CD20 mAb 11B8 light chain is as previously described (SEQ ID NO: 18) and contains a CD-20 specific V of 11B8 fused with the human κ constant domain. L The structural domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 19.

[0329] The daratumumab-WT mAb used in this example comprises the previously described CD38-specific heavy chain polypeptide (https: / / go.drugbank.com / drugs / DB09331) (SEQ ID NO: 20), which contains, in N-terminal to C-terminal order, the CD38-specific V of the daratumumab mAb fused with the CH1-hinge-CH2-CH3 domain of human IgG1. HThe domain 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 as previously described in https: / / go.drugbank.com / drugs / DB09331 (SEQ ID NO: 22), which contains a CD38-specific V of the daratumumab mAb fused with a human κ constant domain. L The structural domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 23.

[0330] The pertuzumab-WT mAb used in this example comprises a HER2-specific heavy chain polypeptide (SEQ ID NO: 24) as previously described at https: / / go.drugbank.com / drugs / DB06366, which contains, in N-terminus to C-terminus, a HER2-specific V-chain peptide fused to the CH1-hinge-CH2-CH3 domain of human IgG1. H The domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 25. Similarly, the polypeptide of the anti-HER2 mAb trastuzumab light chain is as previously described in https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 26), which comprises a HER2-specific V of pertuzumab mAb fused with a human κ constant domain. L The structural domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 27.

[0331] The rituximab-WT mAb used in this example contains a CD20-specific heavy chain polypeptide (as described in https: / / go.drugbank.com / drugs / DB00073) (SEQ ID NO: 28), which contains, in N-terminal to C-terminal order, the CD20-specific V of rituximab fused with the CH1-hinge-CH2-CH3 domain of human IgG1. H The domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 29. Similarly, the peptide of the anti-CD20 mAb rituximab light chain is as previously described in https: / / go.drugbank.com / drugs / DB000723 (SEQ ID NO: 30), which comprises a CD20-specific V of rituximab fused with a human κ constant domain. L The structural domain is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0332] Synthesis of unmodified and mutated heavy chains

[0333] Antibody expression vectors are produced using standard methods known to those skilled in the art. In short, antibody expression vectors consist of synthetic polynucleotide sequences encoding either the heavy or light chain of an antibody, appropriately placed within a plasmid, such as pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors for antibodies with different specificities are produced using existing variable domains (V... H or V L It is produced by cutting at a restrictive site at the boundary. Then, according to the manufacturer's instructions, it is reacted with NEBuilder (New England Biolabs) to incorporate a new synthetic DNA that encodes a new V domain and is side-joined with a sequence (e.g., 25 nucleotides) homologous to the cutting vector.

[0334] According to the manufacturer's instructions, Fc variants are generated by synthesizing a synthetic polynucleotide sequence that encodes the variant, or by cutting the Fc-encoding sequence of an antibody expression plasmid with an appropriate restriction enzyme, and by incorporating new mutagenic synthetic DNA into the reaction using NEBuilder (New England Biolabs).

[0335] Expression of antibody constructs

[0336] Antibody expression was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific). Expi293F cells were cultured in EXPI expression medium (Life Sciences, Inc.), and the cells were divided to a size of 2 x 10⁶ cells 24 hours prior to transfection. 6 A concentration of 1 live cells / ml. On the day of transfection, 7.5 x 10⁻⁶ cells / ml were used. 7One live log-phase cell was centrifuged and resuspended in 25 ml of preheated, antibiotic-free Expi293 expression medium and maintained at 37°C until transfection. Transfection was then performed at room temperature using the Expifectamine transfection kit from LifeScience, as described below. Eighty μL of ExpiFectamine 293 reagent was diluted with 1.5 ml of Opti-MEM-I reduced serum medium (Gibco) and incubated at room temperature for 5 minutes. Thirty µg of DNA (15 µg H-strand DNA and 15 µg L-strand DNA) was diluted in 1.5 ml of Opti-MEM-I reduced serum medium and added to the diluted ExpiFectamine reagent. The mixture was incubated at room temperature for 20–30 minutes and then added dropwise to the Expi293F cell suspension. The cells were then incubated at 37°C for 16–18 hours, at which point 150 µL of manufacturer's enhancer 1 and 1.5 ml of enhancer 2 were added, and the cells were incubated at 37°C for another four days.

[0337] Cell cultures were collected and centrifuged at 2500 rpm for 20–30 minutes. The supernatant was filtered through a 0.2 µm high-flow-rate filter (Sartorius AG, Göttingen, Germany) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0338] Protein A affinity purification of mAb

[0339] mAbs were purified from the supernatant of transfected Expi293F cells using protein A affinity chromatography. In short, a Hi-trap™ protein A high-performance column (GE Healthcare Life Sciences) was washed and equilibrated in binding buffer (20 mM NaH2PO4, pH 7.0), loaded with cell culture supernatant, washed with binding buffer to baseline OD 280 nm, and the bound antibody was eluted with 0.1 M sodium citrate trihydrate dihydrate (pH 3.5). A 1 ml fraction was collected and immediately neutralized with 1 M Tris-HCl pH 9.0, and the fractions containing the antibody were combined.

[0340] Size exclusion chromatography (SEC) of mAb

[0341] Following affinity purification of protein A, the antibody was further purified and characterized using size exclusion chromatography (SEC). The protein A-purified antibody was concentrated to an OD280 nm of 6–8 using a 30 kDa molecular weight cutoff centrifuge (Merck Millipore). A Superose 6 10 / 300GL column (GE Healthcare Life Sciences) was equilibrated in PBS at pH 7.2, and the concentrated protein A-purified antibody was then loaded and separated in PBS at a flow rate of 0.5 ml / min, with a 0.5 ml fraction collected. For some mAbs, SEC was performed at pH 5.0; therefore, the concentrated protein A-purified mAb was 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-equilibrated in the same buffer. The dialyzed antibody was applied to the Superose column for SEC at a flow rate of 0.5 ml / min, and a 0.5 ml fraction was collected from the column.

[0342] Assessment of antigen binding using monoclonal antibodies

[0343] Prior to functional analysis, antigen binding of purified antibodies was tested. Antigen recognition of mAbs that recognize cell surface antigens was tested by flow cytometry, as previously described in Trist et al., *Journal of Immunology* 192(2):792-803, 2014. Binding of anti-CD20 mAbs based on rituximab or 11B8 and anti-CD38 mAbs based on daratumumab to Ramos lymphoma cells expressing CD20 and CD38 was tested. Anti-HER2 mAbs based on trastuzumab and pertuzumab mAbs were tested on the HER2-expressing ovarian cancer cell line SK-OV-3. Briefly, the mAbs were titrated by sequential 2-fold dilutions in 25 µl of FACS buffer (PBS containing 0.5% (w / v) BSA). The mAbs were then converted to 25 µl of 5 x 10⁻⁵ mAbs. 6 / ml of target cells were added to the titrated mAb and incubated on ice for 30 min. Cells were then washed twice in FACS buffer, resuspended in 50 μL of anti-human IgG (Fab')2-Alexa 647 conjugate and incubated on ice for 30 min, washed twice in cold FACS buffer, and then resuspended in 200 µl of FACS buffer. Cells were analyzed by flow cytometry using a BD FACSCanto™ II flow cytometer.

[0344] Flow cytometry detection of C1q binding to cells conditioned with monoclonal antibodies

[0345] The binding of C1q to antibody-mediated cells was assessed by flow cytometry. Ramos lymphoma cells or SK-OV-3 adenocarcinoma cells (5 x 10⁻⁶) were used. 6 Cells (in FACS buffer) were incubated on ice for 30 min with serially diluted antibodies based on either anti-CD20 rituximab or anti-HER2 trastuzumab. Cells were washed twice in FACS buffer, resuspended in normal human serum diluted 1 / 3 in FACS buffer, and incubated on ice for another 30 min. Cells were then washed twice in cold FACS buffer, and C1q binding was detected by resuspending cells in rabbit antiserum (1:500 dilution) for detecting human C1q and incubating on ice for another 30 min. Cells were then washed twice in cold FACS buffer, resuspended in phycoerythrin-conjugated donkey antiserum for detecting rabbit antibodies for 30 min, washed twice more, and resuspended in 200 µL of ice-cold FACS buffer for analysis on a BD FACSCanto™ II flow cytometer (Beddy Medical).

[0346] complement-dependent lysis of cells

[0347] After conditioning target cells with mAbs, CDC was measured by flow cytometry using the Zombie Green Immobilization Viability Kit (Baijin Biotechnology). Starting from the initial concentration indicated on the graph, the mAb of interest was serially diluted two-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). Thus, for each WT mAb or modified mAb (i.e., H429- mutant mAb) or combination thereof, a concentration of 5 x 10⁻⁵ ppm was then achieved in 25 μl of PBS / BSA / G. 6 / ml of target cells were added to the titrated mAb and incubated on ice for 30 min. The cells were then washed twice in buffer and 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 min. After incubation, the cells were washed twice in BSA-free cold buffer and resuspended in 50 µl of Zombie Green (prepared in DMSO according to manufacturer's instructions; Baijin Biotechnology Co., Ltd.) diluted 1 / 500 in PBS and incubated on ice in the dark for another 30 min. These cells were then washed once in buffer, resuspended in 50 µl of 2% paraformaldehyde / BSA / PBS / G, incubated on ice for 30 min, 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 (Beddy Medical).

[0348] MAb collaboration to enhance C1q binding and CDC

[0349] In the presence of a 1 / 3 dilution with normal human serum, 5 x 10⁻⁶ PBS / BSA / G will be added to 25 µl of PBS / BSA / G. 5 Ramos cells / ml were incubated with 2-fold diluted rituximab-WT in the presence of 0.025 µg / ml daratumumab-WT or rituximab-H429F, or 2-fold diluted rituximab-H429F in the presence of 0.025 µg / ml daratumumab-H429F mutant or 0.5 µg / ml 11B8-H429F mutant, at 37°C for 30 min. The cells were washed twice with PBS, incubated on ice with 1 / 500 Zombie Green for 30 min, 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 (Beddy Medical).

[0350] Results and discussion

[0351] Antigen binding as measured by monoclonal antibodies

[0352] Prior to functional analysis, the antigen-binding ability of the purified antibody used in this example was tested and confirmed.

[0353] The purified mAb for detecting cell surface antigens was also tested by flow cytometry against antigen-positive Ramos cells (CD20). + CD38 + ) or SK-OV-3 cells (HER2+ The binding of ) and all mAbs showed easily detectable antigen binding levels ( Figure 14 Furthermore, within each group, modified mAbs carrying mutations in the IgG heavy chain exhibited similar binding activity to their unmodified (WT) forms (e.g., anti-CD20 mAb rituximab-WT, rituximab-H429F, and rituximab-H429Y mutants showed equivalent homogeneous binding to CD20-expressing Ramos cells). Similarly, anti-CD38 daratumumab-WT mAb and its CH3-modified mutant daratumumab-H429F, as well as anti-CD20 11B8-WT and its CH3-modified mutant, all gave binding curves equivalent to their corresponding wild-type forms. Figure 14 Similarly, anti-HER2 mAb trastuzumab-WT and its modified CH3 mutant forms, trastuzumab-H429F and trastuzumab-H429Y, showed the same easily detectable and equivalent binding to SK-OV-3 adenocarcinoma cells as pertuzumab-WT and its modified CH3 mutant. Figure 14 ).

[0354] The effect of the modification at position 429 on antibody properties was further investigated using mAbs that bind to cell surface molecules. Specifically, it was found that the substitution at H429 altered the physical properties of the antibody and selectively promoted antibody oligomerization. For example, using mutants of rituximab antibodies; namely, rituximab-H429F mAb and rituximab-H429Y mAb, in which the incorporated tyrosine and phenylalanine amino acids are structurally similar, it was found that although both rituximab-H429F mAb and rituximab-H429Y mAb eluted as a single homogeneous peak from the protein A affinity column, their elution characteristics were similar to those of unmodified rituximab-WT IgG. Figure 15 A), but purified by size exclusion chromatography (SEC) at pH 7.2 ( Figure 16 A) shows that rituximab-H429Y mAb IgG exhibits a surprisingly different SEC curve (i.e., compared to the curves of rituximab-WT and rituximab-H429F antibodies). In other words, the rituximab-H429Y mAb SEC curve reveals two distinct peaks, one for IgG and one for WT. Figure 16 The peak to the right of the vertical line in A corresponds to a single IgG peak in the SEC of rituximab-WT and rituximab-H429F mAb, and the second peak is considered to contain the preformed oligomer (IgG(oli)) of rituximab-H429Y (see [link to stoichiometry]). Figure 16A (left side of the vertical line), indicating the balance between the formation of oligomeric and non-oligomeric forms. While the presence of both oligomeric and non-oligomeric IgG in rituximab-H429Y mAb is surprising and different from rituximab-WT IgG, it is even more surprising that this is different from what was observed in rituximab-H429F mAb. Therefore, the distinct biophysical properties of rituximab-H429Y and rituximab-H429F mAb are determined solely by the presence of a hydroxyl group in the tyrosine residue at position 429 of rituximab-H429Y mAb.

[0355] SDS-PAGE revealed oligomerization (IgG) observed in the SEC IgG of rituximab-H429Y mAb. oli The non-oligomeric (IgG) forms behaved identically to each other and were identical to rituximab-WT IgG and rituximab-H429F mAb IgG. That is, although rituximab-H429Y IgG... oli It exhibits oligomeric properties, but under non-reducing conditions, it migrates as a single approximately 150 kDa IgG species identical to its non-oligomeric rituximab-H429Y IgG (H2L2). Importantly, this indicates that the oligomerization of mAbs promoted by the H429Y mutation is inherently non-covalent. Figure 17 A). Furthermore, this clearly demonstrates that rituximab-H429Y can function as a preformed oligomer of IgG (IgG, H2L2) and IgG (IgG) at pH 7.2. oli Both are present, resulting from non-covalent association between IgG heavy chains carrying the H429Y mutation. Further analysis under reducing conditions (by dithiothreitol (DTT) reduction) showed that these non-covalent oligomers of rituximab-H429Y, as well as non-oligomeric rituximab-H429Y IgG, decomposed into the expected approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species, identical to the SDS-PAGE characteristics of rituximab-WT and rituximab-H429F mutants.

[0356] Therefore, the H429Y modification endows rituximab with novel properties, in which the mAb exists as a pre-formed non-covalent oligomer of IgG in equilibrium with a single IgG molecule in solution at pH 7.2. In contrast, rituximab-H429F and rituximab-WT mAb exist solely as a single IgG species in solution. Thus, the choice of amino acid at position H429 unpredictably affects the physical properties of rituximab IgG.

[0357] Furthermore, these effects of the mutation at position H429 were found to be independent of the V domain. In other words, equivalence assessments of the H429 substitution in trastuzumab mAbs unrelated to rituximab yielded similar results for detecting cell surface molecules structurally different from CD20 (i.e., HER2) (see [link to relevant documentation]). Figure 15 B, 16B, and 17B). Specifically, elution of trastuzumab-WT IgG from the protein A affinity matrix with citrate buffer (pH 3.0) produced a homogeneous single peak (B, 16B, and 17B). Figure 15 B), the elution of trastuzumab-H429F and trastuzumab-H429Y mutant mAbs was also similar (the single IgG peak was consistent with the peak obtained from unmodified trastuzumab-WT IgG mAb). Then, size exclusion chromatography (SEC) was performed at pH 7.2. Figure 16 B) found that unmodified trastuzumab-WT and trastuzumab-H429F mAb each contained the expected single major IgG species, while trastuzumab-H429Y mAb showed two distinct peaks ( Figure 16 B). The first trastuzumab-H429Y IgG peak was consistent with the single IgG peaks observed in trastuzumab-WT mAb and trastuzumab-H429F mAb. Figure 16 B, to the right of the vertical line), and the second trastuzumab-H429Y IgG peak contains oligomeric IgG (IgG oli )( Figure 16 B, to the left of the vertical line). Additionally, SDS-PAGE ( Figure 17 (B) shows that, under non-reducing conditions (i.e., no disulfide bond reduction), the IgG peaks of both trastuzumab-WT and trastuzumab-H429F mAb migrated with the expected mass of 150 kDa. Furthermore, in the case of trastuzumab-H429Y mAb, SDS-PAGE ( Figure 17 B) shows that, under non-reducing conditions, oligo-trastuzumab-H429Y (IgG) oli The non-oligomeric (IgG H2L2) species of trastuzumab-H429Y IgG migrated identically as a single 150 kDa species. This clearly demonstrates that trastuzumab-H429Y can exist as both a single IgG and a preformed oligomer of IgG at pH 7.2, which is caused by non-covalent association between IgG heavy chains carrying the H429Y mutation.

[0358] Therefore, the amino acid substitution at position 429 in the trastuzumab heavy chain confers the same properties as observed with equivalent substitutions in rituximab-based mAbs, and thus, the effects of the H249Y mutation on the physical properties (particularly oligomerization) of the mutated mAb are independent of antibody specificity, molecular target, epitope, and V. H and V L Structural domain.

[0359] Preformed oligomeric and non-oligomeric H429Y IgG antibodies showed enhanced CDC

[0360] While not wishing to be bound by theory, it is believed that oligomerization, particularly hexamerization, of mAbs provides an optimal basis for C1q binding and activation of the complement cascade, leading to complement-dependent effector responses (e.g., complement-dependent cytotoxic (CDC) phagocytosis or killing of target cells). Such oligomers / hexamers can form in solution or on the target (i.e., “on-target” oligomerization or assembly).

[0361] The effect of H429Y modification of the CH3 domain of the IgG heavy chain on antibody effector function was assessed by complement-dependent cytotoxicity assay (CDC) (see [link to cytotoxicity assay]). Figure 18 For example, using CD20-positive Ramos lymphoma cells as target cells, the effect of the H429 mutation in both oligomeric and non-oligomeric forms of rituximab-H429Y on CDC efficacy was determined. Therefore, both oligomeric rituximab-H429Y (IgG(oli)) and non-oligomeric IgG (H2L2) forms isolated by SEC at pH 7.2 were compared. Figure 18 The CDC efficacy of A) was evaluated and compared with that of unmodified rituximab-WT IgG. In the presence of either rituximab-WT IgG or non-oligomeric rituximab-H429Y IgG (H2L2) Figure 18 A's p1 fraction) or oligorituximab-H429Y IgG H2L2 species ( Figure 18 Ramos cells were incubated with serially diluted mAb (p2 fraction of A). Normal human serum (1 / 3 dilution) was then added as a complement source, and the proportion of Ramos cells killed by CDC at each mAb concentration was determined by flow cytometry. Figure 18 B). Compared with unmodified rituximab-WT, non-oligomeric rituximab-H429Y IgG (H2L2) (P1 fraction) showed a surprising enhancement in CDC potency. Compared with rituximab-WT, rituximab-H429Y (IgG) oliThe non-covalent oligomeric IgG form (p2 fraction) also showed enhanced CDC. Interestingly, both the oligomeric p2 and non-oligomeric p1 forms showed similarly enhanced CDC potency, consistent with the formation of hexamers in a solution at pH 7.2 prior to binding to target cells by the H429Y oligomer, or, in the case of the non-oligomeric IgG form (H2L2), the formation of hexamers on the target cell surface after antigen binding, thereby providing optimal Fc conformation for C1q binding and thus enhancing complement activation, as observed in the mAb-H429Y form of IgG.

[0362] Therefore, replacing H429 with tyrosine not only alters the physical properties of mAbs that allow oligomerization in solution, but also enhances complement-dependent killing of target cells, regardless of whether IgG oligomerizes in solution or on the target.

[0363] Oligomerization of mAbs, including those with the H429Y mutation, is controlled by altering the pH.

[0364] The non-covalent oligomerization properties of mAb modified with H429Y in the CH3 domain were further investigated by changing the pH of the buffer (environment). Figure 18 C). Specifically, trastuzumab-H429Y mutant mAb (e.g., purified by protein A affinity chromatography) was dialyzed into a buffer at pH 7.2 and then subjected to SEC at pH 7.2, or dialyzed into a 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 lowering the buffer pH completely reversed the formation of IgG hexamer oligomers, and only the IgG (H2L2) species were significantly ( Figure 18 (C) Therefore, the H429Y modification of the CH3 domain promotes pH-sensitive non-covalent oligomerization of mAb, and thereby promotes oligomerization / hexamerization in solution or on the target to enhance complement effector potency. Furthermore, the ability to control oligomerization by altering pH may be useful for preparing immunoglobulins carrying this modification.

[0365] The H429F mutation enhances the activation of the serum complement cascade.

[0366] The effect of modification of the CH3 domain at position 429 of the H chain on C1q binding was investigated using flow cytometry on cells treated with an unrelated mAb that recognizes CD20 or HER2 and carries the H429F mutation in the CH3 region of its heavy chain. Results are as follows: Figure 19 As shown in AD. C1q binding was evaluated in CD20 Ramos cells treated with rituximab-WT mAb or rituximab-H429F mAb. Figure 19A, B). Similarly, C1q binding was also measured in HER2-positive SK-OV-3 cells treated with trastuzumab-WT mAb or trastuzumab-H429F mAb. Figure 19 C, D). C1q and Ramos cells treated with rituximab-WT mAb ( Figure 19 A) and SK-OV-3 cells treated with trastuzumab-WT ( Figure 19 The binding of C) was detectably higher than that of background controls. Importantly, compared with rituximab-WT and trastuzumab-WT, C1q binding to Ramos cells treated with rituximab-H429F was significantly higher. Figure 19 B) and SK-OV-3 cells treated with trastuzumab-H429F ( Figure 19 D) Both binding is enhanced. Therefore, the substitution of H429 in the CH3 domain of the heavy chain enhances C1q binding in these unrelated anti-CD20 and anti-HER2 mAbs. Thus, it is equally clear that this functional enhancement of H429 modification is independent of the antibody variable region and the detected molecular targets and epitopes. Furthermore, the enhanced C1q binding is consistent with enhanced antibody-dependent complement activation in the classical complement pathway, which leads to the development of MAC, resulting in cell lysis via CDC.

[0367] 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 with the killing efficacy of unmodified rituximab-WT. Figure 19 E). Specifically, at each of the indicated mAb concentrations, the proportion of Ramos cells killed by CDC was assessed by flow cytometry. Figure 19 E). Similar to the surprising enhancement of C1q binding conferred by the H429F modification of the CH3 domain in rituximab-H429F mAb ( Figure 19 Compared to 19B, the CDC killing efficacy (ECG) of rituximab-WT compared to unmodified rituximab-WT was [missing information]. 50 It also significantly improved (> 10 times) Figure 19 E). The surprising improvement in C1q binding and CDC killing efficacy resulting from the H429F mutation is consistent with the oligomerization (especially hexamerization) of the antibody on the target surface and appears to provide the optimal mAb Fc configuration for binding to the hexamer C1q, as illustrated by the analysis of rituximab-H429F mAb.

[0368] Enhanced complement activation in mAbs with the H429 mutation is epitope-independent.

[0369] As discussed above, the H429 substitution in the anti-CD20 rituximab mAb strongly increased its CDC activity; to assess 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. Results are as follows... Figure 20-22 As shown.

[0370] Type II anti-CD20 mAb 11B8 exhibited naturally poorer CDC activity, but importantly, epitopes different from those of type I anti-CD20 mAb rituximab were also detected (Meyer S et al., British Journal of Hematology 180(6):808-820, 2018). 11B8-WT mAb and its mutants including the H429F substitution were generated as described above and purified separately by protein A affinity chromatography. Figure 20 ). 11B8-WT and 11B8-H429F mAb yielded a single homogeneous IgG peak ( Figure 20 This indicates that the modification failed to alter the purification properties of the modified mAb compared to the unmodified 11B8-WT mAb. Similarly, size exclusion chromatography revealed that 11B8-WT mAb contains a single non-oligomeric IgG (H2L2) species. Figure 21 The same applies to A), 11B8-H429F mAb. SDS-PAGE analysis confirmed this. Figure 21 B), the SDS-PAGE analysis revealed the expected 150 kDa IgG species prior to disulfide bond reduction in wild-type and mutant mAbs, which decomposed into approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in DTT. Figure 21 B).

[0371] 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 cytotoxicity of each mAb concentration against Ramos cells was assessed. 11B8-WT mAb failed to induce significant CDC, but in contrast, Fc-modified 11B8-H429F mAb mediated effective CDC. Figure 22 Therefore, CDC potency is enhanced in two different and unrelated mAbs, rituximab-H429F and 11B8-H429F (comparison). Figure 19(E and 22), indicating that the improvement in CDC resulting from modification of the H429 position can be achieved in different mAbs targeting different epitopes within the same target molecule. This also indicates that the enhanced CDC obtained through CH3 modification is independent of the variable domains of the modified antibody.

[0372] The enhancing effect of H429F is independent of antibody, molecular target, and cell type.

[0373] The enhanced complement activation potency conferred by H429 substitution for other mAbs was evaluated in the antibody daratumumab, which recognizes a fourth and irrelevant molecular target, CD38, a structurally independent cell surface molecule unrelated to CD20 or HER2 (de Weers M et al., *Journal of Immunology* 2011;186:1840-1848, 2011; Overdijk MB et al., *MAb* 7:311-321, 2015). As described above, daratumumab-WT mAb is formatted as a human IgG1 and κ light chain mAb. A mutant of this antibody was engineered in which the H429 residue was replaced with phenylalanine to produce daratumumab-H429F mAb. In each case, the daratumumab-based IgG mAb exhibited equivalent properties and eluted as a single homogeneous peak from the protein A affinity column. Figure 23 Further purification via SEC yielded a single IgG (H2L2) peak for daratumumab-WT mAb and an equivalent peak for the daratumumab-H429F mutant mAb. Figure 24 A). Furthermore, SDS-PAGE confirmed that the SEC-purified mAb contained the expected 150 kDa IgG species (before disulfide bond reduction), and approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in DTT. Figure 24 B).

[0374] The CDC efficacy of daratumumab-H429F mAb was compared with that of daratumumab-WT mAb. Figure 25 In the presence of normal human serum as a complement source, each mAb was titrated individually by sequential 2-fold dilution. For each mAb concentration, the percentage of CD38-expressing Ramos cells killed was assessed, and as shown... Figure 25 As shown. Both daratumumab-WT and daratumumab-H429F mAb achieved approximately 80% killing of Ramos lymphoma cells; however, daratumumab-H429F mAb exhibited greater CDC potency, with an EC50 approximately 15-fold higher. The improved CDC potency of daratumumab-H429F mAb was then investigated using CDC-resistant CD38-positive KMS-12-PE myeloma cells. Figure 26A). Daratumumab-WT mAb showed significantly higher CDC in KMS-12-PE cells with higher lysis rate than background control (approximately 20%) in the presence of complement only (C' without mAb), even at a concentration (5 μg / ml) that is 5 times higher than the concentration required for maximal killing of Ramos lymphoma cells (1 μg / ml) (approximately 80% lysis; see [link to relevant documentation]). Figure 25 In contrast, the effective CDC in KMS-12-PE cells was mediated by daratumumab-H429F mAb. Figure 26 A). In further experiments, the efficacy of CDC was evaluated on SUP-15 acute lymphoblastic leukemia (ALL) cells expressing CD38. (As from...) Figure 26 As shown in B, SUP-15 cells are also resistant to CDC killing by daratumumab-WT mAb, but are easily killed by daratumumab-H429F mutant mAb. Therefore, the substitution at position 429 in the CH3 domain not only enhances CDC against certain targets, but also rescues effective CDC against lysis-resistant targets. Furthermore, the enhanced CDC efficacy against CD38 (which is structurally different from CD20 detected by rituximab and 11B8, and HER2 detected by trastuzumab) indicates that the improved efficacy is independent of the target and the detected epitope, and therefore also independent of the V domain.

[0375] H429 replacement facilitates functional synergy between mAbs targeting different epitopes.

[0376] The functional synergistic effects mediated by mAbs and H429 modification were investigated by determining the degree of C1q binding in a mixture of mAbs (trastuzumab and pertuzumab) targeting individual epitopes in HER2.

[0377] ( Figure 27 and 28 A) illustrates the purification characteristics of pertuzumab-WT mAb and a mutant in which H429 has been replaced by phenylalanine (i.e., pertuzumab-H429F mAb). For each mAb, a single equivalent IgG peak was obtained from protein A affinity chromatography. Figure 27 Furthermore, upon further purification by size exclusion chromatography, single homogeneous non-oligomeric IgG species were observed. Figure 28 A, to the right of the vertical line). SDS-PAGE analysis ( Figure 28 B) confirmed that these peaks contained a 150 kDa IgG (H2L2) species that, upon reduction, decomposed into approximately 50 kDa heavy chains and approximately 25 kDa light chains. Figure 28 B).

[0378] The synergy between irrelevant anti-HER2 mAbs trastuzumab-WT and pertuzumab-WT in detecting different epitopes of HER2 was determined by assessing the extent of C1q binding in the mAb mixture quantified by flow cytometry. Figure 29 A). Ovarian cancer cells SK-OV-3 treated with equal concentrations of HER2 mAb trastuzumab-H429F or pertuzumab-H429F modified with H429F showed binding similar to that observed when the mAb was used alone at the same concentration. Figure 29 A) Compared to pertuzumab-H429F (MFI = 798) or trastuzumab-H429F (MFI = 1739), the C1q binding was enhanced (MFI = 10,877).

[0379] Further evaluation of the collaboration enhancement through C1q combination is conducted as follows ( Figure 29 B): Titrate a pair of individual HER2 mAb or mAb by a two-fold serial dilution, starting at an initial concentration of 5 µg / ml of each individual mAb, or in the mAb mixture at a 1:1 ratio of 2.5 µg / ml:2.5 µg / ml of each mAb in the mixture. Figure 29 B). Trastuzumab-H429F and pertuzumab-H429F synergistically enhance complement activation, especially when the mAb concentration is limiting (e.g., 1.25 μg / ml). Figure 29B is indicated by the arrow). The C1q binding of a mixture of 1.25 μg / ml trastuzumab-H429F and 1.25 μg / ml pertuzumab-H429F (MFI = 6327) 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., 1.25 μg / ml trastuzumab-WT and 1.25 μg / ml pertuzumab-WT MFI = 522) and even greater than that of wild-type mAbs used alone (i.e., at 1.25 μg / ml). The trastuzumab-WT MFI was 258 at 1.25 μg / ml or 405 at 2.5 μg / ml, and the pertuzumab-WT MFI was 424 at 1.25 μg / ml or 483 at 2.5 μg / ml. This improved complement activation potency further indicates that greater functional potency can be achieved through the synergistic effect of mixtures of CH3-domain-modified mAbs that recognize different epitopes.

[0380] Modification of the CH3 domain enables mAbs with different specificities to act independently of the target in CDC.

[0381] The enhanced CDC cleavage of the target leading to enhanced C1q binding was investigated using a mixture of mAbs detecting two different molecular structures (i.e., CD20 and CD38) or two different epitopes within the same molecular structure CD20. Figure 29 This reflects the synergistic effect of mAbs in terms of greater functional efficacy. Results are as follows... Figure 30 As shown.

[0382] The collaboration between antibodies targeting different molecular structures and epitopes in the CDC was determined using a mixture of paired combinations of rituximab-WT or rituximab-H429F mAb targeting CD20 and daratumumab-WT or daratumumab-H429F mAb targeting the unrelated surface molecule CD38. No significant collaboration was observed in the CDC when rituximab-WT mAb was titrated in the presence of 0.25 µg / ml daratumumab-WT mAb. Figure 30 A); that is, the CDC observed at any concentration of rituximab-WT is no greater than that observed in the presence of 0.25 µg / ml daratumumab-WT alone (0 µg / ml rituximab, Figure 30A) or baseline CDC at any concentration of rituximab-WT alone (i.e., titrated in the absence of daratumumab-WT). However, unlike the unmodified wild-type mAb, rituximab-H429F and daratumumab-H429F mAb enhanced the killing effect on CDC cells. Figure 30 B). Specifically, when used with 0.025 µg / ml daratumumab-H429F mAb, synergy between the mAbs in mediating CDC was readily observed within the concentration range of 0.5 µg / ml–0.125 µg / ml rituximab-H429F (Note: enhancement by rituximab-H429F is not explicitly stated in the original text). Figure 30 (Indicated by the upward arrow in B). Rituximab-H429F mAb and daratumumab-H429F mAb mediated CDC significantly greater than H429F mAb alone mediated CDC (rituximab-H429F or 0.025 µg / ml daratumumab; enhancement by H429F mAb alone). Figure 30 (The upward arrow in B indicates this).

[0383] Functional synergy among mAbs detecting different epitopes but within the same target molecule was investigated in a mixture of different CD20 mAbs. Results are as follows: Figure 30 As shown in C. Titration of rituximab-H429F mAb in the presence (0.5 μg / ml) or absence of 11B8-H429F mAb revealed a synergistic effect between the mAbs. When the concentrations of both mAbs were limited (0.25 µg / ml - 0.031 µg / ml rituximab-H429F and 0.05 µg / ml 11B8-H429F), synergy between the mAbs was readily detected, where the CDC was greater than the CDC of the individual mAbs (C). Figure 30 (The enhancement indicated by the upward arrow in C).

[0384] Therefore, the cooperation and synergistic effects of mAbs modified with the antibody H chain at position 429 (specifically H429F) have a wide range of effects. Regardless of the epitope detected by individual mAbs (i.e., whether the epitope is present on the same or different molecular targets), they all contribute to greater functional potency through cooperation and functional synergy in the mixture of mAbs.

[0385] Example 4: Killing B lymphocytes with anti-CD20 therapeutic antibodies containing the H429 mutation

[0386] Monoclonal antibodies are used to treat inflammatory diseases such as autoimmune diseases by targeting normal (i.e., non-malignant) cells. For example, anti-CD20 mAb rituximab is used to treat inflammatory diseases by targeting normal B lymphocytes that are known to express CD20 (Lee DSW et al., Nature Reviews Drug Discovery 20:179-199, 2021).

[0387] Methods and Materials

[0388] Leukocytes isolated from human peripheral blood

[0389] Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated venous blood (Vacutainer ACD-A, Brady Medical) by centrifugation on a Ficoll gradient. The purified cells from the plasma / Ficoll interface were washed in flow cytometry buffer (L-15 medium lacking phenol red and containing 0.5% BSA (L15-BSA)) and resuspended in L15-BSA to 5 x 10⁻⁶ cells / mL. 6 Concentration per ml.

[0390] complement-dependent lysis of cells

[0391] After conditioning cells with mAb, CDC killing of normal peripheral blood B lymphocytes in PBMCs mediated by WT and mutant anti-CD20 mAb was measured by flow cytometry as described above using the Zombie Green Immobilization Viability Kit (Baijin Biotechnology).

[0392] CDC was performed in 96-well plates. Cells (25 μl, 5 x 10⁶ cells / well) were placed in each well. 6The cells were incubated with an equal volume of mAb in L15-BSA on ice for 30 min, then washed in L15-BSA (diluted in 100–200 μl buffer) and centrifuged twice (200 x g, 5 min, 4 °C). The IgG-conditioned cells were then resuspended in 50 μl of human serum diluted 1 / 3 in L15-BSA as a complement source and incubated at 37 °C for 30 min. The treated cells were then washed once in L15-BSA, resuspended in 50 μl of anti-CD19-APC antibody (Baijin Biotechnology) in L15-BSA, and incubated on ice for another 30 min. After washing twice in BSA-free L-15, the cells were resuspended in 50 μl of Zombie Green (1 / 500 dilution in protein-free L-15 medium or PBS according to manufacturer's instructions) and incubated on ice for 30 min. Cells were washed once in L15-BSA and fixed by resuspending in buffer containing 2% paraformaldehyde on ice for 30 minutes, followed by a final wash and resuspending in 200 μl of L15-BSA for flow cytometry analysis. B lymphocytes were identified by CD19 staining, and in PBMC samples treated with negative control mAbs trastuzumab-WT or trastuzumab-H429F antibody, CD19 staining was used to identify B lymphocytes. + Zombie Green + Compared to the background control, the percentage (%) of dead B lymphocytes that will be specifically killed by CDC is listed as CD19. + Zombie Green + Percentage of cells.

[0393] Results and discussion

[0394] The CDC killing effect of rituximab-H429F on normal B lymphocytes was studied by flow cytometry, and compared with the CDC killing effect of unmodified rituximab-WT on normal B lymphocytes. Figure 31 ). Compared to killing only 30% of peripheral blood B lymphocytes ( Figure 31 Compared to the unmodified rituximab-WT (A), which showed significantly lower killing power, rituximab-H429F demonstrated greater potency, killing over 85% of B cells (A). Figure 31 B). Since HER2 is not expressed on B lymphocytes, the negative control anti-HER2 mAb trastuzumab-WT or trastuzumab-H429F did not induce CDC in B lymphocytes. Figure 31 C, D).

[0395] Example 5: H429F modification improves the function of other types of immunoglobulins.

[0396] Histidine 429 is conserved in isomorphic positions across all Ig classes and subclasses. Figure 3 and Figure 4 That is to say, in the CH3 domain of all IgG and IgA subclasses ( Figure 3 In IgD ( Figure 4 ), and is also conserved in IgE and IgM, where 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.

[0397] Methods and Materials

[0398] Antibodies and antibody constructs

[0399] The mAb used in this example contains a heavy chain of either the IgG3 or IgG4 subclass. The IgG3 heavy chain used in this paper contains three amino acid substitutions, N392K, M397V, and R435H, which were introduced to avoid aggregation of the purified antibody (Saito S et al., Prot Sci 28(5):doi:10.1002 / pro, 2019).

[0400] The rituximab-IgG3 CD20-specific heavy chain polypeptide (SEQ ID NO: 38) comprises, in N-terminal to C-terminal order, the CD20-specific VH domain of rituximab fused to the CH1-hinge-CH2-CH3 domain of aggregated anti-human IgG3 (https: / / go.drugbank.com / drugs / DB00073), and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 39. Similarly, the anti-CD20 mAb rituximab light chain polypeptide (SEQ ID NO: 30), as previously described, comprises the CD20-specific VL domain of rituximab fused to the human κ constant domain (https: / / go.drugbank.com / drugs / DB00073), and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0401] The rituximab-IgG4-WT formatted CD20-specific heavy chain polypeptide (SEQ ID NO: 40) comprises, in N-terminal to C-terminal order, the CD20-specific VH domain of rituximab fused to the CH1-hinge-CH2-CH3 domain of human IgG4, and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 41. Similarly, the anti-CD20 mAb rituximab light chain polypeptide (SEQ ID NO: 30) as previously described by reference comprises the CD20-specific VL domain of rituximab fused to the human κ constant domain, and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0402] Modifications to the mAb heavy chain are generated by synthesizing a codon-optimized polynucleotide sequence of the complete antibody chain, including specific modifications encoding, for example, H429F (GeneArt; Thermo Fisher Scientific), and the antibody expression vector is generated using standard methods known to those skilled in the art. In short, the antibody expression vector consists of a synthetic polynucleotide sequence encoding an unmodified antibody heavy chain or a modified / mutated or light chain, appropriately placed within an expression plasmid, such as pcDNA3.4 (Thermo Fisher Scientific).

[0403] Expression of antibody constructs

[0404] Antibody expression was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions as described elsewhere in this specification for rituximab and therapeutic mAbs. For all mAbs, cell cultures were harvested post-transfection and centrifuged at 2500 rpm for 20–30 min, and the supernatant was filtered through a 0.2 µm high-flow-rate filter (Sartorius) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE electrophoresis using the Laemmli method.

[0405] Protein A affinity purification of mAb

[0406] As described in this article, mAb was purified from the supernatant of transfected Expi293F cells using protein A affinity chromatography.

[0407] Size exclusion chromatography (SEC) purification of mAb

[0408] Following affinity purification of protein A, the antibody was further purified and characterized as described herein using size exclusion chromatography (SEC).

[0409] Assessing antigen binding using monoclonal antibodies (mAbs)

[0410] Prior to functional analysis, flow cytometry was used to test antigen binding of the purified antibody on CD20-positive Ramos lymphoma cells.

[0411] complement-dependent lysis of cells

[0412] After conditioning Ramos cells with mAb, mAb-mediated CDC was measured by flow cytometry as described herein using the Zombie Green Immobilization Viability Kit (Baijin Biotechnology Co., Ltd.).

[0413] Results and discussion

[0414] Anti-CD20 mAb rituximab is formatted as follows: unmodified aggregated IgG3 heavy chain (rituximab-IgG3), aggregated IgG3 heavy chain containing the H429F mutation (rituximab-IgG3-H429F), wild-type IgG4 heavy chain (rituximab-IgG4-WT), or IgG4 heavy chain containing the H429F modification (rituximab-IgG4-H429F).

[0415] Rituximab-IgG3 mAb was formatted as an aggregated anti-human IgG3 and κ light chain mAb, and a mutant of this antibody was engineered in which the H429 residue was replaced with phenylalanine to produce rituximab-IgG3(KVH)-H429F mAb. Rituximab-IgG4-WT mAb was also produced and formatted as a human IgG4 and κ light chain mAb. In each case, rituximab-IgG3 and IgG4 mAbs exhibited equivalent properties and eluted as a single homogeneous peak from a protein A affinity column. Further purification by SEC yielded a single IgG (H2L2) peak for each of the mAbs. Furthermore, SDS-PAGE confirmed that the SEC-purified mAbs contained the expected intact IgG species (before disulfide bond reduction), as well as approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in dithiothreitol.

[0416] The CDC potency of rituximab-IgG3-H429F was evaluated by flow cytometry on Ramos cells and compared with that of rituximab-IgG3-WT. Figure 32 A). In the presence of normal human serum as a complement source, each mAb was titrated by successive 2-fold dilutions. Figure 32At each mAb concentration shown in Figure A, the percentage of CD20-expressing Ramos cells was evaluated. Rituximab-IgG3-H429F mAb showed greater CDC potency than rituximab-IgG3 mAb, especially when mAb concentration was limited. These results are important because they demonstrate that the effect of H429 modification can be successfully incorporated into antibodies containing mutations located elsewhere in the heavy chain (e.g., N392K, M397V, and R435H mutations that overcome IgG aggregation). Furthermore, the R435H mutation is known to enhance the in vivo half-life of IgG3, and therefore the enhanced CDC ( ) produced by the H429F mutation. Figure 32 A) This demonstrates that the H429 modification can be successfully incorporated into antibodies with other mutations that affect their in vivo half-life.

[0417] The CDC potency of rituximab-IgG4-H429F was also evaluated on Ramos cells and compared with that of rituximab-IgG4-WT. Figure 32 B). Unmodified rituximab-IgG4-WT failed to kill Ramos cells, but surprisingly, H429-modified rituximab-IgG4-H429F mAb mediated easily detectable CDC.

[0418] Clearly, the functional effects of H429F modification are not limited to human IgG1 and can be applied more broadly to other immunoglobulin types. This is important because histidine 429 is present in all human immunoglobulins (IgG, IgA, IgD, IgE, and IgM; see also...). Figure 3 , Figure 4 It is conserved at its equivalent sites in immunoglobulins of other mammals (e.g., primates).

[0419] Example 6: Antibodies with the H429 mutation also rely on complement to kill CDC.

[0420] The CDC produced by the mutated antibody depends on both the antibody and complement.

[0421] Ramos cells were conditioned with mAbs targeting different molecular targets CD20 and CD38, or targeting epitopes within the same molecular target (CD20). Ramos cells conditioned with daratumumab-H429F or 11B8-H429F were evaluated in the presence of complement (mAb and human complement), in the presence of mAb but without complement (mAb only), or in the absence of mAb but with complement (complement only or C' only). Figure 33Ramos cells showed significant CDC lysis only in the presence of both mAb and complement. Cells conditioned only with mAb (i.e., in the absence of complement) showed little lysis under CDC assay conditions. Similarly, unconditioned cells showed little lysis when incubated with complement only. Therefore, the lysis of conditioned cells in CDC assays depends on both antibody and complement.

[0422] Example 7: H429 modification confers enhanced CDC by monoclonal antibody

[0423] Exatuximab is a monoclonal antibody that recognizes the human CD38 cell surface molecule and exhibits functional properties different from several other anti-CD38 mAbs, such as daratumumab (Deckert J et al., *Clin Cancer Res* 20:4574-4583, 2014). Exatuximab inhibits the ADP-ribosylcyclase activity of the CD38 molecule, and epitopes different from those of daratumumab, which inhibits enzyme activity less effectively, were detected (Deckert J et al., 2014, ibid.). The effect of H429 modification on the CDC activity of functionally different exatuximab on cells resistant to CDC killing was evaluated.

[0424] Methods and Materials

[0425] Antibodies and antibody constructs

[0426] The exatuximab-WT mAb used in this example comprises a CD38-specific heavy chain polypeptide having the amino acid sequence shown as SEQ ID NO: 42, which, in N-terminal to C-terminal order, comprises the CD38-specific VH domain of the exatuximab mAb fused to the CH1-hinge-CH2-CH3 domain of human IgG1, and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 43. The anti-CD38 mAb exatuximab light chain polypeptide comprises the amino acid sequence shown as SEQ ID NO: 44 (comprising the CD38-specific VL domain of the exatuximab mAb fused to the human κ constant domain), and is encoded by codon-optimized DNA having the sequence shown as SEQ ID NO: 45.

[0427] Synthesis of unmodified and mutated heavy chains

[0428] Antibody expression vectors are produced using standard methods known to those skilled in the art. In short, antibody expression vectors consist of synthetic polynucleotide sequences encoding either the heavy or light chain of an antibody, appropriately placed within a plasmid, such as pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors for antibodies with different specificities are produced using existing variable domains (V... H or V L It is produced by cutting at a restrictive site at the boundary. Then, according to the manufacturer's instructions, it is reacted with NEBuilder (New England Biolabs) to incorporate a new synthetic DNA that encodes a new V domain and is side-joined with a sequence (e.g., 25 nucleotides) homologous to the cutting vector.

[0429] According to the manufacturer's instructions, Fc variants are generated by synthesizing a synthetic polynucleotide sequence that encodes the variant, or by cutting the Fc-encoding sequence of an antibody expression plasmid with an appropriate restriction enzyme, and by incorporating new mutagenic synthetic DNA into the reaction using NEBuilder (New England Biolabs).

[0430] Expression of antibody constructs

[0431] Antibody expression was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions described above and elsewhere in this specification for the therapeutic mAbs. For all mAbs, cell cultures were harvested post-transfection and centrifuged at 2500 rpm for 20–30 min, and the supernatant was filtered through a 0.2 µm high-flow-rate filter (Sartorius) prior to purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0432] Purification of mAb and assessment of antigen binding

[0433] As described herein, mAbs were purified from the supernatant of transfected Expi293F cells using protein A affinity chromatography. Following protein A affinity purification, the antibodies were further purified and characterized using size exclusion chromatography (SEC) as described herein. Then, prior to functional analysis, the antigen binding of the purified antibodies on CD38-positive SUP-15 acute lymphoblastic leukemia (ALL) cells was tested by flow cytometry.

[0434] complement-dependent lysis of cells

[0435] After conditioning SUP-15 acute lymphoblastic leukemia (ALL) cells with mAb, mAb-mediated CDC was measured by flow cytometry as described herein using the Zombie Green Immobilization Viability Kit (Baijin Biotechnology Co., Ltd.).

[0436] Results and discussion

[0437] As described above, exatuximab-WT mAb was formatted as a human IgG1 and κ light chain mAb. A mutant of this antibody was engineered in which the H429 residue was replaced with phenylalanine to produce exatuximab-H429F mAb. In each case, the exatuximab-based mAbs exhibited equivalent properties and eluted as a single homogeneous peak from a protein A affinity column. Further purification by SEC also yielded a single IgG (H2L2) peak for each of the mAbs. Furthermore, SDS-PAGE confirmed that the SEC-purified mAbs contained the expected intact IgG species (before disulfide bond reduction), as well as approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in dithiothreitol.

[0438] The CDC efficacy of esatuximab-H429F mAb was compared with that of esatuximab-WT mAb. Figure 34 In the presence of normal human serum as a complement source, each mAb was titrated individually by serial 2-fold dilution. The percentage of CDC potency (killing %) was assessed on SUP-15 acute lymphoblastic leukemia (ALL) cells expressing CD38. (The text abruptly ends here, likely due to an incomplete translation or source material.) Figure 34 As can be seen, SUP-15 cells resisted CDC killing by exatuximab-WT mAb, but were easily killed by exatuximab-H429F mutant mAb. Therefore, the substitution at position 429 in the CH3 domain not only enhanced CDC against certain cellular targets, but also conferred effective CDC against anti-lytic targets. Importantly, the results revealed that lysis of resistance targets can be achieved by H429 modification of irrelevant mAbs, which can also detect different epitopes - exatuximab-H429F ( Figure 34 ) and daratumumab-H429F ( Figure 26 Additionally, the results also indicate that mAbs with other functional properties, such as the ability to inhibit target molecule function, can be endowed with improved CDC efficacy, in this case, the ability of exatuximab to inhibit the ADP-ribosylcyclase activity of CD38.

[0439] Example 8: Generation of anti-death receptor antibodies with H429 mutation

[0440] Methods and Materials

[0441] The mAbs used in this example are listed in Table 3 below. The mAbs were designated as BDR5 and TDR5 and detected two distinct epitopes of human death receptor 5 (DR5; Overdijk MB et al., Molecular Cancer Therapy 19:2126-2138, 2020) and were generated in H2L2 format, with a heavy chain of human IgG1, IgG2 or IgA2 subclass and a light chain of its appropriate human κ class.

[0442] Table 3 - DR5-specific mAbs for detecting different epitopes

[0443]

[0444] Production of DR5-specific antibody constructs with IgG heavy chains

[0445] Immunoglobulin variable and constant region sequences were assembled into complete synthetic DNA molecules using standard molecular biology techniques to encode the complete immunoglobulin H and L chains. These molecules were used in the expression vector pcDNA 3.4 (Thermo Fisher Scientific).

[0446] The unmodified wild-type mAb used in this example is based on the sequences of two different mAbs that specifically bind to the human DR5 molecule (see the DR5-specific mAb described in U.S. Patent No. 10,882,913; the entire disclosure of which is incorporated herein by reference). The mAb was generated as a monoclonal IgG antibody in H2L2 format.

[0447] The resulting unmodified DR5-specific mAbs comprise the specific VH and VL domains of the mAbs indicated in Table 3 and are referred to as wild-type (WT) form; for example, the “wild-ty...

Claims

1. A bispecific immunotherapeutic protein comprising two antigen recognition structures targeting two different epitopes, and comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, said Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)), said one or more polypeptides comprising an amino acid substitution (Eu number) at a position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain.

2. The bispecific immunotherapy protein of claim 1, wherein the Fc region component comprises constant restructure domain 3 (CH3) and constant restructure domain 2 (CH2) of the immunoglobulin heavy chain, optionally combined with lower, core and / or upper hinge sequences.

3. The bispecific immunotherapy protein according to claim 1, wherein the Fc region component comprises only constant restructuring domain 3 (CH3).

4. The bispecific immunotherapy protein according to any one of claims 1 to 3, wherein the Fc region component is derived from IgG or IgA heavy chain polypeptide.

5. The bispecific immunotherapy protein according to any one of claims 1 to 4, wherein the bispecific immunotherapy protein is an immunoglobulin molecule comprising a first immunoglobulin heavy chain polypeptide and a second immunoglobulin heavy chain polypeptide, wherein the first immunoglobulin heavy chain polypeptide and the second immunoglobulin heavy chain polypeptide comprise Fc region components including CH2 and CH3 domains, such that the first polypeptide and the second polypeptide dimerize to form an Fc fragment or an Fc-like fragment.

6. The bispecific immunotherapy protein according to claim 4, wherein the Fc region component is derived from an IgG1 heavy chain polypeptide.

7. The bispecific immunotherapy protein according to claim 4, wherein the Fc region component is derived from IgG2 heavy chain polypeptide, IgG3 heavy chain polypeptide, IgG4 heavy chain polypeptide or IgA heavy chain polypeptide.

8. The bispecific immunotherapy protein according to any one of claims 1 to 7, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is H replaced by an aromatic amino acid or a cyclic amino acid.

9. The bispecific immunotherapy protein according to any one of claims 1 to 7, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is H replaced by a hydrophobic amino acid.

10. The bispecific immunotherapy protein according to any one of claims 1 to 9, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H→Y substitution.

11. The bispecific immunotherapy protein according to any one of claims 1 to 9, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H→F substitution.

12. The bispecific immunotherapy protein according to any one of claims 1 to 11, wherein the bispecific immunotherapy protein is a cell-bridging bispecific immunotherapy protein.

13. The bispecific immunotherapy protein according to any one of claims 1 to 12, wherein the bispecific immunotherapy protein is an antigen-crosslinked bispecific immunotherapy protein.

14. The bispecific immunotherapeutic protein according to any one of claims 1 to 13, wherein the two epitopes are located on different cells.

15. The bispecific immunotherapy protein according to any one of claims 1 to 14, wherein one epitope is located on a cancer cell and the other epitope is located on an immune cell.

16. The bispecific immunotherapeutic protein according to any one of claims 1 to 15, wherein the two epitopes are located on the same cell.

17. The bispecific immunotherapeutic protein according to any one of claims 1 to 16, wherein the two epitopes are located on the same molecule.

18. The bispecific immunotherapy protein according to any one of claims 1 to 17, wherein one or both of the epitopes are located on a molecule selected from: cancer-associated antigens, autoantigens, allergens, antigens associated with inflammatory diseases, antigens from transplanted tissues or organs, or antigens of infectious agents, such as eukaryotes, soluble molecules, bacterial or viral pathogens.

19. The bispecific immunotherapeutic protein of any one of claims 1 to 18, wherein one or both of the epitopes is located on a molecule selected from the group consisting of cluster of differentiation 37 (CD37), programmed death-ligand 1 (PD-L1), cluster of differentiation 3 (CD3), cluster of differentiation 20 (CD20), cluster of differentiation 127 (4-1BB), human epidermal growth factor receptor-2 (HER2), death receptor 5 (DR5) DR5, B-cell maturation antigen (BCMA), FcyRIII, FcyRIIb, FcyRIIa, FcaRI, FcyRI, natural cytotoxicity triggering receptor 1 (NKp46), killer cell lectin-like receptor K1 (NKG2D), natural cytotoxicity triggering receptor (3NKp30), death receptor 5 (DR5), TNP conjugate (2,4,6 tri-nitrophenyl conjugate), biotin conjugate, hapten conjugate, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-17 (IL-17), tumor necrosis factor (TNF), tumor necrosis factor alpha (TNFa), cluster of differentiation 19 (CD19), cluster of differentiation 22 (CD22), cluster of differentiation 27 (CD27), cluster of differentiation 28 (CD28), cluster of differentiation 30 (CD30), cluster of differentiation 33 (CD33), cluster of differentiation 38 (CD38), cluster of differentiation 39 (CD39), cluster of differentiation 47 (CD47), cluster of differentiation 52 (CD52), cluster of differentiation 79 (CD79), cluster of differentiation 123 (CD123), C-type lectin-like molecule-1 (CLL-1), C-type lectin domain family 12 member A (CLEC12A), delta-like 4 (DLL4), cluster of differentiation 135 (FLT-3), Fc receptor-like protein 5 (FcRH5), G protein-coupled receptor class C group 5 member D (GPRC5D), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), prostate-specific membrane antigen (PSMA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), lymphocyte-activation gene-3 (LAG-3), epidermal growth factor receptor (EGFR), tyrosine-protein kinase Met (cMet), glycoprotein 100 (GP100), angiopoietin-2 (Ang-2), vascular endothelial growth factor A (VEGF-A), tumor-associated glycoprotein 72 (Tag72), claudin 18 (CLDN18.2), Cluster of differentiation (CD66), CD300 molecule-like family member F (CD300f), glypican 2 (GPC2), fms-related receptor tyrosine kinase 3 (FLT3), CD276 molecule (B7-H3), cell adhesion molecule (CEA), delta-like canonical notch ligand 3 (DLL3), disialoganglioside 2 (GD2), glycoprotein A33 (gpA33), glypican 3 (GPC3), guanylate cyclase 2C (GUCY2C), major histocompatibility complex, class I, A (HLA-A), mage family member A4 (MAGE-A4), mucin 1, cell surface associated (MUC1), mesothelin (MSLN), mucin 16, cell surface associated (MUC16), mucin 17, cell surface associated (MUC17), New York esophageal squamous cell carcinoma 1 (NY-ESO1), PRAME nuclear receptor transcription regulator (PRAME), prostate stem cell antigen (PSCA), somatostatin receptor 2 (SSTR2), STEAP family member 1 (STEAP1), cluster of differentiation 127 (4-1BB), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), survivin, and SARS-CoV-2 S protein.

20. The bispecific immunotherapeutic protein according to any one of claims 1 to 19, wherein the molecule is a soluble molecule.

21. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are CD37 epitopes.

22. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are DR5 epitopes.

23. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are HER2 epitopes.

24. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are CD3 epitopes.

25. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are CD20 epitopes.

26. The bispecific immunotherapeutic protein according to any one of claims 1 to 19, wherein one or both of the epitopes are CD19 epitopes.

27. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are CD28 epitopes.

28. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are PD-L1 epitopes.

29. The bispecific immunotherapeutic protein according to any one of claims 1 to 19, wherein one or both of the epitopes are 4-1BB epitopes.

30. The bispecific immunotherapeutic protein according to any one of claims 1 to 19, wherein one or both of the epitopes are BCMA epitopes.

31. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are FcγRIII epitopes.

32. The bispecific immunotherapeutic protein according to any one of claims 1 to 19, wherein one or both of the epitopes are FcγRIIb epitopes.

33. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are FcαR1 epitopes.

34. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are FcγR1 epitopes.

35. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein one or both of the epitopes are C5 epitopes.

36. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein the epitope is located on a protein pair selected from the following: PDL-1 and 4-1BB; HER2 and 4-1BB; HER2 and FcγRIII; BCMA and FcγRIII; CD3 and BCMA; CD3 and CD20; CD3 and CD19; FcγRIIb and TNP-BSA; CD3 and CD28; CD3 and CD123; CD3 and CD33; CD3 and CD37; CD3 and CD38; CD3 and CLEC12A; CD3 and FLT-3; CD3 and survivin; CD20 and CD47; CTLA-4 and PD-1; LAG-3 and PD-1; PD-1 and PD-L1; and CD30 and FcγRIII.

37. The bispecific immunotherapy protein according to any one of claims 1 to 19, wherein the bispecific immunotherapy protein is selected from: blinatumomab; emicizumab; amivantamab-vmjw; tebentafusp-tebn; faricimab-svoa; cadonilimab; mosunetuzumab; teclistamab; catumaxomab; odronextamab; and ozoralizumab.

38. The bispecific immunotherapeutic protein according to any one of claims 1 to 37, wherein the bispecific immunotherapeutic protein is generated from a platform selected from: DEKK, ART-Ig, CrossMab, DuoBody, Ortho-Fab, strand exchange engineered domain (SEED), mortise and tenon, DuetMab, κλ body, YBODY, antibody-based bispecific binding to T cell receptors (BEAT), cross-bivariate (CODV), Wuxibody, DVD-Ig, Adaptir, dual-action antigen-binding fragment (Fab) (DAF), FIT-Ig, TsBsIgG, Triomab, and XmAb.

39. The bispecific immunotherapy protein according to any one of claims 1 to 38, wherein the bispecific immunotherapy protein is a microantibody.

40. The bispecific immunotherapy protein according to any one of claims 1 to 39, wherein the bispecific immunotherapy protein forms oligomers by self-association in solution at physiological pH.

41. The bispecific immunotherapy protein of claim 40, wherein the bispecific immunotherapy protein is a hexamer.

42. The bispecific immunotherapy protein according to any one of claims 1 to 39, wherein the bispecific immunotherapy protein forms an oligomer when it binds to an associated target via "on-target" oligomerization.

43. The bispecific immunotherapy protein of claim 42, wherein the bispecific immunotherapy protein forms a hexamer when it binds to an associated target via "on-target" oligomerization.

44. The bispecific immunotherapy protein according to any one of claims 1 to 38, wherein the antibody is produced by a quadroma.

45. The bispecific immunotherapy protein according to any one of claims 1 to 44, wherein said polypeptide comprises an additional mutation selected from one or more of the following: i) An amino acid substitution (Eu number) located at position 274 of the amino acid sequence corresponding to the human IgG1, IgG2, IgG3 or IgG4 heavy chain. ii) Amino acid substitutions (Eu number) located at the position corresponding to amino acid sequence 219 of the heavy chain of human IgG1, IgG2, IgG3 or IgG4. iii) Amino acid substitutions (Eu numbered) located at positions corresponding to amino acid sequence 220 of the human IgG1, IgG2, IgG3, or IgG4 heavy chain; and iv) Amino acid substitutions (Eu number) located at positions corresponding to amino acid sequences 219 and 220 of the heavy chain of human IgG1, IgG2, IgG3 or IgG4.

46. ​​The bispecific immunotherapy protein of claim 45, wherein the additional mutation is a substitution at the position corresponding to C219 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain or a substitution at the position corresponding to C220 (Eu number).

47. The bispecific immunotherapy protein of claim 45, wherein the additional mutation is a substitution selected from the following: K274 replaced by glutamine, C219 replaced by serine, S219 replaced by cysteine, and C220 replaced by serine.

48. An oligomer comprising a bispecific immunotherapeutic protein according to any one of claims 1 to 47.

49. A nucleic acid encoding a bispecific immunotherapy protein according to any one of claims 1 to 47.

50. The nucleic acid according to claim 49, wherein the nucleic acid is selected from RNA, DNA, or combinations thereof.

51. Use of a bispecific immunotherapeutic protein, oligomer, or nucleic acid according to any one of claims 1 to 50 for treating or preventing 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, transplantation symptoms or rejection, infectious diseases, and proliferative diseases.

52. Use of a bispecific immunotherapeutic protein, oligomer, or nucleic acid according to any one of claims 1 to 50, for the preparation of a medicament for the treatment or prevention of a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

53. A method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of a bispecific immunotherapeutic protein, oligomer, or nucleic acid according to any one of claims 1 to 50, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

54. The use according to claim 51 or 52 or the method according to claim 53, wherein the bispecific immunotherapy protein or oligomer removes soluble molecules and / or immune complexes from circulation.

55. The use according to claim 51 or 52 or the method according to claim 53, wherein the bispecific immunotherapy protein enhances the death receptor signaling response.

56. The use according to claim 51 or 52 or the method according to claim 53, wherein the bispecific immunotherapy protein enhancement is based on complement cleavage.

57. A pharmaceutical composition or medicament comprising a bispecific immunotherapeutic protein, oligomer, or nucleic acid according to any one of claims 1 to 50, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

58. The pharmaceutical composition or medicament according to claim 57, wherein the carrier is saline solution at physiological pH.

59. The pharmaceutical composition or medicament according to claim 57, wherein the carrier is a saline solution with a pH less than 6.

5.

60. A kit comprising at least one bispecific immunotherapeutic protein, oligomer, or nucleic acid according to any one of claims 1 to 50.

61. A method for producing a bispecific immunotherapeutic protein according to claims 1 to 50, the method comprising culturing host cells containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant under the following conditions: (i) A weakly acidic pH for recovering bispecific immunotherapeutic proteins in monomeric form; or (ii) A substantially neutral pH for the recovery of bispecific immunotherapeutic proteins in oligomeric form.

62. A method for producing a bispecific immunotherapeutic protein according to any one of claims 1 to 50, the method comprising culturing a host cell containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant using an elution buffer containing arginine at a concentration of less than 130 mM and a pH of less than or equal to 5.0 using a method comprising affinity chromatography.

63. A method for generating / modifying a bispecific immunotherapeutic protein, the method comprising substituting an amino acid (Eu number) located at a position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 heavy chain, wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)).

64. A microantibody comprising one or more immunoglobulin heavy chain polypeptides containing an Fc region component, said Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)), said one or more polypeptides comprising an amino acid substitution (Eu number) at a position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain.

65. The bispecific immunotherapy protein of claim 63 or the microantibody of claim 64, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H→Y substitution.

66. The bispecific immunotherapy protein of claim 63 or the microantibody of claim 64, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide is an H→F substitution.

67. An oligomer comprising a microantibody according to any one of claims 64 to 66.

68. A nucleic acid encoding a microantibody according to any one of claims 64 to 66.

69. The nucleic acid according to claim 68, wherein the nucleic acid is selected from RNA, DNA, or combinations thereof.

70. Use of a microantibody, oligomer, or nucleic acid according to any one of claims 64 to 69 for treating or preventing 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, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

71. Use of a microantibody, oligomer, or nucleic acid according to any one of claims 64 to 69 for the preparation of a medicament for the treatment or prevention of a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

72. A method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of a microantibody, oligomer, or nucleic acid according to any one of claims 64 to 69, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular diseases, neurodegenerative diseases, other inflammatory diseases, transplantation conditions or rejection, infectious diseases, and proliferative diseases.

73. The use according to claim 70 or 71 or the method according to claim 72, wherein the microantibody or oligomer removes soluble molecules and / or immune complexes from circulation.

74. The use according to claim 70 or 71 or the method according to claim 72, wherein the microantibody or oligomer enhances the death receptor signaling response.

75. The use according to claim 70 or 71 or the method according to claim 72, wherein the microantibody or oligomer enhances complement cleavage.

76. The use according to claim 70 or 71 or the method according to claim 72, wherein the microantibody or oligomer enhances T-cell or NK-cell-based lysis.

77. A pharmaceutical composition or medicament comprising a microantibody, oligomer, or nucleic acid according to any one of claims 64 to 69, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

78. A kit comprising at least one microantibody, oligomer, or nucleic acid according to any one of claims 64 to 69.

79. A method for producing a microantibody according to any one of claims 64 to 69, the method comprising culturing a host cell containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant under the following conditions: (i) A weakly acidic pH for recovering bispecific immunotherapeutic proteins in monomeric form; or (ii) A substantially neutral pH for the recovery of bispecific immunotherapeutic proteins in oligomeric form.

80. A method for producing a microantibody according to any one of claims 64 to 69, the method comprising culturing a host cell containing a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant using an elution buffer containing arginine at a concentration of less than 130 mM and a pH of less than or equal to 5.0 using a method comprising affinity chromatography.

81. A method for generating / modifying a microantibody, the method comprising substituting an amino acid (Eu number) located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain, wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) (or at least one constant heavy chain domain 4 (CH4)).

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