Immunotherapeutic proteins comprising fc region components with mutations at position 429

Abstract

An 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) domain (or at least one constant heavy chain domain 4 (CH4) domain), wherein the one or more polypeptides comprise amino acid substitutions (Eu numbering), such as H429Y and H429F, located at a position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain. The immunotherapeutic protein may be in the form of, for example, an antibody, an antibody-like molecule, or a fusion protein. The amino acid substitution may have the effect of enabling the immunotherapeutic protein to be oligomerized (e.g., to be oligomerized into a hexameric form) in solution or upon binding to an associated target.

Description

Technical Field

[0001] The present disclosure relates to immunotherapeutic proteins, including, for example, modified immunoglobulin molecules (ie, antibodies), for use in methods of treating a disease or condition.

[0002] Priority Document

[0003] This application claims priority to Australian Provisional Patent Application No. 2021903609, filed on November 11, 2021, entitled “Modified immunoglobulin and method of use thereof (3)” and Australian Provisional Patent Application No. 2022901632, filed on June 15, 2022, entitled “Modified immunoglobulin and method of use thereof (3)”. The contents of both applications are hereby incorporated by reference in their entirety. Background Art

[0004] Monoclonal antibodies (mAbs) have become one of the most important and successful classes of therapy, revolutionizing the treatment of cancer and inflammatory diseases such as autoimmune diseases. Many mAbs engineered on the IgG antibody class backbone specifically exploit the powerful effector functions of the immune system by engaging both the target antigen through their variable Fab domains and the Fcγ receptors (FcγRs) through their heavy chains (comprising the constant Fc portion (Fc or Fc fragment)), leading to the activation of inflammatory or killer cells. In addition, the Fc of an antibody can also activate blood proteins called complement (involving antibodies coming together in a process called "self-association" or "on-target assembly") to achieve complement-dependent effector functions, such as complement-dependent cytotoxicity (CDC). These different actions act separately, 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, it can also be advantageously exploited through the action of engineered mAbs (i.e., therapeutic mAbs) intended for use in various medical treatments, such as cancer therapy and the treatment of infectious and inflammatory diseases and disorders.

[0005] In view of the above, it is perhaps not surprising that a great deal of research effort has been applied to elucidate the details of Fc interactions (e.g., with FcγRs and complement proteins such as C1q and membrane attack complex (MAC) proteins C4-C9) and to identify modifications (e.g., mutations) that may be made to Fc to potentially enhance the efficacy of therapeutic mAbs. However, to date, few such Fc mutations have been included in therapeutic mAbs; the most well-known are antibodies in which the heavy chain glycans have been engineered to lack fucose and thereby result in enhanced FcγRIII interactions, such as obinituzumab and margetuximab (which include six point mutations in the heavy chain of an IgG1 antibody), as well as other antibodies that are undergoing clinical trials but have not yet been approved, such as various HexaBody TM mAb (GenMab BV; Copenhagen, Denmark) comprising a triple mutation of the Fc fragment of IgG1, i.e., amino acid substitutions at positions 345 (i.e., E345R), 430 (i.e., E430G), and 440 (i.e., S440Y) (referred to as "IgG-RGY") (see International Patent Publication No. WO 2014 / 006217), or a single mutation only at position 430 (i.e., E430G) of the Fc of IgG1 (de Jong RN et al., PLoS Biol 14(1): e1002344, 2016). This E430G mutation has been shown to enhance the ability of IgG1 antibodies to form hexamer on target molecules, and IgG1 mutants comprising the E430G mutation have been reported to show strongly enhanced CDC conditional on antigen binding at target cells (de Jong et al., 2016 supra).

[0013] 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 (eg, mAbs). Summary of the invention

[0006] In the work leading to the present disclosure, the inventors generated a series of mutant antibodies (particularly mutant immunoglobulin G (IgG) molecules) comprising, for example, point mutations at various positions in and around the interface of the CH2 and CH3 domains of the heavy chain polypeptide. Some of the molecules containing mutations at position 429 were found to be a "buried" or inaccessible site within the IgG1 structure (see Figure 1), and is occupied by histidine (His / H) residues to indicate, for example, enhanced activation of the complement system through the classical complement pathway, which includes binding of C1q, formation of a membrane attack complex, and complement-dependent cytotoxicity in complement function assays. In addition, the inventors have obtained evidence that some molecules including Fc region components with mutations at position 429 are able to form oligomers in solution or when bound to relevant targets. The present disclosure relates to immunoglobulins and other molecules comprising an Fc region component (e.g., fusion proteins comprising a partner polypeptide having beneficial functions and / or characteristics), the immunoglobulins and other molecules comprising a mutation at position 429 (or a position corresponding thereto) of the Fc region component, and the potential use of the immunoglobulins and other molecules in therapies for, for example, cancer (e.g., acute lymphoblastic leukemia (ALL) and adenocarcinoma), infections (e.g., COVID-19, emerging viral infections, and parasitic infections such as drug-resistant malaria infections), and inflammatory diseases and disorders (e.g., autoimmune diseases including systemic lupus erythematosus (SLE) and type 1 diabetes (T1D)).

[0007] Thus, in a first aspect, the present disclosure provides an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, wherein the Fc region component comprises at least one CH3 domain (or at least one CH4 domain), wherein the one or more polypeptides comprise an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain (Eu numbering).

[0008] The immunotherapeutic protein may include a dimeric immunotherapeutic protein, such as an immunoglobulin molecule (e.g., an antibody) 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 include an Fc region component comprising a CH2 domain and a CH3 domain (such that the first polypeptide and the second polypeptide can form an Fc fragment or an Fc-like fragment by dimerization). In some embodiments, such a dimeric immunotherapeutic protein can be provided in an oligomeric form of a hexamer, for example, comprising 6 copies of the dimeric immunotherapeutic protein.

[0009] In other embodiments, the immunotherapeutic protein includes a partner polypeptide connected to an Fc region component including at least one CH3 domain (or at least one CH4 domain), so that the immunotherapeutic protein can be in the form of a fusion protein or protein conjugate. In some embodiments, such fusion proteins or protein conjugates may include a dimeric immunotherapeutic protein 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 include an Fc region component including a CH2 domain and a CH3 domain (so that the first polypeptide and the second polypeptide can form an Fc fragment or an Fc-like fragment by dimerization). In some embodiments, such dimeric immunotherapeutic proteins can be provided in an oligomeric form of, for example, a hexameric dimeric immunotherapeutic protein including 6 copies.

[0010] In some preferred embodiments, the immunotherapeutic protein comprises an H→Y or H→F amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain.

[0011] In a second aspect, the present disclosure provides a use of an immunotherapeutic protein as defined in the first aspect 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, other inflammatory diseases, infectious diseases and proliferative diseases.

[0012] In a third aspect, the present disclosure provides a use of an immunotherapeutic protein as defined in the first aspect for the preparation of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases and proliferative diseases.

[0013] In a fourth aspect, the present 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 defined in the first aspect, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

[0014] In a fifth aspect, the present disclosure provides a pharmaceutical composition or medicament comprising the immunotherapeutic protein as defined in the first aspect and a pharmaceutically acceptable carrier, diluent and / or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1An illustration of the structure of the Fc of human IgG1 is provided, showing that the H429 residue is located in an inaccessible position within the Fc fragment. These illustrations are human IgG1-Fc (PDB: 1Fc1) reproduced from Deisenhofer J., Biochemistry 20: 2361-2370, 1981, rendered with space filling. (A) Side view of the Fc, with the A chain shown in black, the B chain shown in light grey, and the N-linked glycans shown in dark grey. The boxed area of ​​the A-chain indicates the area enlarged in panels B and C; (B) The boxed area enlarged in panel A, indicating in grey the solvent accessible amino acids of the chain-B labeled according to Eu numbering of human IgG1: methionine (M428) at position 428, glutamic acid 430 (E430), alanine 431 (A431), leucine 432 (L432), histidine H435, which is located above and masks the histidine 429 (H429) buried in the Fc; (C) A view of panel B after the solvent accessible residues shown in panel B have not been rendered (i.e. removed from view). Only now does the buried H429 shown in grey CPK become clearly visible below these overlaid neighboring solvent accessible residues shown in panel B. The residue accessible surface area (ASA) calculated using PISA (i.e., the Protein Interfaces, Surfaces and Assembly service PISA; http: / / www,ebi.ac.uk / pdbe / prot_int / pistart.htm, and Krissinel E and K Henrick. J Mol Biol 372:774-797, 2007) indicated that H429 was inaccessible and a value of 0.0 was recorded.

[0016] Figure 2 A schematic representation of immunoglobulin (antibody) and antibody-like (Ab-like) molecules is provided, illustrating the modular nature of antibodies:

[0017] Left image:Prototype immunoglobulin structure representation is provided-immunoglobulin comprises the chain of variable (V) and constant (C) domains arranged in the heavy (H) chain of self-assembly and optionally light (L) chain.Domain is further identified by chain (for example, CH3 is the third constant domain of heavy chain).Heavy chain (H) usually further comprises different connection or hinge sequence between Fab and Fc fragment.As shown in the figure (residue H429 is indicated in CH3 domain), some H chains comprise three constant H domains, and other comprise four constant H domains (for example IgM and IgE), wherein CH4 is the equivalent domain of CH3 of other antibodies (for example, IgG and IgA).Antibodies from many species comprise light (L) chain.In the antibody lacking light chain, two H chains dimerize (H2), and generally comprise antibody.On the other hand, in the antibody comprising light (L) chain, two antibody H chains are generally by dimerization, and L chains are associated with each H chain (that is, H2L2). Some antibodies lacking strong H chain interactions (e.g., human IgG4) may sometimes be antibody "half molecules" comprising a monomeric H chain and one L chain (H1L1). The Fc fragment (or Fc) of an immunoglobulin is a dimer formed by covalent and / or non-covalent interactions between portions of each H chain (i.e., two Fc regions of an H chain, each Fc region comprising a hinge, a CH2 domain, and a CH3 domain of a heavy chain).

[0018] Right:Provided is a modular nature showing that immunoglobulin allows for flexible representation in the generation of Ab-like molecules and fusion proteins (comprising immunoglobulin moieties). The molecules shown all comprise "H-like" chains, including at least one CH3 domain. Specifically, the Ab-like molecules described are each composed of a V domain for target antigen recognition connected by a linker sequence with a CH3 domain from the Fc region of, for example, IgG (in the variant of the molecule shown, the V domain can be an equivalent CH4 domain from IgE or IgM). The CH3 domain can carry a mutation, such as the mutation at position 429 as described herein (Eu numbering). The V and CH3 domains can be connected by a sequence that can include a heterologous linker sequence, a synthetic linker sequence, and / or includes other homologous or heterologous sequences or domains connecting the V and CH3 domains (e.g., as depicted in the examples shown in the figure, the V and CH3 domains can be connected by CH1-CH2 or CH2 with or without additional short polypeptide sequences, such as heterologous or synthetic linkers or immunoglobulin hinge sequences), and the CH1, CH2 and hinge sequence modules can be provided in different combinations and orders, not limited to the examples shown. The molecules shown are each in the form of monomers, which can dimerize to form homodimers or heterodimers. Similarly, for the fusion proteins shown, the fusion proteins are each composed of a target recognition portion (depicted as "X") connected by a linker sequence to at least one CH3 domain from the Fc region of, for example, IgG (or an equivalent CH4 domain of IgE or IgM). Likewise, the CH3 domain may comprise a mutation, such as a mutation at position 429 (Eu numbering), and the target recognition portion and the H chain module (e.g., CH3 domain, CH2-CH3 or CH3-CH2 component) are linked by a sequence that may include a heterologous or synthetic linker sequence and / or include other homologous or heterologous sequences or domains linking the V and CH3 domains (e.g., CH1-CH2 or CH2 with or without an additional short polypeptide sequence, such as a heterologous or synthetic linker sequence or an immunoglobulin hinge sequence);

[0019] Figure 3The conservation of domain structure and sequence homology in immunoglobulin G (IgG) and immunoglobulin A (IgA) molecules is shown: a sequence comparison of the hinge and constant domain of the human immunoglobulin heavy (H) chain sequence is provided, thereby depicting the domain-based structure of the H chain (i.e., CH1-hinge-CH2-CH3 defined by IgG1; therefore, the Eu numbering of the first amino acid of each IgG1 domain and hinge is as follows (inverted triangle): CH1 domain, amino acid 118 to 215; hinge amino acid 216 to 230; CH2 domain amino acid 231 to 340; and CH3 domain amino acid 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 subclasses and IgA subclasses is also shown (indicated by arrows). The amino acid sequences were derived from translations of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). The accession numbers for the H chain sequences are: IgG1 H chain, J00228-IGHG1; IgG3 H chain, X03604-IGHG3; IgG4 H chain, K01316-IGHG; IgG2 H chain, J00230-IGHG2; IgA1 H chain, J00220-IGHA1; and IgA2 H chain, J00221-IGHA2. Alignment was performed using Clustal, except for the hinges, which were aligned manually;

[0020] Figure 4The histidine at position 429 in IgG1 is shown to be conserved in all immunoglobulin classes. Comparison of the amino acid sequence of the IgG1 CH3 domain with the corresponding domains of other human immunoglobulin classes shows that the histidine 429 indicated by the arrow is conserved in the CH3 domains of other IgG subclasses IgG2, IgG3 and IgG4 and the IgA subclasses IgA1 and IgA2, IgD and in the equivalent domains (CH4) of IgE and IgM. The IgG1 CH3 domain is defined and numbered according to Eu numbering, and the first amino acid in the CH3 domain is indicated by the inverted triangle above the glycine. The amino acid sequence shown is derived from the translation of the open reading frame of the sequence from the European Nucleotide Archive (https: / / www,ebi.ac.uk / ena / browser / ). The accession numbers for the H chain sequences are: IgG1 CH3 domain, J00228-IGHG1; IgG3 CH3 domain, X03604-IGHG3; IgG4 CH3 domain, K01316-IGHG4; IgG2 CH3 domain, J00230-IGHG2; IgA1 CH3 domain, J00220-IGHA1; IgA2 CH3 domain, J00221-IGHA2; IgE CH4 domain, IGHE-CH4-J00222; IgM CH4 domain, IGHM-CH4-X57331; and IgD CH3 domain, IGHD-K02879;

[0021] Figure 5 The results of purification of ACE2-Fc H429Y fusion protein according to the present disclosure are shown: (A) anion exchange (IEX) chromatography of flACE2-Fc-WT (flow through (ft), eluted fractions and washes), where the ACE2-Fc-containing peak is highlighted with *; (B) SDS-PAGE of flow through (ft) and IEX peak fractions, where flACE2-Fc H429Y migrates above the 250 kDa marker and low molecular weight impurities are marked as (C) Size exclusion chromatography (SEC) of the 1EX fraction containing flACE2-Fc-WT (comparator) using a Superose 6 column, with oligomeric (oli), monomeric (mn), and low mw impurities indicated And (D) SEC of IEX fractions containing flACE2-Fc H429Y, showing a high proportion of oligomeric species. The monomeric (mn) species is considered to be a single molecule (i.e., a monomeric molecule) comprising two copies of the corresponding ACE2-Fc fusion polypeptide self-associated by the Fc region component;

[0022] Figure 6Graphical results showing SARS-CoV-2 RBD binding activity of ACE2-Fc fusion proteins according to the present disclosure are provided. Binding of ACE2-Fc fusion proteins to immobilized RBD-Ig was determined by ELISA: (A) trACE2-Fc-WT, (B) flACE2-Fc-WT and (C) EflACE2-Fc-WT and the following variants thereof:

[0023] F ACE2-Fc protein comprising a mutant H429F Fc region component

[0024] Y oli SEC-purified ACE containing the mutant H429Y Fc region component (oligomeric protein) 2 -Fc protein

[0025] YmnSEC-purified ACE2-Fc protein containing a mutant H429Y Fc region component (monomeric protein; comprising two copies of ACE2-Fc fusion protein dimerized by the Fc region component)

[0026] kiftrACE2-Fc-WT protein produced in the presence of the mannosidase inhibitor kifunensine.

[0027] (D) EC of trACE2-Fc-WT, flACE2-Fc-WT and variant proteins 50 Summary of binding constants, demonstrating that the apparent RBD binding affinity of flACE2-Fc-WT is higher than that of trACE2-Fc-WT and that the binding affinity of the flACE2-Fc-H429Y monomer is weaker (Ymn; i.e., a flACE2-Fc-H429Y fusion protein comprising two copies dimerized by an Fc region component). Welch's unpaired t-test p = 0.0332 (*), < 0.0001 (****);

[0028] Figure 7Results showing that flACE2-Fc H429Y fusion proteins according to the present disclosure form pH-dependent oligomers 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 greater proportion of monomer (mn) fusion protein compared to separation at pH 7.4. (C) Native PAGE of ACE2-Fc H429Y (1 μg). Lane 1, SEC pH 5 oligomers (oli); Lane 2, SEC pH 5 monomers (mn); Lane 3, SEC pH 7.4 oligomers; Lane 4, SEC pH 7.4 monomers; Lane 5, SEC pH 5 monomers were redialyzed against PBS pH 7.4 and purified by SEC, and the mn fractions collected as in Figure D. (D) SEC chromatogram of the pH 5.0 oligomer analyzed in lane 1 of panel C was redialyzed against PBS pH 7.4 and SEC was performed in PBS pH 7.4. (E) flACE2-Fc-H429Y monomer (Y mn ) has improved RBD-Ig binding activity. A monomer is considered to be a single molecule (i.e., a monomeric molecule) comprising two copies of the corresponding ACE2-Fc fusion protein dimerized by the Fc region component;

[0029] Figure 8 Results are provided showing that the SARS-CoV-2 neutralization potency of ACE2-Fc fusion proteins according to the present disclosure is affected by the ACE2 scaffold (i.e., truncated or full-length) and Fc mutations. The neutralization potency of ACE2 polypeptides and three groups of ACE2-Fc-WT fusion proteins and variant proteins was determined by titration to the cytopathic effect (CPE) endpoint in a microneutralization assay. The fusion proteins are trACE2-Fc, flACE2-Fc, and EflACE2-Fc WT and Fc variants, H429F, F; H429Y oligomers, Y on SEC oli and H429Y monomer on SEC, Y mn (Comprising two copies of the fusion protein dimerized by the Fc region component). The trACE2-Fc fusion protein comprises a glycan-modified trACE2-Fc-kif. Mean ± SEM, ANOVA of the neutralization endpoint using Dunnett's multiple comparison test compared to ACE2 and ACE2-Fc WT. p = 0.1234 (not significant), 0.0332 (*), 0.0021 (**), 0.0002 (***), < 0.0001 (****);

[0030] Fig. 9 The results of assays evaluating the interaction of ACE2-Fc fusion proteins with FcγRs according to the present disclosure are provided. Ramos-S cells were conditioned with ACE2-Fc WT fusion proteins and variant proteins (5 μg / ml). Biotinylated (A) dimeric rsFcγRIIa or (B) dimeric rsFcγRIIIa probes, followed by streptavidin-APC, were bound to the conditioned cells, and the binding was described as the median fluorescence intensity (median FI) determined by flow cytometry. The FcR binding activity of the ACE2-Fc fusion protein was easily detected, except for the ACE2-Fc H429Y fusion protein, which had greatly reduced FcR binding activity (3 replicates, mean ± SEM);

[0031] Fig.10 Provided are assay results for evaluating the ability of ACE2-Fc fusion proteins according to the present disclosure to mediate cell activation by FcγRIIIa. The results show that, except for the inability of Fc H429Y mutants in any ACE2 format to stab, flACE2-Fc protein is an effective activator of FcγRIIIa. Defucosylated trACE2-Fc-kif is also an effective activator of FcγRIIIa. Ramos-S target cells were opsonized with the following: (A) trACE2-Fc, (B) flACE2-Fc and (C) EflACE2-Fc, WT and variant proteins (including H429F, F); H429Y, oligomers, Yoli; H429Y monomers, Ymn (including two copies of fusion proteins dimerized by Fc region components); or trACE2-Fc kif produced by trACE2-Fc WT in Expi293 cells in the presence of the mannosidase inhibitor kif. In some experiments, Ramos-S target cells were opsonized with the anti-CD20 mAb rituximab RIT. These opsonized targets were incubated with FcγRIIIa-NF-κB-RE nanoluciferase reporter cells, and activation of FcγRIIIa was measured by induction of nanoluciferase (RLU). The data were fitted to the agonist response curve to estimate the EC 50 (D) shows the EC from the curve fit. 50 (nM) values. Mean ± SEM, ANOVA with Dunnett's multiple comparison test, n ≥ 4, compared with trACE2-Fc WT. p = 0.0021 (**), 0.0002 (***), < 0.0001 (****);

[0032] Fig.11Results are provided showing that ACE2-Fc fusion proteins including Fc region components with H429F and H429Y mutations according to the present disclosure strongly fix complement of Ramos-S target cells and direct complement-dependent cytotoxic killing; as determined using 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 captured by plate-bound avidin: Binding of C1q to ACE2-Fc fusion protein variants (2.0 μg / ml) at different concentrations of avidin-captured RBD on the plate (A, B); Titration of C1q (C, D) or C5b-9 (E, F) binding by serially diluted ACE2-Fc fusion protein variants bound to avidin-captured RBD-biotin (2.5 μg / ml) (mean ± SEM); two independent ELISA experiments. (G) Flow cytometric analysis of complement-dependent cytotoxicity (CDC) (% killing) of opsonized Ramos-S cells in the presence of 1 / 3 diluted normal human serum as a complement source (EC from curve fitting is shown). 50 (nM) value);

[0033] Fig.12 Results of assays showing that IgG1 antibodies comprising an Fc region component with a H429F mutation of the CH3 domain strongly fix complement and show the same antigen binding are provided. (A, B) TNP-BSA antigen at different concentrations (20 μg / ml-0.625 μg / ml) was adsorbed to wells of an ELISA plate and reacted with a single concentration (2 μg / ml) of chimeric anti-TNP human IgG1 and IgG2 mAbs comprising unmodified wild-type (WT) heavy chains or with 2 μg / ml of an anti-TNP mAb comprising an IgG1 heavy chain comprising the Fc component mutation H429F (TNP-IgG1-H429F). Fixation of C1q with (A) purified human C1q and detected with anti-C1q rabbit polyclonal antibody and (B) antibody-opsonized TNP-BSA coated wells treated with human serum as a complement source and formation of membrane attack complex (C5b-C9) detected with anti-C5b-C9 rabbit polyclonal antibody. (C) provides the results of ELISA showing the antigen binding activity of the chimeric anti-TNP mAbs used in Figures A and B, including unmodified (TNP-IgG1-WT and TNP-IgG2-WT mAbs) and mAbs modified with H429, i.e., TNP-IgG1-H429F. ELISA plates coated with TNP-BSA were then titrated against anti-TNP mAbs, and antibody binding was detected with HRP-conjugated anti-human IgG. All mAbs showed similar antigen binding activity;

[0034] Fig.13 Results showing C1q fixation and MAC (C5b-9) formation determined by mAbs including IgG1 heavy chains modified with glutamic acid, glutamine or serine at position 429 of the heavy chain are provided. TNP-BSA (20 μg / ml) is adsorbed into wells and reacted with chimeric human anti-TNP mAbs, titrated across a concentration range of 4 μg / ml-0.125 μg / ml. The mAbs tested include unmodified wild-type (WT) heavy chains of IgG1 (TNP-IgG1-WT) (Figure AD) or IgG2 (TNP-IgG2-WT) (Figure AC) or IgG1 H chains including mutations TNP-IgG1-H429Q or TNP-IgG1-H429E (A, C) or TNP-IgG1-H429S (B, D). Wells coated with antibody-opsonized TNP-BSA are treated with human serum as a complement source. Immobilization of C1q (A, B) was detected with anti-C1q rabbit polyclonal antibody, and formation of membrane attack complex (C5b-C9) was detected with rabbit anti-C5b-C9 polyclonal antibody (C, D);

[0035] Fig.14 Results of flow cytometric quantification of SEC-purified mAb binding to target cell surface antigens are provided. 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 (SEC (IgG H2L2) peak fraction p1 and IgG oli The binding activity of unmodified trastuzumab-WT, pertuzumab-WT and its CH3 heavy chain mutant carrying mutation H429F was evaluated on SK-OV-3 cells expressing HER2;

[0036] Fig.15 Elution chromatograms of protein A affinity purification characteristics of rituximab-WT, trastuzumab-WT and its mutants carrying modifications in the CH3 of the heavy chain are provided. IgG was recovered from the column after elution with sodium citrate buffer pH 3.0: (A) Elution profile of anti-CD20 rituximab-based mAb. Rituximab-WT IgG and each mutant IgG eluted as a single homogenous peak; (B) Elution profile of anti-HER2 trastuzumab-based mAb. Trastuzumab-WT IgG and each mutant IgG eluted as a single homogenous peak;

[0037] Fig.16Provided are size exclusion chromatography (SEC) curves that reveal that the H429 mutation can change the physical properties of IgG. After protein A affinity purification, mAbs were further purified by SEC at pH 7.2: (A) SEC curves of unmodified rituximab-WT (WT) and rituximab-H429F (H429F) mutants consist of a single homogenous IgG peak (fractions to the right of the vertical dashed line) and minimal oligomeric species (fractions to the left of the vertical dashed line). Thus, for example, the SEC curve of unmodified rituximab-WT shows a single major species corresponding to IgG (H2L2) as expected, as confirmed by SDS-PAGE analysis ( Fig.17 A), wherein it migrates with an expected unreduced IgG of about 150 kDa mass and is decomposed into its about 50 kDa heavy (H) chain and about 25 kDa light (L) chain species after reduction in DTT. On the other hand, rituximab-H429Y mAb includes both non-oligomeric IgG and oligomeric IgG; (B) SEC curves of protein A-purified anti-HER2 trastuzumab wild type (WT) (i.e., unmodified form of trastuzumab) and its mutants containing amino acid substitutions of H429F or H429Y. For trastuzumab-WT and H429F mutant mAbs, a single peak of non-oligomeric IgG is observed on the right side 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 equivalent rituximab-based mAb in Figure (A). The SEC profile of oligomeric IgG in trastuzumab-H429Y is consistent with the oligomeric IgG in rituximab-H429Y in Figure (A);

[0038] Fig.17 Images obtained from SDS-PAGE of SEC-purified mAbs 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-reducing conditions, the non-oligomeric IgG peaks of rituximab-WT (WT IgG) and rituximab-H429F (HF IgG) migrated as a single species with the expected IgG (i.e., H2L2) molecular size of approximately 150 kDa. After reduction in DTT, the 150 kDa IgG was resolved into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains as expected. Before reduction, the non-oligomeric IgG (HY IgG (H2L2)) and oligomeric (HY IgG (oli)) forms of rituximab-H429Y mAb (see Fig.16) migrate identically as a single 150 kDa IgG, i.e., H2L2 species. After reduction, both forms migrate 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) migrate as a single species with the expected molecular size of approximately 150 kD IgG, i.e., H2L2. After reduction with dithiothreitol (DTT), the 150 kDa IgG is resolved into approximately 50 kDa heavy (H) chains and approximately 25 kDa light (L) chains as expected. Before reduction, non-oligomeric IgG (HY IgG (H2L2)) and oligomeric (HYIgG (oli)) forms of trastuzumab-H429Y mAb migrate identically under non-reducing conditions and after reduction as a single 150 kDa IgG species with an expected ~50 kDa heavy (H) chain and ~25 kDa light (L) chain; WT = wild type, HF = H429F, HY = H429Y; M = molecular weight marker, whose mass (kD) is shown on the left;

[0039] Fig.18 Experimental results are provided showing that oligomeric and non-oligomeric forms of H429Y-modified IgG antibodies show equivalent CDC potency and that their formation is pH sensitive: (A) SEC of rituximab-H429Y at pH 7.2 shows the presence of two major IgG forms (see also Fig.16A). Non-oligomeric IgG (H2L2) (right side of vertical dashed line, indicated as p1) and oligomeric forms of IgG (IgG (oli)) (left side of vertical dashed line, indicated as p2) were collected separately to further evaluate complement-dependent cytotoxicity (CDC) efficacy; (B) Oligomeric (p2) and non-oligomeric (p1) IgG forms from Figure A and SEC-purified unmodified rituximab-WT IgG were titrated, and CDC efficacy was determined by flow cytometry using Ramos lymphoma cells and normal human serum diluted 1 / 3 as complement sources. The degree of killing of Ramos cells opsonized with non-oligomeric rituximab H429Y p1 (open squares), oligomeric rituximab-H429Y p2 (triangles) or unmodified rituximab-WT (solid circles) was determined. The control background CDC of complement in the absence of mAb (no mAb only C') is shown as a filled diamond; and (C) The formation of IgG oligomers is sensitive to pH. Protein A purified trastuzumab-H429Y was analyzed by size exclusion chromatography (SEC) at pH 7.2 (left) or pH 5.0 (right). At pH 7.2, both oligomeric (IgG(oli)) and non-oligomeric IgG were present, whereas at pH 5.0, only a single peak corresponding to non-oligomeric IgG was present;

[0040] Fig.19 Results showing that the H429F mutation in the CH3 domain of the IgG H chain effectively promotes C1q binding and complement-dependent cell-mediated cytotoxicity are provided. Flow cytometry detection of C1q binding (fluorescence intensity) of Ramos lymphoma cells conditioned with unmodified rituximab-WT (A) or with modified rituximab-H429F mAb (B), or SK-OV-3 ovarian cancer cells conditioned with trastuzumab-WT (C) or with modified trastuzumab-H429F mAb (D) is depicted. Cells conditioned with mAb were treated with normal human serum as a source of complement, and the binding of C1q was detected by staining with anti-C1q rabbit polyclonal antibody. The binding of C1q to cells conditioned with mAb is shown in a histogram without shading, and the C1q background binding control (i.e., cells treated with serum complement in the absence of mAb) is shown in a histogram with gray shading. The median fluorescence intensity (MFI) of each histogram is indicated in brackets. (E) Complement-dependent cytotoxicity (CDC; i.e., % killing detected using Zombie Green) of Ramos lymphoma cells opsonized with rituximab WT (filled circles) or its mutant variant mAb rituximab-H429F (filled squares) was determined by flow cytometry using normal human serum as a complement source. The mutation at position 429 in rituximab-H429F greatly enhanced CDC compared to rituximab-WT mAb;

[0041] Fig. 20 Results of protein A affinity chromatography of type II anti-CD20 11B8-WT mAb and mutant mAbs containing H429 substitutions are shown. mAb is 11B8-WT mAb (WT) produced with wild-type human IgG1 heavy chain. 11B8-H429F (H429F) is produced with a modified IgG1 heavy chain in which the amino acid histidine at position 429 in the CH3 domain of the Ig heavy chain is substituted with phenylalanine;

[0042] Fig.21 The results of size exclusion chromatography (SEC) purification and SDS PAGE analysis of 11B8-WT mAb and 11B8-H429F (H429F) mutant 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 monomeric IgG mAb purified by SEC from Figure A. Before reduction (non-reducing), all SEC-purified antibodies migrated with the expected molecular size of about 150 kDa for IgG, i.e. H2L2. After reduction (reducing) with DTT, all antibodies were resolved into about 50 kDa heavy (H) chains and about 25 kDa light (L) chains as expected. WT = wild type, HF = H429F, M = molecular weight marker, whose mass (kD) is shown on the left;

[0043] Fig. 22 Results showing that the H429 modification of the CH3 domain confers CDC potency to type II anti-CD20 mAbs are provided. CDC produced by 11B8-WT mAb (filled circles) or CH3-mutated 11B8-H429F mAb (filled squares). Complement-dependent lysis was determined using Ramos lymphoma cells opsonized with the indicated concentrations of mAb and normal human serum diluted 1 / 3 as a complement source. CDC (% killing) was determined by flow cytometry using Zombie Green. The control background CDC of complement in the absence of mAb (no mAb only C') is shown as a filled diamond. 11B8-WT mAb failed to mediate CDC, while 11B8-H429F mutant mAb mediated effective complement-dependent lysis of lymphoma cells (WT = wild type);

[0044] Fig.23 Results of protein A affinity chromatography of anti-CD38 daratumumab-WT mAb and daratumumab-H429F mAb are shown. The elution chromatograms show that each of the antibodies eluted as a single consistent homogenous peak;

[0045] Fig.24 Results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of daratumumab-WT mAb and mutant mAb daratumumab-H429F are shown. (A) Chromatograms of SEC-purified mAbs. 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 of SEC-purified IgG from Figure A (5-15% gradient gel). Before reduction (non-reducing), all antibodies migrated with the expected molecular size of about 150 kDa for IgG (i.e., H2L2), and after reduction with DTT (reducing), all antibodies were resolved into about 50 kDa heavy (H) chains and about 25 kDa light (L) chains as expected. WT = wild type, HF = H429F, M = molecular weight marker, whose mass (kD) is shown on the left;

[0046] Fig.25 Results showing that the H429F substitution significantly enhances complement-dependent lysis of lymphoma cells by daratumumab-WT are provided. The figure shows CDC levels (% killing) of daratumumab-WT mAb (solid circles) and daratumumab-H429F (solid squares). Background lysis (solid triangles) is measured in the absence of mAb but only in the presence of complement. CDC is measured by flow cytometry using Zombie Green. Complement-dependent lysis of lymphoma cells mediated by daratumumab-H429F mAb is more effective than complement-dependent lysis of lymphoma cells mediated by unmodified daratumumab-WT;

[0047] Fig.26 Results showing that the H429F substitution confers resistance to lysis by anti-CD38 mAbs for myeloma and leukemia cells are provided. CDC generated by complement-dependent lysis was determined using the following assays: (A) KMS-12-PE myeloma cells conditioned with the indicated concentrations of daratumumab-WT mAb (filled circles) or daratumumab-H429F mutant mAb (filled squares). Background lysis (filled triangles) was determined in the absence of mAb but only in the presence of complement; and (B) SUP-1 5 acute lymphoblastic leukemia (ALL) cells conditioned with the indicated concentrations of daratumumab-WT mAb (filled circles) or daratumumab-H429F mutant mAb (filled squares). Background lysis (filled triangles) was determined in the absence of mAb but only in the presence of complement. CDC (% killing) was determined by flow cytometry using Zombie Green;

[0048] Fig. 27Results of protein A affinity chromatography of the anti-HER2 mAb Pertuzumab-WT and CH3 variants are shown. The elution chromatograms show that each of the antibodies eluted as a single consistent homogenous peak;

[0049] Fig.28 The results of size exclusion chromatography (SEC) purification and SDS-PAGE analysis of anti-HER2 mAb Pertuzumab-WT and CH3 H429F variants are shown. (A) Chromatogram of SEC-purified mAb. After protein A affinity chromatography, mAb was further purified by size exclusion chromatography (SEC) at pH 7.2. Chromatographic curves of unmodified Pertuzumab-WT and Fc-modified variant Pertuzumab-H429F (H429F) are shown. In each case, the mAb purified by protein A contains a single non-oligomeric IgG peak (fraction on the right side of the vertical dashed line) and there is no oligomeric species (fraction on the left side of the vertical dashed line). (B) SDS-PAGE analysis (5-15% gradient gel) of SEC-purified IgG from Figure A. Before reduction (non-reduction), all SEC-purified antibodies migrated with an expected molecular size of about 150kDa for IgG (i.e., H2L2). After reduction with DTT (reduction), all antibodies resolved 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, which is shown in kilodaltons (kD);

[0050] Fig.29Results showing cooperation and functional synergy in a mixture of mAbs comprising an amino acid substitution at position H429 are provided; specifically, the results show that mAbs modified with H429F can bind to enhance binding of C1q to target cells: (A) Flow cytometry histograms of C1q binding to HER2-expressing SK-OV-3 cells opsonized with trastuzumab-H429F alone, pertuzumab-H429F alone, or a 1:1 mixture of trastuzumab-H429F and pertuzumab-H429F (unfilled histograms). The filled histograms show background binding of C1q to unopsonized cells. The 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 cooperation. SK-OV-3 cells were opsonized with titrated anti-HER2 mAbs alone, 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. The double-headed vertical arrow illustrates extensive cooperation in the mixture of trastuzumab-H429F and pertuzumab-H429F, where antibody concentration was limiting compared to C1q binding to either mAb alone;

[0051] Fig.30Graphical results showing the cooperation and functional synergy of H429-modified mAbs enhance complement-dependent killing (CDC) of target cells are provided. The indicated rituximab-based mAbs were titrated alone on Ramos cells (light grey bars) or in the presence of fixed concentrations of the indicated anti-CD38 or anti-CD20 mAbs (dark bars): (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 (% killing) was determined by flow cytometry using Zombie Green. The horizontal dashed lines in (B, C) indicate the percentage of CDC killing obtained in the absence (0 μg / ml) of rituximab-based mAbs but only in the presence of (B) 0.025 μg / ml daratumumab-H429F or (C) 0.5 μg / ml 11B8-H429F. The black arrows in (B, C) indicate that the enhanced CDC of the mAb mixture is higher than the enhanced CDC of the mAb alone (i.e., CDC of rituximab-H429F alone (light bars) or 0.025 μg / ml daratumumab-H429F or 0.5 μg / ml 11B8-H429F (dark bars), CDC of 0 μg / ml rituximab) at the indicated concentrations);

[0052] Fig.31 Provided is a rituximab antibody comprising an H429F mutation for normal peripheral blood CD19 + The flow cytometry analysis results of the CDC efficacy of B lymphocytes.The results show that, compared with unmodified rituximab-WT, rituximab-H429F mAb shows more effective CDC killing to normal peripheral blood B cells.After peripheral blood mononuclear cells are treated with indicated mAb and human complement, the ratio of live cells or dead B cells is determined by staining with anti-CD19 to identify B cells and also with Zombie Green (ZG) to identify dead cells.Cell histograms show the ZombieGreen median fluorescence intensity (ZG MFI) (CD19 median fluorescence intensity (CD19 MFI)) of gated CD19 B cells. In each cell histogram, the % of dead cells (ZG positive) is shown in the upper section 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 % of live B cells is shown in the lower section Q2;

[0053] Fig.32 Results are provided showing that the effect of modification of H429 on antibody function is not limited to IgG1, and extends to other immunoglobulin classes. Here, rituximab antibodies formatted with (A) IgG3 or (B) IgG4 subclass heavy chains modified with wild-type (WT) or H429F (HF) were evaluated for CDC efficacy against Ramos lymphoma cells;

[0054] Fig.33 Results are provided showing that effective CDC of target cells mediated by mAbs is dependent on the presence of both the monoclonal antibody and serum complement. CDC (% killing) of rituximab-H429F, daratumumab-H429F or 11B8-H429F against Ramos cells or daratumumab-H429F against SUP-15 cells is shown. A = Percent killing in the presence of the indicated mAbs and human serum as a source of complement; B = Lysis in the presence of mAbs only at the same concentration as in A; C = Lysis in the presence of complement only;

[0055] Fig.34 Results showing that the H429F substitution confers complement-dependent lysis of leukemic cells resistant to lysis by unmodified anti-CD38 mAb isatuximab are provided. CDC generated by complement-dependent lysis was determined using SUP-15 acute lymphoblastic leukemia (ALL) cells, which were opsonized with indicated concentrations of isatuximab-WT mAb (dashed line with filled circles) and isatuximab-H429F mutant mAb (solid line with filled squares). Background lysis (unfilled circles) was determined in the absence of mAb but only in the presence of complement (C' only, no mAb). Background lysis induced by 5 μg / ml of mAb in the absence of complement was determined for isatuximab-WT mAb (inverted unfilled triangles) and isatuximab-H429F mutant mAb (hollow unfilled squares). CDC (% killing) was determined by flow cytometry using Zombie Green;

[0056] Fig.35Results are provided for flow cytometric quantification of binding of purified DR5-specific mAbs comprising unmodified wild-type (WT) H chains of the human IgG1 or IgG2 subclass or comprising heavy chains of the human IgG1 or IgG2 subclass that have been modified by replacement of histidine 429 with phenylalanine (H429F) to Colo205 colorectal cells. The graphs show: (A) binding of BDR5-1WT comprising a wild-type H chain of the human IgG1 subclass or binding of BDR5-1HF mAb comprising an IgG1 heavy chain containing the H429F modification, (B) binding of BDR5-2WT comprising an unmodified heavy chain of the human IgG2 subclass or binding of BDR5-2HF mAb comprising an IgG2 heavy chain containing the H429F modification, (C) binding of TDR5-1WT mAb comprising an unmodified heavy chain of the human IgG1 subclass or binding of TDR5-1HF mAb comprising an IgG1 heavy chain containing the H429F modification, and (D) binding of TDR5-2WT comprising an unmodified heavy chain of the human IgG2 subclass or binding of TDR5-2HF mAb comprising an IgG2 heavy chain containing the H429F modification. mAbs were titrated 2-fold serially and binding activity on Colo205 cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. In all figures, the level of nonspecific binding of the fluorescent conjugate to the cells is shown (▲ conjugate); MFI = median fluorescence intensity;

[0057] Fig.36Results are provided for flow cytometric quantification of binding of purified DR5-specific mAbs comprising unmodified wild-type (WT) H chains of human IgG1 or human IgG2 subclasses or comprising human IgG1 or human IgG2 heavy chains that have been modified by replacing histidine 429 with phenylalanine (H429F) to target Ramos lymphoma cells. The graphs show: (A) binding of BDR5-1WT comprising an unmodified heavy chain of the human IgG1 subclass or binding of BDR5-1HF mAb comprising an IgG1 heavy chain modified with H429F; (B) binding of BDR5-2WT comprising an unmodified heavy chain of the human IgG2 subclass or binding of BDR5-2HF mAb comprising an IgG2 heavy chain modified with H429F; (C) binding of TDR5-1WT comprising an unmodified heavy chain of the human IgG1 subclass or binding of TDR5-1HF mAb comprising an IgG1 heavy chain modified with H429F; and (D) binding of TDR5-2WT comprising an unmodified heavy chain of the human IgG2 subclass or binding of TDR5-2HF mAb comprising an IgG2 heavy chain modified with H429F. mAbs were titrated serially 2-fold and binding to Ramos cells was quantified by flow cytometry using an anti-IgG secondary reagent labeled with goat anti-hIgG Fc FITC. In all figures, the level of nonspecific binding to cells was determined using an irrelevant IgG antibody (negative IgG), and background binding of fluorescent anti-IgG conjugates to cells is shown (▲ conjugates); MFI = median fluorescence intensity;

[0058] Fig.37 The results of flow cytometry quantification of the binding of purified DR5-specific mAb to target KMS-12-PE myeloma cells, including unmodified wild-type (WT) H chains of human IgG1 or human IgG2 subclasses or human IgG1 or human IgG2 heavy chains modified by replacing histidine 429 with phenylalanine (H429F), are provided. The binding activity of DR5-specific mAb, BDR5 and TDR5 is shown, including unmodified wild-type H chains of human IgG1 subclasses (BDR5-1WT, TDR5-1WT) or human IgG2 subclasses (BDR5-2WT, TDR5-2WT), or Fc mutations of IgG1 subclasses (BDR5-1HF, TDR5-1HF) or IgG2 subclasses (BDR5-2HF, TDR5-2HF) containing H249F mutations. KMS12-PE cells are 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 controls for anti-IgG conjugate binding alone (Conjugate) are shown along with background fluorescence of cells alone (Cells);

[0059] Fig.38 Results showing the survival of Colo205 colorectal cells in the presence of purified DR5-specific mAbs comprising an unmodified wild-type H chain of the human IgG1 or human IgG2 subclass or an Fc-mutated H chain of the human IgG1 or human IgG2 subclass containing the H429F mutation are provided. Ten thousand cells were cultured in each well of a 96-well plate in the presence of 20 μg / ml of each of the following mAbs: TDR5-1WT mAb comprising an unmodified wild-type H chain of the human IgG1 subclass, or TDR5-1HF mAb comprising an H chain of the human IgG1 subclass containing the H429F mutation, or TDR5-2WT mAb comprising an unmodified wild-type H chain of the human IgG2 subclass, or TDR5-2HF mAb comprising an H chain of the human IgG2 subclass containing 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 mAbs (TDR5-2HF+BDR5-2HF), both of which included a human IgG2 H chain with an Fc mutation carrying the H429F mutation. Cell viability was quantified using a CCK8 colorimetric cell viability assay, in which the absorbance (Abs450nm) of the cell culture supernatant was a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibody (no ab). Survival was shown as: [Abs450 nm-background Abs450 of experimentally determined mAb or control treatment-cell culture medium], and the average Abs450 and four replicate values ​​were shown;

[0060] Fig.39Results showing the survival of Ramos lymphoma cells in the presence of purified DR5-specific mAbs comprising unmodified wild-type or Fc-mutated H chains of human IgG1 or IgG2 subclasses containing the H429F mutation are provided. Ten thousand cells were cultured in each well of a 96-well plate in the presence of 20 μg / ml of each of the following mAbs: TDR5-1WT mAb comprising unmodified wild-type H chains of human IgG1 subclass, or TDR5-1HF mAb comprising H chains of human IgG1 subclass containing the H429F mutation, TDR5-2WT mAb comprising unmodified wild-type H chains of human IgG2 subclass, or TDR5-2HF mAb comprising H chains of human IgG2 subclass containing 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 mAbs (TDR5-2HF+BDR5-2HF), both of which included a human IgG2 H chain with an Fc mutation carrying the H429F mutation. Cell viability was quantified using a CCK8 colorimetric cell viability assay, in which the absorbance (Abs450 nm) of the cell culture supernatant was a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibody (no ab). Survival was shown as: [Abs450 nm-background Abs450 of experimentally determined mAb or control treatment-cell culture medium], and the average Abs450 and four replicate values ​​were shown;

[0061] Fig.40Results showing the viability of Colo205 colorectal cells in the presence of a mixture of purified DR5-specific mAbs are provided, wherein the mAbs include 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 of IgG1 or IgG2 subclasses containing the H429F modification. Colo205 cells (30,000 cells / well of a 96-well plate) were cultured in the presence of serial 2-fold dilutions of a 1:1 mixture of mAbs containing the same H chain. A starting concentration of 1 μg / ml contained 0.5 μg / ml of each mAb in the mixture. Therefore, the mixture used was: BDR5-1WT mAb including an unmodified wild-type IgG1 H chain mAb was mixed with TDR5-1WT including an unmodified wild-type IgG1 H chain (BDR5-1WT+TDR5-1WT); BDR5-2WT mAb including an unmodified wild-type IgG2 H chain mAb was mixed with TDR5-2WT including an unmodified wild-type IgG2 H chain (BDR5-2WT+TDR5-2WT); BDR5-1HF mAb including an IgG1 H chain modified with H429F was mixed with TDR5-1HF mAb including an IgG1 H chain modified with H429F (BDR5-1HF+TDR5-1HF); BDR5-2HF mAb including an IgG2 H chain modified with H429F was mixed with TDR5-2HF mAb including an IgG2 H chain modified with H429F (BDR5-2HF+TDR5-2HF). 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 antibody (no ab), and maximum death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability was expressed as: [Abs450nm of experimentally determined mAb or control treatment - background Abs450 of cell culture medium];

[0062] Fig.41Results showing the survival of Colo205 colorectal cells in the presence of different pairwise combinations of purified DR5-specific mAbs are provided, wherein the mAbs include wild-type or Fc-mutated H chains of different IgG subclasses. The mAbs include H chains of human IgG1 subclass or human IgG2 subclass comprising a wild-type heavy chain or a heavy chain modified by the H429F modification. Colo205 cells (10,000 cells / well of a 96-well tissue culture plate) were cultured in the presence of a 1:1 mixture of mAbs serially diluted 2-fold from 1 μg / ml. The starting concentration of 1 μg / ml included 0.5 μg / ml of each mAb in the mixture. The mixtures of wild-type mAbs used were: BDR5-2WT including wild-type human IgG2 H chain mixed with TDR5-1WT including unmodified wild-type human IgG1 H chain (BDR5-2WT+TDR5-1WT); BDR5-1WT including wild-type human IgG1 H chain mixed with TDR5-2WT including unmodified wild-type human IgG2 H chain (BDR5-1WT+TDR5-2WT); and a control mixture of BDR5-2WT including unmodified wild-type human IgG2 H chain mixed with TDR5-2WT including unmodified wild-type human IgG2 H chain (BDR5-2WT+TDR5-2WT) as described in Fig.40 The mixture of DR5 mAbs including the H chain with the H429F Fc-mutation was: BDR5-2HF including the human IgG2 H chain with the H429F Fc-mutation was mixed with TDR5-1HF including the human IgG1 H chain with the H429F Fc-mutation (BDR5-2HF+TDR5-1HF); BDR5-1HF including the human IgG1 H chain with the H429F Fc-mutation was mixed with TDR5-2HF including the human IgG2 H chain with the H429F Fc-mutation (BDR5-2HF+TDR5-2HF); a positive killing control mixture (BDR5 2HF+TDR5-2HF) containing the human IgG2 H chain with the H429F Fc-mutation and the IgG2 H chain with the H429F Fc-mutation was mixed with BDR5-2HF including the H429F Fc-mutation Fig.38 and Fig.40 Observed in. Cell viability was quantified using a CCK8 colorimetric cell viability assay, in which 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 antibody (no ab), and maximum death was determined by culturing cells with sodium dodecyl sulfate (SDS). Viability is presented as: [Abs450 nm of experimentally determined mAb or control treatment - background Abs450 of cell culture medium];

[0063] Fig.42 Provided are results showing the viability of Colo205 cells in the presence of paired mixtures of BDR5 and TDR5 mAbs at different ratios. In the presence of a single mAb BDR5-1WT or TDR5-1WT including a wild-type IgG1 H chain or a single mAb BDR5-1HF or TDR5-1HF including an H chain modified with H429F, or a mixture of BDR5-1HF and TDR5-1HF mAbs, 10,000 Colo205 cells were cultured for 48 hours 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 viability in the absence of mAb (no ab) or the maximum death control in the presence of sodium dodecyl sulfate (SDS). Cell viability was quantified using a CCK8 colorimetric cell viability assay, in which the absorbance (Abs450 nm) of the cell culture supernatant is a measure of cell viability and viability. Viability is expressed as: [Abs450 nm of experimentally determined mAb or control treatment - background Abs450 of cell culture medium];

[0064] Fig.43 Provided are results showing the survival rate of Ramos lymphoma cells in the presence of paired mixtures of BDR5 and TDR5 mAbs of different ratios. In the presence of a single mAb BDR5-1WT or TDR5-1WT including a wild-type H chain or a single mAb BDR5-1HF or TDR5-1HF including an H chain modified with H429F, or a mixture of BDR5-1HF and TDR5-1HF mAb, 10,000 Ramos cells were cultured for 48 hours at the following ratios: 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixture shown in the figure, BDR5-1HF mAb is designated as B-1HF, and TDR5-1HF is designated as T-1HF. CCK8 colorimetric cell viability assay is used to quantify cell viability, wherein the absorbance (Abs450 nm) of the cell culture supernatant is a measure of cell viability and viability. Maximal cell viability was determined by culturing cells in the absence of antibody (no ab), and maximal cell death was determined by culturing cells with sodium dodecyl sulfate (SDS). Survival was expressed as: [Ab450 nm of experimentally determined mAb or control treatment - background Ab450 of cell culture medium];

[0065] Fig.44Results of quantitative detection of different epitopes and binding of DR5-specific mAbs to Colo205 cells including wild-type or Fc-mutated heavy chains of human IgA2 subclasses are provided. mAbs used as tissue culture supernatants from appropriately transfected Expi293 cells include unmodified wild-type IgA2 heavy chains (BDR5-A2WT) and (TDR5-A2WT) or Fc-mutated IgA2H chains carrying H429F mutations (BDR5-A2HF) and (TDR5-A2HF) of human IgA2 subclasses. Background fluorescence (no ab) determined by measuring the binding of fluorescent conjugates alone is shown together with nonspecific fluorescence of cells alone (cells);

[0066] Fig.45 Results showing the survival of Colo205 cells in the presence of two different DR5-specific mAbs comprising a heavy chain of the human IgA2 subclass are provided. Colo205 cells (10,000 cells / well of a 96-well plate) were cultured for 48 hours in the presence of serial 2-fold dilutions of tissue culture supernatant from Expi293 cells to generate Fig.44 The DR5 IgA mAbs used in the assay were: BDR5-A2WT comprising a wild-type heavy chain of the human IgA2 subclass; TDR5-A2WT comprising a wild-type heavy chain of the human IgA2 subclass; BDR5-A2HF comprising a heavy chain of the human IgA2 subclass carrying the H429F modification; TDR5-A2HF comprising a heavy chain of the human IgA2 subclass carrying the H429F modification. Cell viability was quantified using a CCK8 colorimetric cell viability assay, in which the absorbance (Abs450 nm) of the cell culture supernatant is a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibody (no ab), and maximum death was determined by culturing cells with sodium dodecyl sulfate (SDS). Survival is expressed as: [experimentally determined Abs450 nm-background Abs450 of the cell culture medium];

[0067] Fig.46Results showing enhanced killing of Ramos lymphoma cells by a mixture of H429F-modified mAbs detecting different molecular targets are provided. Ramos cells were cultured at 10,000 / well alone or with their Fc-modified counterparts, including isatuximab carrying an H429F-modified H chain (Isa-HF) and BDR5-1 carrying an H429F-modified H chain (BDR5-1HF) or a 1:1 mixture of the two mAbs (Isa-WT+BDR5-1WT) in the presence of anti-CD38 mAb isatuximab comprising an IgG1 WT H chain (Isa-WT) or anti-DR5 mAb BDR5-1 comprising an IgG1 WT H chain (BDR5-1WT) or a 1:1 mixture of the two mAbs (Isa-WT+BDR5-1WT), as described in Example 7. All mAbs are present in the culture at 10 μg / ml, and for the mixture, each mAb is 10 μg / ml, and the total mAb concentration is 20 μg / ml. After 48 hours of cultivation, quantitative cell viability is determined using CCK8 colorimetric cell viability assay, wherein the absorbance (Abs450 nm) of the cell supernatant processed by CCK8 is a measure of cell viability and viability. Maximum cell viability is determined by culturing cells in the absence of antibody (no Ab), and maximum cell death is determined by incubating cells with SDS before CCK8 treatment cells. Survival rate is shown as: [abs450 nm-background Abs450 of cell culture medium determined experimentally], and average Abs450 and four replica values ​​are shown;

[0068] Fig.47 Results are provided showing that the H429F substitution confers complement-dependent lysis (CDC) of leukemic cells resistant to lysis by the unmodified anti-CD38 mAb mezagitamab. CDC resulting from complement-dependent lysis was determined using Ramos lymphoma cells specifically opsonized with the indicated concentrations of mezagitamab-WT mAb or mezagitamab-H429F mutant mAb. Background lysis was determined in the absence of mAb but in the presence of complement only (C' only). CDC (% killing) was determined by flow cytometry using Zombie Green;

[0069] Fig.48Results are provided showing that additional mutations of K439E or S440K in the Fc region component can inhibit the enhanced CDC of H429F-modified rituximab, and that the combination of rituximab-H429F / K439E and rituximab-H429F / S440K restored effective CDC killing of Ramos cells. (A) Ramos lymphoma cells were opsonized with rituximab-WT (WT) or rituximab-H429F (H429F) mAbs alone or with rituximab-H429F including the additional H chain mutation K439E (H429F / K439E) or with rituximab-H429F including the additional H chain mutation S440K (H429F / S440K) or with paired mixtures of rituximab-H429F / S440K and rituximab-H429F / K439E (H429F / K439E+H429F / S440K). Background lysis was determined in the absence of mAb but in the presence of complement only (no mAb C' only). CDC (% killing) of mAbs was measured by flow cytometry using Zombie Green in the presence of 1 / 3 diluted human serum. Enhanced CDC mediated by the H429F modification of rituximab was inhibited by the inhibition of Fc:Fc interactions with either the K439E or S440K mutations, but was found to be fully restored by a mixture of both IgG mutants. (B) For binding analysis, proteins were titrated two-fold serially and binding activity was quantified by flow cytometry (on Ramos cells) using a FITC-conjugated anti-hIgG-Fc secondary reagent. Nonspecific background binding levels of the fluorescent conjugates to cells are shown (conjugate only); MFI = median fluorescence intensity. All mAbs bound to CD20 almost identically;

[0070] Fig.49 Results of flow cytometric analysis of binding (A, C) and CDC (B, D) of purified flACE2-Fc fusion protein (A, B) and SARS-CoV spike-specific mAb (C, D) using Ramos-S cells are provided. For binding analysis, the protein was titrated two-fold continuously, and the binding activity (on Ramos cells) was quantified by flow cytometry using a FITC-conjugated anti-hIgG-Fc secondary reagent. The level of nonspecific background binding of the fluorescent conjugate to the cell is shown (conjugate); MFI = median fluorescence intensity. For CDC analysis, the protein was titrated two-fold continuously, and human serum was used as a complement source. CDC lysis was quantified using Zombie Green. Background lysis by complement in the absence of antibody (no mAb only C') was determined and displayed; and

[0071] Fig.50Graphical results from flow cytometry analysis are provided showing that the cooperation and functional synergy of H429F-modified flACE2-Fc fusion protein and H429F-modified anti-SARS-CoV-2 mAb further enhances complement-dependent killing (CDC). (A) mAb S2P6-H429F (S2P6-HF) or S2P6-WT was titrated alone or in the presence of a fixed concentration of flACE2-Fc-H429 fusion protein (1 μg / ml final concentration) (S2P6-HF+flACE2-Fc-HF), when used alone, mediated 23.4% CDC killing (indicated by open diamond symbols), or titrated in the presence of a fixed concentration of flACE2-Fc-WT (final 1 μg / ml) (S2P6-WT+flACE2-Fc-WT), when used alone, mediated 5.0% killing % (filled diamonds). Background lysis by complement in the absence of mAb or Fc fusion protein is shown (C' only); arrows show examples of maximum synergy; (B) CDC killing efficacy of flACE2-Fc modified by H429F was evaluated on Ramos-S cells. mAbs 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 killing % and background lysis by complement in the absence of mAb or Fc fusion protein (C' only). Four replicate values ​​and SEM are shown. The average killing % value is shown above each column;

[0072] Fig.51The results provided show that ACE2-Fc fusion proteins in three formats, namely trACE2-Fc, flACE2-Fc and EflACE2-Fc, which include Fc region components with wild-type sequences or H429F mutations, bind to Ramos-S target cells in an isopotent manner; as determined by flow cytometry using anti-IgG secondary reagents 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 figures, the level of nonspecific binding of the fluorescent conjugate to the cells (conjugate only) is shown; MFI = median fluorescence intensity. EC50 (nM) values ​​from curve fitting are shown;

[0073] Fig.52 The results provided show that the ACE2-Fc fusion proteins (trACE2-Fc, flACE2-Fc and EflACE2-Fc) in three formats, including Fc region components with wild-type sequences or H429F mutations, strongly fix complement and direct complement-dependent cytotoxicity (CDC) of Ramos-S target cells; as determined by flow cytometry analysis of CDC using opsonized Ramos-S cells using diluted normal human serum as a 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 figures, in the absence of ACE2-Fc fusion proteins, only the non-specific complement killing level of complement (only C') is shown. The EC50 (nM) value of the curve fit from the H429F protein is shown. The fit of the WT protein could not be determined (nd);

[0074] Fig.53 A schematic representation of immunoglobulins (antibodies) and immunoglobulin (antibody)-like molecules is provided, illustrating the modular nature of antibodies:

[0075] (A) Left: Immunoglobulin chains and components are defined as in Figure 2 In the left figure, and are also defined therein.

[0076] Middle panel: An example of an Ab-like fusion protein is provided, demonstrating that the modular nature of immunoglobulins allows flexibility in the generation of Ab-like molecules (e.g., Figure 2In the specifically described Ab-like fusion proteins, the target recognition structure (shown as X1) is the same in all chains, as in the case of the Ab-like molecules described in Example 14 (wherein the EflACE2 polypeptide is linked to both the H chain and L chain constant domains at the CH1 domain, respectively, to enable assembly into an H2L2 Ab-like fusion protein).

[0077] Right: describes possible Ab-like fusion proteins including 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", "X1 X1X2 X3", "X1 X2 X3 X4"; wherein "X1", "X2", "X3" and "X4" represent different target recognition structures (or enzymes or reporter molecules));

[0078] (B) SDS-PAGE analysis of EflACE2-Ab-WT fusion proteins eluted from protein A affinity matrix with 0.4M arginine (pH 4) demonstrated that fully disulfide-bonded molecules (lane 1) consistent with the H2L2 Ab-like configuration were obtained. This molecule includes an EflACE2 polypeptide fused to an immunoglobulin constant heavy chain (EflACE2-CH), which is self-assembled with an equivalent EflACE2 polypeptide fused to an immunoglobulin light chain constant domain (EflACE2-CL). When reduced with dithiothreitol, the two chains (i.e., EflACE2-CH (ACE2-CH) and EflACE2-CL (ACE2-CL); Lane 2) are individually decomposed.

[0079] (C, D) The results provided show that EflACE2-Ab-like-H429F fusion protein (H429F) strongly directs CDC of Ramos-S target cells, while the corresponding fusion protein of the H chain with wild-type sequence (WT) is ineffective. The CDC of opsonized Ramos-S cells was determined in the presence of 1 / 3 diluted normal human serum as a complement source. Compared with EflACE2-Ab-like-WT fusion protein, this effective CDC mediated by EflACE2-Ab-like-H429F fusion protein cannot be attributed to different levels of opsonization, because the binding of these two proteins to Ramos cells expressing SARS-CoV-2 spikes is comparable. Anti-IgG secondary reagents labeled with goat anti-hIgG Fc FITC were used to determine binding using flow cytometry analysis. MFI = median fluorescence intensity; background binding of anti-Ig fluorescent conjugates shown as (conjugate only);

[0080] Fig.54The results of purifying EflACE2-Ab-like-Fc-H429F fusion protein using protein A and eluting with arginine are shown. (A) Using Hitrap TM (A) Protein A chromatography on a Protein A column with a gradient elution from 30 mM arginine (pH 4) to 35% 130 mM arginine (pH 4); (B) Size exclusion chromatography (SEC) of the pooled and concentrated Protein A fractions containing EflACE2-Ab-like-Fc-H429F using a Superose 6 Increase 10 / 300 column with the indicated oligomeric material (HMW); and (C) SDS-PAGE analysis of the pooled Protein A eluates and the pooled SEC monomer fractions under non-reducing (without DTT, dithiothreitol) and reducing (with DTT) conditions; and

[0081] Fig.55 Results showing the survival of Colo205 colorectal cells in the presence of purified DR5-specific mAbs comprising IgG1 H chains with a single H429F mutation (HF) or L234A, L235A and H429F (LA / LA / HF) mutations are provided. mAbs were titrated individually or mixed and titrated (BDR5-1LA / LA / HF+TDR5-1LA / LA / HF) and compared to a mixture of BDR5-1HF+TDR5-1HF, wherein both mAbs in the mixture comprised an IgG1 H chain with only a H429F modification; Colo205 cells (10,000 cells / well of a 96-well plate) were cultured in the presence of serial two-fold dilutions of the indicated mAbs. Individual mAbs were titrated starting at 1 μg / ml. For titrations of the mixture, a starting concentration of 1 μg / ml included 0.5 μg / ml of each mAb in the mixture. CCK8 colorimetric cell viability assay was used to quantify cell viability, wherein the absorbance (Abs450nm) of the cell culture supernatant was a measure of cell viability and viability. Maximum cell viability was determined by culturing cells in the absence of antibody (no Ab), and maximum death was determined by culturing cells with sodium dodecyl sulfate (SDS). Survival was expressed as: [Ab450 nm determined by the experiment, mAb or control treated Abs450 nm-background Abs450 of the cell culture medium]. DETAILED DESCRIPTION

[0082] The present disclosure relates to immunotherapeutic proteins comprising one or more polypeptides including an Fc region component, wherein the one or more polypeptides comprise an amino acid substitution (mutation) at a position corresponding to H429 (within the CH3 domain) of the amino acid sequence of a human immunoglobulin G1 (IgG1) heavy (H) chain (Eu numbering).

[0083] The amino acid numbering used herein is based on the so-called Eu numbering system, which is related to the sequence numbering used in describing the human IgG1 myeloma protein called Eu (Edelman GM et al., Proc Natl Acad Sci USA 63(1):78-85, 1969). According to this system, for example, H429 of IgG1 (i.e., histidine at position 429) occurs at position 429 of the Eu sequence. Thus, in any immunoglobulin molecule such as an antibody or fragment thereof or antibody-like molecule, the amino acid in a given position number refers to or corresponds to the corresponding amino acid residue position number in the Eu sequence.

[0084] 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 to consist of a dimeric protein comprising two monomeric heavy (H) chain polypeptides each optionally associated with a light (L) chain, and thus exists in the H2L2 format (see Figure 2 However, other formats can be formed that include immunoglobulin molecules in which one heavy chain (H1) associates with one light (L1) chain in an H1L1 format, as well as other formats in which the two heavy (H) chains can form dimers in the absence of light (L) chains to produce a heavy chain complex in an H2 format ( Figure 2 ). When the light (L) chain and the heavy (H) chain associate in the form of H1L1, the molecule can dimerize in this configuration to produce a typical immunoglobulin structure designated as H2L2. This is exemplified by the typical human IgG class of immunoglobulins, in which two heavy (H) chains are covalently bound to each other and a light (L) chain is covalently bound to each heavy (H) chain.

[0085] The dimeric 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 is also the basis of most mammalian immunoglobulin classes, noting that exceptions to this format are known (e.g., camelid immunoglobulins can form heavy chain dimers (abbreviated H2) without light chains). Thus, for example, a human immunoglobulin G (IgG) molecule physiologically exists in solution as a single H2L2 unit. However, other covalently bound higher order oligomers of the basic H2L2 unit do exist in nature and are common, particularly for IgM and IgA.

[0086] For example, IgM can form covalent pentamer or hexamer rings of the H2L2 format, in which each H2L2 unit has a disulfide bond with an adjacent H2L2 unit to form a pentamer oligomer (H2L2)5 or a hexamer oligomer (H2L2)6 (Eskeland T and TB Christensen, Scand J Immunol 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 of the soluble hexameric protein C1q (Eskeland T and TB Christensen, 1975 supra; Randall TD et al., Proc. Natl. Acad. Sci. USA 89:962-966, 1992; Hughey CT et al., J Immunol. IgM is also recognized as the most effective lectin because of its covalent oligomerization, which results in the presence of 10-12 specific and identical antigen-binding variable domains in each IgM pentamer or hexamer. Since individual antigen recognition structures (e.g., Fab fragments) have a defined affinity for a target antigen site or epitope, the strength of which is defined by its monovalent binding to one target structure, the presence of multiple antigen recognition structures in one molecule (e.g., 12 antigen-binding variable domains per IgM hexamer) confers stronger or more affinity binding to oligomeric immunoglobulins. Therefore, oligovalent binding comes from the combined strength of the individual antigen recognition interactions of 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 of these two antigen recognition structures.

[0087] Immunoglobulins can also be viewed as modular multifunctional proteins, in which the target recognition structure is connected to the functional activation structure by a flexible linker (i.e., the antigen recognition structure provided by the V domain of Fab is connected to the Fc region by a flexible hinge), and in which, in the case of a typical immunoglobulin, each H chain and each L chain comprises different domains, which can be viewed as structural or functional modules ( Figure 2 The H chain specifically contains a variable sequence antigen target recognition domain (V Hdomain), followed by a series of constant domains that are specific to the heavy chains of the Ig classes (e.g., IgG or IgA), and sequence analysis has shown that these constant domains are related between the immunoglobulin classes (see Figure 3 and 4 ). Therefore V H The first constant domain (CH1) is followed by the first constant domain. This first constant domain is connected to the second constant domain (CH2) by a flexible polypeptide, which acts as a linker called the hinge region. The second constant domain is followed in turn by the third constant domain (CH3), so that a typical H chain consists of discrete structural molecules starting from the amino terminus (i.e., NH2-terminal V H -CH1-Hinge-CH2-CH3( Figure 2 )) and is exemplified by the human IgG1 H chain sequence (SEQ ID NOs: 3 and 114) and is conserved across all human heavy chains ( Figure 3 , 4 ), which are exemplified by human IgG3 (SEQ ID NOs: 4 and 115), IgG4 (SEQ ID NOs: 5 and 116), IgG2 (SEQ ID NOs: 6 and 117), IgA1 (SEQ ID NOs: 7 and 118), IgA2 (SEQ ID NOs: 8 and 119), and IgE (SEQ ID NO: 9), IgM (SEQ ID NO: 10), and IgD (SEQ ID NO: 11), as well as mammalian immunoglobulins in general. Similarly, the light (L) chain contains a variable antigen target recognition domain (V L domain), followed by a constant domain specific for the light chain class.

[0088] Given their modular nature, immunoglobulins provide a general platform for creating a variety of immunotherapeutic or diagnostic molecules, including bivalent antibodies such as classical hybridoma-derived mAbs, heavy chain antibodies comprising dimers of heavy chains (e.g., camelid antibodies; Hamers-Casterman C et al., Nature 363:446, 1993), antibody-like (Ab-like) molecules (including some fusion proteins comprising at least one CH3 domain (or at least one CH4 domain)) and other immunoglobulin derivatives known to those skilled in the art as summarized in the "Periodic Table of Antibodies"; https: / / absoluteantibody.com / periodic-table-of-antibodies / #, the entire disclosure of which is incorporated herein by reference). Other specific examples include antibodies and antibody fragments, 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 (WO2007110205); dual variable domain immunoglobulins (US 7,612,181), knob-in-hole antibody formats (WO 1998 / 050431), diabody molecules (WO 2011 / 131746), IgG-like bispecifics (Shen J et al., J Immunol Methods 318(1-2):65-74, 2007), and fusion proteins comprising Fc or Fc region components, such as scFv fusions and double scFv fusions.

[0089] In this specification, many terms well known to those skilled in the art are used. Nevertheless, for the sake of clarity, many of these terms are defined below.

[0090] As used herein, the term "antibody" should be understood to include (unless otherwise specifically indicated) polyclonal antibodies (pAb), monoclonal antibodies (mAb), chimeric antibodies, humanized antibodies, antibody mixtures (e.g., recombinant polyclonal antibodies), such as methods well known to those skilled in the art for producing multiple antibodies with different specificities from a single host cell line, such as methods for producing antibodies (e.g., Technology: Merus BV, Utrecht, The Netherlands) or transgenic animals. Further, it will be understood that the antibody can be of any immunoglobulin class (i.e., isotype) or allotype. Thus, for example, the antibodies disclosed herein can be an isotype selected from well-known immunoglobulin isotypes, or include components from more than one isotype (e.g., some chimeric antibody types may be the case). Likewise, the antibody can include "mixed" chains; for example, an antibody comprising an H2 or H2L2 unit, wherein the H chains are different (i.e., the H x and H y (H x H y or L x H x H y L y )).

[0091] The term "antibody-like molecule" (Ab-like molecule) is understood to refer to a protein that is not an antibody and minimally includes a target recognition structure (e.g., a receptor or ligand, such as an antigen recognition sequence (e.g., a variable (V) domain or a complementarity determining region (CDR) of an immunoglobulin)) connected to at least one CH3 domain (or CH4 domain) of an immunoglobulin heavy (H) chain (providing the molecule with an "H-like" chain), and includes, for example, a receptor fusion protein that includes at least one CH3 domain (or at least one CH4 domain) of an H chain. Examples of Ab-like molecules include the Ab-like molecules mentioned above (e.g., Figure 2 , Fig.53 A and / or Ab-like molecules described in the antibody periodic table). In some examples, an H-like chain including at least one CH3 domain is considered to include an Fc region component, and may be, for example, a complete 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. H-like chains may enable dimerization, such that antibody-like molecules may be provided, for example, in the form of homodimers or heterodimers. As described in Fig.53 As depicted in , in some embodiments, Ab-like molecules can include an H2L2 format and can include, for example, fusion proteins comprising the same or different target recognition structures (or enzymes and / or reporter molecules), such as "X1 X1 X1 X1", "X1 X1 X1X2", "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).

[0092] 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 viewed as 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 (e.g., a variable (V) domain or a complementary determining region (CDR) of an immunoglobulin) linked to at least one CH3 domain (or at least one CH4 domain) of an immunoglobulin heavy (H) chain (see Figure 2 ). 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, an Fc fragment, wherein the Fc regions (chains) are different (e.g., Fc1x and Fc2y), and wherein each of the Fc regions may optionally be connected to different target recognition structures (e.g., ligand 1 and ligand 2). Those skilled in the art will appreciate that the target or antigen recognition structure may be connected to a polypeptide comprising at least one CH3 domain (or an equivalent CH4 domain of IgE or IgM), which may, for example, include a complete H-chain or include an H-like chain, which may include, for example, a complete 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 connecting a target or antigen recognition structure.

[0093] As used herein, the term "Fc fragment" (Fc or Fc part) refers to a dimer formed by covalent and / or non-covalent interactions between portions of immunoglobulin heavy (H) chains (i.e., a dimer formed between two Fc regions of an H chain (each of which includes a CH2 domain and a CH3 domain of a heavy chain and optionally a hinge sequence)), and which is responsible for the bulk of the activation of the immune effector functions of immunoglobulins: in particular, cell-based effector responses initiated by antibodies and Fc receptors located on the cell surface, such as through antibody-dependent cell-mediated cytotoxicity (ADCC) or Antibody-dependent cell-mediated phagocytosis (ADCP) or phagocytosis kills target cells, and this also leads to the modulation and inhibition of the activity of innate and adaptive immune cells by inhibitory Fc receptors; and the effector response of the innate immune system initiated by antibodies activating the complement system through 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 of targets by direct lysis through complement-dependent cytotoxicity (CDC) and / or by phagocytosis of targets through specific receptors for complement components (C'ADCP). The Fc fragment also provides an association site between immunoglobulin molecules, thereby allowing the immunoglobulin molecules to assemble (self-associate) into higher order oligomers (e.g., through covalent binding between domains in the Fc region of the H chain of one immunoglobulin and domains in the Fc region of the H chain of an adjacent immunoglobulin (e.g., as seen in the pentamer and hexamer forms of IgM) and non-covalent self-association (as seen in IgG), which, when bound to an antigen, results in oligomerization including hexamer formation (Diebolder CA et al., Science 343(6176): 1260-1263, 2014) and certain effector functions, among other properties).

[0094] As used herein, the term "Fc region component" is understood to refer to a portion of the Fc region of an immunoglobulin heavy (H) chain in which H429 is located, the Fc region component comprising at least one CH3 domain (or at least one CH4 domain), but preferably comprising a CH2 domain and a CH3 domain, and optionally further comprising an immunoglobulin hinge sequence (which may include all or part of the lower hinge, core hinge, and upper hinge sequences in sequence), which is capable of forming (e.g., by dimerization) an Fc fragment or an Fc-like fragment. "Fc-like fragment" is understood to refer to an Fc fragment-like structure, but the Fc-like fragment includes a fragment or component of the Fc region (e.g., a CH3 domain (or CH4 domain) alone or a combination of a CH2 domain and a CH3 domain, and optionally further comprising an immunoglobulin hinge sequence (which may include all or part of the lower hinge, core hinge, and upper hinge sequences in sequence).

[0095] As used herein, the term "treating" encompasses treating as well as alleviating established symptoms of a disease or condition. Thus, the act of "treating" a disease or condition includes: (1) preventing or delaying the onset of clinical symptoms of an ongoing disease or condition in a subject suffering from or susceptible to the disease or condition; (2) inhibiting the disease or condition (i.e., in the case of maintenance treatment, arresting, reducing or delaying the development of the disease or condition or its recurrence, or arresting, reducing or delaying at least one clinical or subclinical symptom thereof); and (3) relieving or alleviating the disease or condition (i.e., causing regression of at least one clinical or subclinical symptom of the disease or condition or its clinical or subclinical symptoms).

[0096] As used herein, the phrase "the preparation of a medicament" encompasses the use of one or more immunotherapeutic proteins as defined in the first aspect directly as a medicament or at any stage in the preparation of a medicament comprising one or more immunotherapeutic proteins as defined in the first aspect.

[0097] The term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. The effective amount can be administered in one or more administrations. Typically, the effective amount is sufficient to treat a disease or condition or otherwise alleviate, improve, stabilize, reverse, slow down or delay the development of a disease or condition. By way of example only, an effective amount of an immunotherapy protein such as a mutant IgG1 antibody may include about 0.1 to about 250 mg / kg body weight / day, more preferably between about 0.1 to about 100 mg / kg body weight / day, and still more preferably between about 0.1 to about 25 mg / kg body weight / day. However, despite this, it will be understood by those skilled in the art that the effective amount may vary and depend on a variety of factors, including the age, weight, sex and / or health status of the subject being treated, the activity of a specific immunotherapy protein, the metabolic stability and length of action of a specific immunotherapy protein, the route and time of administration of a specific immunotherapy protein, the excretion rate of a specific immunotherapy protein and, for example, the severity of the disease or condition being treated.

[0098] As shown in the examples provided below, it has been found that mutant antibodies comprising point mutations at the H429 position can confer significant functional changes. For example, IgG mutants with H429F substitutions show the ability to activate complement-dependent cytotoxicity (CDC) in complement assays (i.e., assays of complement function). Similarly, it has been found that the fusion protein (which can act as a "bait" to block viral interactions and coronaviruses from entering host cells) of the Fc region components at position 429 fused to the angiotensin-converting enzyme 2 (ACE2) extracellular domain can provide the ability to provide enhanced antiviral effects by the CDC of infected cells, while similar fusion proteins with H429Y substitutions show enhanced virus neutralization, with little or no complement activation (because the immunotherapy protein including the Fc component modified by H429Y shows the FcγR binding and activation eliminated, especially the binding and activation with FcγRIIIa). While not wishing to be bound by theory, it is believed that these effects may result from oligomerization of the protein into oligomers upon binding to a relevant target molecule (e.g., the antigen to which the antibody mutant is directed) either by self-association of the antibody / fusion protein in solution or by "on-target" oligomerization.

[0099] Additionally, in this regard, it should be noted that although monomeric IgG1 and IgG3 can deliver CDC, the levels of CDC can be considered low or "poor" compared to the levels achieved by native pentameric or hexameric IgM. Furthermore, unlike IgM, IgG antibodies such as IgG1 and IgG3 are also relatively poor agglutinins due to their bivalency. Thus, by achieving oligomerization (through the potential to enhance the known poor intrinsic self-association ability of the Fc of immunoglobulins such as IgG, particularly after the immunoglobulin has bound to the antigen (i.e., "on-target" oligomerization), the immunotherapeutic proteins according to the present 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 specifically 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 the CH3 residues form an interface between adjacent IgG:IgG molecules, thereby 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.

[0100] In addition, many cell molecules including cell surface molecules need to be clustered in large quantities by ligands (which may be soluble or another cell surface molecule) to induce signals, thereby inducing cell responses, and therefore the dimerization usually achieved with antibodies may not be enough to induce signals that lead to meaningful cell responses. However, it is known that the intensity of the signal can be increased by, for example, increasing the clustering of target molecules by methods involving the addition of other entities of cross-linked ligands (Chenoweth AC et al., "Immunology and Cell Biology (Immunol Cell Biol)" 98: 287-304, 2020), and therefore, in the case where antibodies (such as mAbs) are bound to their target molecules, such "super cross clustering" or "super clustering" of target molecules can be achieved by using cross-linking other anti-immunoglobulin antibodies of mAbs bound to target molecules. Although it is not desired to be bound by theory, it is believed that proteins, such as mutant antibodies comprising H429F point mutations as mentioned above, can similarly achieve super cross clustering or super clustering of target molecules by self-association when bound to target molecules. Depending on the nature of the target molecule, such clustering may induce an enhanced signaling response that may result in, for example, cell proliferation (e.g., when the target molecule is, for example, CD3 or CD28), stimulation of inhibitory pathways to inhibit or reduce cellular responses (e.g., immune checkpoints; reviewed in Chenoweth et al., supra 2020), or stimulation of pathways that induce cell death, such as programmed cell death including apoptosis (see, e.g., Ashkenazi A., Nat Rev Drug Discov 7(12): 1001-1012, 2008). Stimulation of cellular pathways by mAbs to develop therapeutic molecules is an approach used to treat a range of diseases, including cancer, inflammatory and autoimmune diseases, infections, cardiovascular disease, and other diseases (Ashkenazi, supra 2008).

[0101] Thus, in a first aspect, the present disclosure provides an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, wherein the Fc region component comprises at least one CH3 domain (or at least one CH4 domain), wherein the one or more polypeptides comprise an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain (Eu numbering).

[0102] In some embodiments, the immunotherapeutic protein of the first aspect comprises a dimeric immunotherapeutic protein, 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 include an Fc region component comprising a CH2 domain and a CH3 domain (such that the first polypeptide and the second polypeptide 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 polypeptide and the second polypeptide includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain (Eu numbering). Thus, the immunotherapeutic protein of such embodiments can be an immunoglobulin molecule, such as an antibody or antibody-like molecule, such as a dimeric polypeptide comprising a pair of single-chain Fv polypeptides (i.e., scFv-Fc) connected by an Fc fragment, or a miniantibody (i.e., a protein comprising a pair of scFv polypeptides connected by a CH3 domain (see, e.g., Olafsen T et al., Generation of Antibody Fragments and Their Derivatives, Antibody Engineering, 2nd ed., pp. 69-84, 2010) for a discussion of multivalent scFv-Fc and miniantibodies).

[0103] In other embodiments, the immunotherapeutic protein of the first aspect comprises a partner polypeptide connected 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 numbering) at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain. Therefore, the immunotherapeutic protein of such embodiments can be provided in the form of a fusion protein or a protein conjugate. Those skilled in the art will appreciate that in a fusion protein, the partner polypeptide will be covalently linked (i.e., "fused") to the Fc component (i.e., as a fusion partner) via a peptide bond or linker sequence (e.g., a short peptide linker sequence such as an immunoglobulin hinge sequence or a well-known glycine-serine linker such as GGGGS) at the N-terminus or C-terminus of the fusion partner (i.e., Fc component), whereas in a protein conjugate, the partner polypeptide will be covalently linked (i.e., "fused") to the Fc component (i.e., as a fusion partner) via a chemical bond such as a disulfide bond (e.g., through one or more cysteine ​​(C) residues) or a cross-linker compound such as a bifunctional cross-linker such as disuccinimidyl suberate (DSS) (e.g., bis(sulfosuccinimidyl) suberate (BS 3)); Thermo Fisher Scientific, Waltham, MA, United States of America, is covalently or non-covalently linked to the Fc region component (i.e., as a conjugate partner), or disuccinimidyl tartrate (DST) with an amine or heterobifunctional cross-linker, such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MDS) and N-(ε-maleimidopropionyloxy) succinimide ester (EMCS), or by other non-covalent binding such as hydrogen bonding. When the Fc component is a conjugate partner, the immunotherapy protein conjugate can be considered a cross-linked protein, and the Fc component can be conjugated to the partner polypeptide at the N-terminus or C-terminus, but can also be conjugated in other ways at any other suitable site on the partner polypeptide. Alternatively, the Fc component (as a conjugate partner) may be otherwise conjugated to a partner polypeptide at the N-terminus or C-terminus of the Fc component or at any other suitable site on the Fc component (e.g., within the CH1 or upper hinge sequence, if these are included in the Fc region component).

[0104] The Fc region component of the immunotherapeutic protein (hereinafter also referred to as the "Fc component") can be derived from one or more immunoglobulin types (e.g., IgG or IgA), and can include a full-length (i.e., "intact") Fc region, for example, 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 the upper hinge sequence to produce an Fc region, wherein the Fc region includes constant heavy domain 2 (CH2; amino acids A231 to K340 of a human IgG1 heavy chain polypeptide (Eu numbering)), constant heavy domain 3 (CH3; amino acids G341 to G446 or K447 of a human IgG1 heavy chain polypeptide (Eu numbering)) and a lower hinge sequence (also referred to as the hinge-proximal sequence of CH2; amino acids P232 to P238 (Eu numbering)) and a core hinge sequence. (amino acids C226 to C229) and similar heavy chain polypeptide fragments that can be prepared by digesting immunoglobulin heavy chain polypeptides with plasmin and human neutrophil elastase (NHE). Additional examples of suitable Fc region components may include fragments of heavy chain polypeptides that, in addition to the CH2 domain and the CH3 domain and the lower and core hinge sequences, also include all or part of the upper hinge sequence and constant heavy domain 1 (CH1). On the other hand, other suitable Fc region components may include fragments of heavy chain polypeptides that include only the CH3 domain (e.g., amino acids G341 to G446 or K447 (Eu numbering) of a human IgG1 heavy chain polypeptide) or fragments thereof. In addition, suitable Fc region components may include heterologous ("hybrid") CH3 domains, such as those derived from IgG1 The strand exchange engineered domain (SEED) format of the CH3 domain and CH3 domains of fragments of other proteins such as IgA (Davies et al., Prot Eng Des Sel 23(4): 195-202, 2009).

[0105] In some embodiments, the Fc region component is derived from a human immunoglobulin heavy chain polypeptide (e.g., Figure 3 and 4 Human immunoglobulin heavy chain polypeptide shown in ).

[0106] In other embodiments, the Fc region component is derived from an IgG heavy chain polypeptide, preferably an IgG1 or IgG3 (eg, human IgG1 or IgG3) heavy chain polypeptide, and more preferably IgG1.

[0107] The Fc region component includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide (Eu numbering). When the Fc region component is derived from another immunoglobulin type or isotype (or from an immunoglobulin from another species), it is understood that a person skilled in the art can easily determine the position corresponding to H429 of a human IgG1 heavy chain polypeptide IgG1 by, for example, conventional sequence alignment (e.g., Figure 3 and 4 shown).

[0108] Suitable mutations at position 429 include:

[0109] H→X, wherein X is selected from tyrosine (H429Y), phenylalanine (H429F), tryptophan (H429W), glutamate (H429E), aspartate (H429D), glutamine (H429Q), serine (H429S), asparagine (H429N), and threonine (H429T).

[0110] Some preferred amino acid substitutions at the position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide include H→Y (eg, H429Y) substitutions and H→F (eg, H429F) substitutions.

[0111] The amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide may be the only mutation in the Fc component. However, in some embodiments, the Fc component may include one or more additional mutations (eg, amino acid substitutions). For example, the Fc component may include one or more sequence mutations known to those skilled in the art (see, e.g., Wang X et al., Protein Cell 9(1):63-73, 2018, for examples listed in Table 1; the entire disclosure of which is incorporated herein by reference), such as: mutations that may modulate FcγR binding (e.g., S239D / I332E of IgG1 that increases FcγRIIIa binding (Eu numbering)); mutations that increase antibody-dependent cellular cytotoxicity (ADCC), such as S239D / I332E (Lazar GA et al., Proc. Natl. Acad. Sci. USA 103(11):4005-4010, 2006), opsonophagocytosis (e.g., G236A / S239D / I332E; Richards JO et al., Mol. Cancer Therapy 103(12):401-402, 2008); Ther (Ther) 7: 2517-2527, 2008) or complement activation (e.g., S267E / 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); mutations that may increase inhibition by FcγRIIb co-engagement (S267E / L328F; Chu SY et al., Mol Immunol (Mol Immunol 200(1): 16-26, 2000); and mutations that confer enhanced binding to the 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).

[0112] Other mutations that may be included in the Fc region component include mutations for enhancing complement activation, such as amino acid mutations at positions corresponding to K447 of the amino acid sequence of a human IgG1 heavy chain polypeptide (Eu numbering); specifically K447X, wherein X is selected from null (i.e., K447del; amino acid deletion or truncation of the Fc component) and glutamic acid (i.e., K447E) (see van der Bremer ETJ et al., mAb 7(4): 672-680, 2015). Further, the Fc region component may also include mutations that regulate glycosylation (e.g., mutations at positions corresponding to Asn297 of the amino acid sequence of a human IgG1 heavy chain polypeptide (Eu numbering), such as N297A, N297Q or N297G (Wang et al., 2018 supra)) to provide a site with modified glycosylation (e.g., lacking a glycan at position 297) to eliminate FcγR and complement C1 binding and / or activation.

[0113] Alternatively, the Fc region component can also be manipulated to achieve modified glycosylation by producing the immunotherapeutic protein in the presence of kifnosine (a mannosidase inhibitor that prevents normal maturation of N-linked glycans, including core fucosylation of N-linked glycans); this is a modification that specifically enhances activity through FcγRIIIa. Further, Fc components lacking core fucosylation of the Asn297 glycan can also be achieved by culturing host cells expressing immunotherapeutic proteins with inhibitors of fucosylation (e.g., 2-fluoroperacetylated fucose or the like) or by expressing enzymes that modify glycosylation pathways (e.g., GDP-6-deoxy-D-lyso-4-hexulose reductase; Neha M et al., J Biotech 5:100015, 2020) or by genetic inhibition of these pathways (e.g., siRNA silencing of the α-1,6-fucosyltransferase 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).

[0114] The immunotherapeutic protein of the first aspect may be monomeric, dimeric or oligomeric in solution (eg, in saline at a neutral pH, such as a physiological pH of about 7.4).

[0115] For example, in some embodiments, the immunotherapeutic protein comprises monomers in saline, wherein each monomer comprises one copy of the Fc region component, while in other embodiments, the immunotherapeutic protein comprises dimers in saline, wherein the Fc region components self-associate (i.e., form dimers) through non-covalent bonding such as hydrogen bonding or through disulfide bond formation by one or more cysteine ​​(C) residues, particularly within the hinge sequence, particularly the core hinge sequence (if present) (Yoo EM et al., Journal of Immunology 170:3134-3138, 2003). Thus, in some embodiments, the Fc region component includes a core hinge sequence so that the immunotherapeutic protein can self-associate (i.e., form a dimer) by forming an interchain disulfide bond between one or more cysteine ​​(C) residues of the core hinge sequences of the two Fc region components, while in some other embodiments, the Fc region component includes a CH3 domain so that CH3:CH3 can self-associate by non-covalent interactions, or the Fc region component includes a CH3 domain and a CH2 domain so that self-association can be achieved by CH2:CH2 and CH3:CH3 non-covalent interactions. It should be noted that in the examples and figures below, the dimeric form of the immunotherapeutic protein is considered to be a single molecule (i.e., each molecule includes two copies of the protein dimerized by the Fc region component) and is referred to as a monomer (monomeric).

[0116] In other embodiments, the immunotherapeutic protein includes oligomers in saline (at neutral pH, such as at a physiological pH of about 7.4), wherein the immunotherapeutic protein includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide, which enables the Fc or Fc-like fragment comprising the mutated Fc component to self-associate into a soluble oligomeric form in saline (e.g., an oligomeric form comprising, for example, 3 copies, 4 copies, 5 copies, 6 copies, or 12 copies assembled from, for example, a dimeric form of the immunotherapeutic protein, such that in some specific embodiments, the oligomeric form of the immunotherapeutic protein may include six dimeric proteins (i.e., a hexameric form), including a total of 12 copies of the immunotherapeutic protein). In such embodiments, the amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide may be:

[0117] H429X 1 , where X 1 selected from the group consisting of tyrosine (i.e., H429Y), methionine, isoleucine, leucine, tryptophan, and valine;

[0118] Preferably, however, the immunotherapeutic protein comprises a H→Y (ie, H429Y) substitution.

[0119] In yet other embodiments, the immunotherapeutic protein can form oligomers when bound to a target of interest by "on-target" oligomerization. Such oligomerization can occur with an immunotherapeutic protein that is a monomer or dimer in saline (at a neutral pH, such as at a physiological pH of about 7.4), and includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide, which enables oligomerization on the target into an oligomeric form (e.g., a form comprising 3, 4, 5, 6, or 12 copies of the immunotherapeutic protein (e.g., a hexamer in dimer form). In such embodiments, the amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide can be:

[0120] H429X 2 , where X 2 Selected from the group consisting of phenylalanine (ie, H429F), glutamic acid, glutamine and serine; however, preferably, the immunotherapeutic protein includes a H→F (ie, H429F) substitution.

[0121] Alternatively, oligomeric forms of the immunotherapeutic protein of the first aspect may be produced by using other techniques well known to those skilled in the art, such as using Fc multimer forms (stradomers TM ), which includes a multimerization domain (MD) sequence linked from the hinge region of human IgG2 or isoleucine zipper (ILZ) to the N-terminus or C-terminus of murine IgG2a (Fitzpatrick EA et al., Front Immunol 11, Chapter 496, 2020), as well as using a multimerization sequence from IgM (Melcheil et al., Sci Rep 1:124 doi:10.1038 / srep0012, 2011), or docking and dimerization sequences from unrelated proteins such as cyclic adenosine monophosphate-dependent protein kinase and A-kinase anchor protein (Rossi EA et al., Bioconjug Chem 23(3):309-323, 2012).

[0122] It is further found that the immunotherapeutic protein of the first aspect can show enhanced neonatal Fc receptor (FcRn) binding by amino acid substitution (Eu numbering) at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain. It is expected that this will mean that the immunotherapeutic protein will show a longer half-life in vivo (i.e., relative to an equivalent immunotherapeutic protein without a mutation at the H429 position), and thereby improve pharmacokinetics (PK), because FcRn is known to "recycle" antibodies in order to control how long they last in vivo (Ward ES and RJ Orber, Trends Pharmacol Sci 39(10): 892-904, 2018).

[0123] Dimeric immunotherapeutic proteins (e.g., immunoglobulin molecules)

[0124] The immunotherapeutic protein of the first aspect can be a dimeric immunotherapeutic protein comprising an immunoglobulin molecule, wherein the immunoglobulin molecule comprises a first immunoglobulin heavy chain polypeptide and a second immunoglobulin heavy chain polypeptide, each comprising an Fc region component comprising at least one CH3 domain (or at least one CH4 domain). The Fc region components of the first immunoglobulin heavy chain polypeptide and the second immunoglobulin heavy chain polypeptide can self-associate (i.e., form a dimer) by non-covalent bonding such as hydrogen bonding or by forming disulfide bonds through one or more cysteine ​​(C) residues, particularly at a hinge sequence, especially a core hinge sequence (if present) (Yoo EM et al., Journal of Immunology 170: 3134-3138, 2003), or are otherwise connected (e.g., by cross-linking the CH3 domains of the Fc region components) by, for example, a cross-linker compound as mentioned above. Thus, the immunotherapeutic protein may be an immunoglobulin molecule, such as an antibody or antibody derivative, such as scFv-Fc (in which dimers of scFv polypeptides are formed by dimers of Fc region components), miniantibodies (in which dimers of scFv polypeptides are formed by linking CH3 domains / CH4 domains), or any other suitable Fc-containing antibody or derivative known to those skilled in the art (e.g., as outlined in the "Periodic Table of Antibodies" mentioned above).

[0125] Thus, the dimeric immunotherapeutic protein may further include at least one antigen recognition structure, or in other words, an antigen binding region (e.g., a scFv or a V L and V HThe antigen binding region can specifically bind to an antigen or epitope of therapeutic interest; for example, a cancer-associated antigen, such as a cancer antigen present on the surface of a cancer cell (e.g., a cell surface antigen differentially expressed and / or present in cancer cells, such as the 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), an autoantigen (e.g., an autoantigen associated with SLE or multiple sclerosis (MS)), an allergen (e.g., bee venom), an antigen associated with other inflammatory diseases such as immune complex vasculitis, an antigen from a transplanted tissue or organ, or an antigen from an infectious source such as an antigen of a bacterial, yeast, parasitic, or viral pathogen (e.g., an antigen of SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), respiratory syncytial virus (RSV), or dengue virus). In other examples, the antigen binding region can specifically bind to a cell surface molecule known to induce cell proliferation (i.e., the cell surface molecule can be, for example, CD3 or CD28) and / or stimulate an inhibitory pathway to inhibit or reduce a cellular response (i.e., the cell surface molecule can be, for example, an immune checkpoint molecule, such as 4-1BB (CD137), cluster of differentiation (CD40, CD154), OX40 receptor (TNFRSF4, CD134), tumor necrosis factor receptor type II (TNFR2, CD120b), glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), cluster of differentiation 27 (CD27), T cell immunoglobulin and mucin domain-containing 3 (TIM-3), B and T lymphocyte attenuator (BTLA, CD272), lymphocyte activation gene-3 (LAG3, CD223), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD1 52), inducible T cell co-stimulation (ICOS, CD278), cluster of differentiation 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); and wherein oligomerization of the immunotherapeutic protein can produce enhanced cross-linking (e.g., super clustering), and thereby lead to enhanced signal transduction to induce, for example, enhanced cell proliferation or inhibited / reduced cellular responses (i.e., enhanced stimulation through inhibitory pathways). In yet other examples, the antigen binding region can specifically bind to a cell surface molecule known to be used to induce a cellular response selected from stimulating an inhibitory pathway to inhibit or reduce the response. In still yet other examples, the antigen binding region can specifically bind to a cell surface molecule that, when bound by a ligand or other molecule such as an agonist mAb, induces cell death.Such cell surface molecules include, for example, cell surface molecules of the TNF-receptor superfamily (also known as TNFRSF), including tumor necrosis factor receptor 1 (TNFR1, TNFRSF1A, CD120a), Fas (CD95, APO-1), death receptor 3 (DR3, TNFRSF25), death receptor 4 (DR4, CD261, TNFRSF10A, TRAILR1), death receptor 5 (DR5, CD262, TNFRSF10B, TRAILR2) and death receptor 6 (CD358, TNFRSF21). For example, under physiological conditions, binding of TNF-related apoptosis-inducing ligand (TRAIL) to its receptor DR5 (Carneiro BA et al., Nat Rev Clin Oncol 17(7): 395-417, 2020) or binding of FAS ligand to its receptor FAS (CD95) on the cell surface induces oligomerization that initiates signaling leading to cell death (Leukocyte and Stromal Cell molecules: The CD Markers, from Zola H et al., page 195, John Wiley & Sons, 2007). Other molecules (independent of TNFRSF) that can induce apoptotic signals leading to cell death when cross-linked, for example, by mAbs or fragments thereof, include CD38 (see Gambles MT et al., Molecules 26(15):4658, 2021), CD20 (see Shan D et al., Cancer Immunol Immunother 48:673-683, 2000; and Cardarelli PM et al., Cancer Immunol Immunother 51:15-24, 2002), and CD52 (see Rowan W et al., Immunology 95:427-436, 1998). These molecules can also be advantageously targeted by dimeric immunotherapeutic proteins, such that oligomerization may result in enhanced cross-linking and induction of apoptotic signals, leading to potentially desirable cell death in target cells.

[0126] In some embodiments, an immunoglobulin molecule according to the present disclosure may include two antigen binding regions, each antigen binding region specifically binding to a different antigen or epitope.

[0127] In some embodiments, the immunoglobulin molecules according to the present disclosure are antibodies, which may be, for example, of IgD, IgE or IgM isotypes, but will preferably be of IgA or IgG isotypes, such as antibodies of any of the human IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 subtypes.

[0128] The first immunoglobulin heavy chain polypeptide and the second immunoglobulin heavy chain polypeptide of the immunotherapeutic protein comprising a dimeric immunoglobulin molecule can be the same (ie, a homodimer) or different (ie, a heterodimer).

[0129] In some embodiments, the immunoglobulin molecules according to the present disclosure are antibodies, which can be antibodies that form oligomers by self-association of antibodies in solution (e.g., in physiological saline at neutral pH, such as a physiological pH of about 7.4), by oligomerization into oligomers (e.g., hexamers), or by "on-target" oligomerization when bound to a relevant target (e.g., an antigen against which the antibody mutant is directed).

[0130] For example, and although not wishing to be bound by theory, it is believed that immunoglobulin molecules that are antibodies comprising the H429Y amino acid substitution in the Fc region component can form oligomers in solution (e.g., in saline at physiological pH), which may result in an overall increase in binding strength (e.g., enhanced affinity) with a target binding partner (e.g., a cancer antigen, an antigen of a bacterial or viral pathogen, or other soluble target molecule or molecular complex). Thus, in the context of antibodies against cancer antigens present on the surface of cancer cells, antibodies according to the present disclosure (in the form of oligomers in solution) can show an enhanced ability to bind to cancer cells, thereby causing the cancer cells to be destroyed by, for example, CDC through 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 the present disclosure (in the form of oligomers in solution) can show an enhanced ability to bind to soluble target molecules or molecular complexes, thereby causing the soluble target molecules or molecular complexes to be removed by phagocytosis of macrophages.

[0131] On the other hand, it is believed that immunoglobulin molecules as antibodies comprising the H429F amino acid substitution in the Fc region component can form oligomers when bound to relevant targets, so that the Fc component assembled "on target" presents a stable optimal binding platform, wherein, for example, the affinity with the C1q complement protein complex is enhanced, and thereby leads to complement activation, and in turn to complement dependent cytotoxicity (CDC). Therefore, in the context of antibodies against cancer antigens present on the surface of cancer cells, the antibodies according to the present disclosure can show an enhanced ability to activate complement on cancer cells, thereby causing the cancer cells to be destroyed.

[0132] In some embodiments, an immunoglobulin molecule according to the present disclosure may be provided or used as a first immunoglobulin molecule having a first antigen binding region for a first antigen in combination with a second immunoglobulin molecule (according to the present disclosure) having a second antigen binding region for a second antigen. By way of example only, in such a combination, the first antigen may be a cancer antigen present on the surface of a cancer cell (e.g., CD38 found on the surface of a CLL cell), and the second antigen may be a death receptor (e.g., DR5). Oligomerization of the first immunoglobulin molecule and the second immunoglobulin molecule may result in the formation of hetero-oligomers, such as hetero-hexamers, in which different cell surface targets are integrated into clusters, which may result in, for example, enhanced induction of apoptotic signals leading to cell death (e.g., in the case where a death receptor has been targeted), wherein target cell specificity is achieved by binding to, for example, a cancer antigen present on a cancer cell.

[0133] Fusion / conjugate immunotherapeutic proteins

[0134] The immunotherapeutic protein of the first aspect may include a fusion protein or protein conjugate comprising a partner polypeptide linked to an Fc region component, the Fc region component including at least one CH3 domain (or at least one CH4 domain).

[0135] Fusion proteins or protein conjugates according to the present disclosure may be monomeric, dimeric or oligomeric. For example, in some embodiments, the fusion protein or protein conjugate includes a monomer, wherein each monomer includes a copy of a partner polypeptide (or a fragment thereof) and a copy of an Fc region component, while in other embodiments, the immunotherapeutic protein includes a dimer, wherein the Fc region component self-associates (i.e., forms a dimer) by non-covalent binding such as hydrogen bonding or by forming a disulfide bond through 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). In such dimers, the immunotherapeutic protein includes 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 to be divalent relative to the partner polypeptide (or its fragment). The two fusion / conjugation partner polypeptides (or two fragments thereof) of a dimeric fusion protein or protein conjugate can be the same (i.e., homodimers) or different (i.e., heterodimers). In some embodiments of such dimers, such as in Fig.53As depicted in A, the fusion protein is antibody-like (Ab-like) and can have, for example, an H2 format or an H2L2 format (wherein the monomeric fusion protein is produced by a light (L) chain, which in turn can be optionally fused / conjugated to a partner polypeptide (such as a target recognition structure). It can be easily 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", wherein X1, X2, X3 and X4 each represent a different target recognition structure. Similarly, one or more of the target recognition structures can be replaced by alternative partner polypeptide types such as enzymes or reporter molecules.

[0136] Partner polypeptides may provide beneficial functions and / or characteristics to immunotherapeutic proteins.

[0137] In some embodiments, the partner polypeptide can be a cell surface receptor polypeptide (or a fragment thereof) or a co-receptor polypeptide (or a fragment thereof).

[0138] For example, the partner polypeptide can be a cell surface molecule, such as a cell surface receptor polypeptide (or fragment thereof) that can bind to the structural protein of the virus, so that the immunotherapeutic protein can act as a "bait" to block viral interaction and viral entry into the host cell. Therefore, some examples of such cell surface receptor polypeptides (or fragments thereof) that can include partner polypeptides include angiotensin converting enzyme 2 (ACE2) extracellular domain (ACE2 is a cell entry receptor for SARS-CoV-2), nucleolin (cell entry receptor for RSV), dipeptidyl peptidase 4 (DPP4, CD26), Hsp70 (cell entry receptor for Japanese encephalitis virus), hepatitis A virus cell receptor 1 (HAVCR1 / TIM-1; cell entry receptor for hepatitis A virus and Ebola virus), cluster of differentiation 155 (CD155; polio virus ), glucose transporter 1 (GLUT1; cell entry receptor for human T-cell leukemia virus 1), proto-oncogene tyrosine protein kinase MER (MERTK; host factor that promotes entry of classical swine fever virus), TYRO3 protein tyrosine kinase (TYRO3; cell surface protein associated with lymphocytic choriomeningitis (LMCV) infection), AXL (cell surface protein associated with lymphocytic choriomeningitis (LMCV) infection) and cluster of differentiation 4 receptor (CD4 receptor; cell entry receptor for human immunodeficiency virus (HIV)), to name a few. In embodiments where the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof) that is capable of binding to a structural protein of the virus, a point mutation (Eu numbering) at a position of the Fc region component corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain can, for example, provide the immunotherapeutic protein with an enhanced ability to provide an antiviral effect through CDC of infected cells (e.g., the mutation is H429F) or confer enhanced viral neutralization to the immunotherapeutic protein (e.g., the mutation is H429Y). In other examples, the partner polypeptide can be a ligand of a cell surface molecule known to induce cell proliferation (i.e., the cell surface molecule can be, for example, CD3 or CD28) and / or stimulate an inhibitory pathway to inhibit or reduce a cellular response (i.e., the cell surface molecule can be, for example, an immune checkpoint molecule 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 immunotherapeutic protein can result in enhanced cross-linking (e.g., super cross-linking clustering), and in turn, enhanced signaling to induce, for example, enhanced cell proliferation or an inhibited / reduced cellular response (i.e., by enhanced stimulation of an inhibitory pathway).In yet other examples, the partner polypeptide can be a ligand for a cell surface molecule that induces cell death when engaged by a ligand or other molecule, such as agonistic mAbs (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., supra 2000; and Cardarelli et al., supra 2002) and CD52 (Rowan et al., supra 1998). Therefore, in some embodiments, the immunotherapeutic protein of the first aspect may include a dimeric fusion protein or protein conjugate comprising, for example, TRAIL (i.e., a ligand of DR4 or DR5) as a partner polypeptide linked to an Fc region component, the Fc region component comprising at least one CH3 domain comprising an H429F amino acid substitution. When the ligand binds to a cell surface molecule, oligomerization of the immunotherapeutic protein of these embodiments may result in enhanced cross-linking and induction of apoptotic signals, thereby resulting in cell death that may be desired in the target cell.

[0139] When the partner polypeptide is a co-receptor polypeptide (or a fragment thereof), the co-receptor polypeptide (or a fragment thereof) can 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 to phosphatidylserine displayed on viruses, including West Nile virus, Zika virus and Ebola virus, and promote the entry of such viruses into host cells), etc.

[0140] In another example, the partner polypeptide can be a cell surface receptor polypeptide (or its fragment) that can be combined with a cell surface receptor ligand. Therefore, some examples of such polypeptides (or its fragment) include cytotoxic T lymphocyte-associated protein 4 (CTLA4) (or its soluble extracellular fragment). CTLA4 plays a role as an immune checkpoint and lowers the immune response. The immunotherapy protein including the fusion protein or protein conjugate (for example, CTLA4-Fc component fusion protein) comprising the CTLA4 partner polypeptide can be shown by forming a dimer or oligomer to enhance the combination with the CTLA4 ligand, and thus act as a bait to produce various therapeutic effects for the potential treatment of tumors (for example, melanoma and colorectal cancer) and various autoimmune diseases such as SLE and rheumatoid arthritis (RA). Additional examples of suitable cell surface receptor polypeptides (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 interleukin-1 receptor (IL-1R), interleukin-6 receptor (IL-6R), tumor necrosis factor receptor-2 (TNFR2, also known as CD120b) or receptors for cytokines of the TGF-β superfamily (see Czajkowsky DM et al., Mol Med 4(10): 1015-1028, 2012 for review). Immunotherapeutic proteins including fusion proteins or protein conjugates comprising IL-1R (or fragments thereof) can potentially be used in anti-IL-1 therapy to treat, for example, type 2 diabetes, and immunotherapeutic proteins including fusion proteins or protein conjugates comprising IL-6R (or fragments thereof) can potentially be used in anti-IL-6 therapy to treat, for example, tumors and RA. Immunotherapeutic proteins including fusion proteins or protein conjugates comprising TNF-R2 or its extracellular domain can potentially be used to treat RA or other inflammatory diseases or conditions.

[0141] Although not wishing to be bound by theory, it is believed that the fusion proteins or protein conjugates according to the present disclosure comprising the H429Y amino acid substitution in the Fc region component (whether in monomeric or dimeric form) can form oligomers in solution (e.g., in physiological saline at neutral pH, such as a physiological pH of about 7.4), which may result in an overall increase in binding strength (e.g., enhanced avidity) to a target binding partner (e.g., a structural protein of a virus or a ligand of a cell surface receptor, or other soluble target molecule or molecular complex). Thus, in the context of a fusion or conjugated protein comprising a cell surface receptor polypeptide or fragment thereof capable of binding to a structural protein of a virus, an enhanced avidity for binding to the virus is resulted to provide enhanced virus neutralization (possibly imparted by cross-linking and / or aggregating virus particles (virions)). However, it is believed that, in at least some embodiments, such soluble oligomers are substantially incapable of binding to Fc receptors, and are therefore capable of virus neutralization with little or no complement activation.

[0142] On the other hand, and again without wishing to be bound by theory, it is believed that fusion proteins or protein conjugates comprising the H429F amino acid substitution in the Fc region component (whether in monomeric or dimeric form) can form oligomers upon binding to relevant targets (e.g., structural proteins of a virus or ligands of cell surface receptors) such that the "on-target" assembled Fc component presents a stable optimal arrangement for binding to the C1q complement protein complex (e.g., with enhanced avidity), and thereby leading to complement activation, and in turn enhancing complement-based effector functions, such as complement-dependent cytotoxicity (CDC).

[0143] Immunotherapeutic proteins according to the present disclosure can be produced according to any standard method known to those skilled in the art. For example, one skilled in the art can easily prepare immunotherapeutic fusion proteins by: using standard molecular biology techniques to produce a construct comprising a polynucleotide sequence encoding a fusion protein; introducing the construct into a suitable host cell (e.g., a human kidney (HEK) host cell or a derivative thereof, such as Expi293 cells (Thermo Fisher Scientific)) to express the fusion protein; culturing the host cells according to a standard culture protocol; and using any method such as a known suitable purification method (e.g., affinity chromatography (e.g., protein A), ion exchange chromatography (IEX), size exclusion chromatography (SEC), and combinations thereof) to recover the expressed fusion protein from the culture supernatant. Similar methods can be used to prepare immunotherapeutic proteins, which are immunoglobulin molecules, such as antibodies or Ab-like molecules. That is, one skilled in the art can easily prepare mutant antibodies (i.e., antibodies comprising an amino acid substitution (Eu numbering) at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain) by producing a construct comprising a variable heavy (V H ) and light (V L ) region sequence (e.g., a polynucleotide sequence comprising an antigen binding region that binds to an antigen of interest) and a constant weight (CH) region from, for example, an IgG1 antibody, and a polynucleotide sequence encoding a CH3 region is incorporated by standard molecular biology techniques such as site-directed mutagenesis, and the polynucleotide sequence is changed to encode a mutation at H429 (e.g., H429F and H429Y). As with the preparation of immunotherapeutic fusion proteins, the construct can be introduced into a suitable host cell (e.g., a human kidney (HEK) host cell or a derivative thereof), cultured according to a standard culture protocol, and the expressed mutant antibody is purified from the culture supernatant using, for example, any of the known suitable purification methods such as affinity chromatography. Further, it has been found that in the case of using affinity chromatography (e.g., protein A) in purification, especially in the case of an immunotherapeutic protein in the form of an Ab-like molecule, it is advantageous to use mild elution conditions, such as using an elution buffer comprising a low concentration of arginine (e.g., less than 130 mM) and a pH less than or equal to 5, so as to inhibit the formation of aggregates. This can be achieved, for example, by a standard liquid chromatography system which 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.

[0144] In some preferred embodiments, the recovery of the expressed immunotherapeutic protein according to the present disclosure is:

[0145] (i) preferably at a weakly acidic pH (e.g., a pH less than neutral pH, such as pH 6.5, preferably pH 5.5, and more preferably pH 5.0), wherein it is desired to provide the immunotherapeutic protein in a monomeric form; or

[0146] (ii) where it is desired that the immunotherapeutic protein be provided in an oligomeric form (e.g., as a hexamer), preferably under conditions of substantially neutral pH (e.g., a pH in the range of 7.0 to 8.5, preferably 7.5 to 8.0, or more preferably a physiological pH of about 7.4); or

[0147] (iii) is preferably performed using a method comprising affinity chromatography using an elution buffer comprising 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), particularly where it is desired that the immunotherapeutic protein be provided as an antibody-like molecule.

[0148] In some specific embodiments, the recovery of the expressed immunotherapeutic protein according to the present disclosure comprises recovery by size exclusion chromatography (SEC), for example under weakly acidic pH conditions for producing a monomeric form of the immunotherapeutic protein, or under substantially neutral pH conditions for producing an oligomeric form of the immunotherapeutic protein. If desired, SEC can be performed after a recovery stage including ion exchange chromatography (IEX).

[0149] In a second aspect, the present disclosure provides a use of an immunotherapeutic protein as defined in the first aspect 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, other inflammatory diseases, infectious diseases and proliferative diseases.

[0150] In a third aspect, the present disclosure provides a use of an immunotherapeutic protein as defined in the first aspect for the preparation of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases and proliferative diseases.

[0151] In a fourth aspect, the present 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 defined in the first aspect, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

[0152] Among the diseases and conditions that can be treated or prevented by the method of the fourth aspect 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 (LPDs), including leukemias (e.g., acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL)), adenocarcinomas and lymphomas, as well as multiple myeloma (MM) and X-linked proliferative diseases.

[0153] The method of the fourth aspect of the present disclosure will typically be applied to treat a disease or condition in a human subject. However, the subject may also be selected from, for example, livestock 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.).

[0154] In some embodiments, wherein the immunotherapeutic protein comprises a fusion protein comprising a partner polypeptide connected to an Fc region component, the Fc region component comprises at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain), and the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof), which is capable of binding to the structural proteins of the virus, so that the immunotherapeutic protein can act as a "bait" to block viral interactions and viral entry into host cells, the method of the fourth aspect may further include administering antibodies against the virus (i.e., target virus). For example, where the immunotherapeutic protein includes an ACE2 polypeptide (or a fragment thereof) that binds to the RBD of the CoV-2 spike protein, the antibody can be selected, for example, from antibodies that are: 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 without neutralizing 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 can, for example, target an epitope of a SARS-CoV-2 structural protein other than the spike protein (S), such as the envelope protein (E), the membrane protein (M), or the nucleocapsid protein (N). In some other specific embodiments, the antibody can target, for example, an epitope on the spike protein (S) but at a site different from the RBD. More generally, in the case where the immunotherapeutic protein includes a cell surface receptor polypeptide or a co-receptor polypeptide or a fragment thereof other than the ACE2 polypeptide, the antibody against the target virus can be selected from, for example, an antibody with broad neutralization to a class of viruses / virus families (e.g., human immunodeficiency virus (HIV)), or an antibody with broad reactivity to a class of viruses or virus families, an antibody with broad neutralization to strains of a specific virus type (e.g., bNmAb that can neutralize multiple HIV-1 strains) and mAbs with broad reactivity to specific virus types. As shown below, it has been found that the immunotherapeutic proteins of the present disclosure and antibodies against target viruses can work synergistically to enhance CDC killing of cells (e.g., virus-infected cells).

[0155] Similarly, when the immunotherapeutic protein includes a fusion protein, the fusion protein includes a partner polypeptide connected to an Fc region component, the Fc region component includes at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain), and the partner polypeptide is, for example, directed against a target of therapeutic significance (for example, the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof), which is capable of binding to CTLA4 that acts as an immune checkpoint and downregulates the immune response, and the method of the fourth aspect may further include administering an antibody against the target (e.g., an antibody that binds to CTLA4), for example, to provide an enhanced response, such as enhanced CDC killing of cells (e.g., cancer cells).

[0156] Where the method of the fourth aspect comprises administering an antibody in addition to administering a fusion protein (i.e., as described in the preceding two paragraphs), preferably, the antibody comprises an Fc region component comprising an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide (EU numbering), e.g., H429X 2 Amino acid substitution, where X 2 Selected from phenylalanine (H429F), glutamic acid (H429E), glutamine (H429Q) and serine (H429S).

[0157] In a fifth aspect, the present disclosure provides a pharmaceutical composition or medicament comprising the immunotherapeutic protein as defined in the first aspect and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0158] Immunotherapeutic proteins can be administered in combination with one or more additional agents used to treat the specific disease or condition being treated. For example, in the context of treating a proliferative disease, an immunotherapeutic protein can be administered in combination with other drugs used to treat cancer (e.g., anti-tumor drugs such as cis-platin, gemcitabine, cytosine arabinoside, doxorubicin, epirubicin, taxoids including paclitaxel, topoisomerase inhibitors such as etoposide, cytostatics such as tamoxifen, aromatase inhibitors (e.g., anastrozole), and inhibitors of growth factor function (e.g., antibodies such as the anti-erbB2 antibody trastuzumab). TM))) used in combination. In some embodiments, the immunotherapeutic protein can be administered with one or more additional agents, which can also be immunotherapeutic proteins according to the present disclosure. For example, when the first immunotherapeutic protein according to the present disclosure includes an ACE2 polypeptide (or a fragment thereof) that binds to the RBD of the CoV-2 spike protein, the second immunotherapeutic protein of the present disclosure can be an antibody against the target CoV-2 virus, in particular an antibody that targets different structural proteins (e.g., envelope protein (E), membrane protein (M), or nucleocapsid protein (N)) or an epitope different from the RBD site on the same structural protein (i.e., spike protein (S)). As shown below, it has been found that combinations of such immunotherapeutic proteins can work synergistically to enhance CDC killing of cells (e.g., virus-infected cells).

[0159] When used in combination with other agents, the immunotherapeutic protein can be administered in the same pharmaceutical composition or in a separate pharmaceutical composition. If administered in a separate pharmaceutical composition, the immunotherapeutic protein and the other agent can be administered simultaneously or sequentially in any order (e.g., within seconds or minutes or even within hours (e.g., 2 to 48 hours)).

[0160] Immunotherapeutic proteins can be formulated into pharmaceutical compositions together 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 in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1995. Examples of suitable excipients for 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 carried out according to the intended route of administration and standard pharmaceutical practice.

[0161] The pharmaceutical composition comprising the immunotherapy protein as described in the first aspect may further include any suitable adhesive, lubricant, suspending agent, coating agent and solubilizing agent. Examples of suitable adhesives include starch, gelatin, natural sugars, such as glucose, anhydrous lactose, free-flowing lactose, beta-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 can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and parahydroxybenzoates. Antioxidants and suspending agents can also be used.

[0162] The pharmaceutical composition comprising the immunotherapeutic protein as described in the first aspect will generally be suitable for intravenous or subcutaneous administration. Thus, the pharmaceutical composition may comprise a solution or emulsion that can be injected into a subject and is prepared from a sterile or sterilizable solution. The pharmaceutical composition may be formulated into a unit dosage form (i.e., in the form of discrete portions containing a unit dose or multiples or subunits of a unit dose).

[0163] The immunotherapeutic proteins, uses and pharmaceutical compositions of the present disclosure are further described below with reference to the following non-limiting examples.

[0164] Examples

[0165] Example 1 ACE-2-Fc fusion protein including H429 mutation and activity analysis

[0166] Methods and Materials

[0167] Constructs and fusion proteins

[0168] The amino acid sequence of human ACE2 polypeptide can be obtained from the European Nucleotide Archive (ENA, European Molecular Biology Laboratory) under accession number BAB40370. The extracellular domain of the protein (amino acids 19 to 740; shown as SEQ ID NO: 1 in Table 1) contains a catalytic domain and a collectrin domain. In this example, different forms of the ACE2 extracellular domain were produced and studied; in particular, a truncated (tr) ACE2 extracellular domain (which includes amino acids 19 to 615 of the mature ACE2 polypeptide (named trACE2, shown as SEQ ID NO: 2 in Table 1) and does not include the collectrin domain), a full-length (fl) ACE2 extracellular domain to (flACE2; including amino acids 19 to 740 of the mature ACE2 polypeptide; SEQ ID NO: 1 in Table 1), and an enhanced flACE2 extracellular domain (EflACE2) (which includes triple mutations within the ACE2 polypeptide that have been reported to increase its binding affinity to the S protein) (Chan et al., 2020 supra). These proteins were produced as fusion proteins according to standard techniques, with an Fc region component derived from human IgG1 to produce trACE2-Fc, flACE2-Fc and EflACE2-Fc (see Table 2).

[0169] For example, by combining a polynucleotide sequence encoding the extracellular domain of trACE2 with a synthetic sequence encoding a linker and a sequence encoding human IgG1 Fc (specifically, an IgG1 Fc (immunoglobulin gamma 1 constant region, part [Homo sapiens (Homo sapiens)] having an amino acid sequence of accession number AXN93652.1) sapiens)]; National Center for Biotechnology Information (NCBI) database) was connected to prepare a construct encoding the trACE2 extracellular domain in pcDNA3.4 (Thermo Fisher Scientific). To generate the flACE2-Fc expression construct, KpnI digestion of the trACE2 construct was performed, followed by insertion of a codon-optimized polynucleotide sequence encoding the ACE2collectrin domain (GeneArt, Thermo Fisher Scientific). To generate the EflACE2-Fc expression construct, a synthetic polynucleotide sequence equivalent to the synthetic polynucleotide sequence encoding flACE2-Fc but with three mutations (i.e., T27Y, L79T, and N330Y; Chan et al., 2020 sACE2.v2.4 described above) was used. In addition, according to the manufacturer's instructions, the IgG Variants of the fusion protein were generated by cleavage at a unique AfeI site within the Fc coding sequence, H429F and H429Y mutations incorporated into the Fc component, and subsequent insertion of appropriate mutagenic oligonucleotides using NEBuilder (New England Biolabs, Ipswich, MA, United States of America).

[0170] The H429 residue occupies a "buried" site within the IgG1 Fc structure (see Figure 1 ) and is occupied by a histidine (His / H) residue that is also found in the corresponding position of the Fc fragment of all other human immunoglobulin classes ( Figure 3 , Figure 4 ), and is 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 supra) indicated that H429 is not a surface accessible residue ( Figure 1 ), and in the space-filling representation of Fc, H429 is “visible” only when the overlapping residues are presented in a non-space-filling manner, e.g. Figure 1 C. Similarly, analysis of residues adjacent to H429 indicated that H429 was located below the side chains of these residues, and calculation of the accessible surface area (ASA) of Fc residues indicated that H429 was inaccessible to the solvent (0% ASA Figure 1 C). H429 is also not a surface accessible residue in other Fc structures that are included within the structure of an anti-HIV mAb that forms a hexameric ring structure (PDB: 1HZH; Saphire et al., supra), but H429 does not form part of the interface. Similarly, analysis of the Fc structure of an anti-Lewis Y mAb (Ramsland et al., Journal of Immunology 187(6):3208-3217, 2011) suggests that H429 is also buried in this antibody.

[0171] Table 1

[0172]

[0173]

[0174]

[0175] The transient transfection of Expi293 cells (Thermo Fisher Scientific) was used to express the fusion protein. First, all expressed fusion proteins were purified from the culture supernatant by ion exchange chromatography (IEX), and then further purified by size exclusion chromatography (SEC). Specifically, the supernatant of Expi293 cells (wherein the Fc region is according to the wild type (WT) hIgG1 Fc mentioned in the preceding paragraph) of transient transfection to express the corresponding ACE2-Fc fusion protein was extensively dialyzed for 10mM Tris-HCl pH 8.0, and applied to High-Q columns (BioRad Laboratories, Hercules, CA, United States of America, California, Hercules). The bound protein was eluted with a linear gradient into a buffer A containing 0.4M NaCl. Fractions were examined by SDS-PAGE, and those containing flACE2-Fc WT fusion protein were pooled and concentrated using a 30 kDa cutoff filter device (Pall Corporation, Port Washington, NY, United States of America) and separated by size exclusion chromatography (SEC) using a Superose 6 column (GE Life Sciences, Chicago, IL, United States of America).

[0176] SARS-CoV-2 RBD-Ig and RBD AviTag have been described previously. RBD AviTag was biotinylated in situ using Expi293BirA cells (Wines BD et al., J. Immunol. 197(4): 1507-1516, 2016).

[0177] Lamelli native PAGE (N-PAGE), 150 V, 2.5 hours, 4° C., according to Wines BD et al., J. Immunol. 162(4): 2146-2153, 1999.

[0178] Virus neutralization assay

[0179] Antiviral titers were determined using SARS-CoV-2 (CoV / Australia / VIC01 / 2020) in a microneutralization assay as previously described (Juno JA et al., Nat Med 26(9): 1428-1434, 2020).

[0180] Biolayer Interferometry

[0181] The affinity of ACE2-Fc fusion protein to CoV-2 S RBD was measured on Octet RED96e (FortéBio, Fremont, CA, United States of America). All assays were performed at 25°C using anti-human IgG Fc capture (AHC) biosensor tips (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 (TWEEN-20)). After a 60-second (60s) biosensor baseline step, the fusion protein (20 mg / mL) was loaded onto the anti-human IgG Fc capture (AHC) biosensor by immersing the sensor tip for 200 seconds and then washing in kinetic buffer for 60 seconds. For most fusion proteins, association measurements were performed by immersing them in a two-fold dilution series of 16-250 or 500 nM SARS-CoV-2 RBD for 180 seconds and then measuring dissociation in kinetic buffer for 180 seconds. For EflACE2-Fc WT, a two-fold dilution series of 2-31 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 and 5 seconds in kinetic buffer, and baseline drift was corrected by subtracting the average offset of sensors loaded with fusion proteins that were not incubated with SARS-CoV-2 RBD and unloaded sensors incubated with SARS-CoV-2 RBD. Curve fitting analysis was performed using Octet Data Analysis 10.0 software using a global fit 1:1 model to determine K D Values ​​and kinetic parameters. Curves that could not be fitted were excluded from the analysis. The reported kinetic constants are representative of two independent experiments.

[0182] ACE2-Fc fusion protein and recombinant dimeric rsFcγR binding determined by flow cytometry

[0183] ACE2-Fc fusion protein or rituximab (a chimeric mAb targeting CD20) at 5 μg / ml or the indicated concentrations were incubated with Ramos cells expressing the transfected spike protein (Ramos-S cells; WS et al., medRxiv doi: 10.1101 / 2020.12.13.20248143, 2020) in 25 μl of PBS containing 0.5% (w / v) BSA, 1 mM glucose (PBS / BSA / G) at a rate of 5 × 10 6Cells / ml were incubated on ice for 30 min and then washed twice with PBS / BSA / G, incubated with PE- or FITC-conjugated anti-human IgG-Fc on ice for 30 min, then washed again and resuspended in 25 μl of PBS / BSA / G.

[0184] Binding of dimeric recombinant soluble FcγR (rsFcγR) was assessed as previously described (Wines et al., 2016 supra). ACE2-Fc opsonized Ramos-S cells or rituximab opsonized cells were resuspended in 0.5 μg / ml of biotinylated dimeric rsFcγRIIa (H131 allele form) or dimeric rsFcγRIIIa (V158 allele form) or BSA / PBS / G and incubated on ice for 30 minutes, followed by incubation on ice for 20 minutes with 1 / 500 streptavidin-APC (or anti-hIgG-Fc FITC for confirmation of ACE2-Fc opsonization). Cells were washed, resuspended in PBS / BSA / G, and incubated on Canto TM The cells were analyzed on a Flow Cytometer II (Becton Dickinson, Franklin Lakes, NJ, United States of America).

[0185] Complement-dependent cytotoxicity (CDC) assay

[0186] CDC was measured using Ramos-S cells opsonized with ACE2-Fc functional protein or control mAb rituximab and then incubated with human serum as a complement source. Therefore, Ramos-S cells were first incubated with fusion protein or rituximab as described above (5x10 6 Cells / ml were added in 25 μl of PBS / BSA / G on ice for 30 min) and then washed before resuspension in 1 / 3 diluted normal human serum for 30 min at 37° C. Cells were washed twice with PBS and then enumerated for dead cells by staining with 1 / 500 Zombie Green (from Zombie Green Fixable Viability Kit, BioLegend, San Diego, CA, United States of America, according to the manufacturer's instructions) and then fixed with 2% paraformaldehyde in PBS and incubated in Canto TM The cells were analyzed on a Flow Cytometer II (Bedi Medical).

[0187] Complement fixation immunoassay for ACE2-Fc fusion protein

[0188] Ninety-six-well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated overnight with 5 μg / ml avidin in PBS, blocked, and then incubated with serial two-fold dilutions or a single concentration (2.5 μg / ml) of biotinylated RBD (Hartley et al., Science Immunology 5(54) doi: 10.1126 / sciimmunol.abf8891, 2020) in 0.1% casein at room temperature for 1 hour. ACE2-Fc fusion proteins were then added within the indicated concentration range. To measure C1q immobilization, plates were incubated with 10 μg / ml purified human C1q (Merck Millipore, Burlington, MA, United States of America) for 30 minutes at room temperature, followed by incubation with 1 / 2000 diluted rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:452019) for 1 hour at room temperature. In the experiment of measuring C5b-C9 fixation, the plate was incubated with 10% fresh human serum for 30 minutes at room temperature, followed by incubation with rabbit anti-C5b-C9 (Merck Millipore) at 1 / 2000 dilution at room temperature for 1 hour, then incubated with goat anti-rabbit IgG (Merck Millipore) conjugated with HRP at 1 / 2000 dilution at room temperature for 1 hour, followed by incubation with TMB substrate (Life Technologies Corporation, Carlsbad, CA, United States of America) at room temperature for 15-20 minutes. Reactivity was stopped using 1M sulfuric acid, and absorbance was measured at OD450 nm. Test samples and reagents were prepared in PBS supplemented with 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 in which ACE2-Fc protein was omitted. Means and SEMs from independent experiments are shown.

[0189] RBD variant multiplex assay

[0190] Custom multiplex arrays were generated with SARS-1 S1 subunit (ACRO Biosystems, 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 (BPS Bioscience, San Diego, CA, United States of America), and hexa-histidine-tagged RBD WT protein (amino acids 19-613) and 21 variants identified from the GISAID RBD monitoring library expressed from pcDNA3 in Expi293 cells and purified by affinity chromatography. FlexMap3D TM Bead coupling, washing steps and data acquisition on the analyzer (Luminex Corporation, Austin, TX, United States of America) were as previously described (Lee et al., 2020 supra). Briefly, 25 μl of 1.3 μg / ml anti-human IgG R-phycoerythrin conjugate (Southern Biotech, Birmingham, AL, United States of America) was used to detect direct binding of ACE2-Fc fusion proteins trACE2-Fc, flACE2-Fc and EflACE2-Fc at 0.5 to 250 nM. The data were fitted to a 3-parameter agonist-to-response curve (r 2 >0.85) to determine each EC 50 In the competition assay, RBD-coupled beads or S1-coupled beads were incubated simultaneously with 20 μl of 25 μg / ml biotinylated, AviTagged ACE2 at 1 to 280 nM for 2 h at room temperature in the presence of various non-biotinylated ACE2-Fc fusion protein "competitors". Binding of biotinylated ACE2 (aa19 to 615) was performed first using 4 μg / ml of streptavidin, R-phycoerythrin conjugate (SAPE) (Thermo Fisher Scientific) (1 h) followed by 10 μg / ml of R-phycoerythrin, biotin-XX conjugate (Thermo Fisher Scientific) (1 h).

[0191] FcγRIIIa-NF-κB-RE nanoluciferase reporter gene assay

[0192] FcγRIIIa-NF-κB-RE nanoluciferase reporter gene assay was 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 was performed essentially as previously described (Lee et al., 2020 supra). Briefly, Ramos cells expressing spike-IRES-orange2 were used as target cells and incubated with agonists and FcγRIIIa / NF-κB-RE reporter cells for 5 hours, and then the induced nanoluciferase was measured with Nano-Glo substrate (Promega Corporation).

[0193] Results and discussion

[0194] ACE2-Fc fusion protein construction and production

[0195] A series of ACE2-Fc fusion proteins were generated (Table 2) and analyzed for their ability to neutralize SARS-CoV-2 infection and mediate Fc-dependent effector functions, which are generally attributed to the mechanism of action of antibodies.

[0196] Table 2

[0197]

[0198]

[0199] *N / A: Not applicable as no Fc present (i.e., only truncated extracellular domain)

[0200] In an attempt to enhance avidity for binding to the SARS-CoV-2 spike protein or to confer and improve Fc-dependent effector functions, three versions of the ACE2 extracellular domain were fused to the Fc region of IgG1 that was either unmodified or altered by mutation (i.e., substitution of histidine 429 with phenylalanine (H429F) or tyrosine (H429Y)) or modified glycosylation (i.e., lack of core fucose; trACE2-Fc-kif).

[0201] ACE2-Fc fusion protein purification

[0202] The protein was 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 that 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 major species of approximately 270 kDa ( Figure 5 B). SEC analysis and purification confirmed the presence of a major monomeric species, except for those fusion proteins including the H429Y mutation ( Figure 5 C) (Note: the monomeric species was considered to be a single molecule (i.e., a monomeric molecule) comprising two (dimerized) copies of the corresponding ACE2-Fc fusion protein), which was collected for further analysis, and only trace amounts of high molecular weight oligomers and other impurities were evident, such as flACE2-Fc-WT ( Figure 5 In fact, the presence of similar monomeric species was also evident after IEX purification of all fusion proteins with either unmodified Fc-WT or Fc region components containing the H429F modification (not shown). For fusion proteins including the H429Y mutation, SEC analysis ( Figure 5 D) (i.e., the major IEX peak from the flACE2-Fc H429Y mutant, not shown), as well as the major IEX peaks from the trACE2-Fc H429Y and EflACE2-Fc H429Y proteins (not shown), revealing a large number of oligomeric (Y oli ) and monomer (Y mn ) species, which were then individually assessed for their functional activity.

[0203] ACE2-Fc fusion protein binding to SARS-CoV-2 S RBD by ELISA

[0204] 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 0.35nM, 0.27nM and 0.25nM respectively; Figure 6 D), where the enhanced intrinsic affinity of EflACE2-Fc was less pronounced in binding to this bivalent form of the RBD when fused to mouse IgG1 Fc (RBD-Ig). The binding activity of the various fusion proteins with mutated Fc components was also equivalent, except for slightly lower affinity (EC 500.48 nM) of monomeric flACE2-Fc-H429Y ( Figure 6 D) and other H429Y Fc variants that also trended toward lower affinity levels ( Figure 6 ).

[0205] Oligomerization of ACE2-Fc H429Y fusion protein is pH-dependent

[0206] Oligomerization of the fusion protein containing the mutant H429Y Fc component was examined by SEC separation at pH 5.0 of flACE2-Fc-H429Y prepared by IEX. Figure 5 D and Figure 7 A) compared to SEC, at pH 5.0 ( Figure 7 B) reveals 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). The DNA was purified by dialysis at pH 7.4 followed by N-PAGE ( Figure 7 C) and by SEC at pH 7.4 ( Figure 7 D) The two reacted with purified monomeric flACE2-Fc-H429Y mn , pH 5.0 and re-analyzed. Re-exposure to pH 7.4 produced oligomer Y oli and monomer Y mn A mixture of species ( Figure 7 C; Lane 2 pH 5.0 compared to Lane 5 pH 7.4), indicating that some equilibrium occurs between these forms at neutral pH. flACE2-Fc-H429Y prepared at pH 5.0 mn Shows equivalent binding to RBD-Ig as other flACE2-Fc WT fusion proteins and Fc variants ( Figure 7 E) It is also apparent that the prevalence of oligomers is associated with the tyrosine substitution of histidine 429, as this was not observed in fusion proteins with the H429F Fc region component substituted with phenylalanine.

[0207] Evaluation of virus neutralization potency

[0208] The antiviral activity of ACE2-Fc fusion protein was determined in a microneutralization assay of SARS-CoV-2 infection in Vero cells ( Figure 8 ), where EC 50 The endpoint corresponds to neutralization of approximately 99% of the inoculum virus particles (Khoury DS et al., Nat Rev Immunol 20(12): 727-738, 2020).

[0209] The SARS-CoV-2 neutralization endpoint of the truncated extracellular domain trACE2 (2.70 μM) was enhanced by approximately 10-fold by fusion with the wild-type Fc region of IgG1 (trACE2-Fc WT, 283 nM). Figure 8 ). The increase in potency is consistent with the increase in affinity for binding to the SARS-CoV-2 spike RBD, because the bivalency of ACE2-Fc generated by the fusion of the ACE2 extracellular domain with the IgG Fc region is also similar to that of flACE2-Fc WT. EflACE2-Fc-WT showed a further increase of about 20-fold (11 nM) over flACE2-Fc WT and trACE2-Fc WT fusion proteins, and about 200-fold over unfused trACE2.

[0210] Analysis of Fc modifications revealed several interesting differences. First, the H429Y mutation in trACE2-Fc and flACE2-Fc increased their virus neutralization potency ( Figure 8 ); this is surprising because the neutralizing function of the ACE2-Fc fusion protein occurs away from the modification at the H429 position (i.e., in the fusion protein, the ACE2 polypeptide is expected to neutralize the virus, but the H429 residue is located distal to the ACE2 present in the CH3 domain of the Fc component). Second, it was found that trACE2-Fc-H429Y separated by SEC at pH 7.4 oli The neutralizing activity of the oligomeric form of flACE2-Fc-H429Y (21.9 nM) was 13-fold higher than that of trACE2-Fc WT. oli and monomer Y mn Both forms include two (dimerized) copies of the ACE2-Fc fusion protein ( Figure 5 B) (end points were 10.0 and 20.9 nM, respectively; Figure 8 ) showed a stronger potency than the flACE2-Fc WT (124 nM) fusion protein and similar virus neutralization to that achieved with EflACE2-Fc WT (10.6 nM), but EflACE2-Fc-H429Y oli It appears to be more potent (4 nM), which means that the neutralizing activity of EflACE2-Fc WT (10.6 nM) is more than 200 times that of unfused trACE2, or in the case of EflACE2-Fc-H429Y (4 nM), more than 600 times ( Figure 8). Thus, the Fc-H429Y mutation increases SARS-CoV-2 neutralization in both trACE2-Fc and flACE2-Fc fusion protein formats and also tends to higher levels of potency when combined with the triple mutation of ACE2 with the inherently higher affinity EflACE2-Fc.

[0211] Phenylalanine substitution of histidine 429 (H429F) of the ACE2-Fc fusion protein did not enhance neutralization.

[0212] Interaction between ACE2-Fc fusion protein and FcγR

[0213] The interaction of FcγRIIa and FcγRIIIa with ACE2-Fc fusion proteins was assessed by flow cytometry using ACE2-Fc opsonized Ramos-S cells and dimeric recombinant soluble FcγR (Wines et al., 2016 supra). trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins all bound to FcγRIIa and FcγRIIIa ( Fig. 9 A, 9B), however, variant proteins including the mutated H429Y Fc component largely abolished binding to both Fc receptor types.

[0214] Activation of FcγRIIIa by ACE2-Fc fusion protein

[0215] Antibody-dependent cellular 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 (J Exp Med) 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 including wild-type (WT) Fc regions all initiated FcγRIIIa activation ( Fig.10). However, surprisingly, the flACE2-Fc fusion protein induced higher levels of activation than trACE2-Fc, indicating that the collectrin domain contained in the ACE2 component of the fusion protein significantly and surprisingly increased the potency of the ACE2-Fc fusion protein to activate FcγRIIIa. Further, it was found that the increased affinity of the EflACE2-Fc fusion protein for the SARS-CoV-2 spike protein did not change the level of FcγRIIIa activation, which was comparable to that of flACE2-Fc ( Fig.10 D).

[0216] Production of a trACE2-Fc fusion protein in the presence of kifunine (van Berkel PHC et al., Biotechnology and Bioengineering 105(20:350-357, 2010), trACE2-Fc-kif, also enhanced the modest activation levels of FcγRIIIa shown by trACE2-Fc to levels comparable to those of flACE2-Fc and EflACE2-Fc fusion proteins ( Fig.10 D) and is close to CD20 + Levels of therapeutic anti-CD20 mAb rituximab on Ramos-S cells ( Fig.10 A, 10D). Kifusine 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 of the United States of America 108 (31): 12669-12674, 2011). Therefore, it is assumed that similar treatment or amino acid residue substitution of flACE2-Fc and EflACE2-Fc fusion proteins to increase affinity for FcγRIIIa (Wang et al., 2018 supra) may further improve their FcγRIII activation potency.

[0217] Modification of trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins by inclusion of the H429F Fc component mutation did not affect activation of FcγRIII by opsonized Ramos-S cells ( Fig.10 ). In contrast, inclusion of the mutated H429Y Fc component in all ACE2-Fc fusion proteins abolished cellular FcγRIIIa activation, consistent with the aforementioned inability to bind to Fc receptors, particularly FcγRIIIa ( Fig. 9B). Thus, while enhancing virus neutralization, the H429Y-modified Fc component in trACE2-Fc, flACE2-Fc, and EflACE2-Fc fusion proteins had no activity against FcγR binding ( Fig. 9 ), and activates cells via FcγRIIIa ( Fig.10 ). The hierarchy of FcγRIIIa activation by fusion proteins is flACE2-Fc WT = EflACE2-Fc > trACE2-Fc, thus emphasizing that the presence of the collectrin domain is a key component for optimal FcγRIIIa activation by ACE2-Fc fusion proteins.

[0218] Complement fixation, activation, and lysis of SARS-CoV-2 spike cells by ACE2 Fc protein The ability of ACE2-Fc fusion proteins including mutated Fc region components to fix complement components C1q and C5-C9 was examined in an ELISA-based assay using avidin-immobilized RBD-biotin ( Fig.11 AF) and importantly its ability to mediate complement-dependent killing of cells expressing the SARS-CoV-2 spike protein ( Fig.11 G). When RBD is restrictive ( Fig.11 A, 11B), the activity of the fusion protein including the mutant H429F Fc component was surprisingly enhanced compared to the activity of the counterpart including the wild-type Fc region. Fig.11 E, 11F) binding was equivalent to that of a fusion protein including a mutated H429FFc region component. Further, despite oligomerization ( Figure 6 and 7 )(associated with its superior CoV-2 neutralization activity( Figure 8 )), but both trACE2-Fc-H429Y and flACE2-Fc-H429Y fusion proteins showed little difference in C1q or C5-9 immobilization compared to their counterparts including the wild-type Fc region ( Fig.11 AF). In addition, it is clear that the trACE2-Fc fusion protein is more effective than flACE2-Fc in fixing complement C1q (compared to Fig.11 C, 11E and 11D, 11F); it 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 worth noting that this difference is greatly reduced by the H429F Fc mutation containing trACE2-Fc-H429F and flACE2-Fc-H429F fusion proteins; it is particularly evident in the fixation of the C5b-C9 complex ( Fig.11 E, 11F).

[0219] Surprisingly, the fusion protein including the mutant H429F Fc region component was the only highly active ACE2-Fc fusion protein in serum complement-dependent cytotoxicity (CDC) of Ramos-S cells ( Fig.11 G). Although trACE2-Fc WT, trACE2-Fc-kif fusion protein and flACE2-Fc WT have the ability to fix C1q and C5b-9 in ELISA assay ( Fig.11 A, 11C, 11E), but despite having a higher affinity for the SARS-CoV-2 spike protein, it still failed to induce complement-mediated cell death, and neither did EflACE2-Fc WT. trACE2-Fc-H429Y mn The fusion protein is also active, albeit weakly, in complement-mediated killing.

[0220] Since it is well known that activation of the complement cascade may also lead to phagocytosis of cells, microorganisms or particles opsonized, for example, with C1q (or other complement fragments, such as C3b or C3bi; Ricklin D et al., Immunol Reviews 274(1):33-58, 2016) bound to specific cell surface receptors on phagocytes such as CR1 or CR3 (Vandendriessche S et al., Front Cell Dev Biol 9:624025, 2021), it is believed that the observed enhancement of Fc-dependent complement lysis by ACE2-Fc fusion protein containing the H429Y mutation will also be reflected in a similar enhancement of complement-dependent phagocytosis of ACE2-Fc-coated targets (e.g., viral particles) through complement receptors.

[0221] in conclusion

[0222] It has been found that immunotherapeutic proteins in the form of fusion proteins provide considerable potential for treating or preventing coronavirus infections, the fusion proteins comprising angiotensin converting enzyme 2 (ACE2) polypeptides (or fragments thereof) fused to Fc region components including H429 mutations. For example, the selection of full-length or truncated ACE2 (i.e., fragments) and various modifications to the Fc component can also enable considerable "adjustment" of antiviral agents to modify the effect of achieving antiviral effects. For example, by including an Fc component in an immunotherapeutic protein including an H429Y substitution, oligomeric immunotherapeutic proteins can be produced, which show increased viral neutralization and eliminated FcγR binding and activation. On the other hand, by including an Fc component in an immunotherapeutic protein including an H429F substitution, antiviral effects can be achieved, including complement dependent cytotoxicity (CDC) of cells expressing spike proteins (e.g., infected cells). Therefore, these unique Fc mutations enable a complementary approach to modulate the function of the Fc region components, which may help to develop ACE2-Fc fusion proteins and other therapeutically significant fusion proteins by allowing the selection of desired functional curves.

[0223] Example 2 Chimeric anti-TNP IgG1 antibodies comprising H429 mutation and activity analysis

[0224] Methods and Materials

[0225] Generation of anti-TNP human IgG and mutant anti-TNP human IgG plasmid constructs The variable weight (V H ) and light (V L Chimeric anti-TNP human IgG antibody constructs consisting of sequences from the constant heavy (CH) region of the human IgG subclass have been described in detail previously - hIgG1 (Patel D et al., Journal of Immunology 184(11): 6283-6292, 2010), hIgG2 and hIgG4 (Wines et al., 2016 supra). All chimeric antibody sequences were subcloned into the pCR3 vector. The mutations of H429F, H429Q, H429E, H429S, H433A and H435A substitutions were introduced into the cDNA sequence encoding the Fc region component using standard molecular biology techniques.

[0226] Expression and production of IgG antibodies by Expi293 cells

[0227] As previously described, IgG antibodies were produced in Expi293 human embryonic kidney cells (Wines et al., 2016 supra). In brief, for both cell growth and protein production, Expi293 cells were maintained in Expi293 expression medium (Gibco, Waltham, MA, United States of America). Using the Expifectamine transfection kit (Life Technologies), cells were transfected simultaneously with IgG heavy chain plasmids (15ug) and light chain plasmids (15μg) 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, followed by affinity chromatography using a Hi-Trap HP protein A column (GE Healthcare Life Sciences, Marlborough, MA, United States of America), eluted with 0.1 M citric acid, pH 3.5, then neutralized with 1 M Tris-HCl, pH 9.0, and dialyzed against PBS pH 7.5. Aggregates were subsequently removed by gel filtration on a Superose 610 / 300GL column (GE Healthcare Life Sciences), and monomeric IgG peak fractions were collected. As described, the antigen binding activity of all antibody preparations was tested on BSA-TNP by ELISA (Wines et al., 2016 supra).

[0228] Evaluation of antigen binding activity by monoclonal anti-TNP mAb

[0229] Antigen binding activity of anti-TNP mAbs was assessed by ELISA by binding to TNP haptenated BSA (TNP-BSA) as previously described in Wines et al., 2016 supra.

[0230] ELISA immunoassay for detection of complement fixation by mAb

[0231] Ninety-six-well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated with 20 μg / ml TNP-BSA in PBS at 4°C overnight. The next morning, the plates were blocked with 0.1% (w / v) casein in PBS at 37°C for 2 hours and then incubated with anti-TNP mAb (concentrations between 4 and 0.125 μg / ml) at room temperature for 2 hours. To measure C1q immobilization, TNP: anti-TNP plates were incubated with 10 μg / ml purified human C1q (Merck Millipore) for 30 minutes at room temperature, followed by incubation with rabbit anti-C1q IgG (Kurtovic L et al., BMC Medicine 17:452019) diluted 1 / 2000 for 1 hour at room temperature. In experiments measuring C5b-C9 fixation, TNP: anti-TNP plates were incubated with 10% fresh human serum for 30 minutes at room temperature, followed by incubation with rabbit anti-C5b-C9 (Merck Millipore) at a dilution of 1 / 2000 for 1 hour at room temperature, then washed, and then incubated with goat anti-rabbit IgG conjugated to HRP (Merck Millipore) at a dilution of 1 / 2000 for 1 hour at room temperature, followed by incubation with TMB substrate (Life Technologies) for 15-20 minutes at room temperature. Reactivity was stopped using 1M 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 plates were 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 in which the antibody was omitted.

[0232] Results and discussion

[0233] Anti-TNP antibody constructs

[0234] The TNP-WT mAb used in this example includes either of two human heavy chain isotypes; first, an IgG1 isotype polypeptide (SEQ ID NO: 32) comprising, in N-terminal to C-terminal order, the TNP-specific V domain of the mouse monoclonal antibody TIB142 fused to the CH1-hinge-CH2-CH3 domain of human IgG1 encoded by cDNA (SEQ ID NO: 33). H domain (Patel D et al., Journal of Immunology 184:6283-6292, 2010); and secondly, an IgG2 isotype polypeptide (SEQ ID NO: 34) comprising, in order from N-terminus to C-terminus, the TNP-specific V domain of mouse monoclonal antibody TIB142 fused to the CH1-hinge-CH2-CH3 domain of human IgG2 Hdomain and is encoded by a codon-optimized cDNA having the sequence shown as SEQ ID NO: 35. The TNP-specific light chain polypeptide (SEQ ID NO: 36) comprises the TNP-specific V domain of mAb TIB142 fused to the human kappa chain constant domain. L domain, encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO:37.

[0235] Modification of IgG enhances complement fixation

[0236] When an antibody, such as a mAb, binds to its target antigen, the classical pathway of complement may be activated, resulting in physiological effects that include destruction of target cells by complement-dependent lysis (CDC), or complement-mediated antibody-dependent phagocytosis (C'ADCP) by specific phagocytes through specific receptors for complement proteins and fragments. The activation of this major amplification effector system of innate immunity is primarily dependent on and initiated by the binding of the hexameric complement protein C1q, which is part of the C1 complex. This binding of C1q to antibodies bound to their target antigen ultimately leads to the formation of a membrane attack complex (MAC) that includes other complement components (C5b, C6, C7, C8, C9).

[0237] Since the H429F Fc region component mutation confers potent CDC in ACE2-Fc fusion proteins (see Example 1), these ( Fig.12 ) and several other mutations ( Fig.13 ). In the context of an intact (chimeric) IgG1 antibody with a mouse V domain and a human IgG1 constant domain recognizing the TNP hapten, complement fixation by mutations in the Fc component was examined in an ELISA. Surprisingly, substitution of the histidine at position 429 in the TNP-IgG1-H429F mAb resulted in enhanced C1q binding compared to C1q binding of the unmodified TNP-IgG1-WT mAb ( Fig.12 Next, the production of membrane attack complex proteins by TNP-specific mAbs was evaluated ( Fig.12). Similarly, TNP-IgG1-H429F mAb showed enhanced complement activation, as seen by increased formation of MAC proteins (C5b, C6, C7, C9), and importantly, this was entirely consistent with the observed enhanced ability to bind to C1q conferred by the H429F mutation. Thus, modification at position 429 can unexpectedly alter the properties of an antibody. This enhanced function is surprising because amino acid 429 is buried within the CH3 domain of IgG1 and is far away from the complement C1q binding surface and the binding site of the leukocyte Fc receptor in the CH2 of the H chain (Hogarth PM and Pietersz GA. Nature Reviews Drug Discovery 11: 311-331, 2012; Chenoweth et al., 2020 supra).

[0238] The H429F mutation in IgG1 antibodies enhances C1q( Fig.12 A) and C5b-C9 fixation ( Fig.12 B) Both. The complement-enhancing activity of the mutant IgG1 antibodies including the H429F mutation, particularly in terms of fixation of C1q, was most pronounced when the lower density TNP:BSA antigen was opsonized by the mutant IgGs ( Fig.12 ). Thus, in the context of ACE2-Fc fusion protein and intact IgG1 antibody, the H429F Fc component mutation enhanced complement activation.

[0239] Antigen binding activity of anti-TNP mAbs, TNP-IgG1-WT, TNP-IgG2-WT and modified (mutant) mAb, TNP-IgG1-H429F, bound to TNP haptenized BSA (TNP-BSA) was assessed by ELISA as described (Wines et al., 2016 supra). All mAbs showed substantially equivalent binding activity to TNP-BSA ( Fig.12 C), indicating that the enhanced complement activation of the Fc-modified anti-TNP antibody is due to the Fc mutation rather than to differences in antigen binding activity.

[0240] in conclusion

[0241] The data obtained in this example demonstrate that the IgG-H429F mutant is able to enhance the immobilization of C1q and C5b-C9 to enable increased complement activation and can therefore be expected to be able to induce useful complement-based effector functions.

[0242] Example 3 Generation and analysis of modified forms of therapeutic antibodies comprising H429 mutations

[0243] In this example, the effect of modifying position H429 (i.e., by amino acid substitution) on antibody properties was evaluated using a number of unrelated chimeric mAbs containing different antigen-binding variable domains that bind to unrelated epitopes in a range of different target molecules on different cell types. These antibodies are "based on" various commercially used mAbs, and in some cases, mAbs of clinical significance. Specifically, the experiments were designed to determine the possible functional effects of amino acid substitutions at position 429 in antibodies carrying the V domains of the anti-HER2 mAbs trastuzumab and pertuzumab; anti-CD20 mAbs carrying the V domains of rituximab or 11B8; and the V domains of the anti-CD38 mAb daratumumab.

[0244] Methods and Materials

[0245] Antibodies and Antibody Constructs

[0246] DNA sequences obtained by RT-PCR or synthetic DNA corresponding to immunoglobulin variable and constant sequences were assembled by standard molecular biology techniques including ligation and Gibson assembly (NEBuilder, New England Biolabs) or as complete synthetic DNA encoding complete immunoglobulin H and L chains. These sequences were used in expression vectors such as pcDNA3.1, pcDNA 3.4 (Thermo Fisher Scientific) and pCIneo (Promega).

[0247] The unmodified mAbs used in this example were formatted on human IgG heavy chains and generated with human kappa light chains. The unmodified mAbs contained the specific V H and V L domains, and are referred to as wild-type (WT) forms; for example, as previously described, the "WT" trastuzumab antibody used in this example includes the wild-type (WT) HER2-specific V domain of trastuzumab. H and V L domain (https: / / go.drugbank.com / drugs / DB00072).

[0248] More specifically, the trastuzumab WT mAb used in this example includes a HER2-specific heavy chain polypeptide described at https: / / go.drugbank.com / drugs / DB00072 (which includes, in order from N-terminus to C-terminus, the HER2-specific V domain of trastuzumab fused to the CH1-hinge-CH2-CH3 domain of human IgG1). Hdomain) (whose amino acid sequence is provided as SEQ ID NO: 12), and is encoded by a codon-optimized DNA having a sequence shown as SEQ ID NO: 13. Similarly, the polypeptide of the anti-HER2 mAb trastuzumab light chain is as described at https: / / go.drugbank.com / drugs / DB00072 (SEQ ID NO: 14), and includes the HER2-specific V domain of trastuzumab fused to a human kappa constant domain. L domain, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO:15.

[0249] The 11B8-WT mAb used in this example includes the previously described CD20-specific heavy chain polypeptide (which, in N-terminal to C-terminal order, includes the CD20-specific V domain of 11B8 fused to the CH1-hinge-CH2-CH3 domain of human IgG1). H Domain) (U.S. Pat. No. 8,529,902) (SEQ ID No: 16), and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 17. Similarly, the polypeptide of the anti-CD20 mAb 11B8 light chain is as previously described (SEQ ID NO: 18), and comprises the CD20-specific V domain of 11B8 fused to a human kappa constant domain. L domain, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO:19.

[0250] The daratumumab-WT mAb used in this example comprises the previously described CD38-specific heavy chain polypeptide (which, in N-terminal to C-terminal order, comprises the CD38-specific V domain of daratumumab mAb fused to the CH1-hinge-CH2-CH3 domain of human IgG1). H Domain) (https: / / go.drugbank.com / drugs / DB09331) (SEQ ID NO: 20) and is encoded by a codon-optimized DNA having a sequence shown as SEQ ID NO: 21. Similarly, the polypeptide of the anti-CD38 mAb daratumumab light chain is as previously described at https: / / go.drugbank.com / drugs / DB09331 (SEQ ID NO: 22), which includes the CD38-specific V domain of the daratumumab mAb fused to a human kappa constant domain. L domain, encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO:23.

[0251] The Pertuzumab-WT mAb used in this example includes a HER2-specific heavy chain polypeptide (SEQ ID NO: 24) as previously described at https: / / go.drugbank.com / drugs / DB06366 (which includes, in N-terminal to C-terminal order, a HER2-specific V domain fused to the CH1-hinge-CH2-CH3 domain of human IgG1). H domain) and is encoded by a 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 at https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 26), which includes the HER2-specific V domain of the pertuzumab mAb fused to the human kappa constant domain. L domain, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 27.

[0252] The rituximab-WT mAb used in this example includes a CD20-specific heavy chain polypeptide (which includes, in N-terminal to C-terminal order, the CD20-specific V domain of rituximab fused to the CH1-hinge-CH2-CH3 domain of human IgG1). H Domain) (as described at https: / / go.drugbank.com / drugs / DB00073) (SEQ ID NO: 28) and is encoded by a codon-optimized DNA having a sequence shown as SEQ ID NO: 29. Similarly, the polypeptide of the anti-CD20 mAb rituximab light chain is as previously described at https: / / go.drugbank.com / drugs / DB000723 (SEQ ID NO: 30), which includes the CD20-specific V domain of rituximab fused to a human kappa constant domain. L domain, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO:31.

[0253] Synthesis of unmodified and mutated heavy chains

[0254] Antibody expression vectors are produced by standard methods known to those skilled in the art. Briefly, antibody expression vectors consist of synthetic polynucleotide sequences encoding antibody heavy or light chains, appropriately placed in plasmids, such as pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors with antibodies of different specificities are generated by adding a variable domain (V H or V L) borders. Newly synthesized DNA encoding the new V domain and flanked by sequences homologous to the cleaved vector (e.g., 25 nucleotides) is then incorporated by reaction with NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0255] Fc variants were generated by synthesizing synthetic polynucleotide sequences encoding the variants or by cleaving the Fc coding sequence of an antibody expression plasmid with appropriate restriction enzymes and incorporating the newly mutagenized synthetic DNA by performing a reaction using NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0256] Expression of antibody constructs

[0257] Transient transfection of Expi293F cells (Thermo Fisher Scientific) was used for antibody expression. Expi293F cells were cultured in EXPI expression medium (Life Technologies) and split to 2×10 cells 24 hours before transfection. 6 On the day of transfection, 7.5x10 7 Live logarithmic phase cells were centrifuged and resuspended in 25 ml of preheated, antibiotic-free Expi293 expression medium and maintained at 37°C until transfection. The cells were then transfected at room temperature using the Life Technologies Expifectamine transfection kit as described below. Eighty microliters of ExpiFectamine293 reagent were diluted with 1.5 ml Opti-MEM-I reduced serum medium (Gibco) and incubated at room temperature for 5 minutes. Thirty μg of DNA (15 μg of H-chain DNA and 15 μg of L-chain DNA) were diluted in 1.5 ml of Opti-MEM-I reduced serum medium, then added to the diluted ExpiFectamine reagent, incubated at room temperature for 20-30 minutes, and then added dropwise to the Expi293F cell suspension, which was then cultured at 37°C for 16 to 18 hours, at which time 150 μL of the manufacturer's enhancer 1 and 1.5 ml of enhancer 2 were added, and the cells were cultured for another four days at 37°C.

[0258] Cell cultures were harvested and centrifuged at 2500 rpm for 20-30 min and filtered through a 0.2 μm high flow filter (Sartorius AG, Göttingen, Germany) prior to purification. The supernatant was filtered by SDS-PAGE to confirm the presence of the expected antibody in the supernatant.

[0259] Protein A affinity purification of mAbs

[0260] mAbs were purified from supernatants of transfected Expi293F cells by protein A affinity chromatography. TM A Protein A high performance column (GE Healthcare Life Sciences) was washed and equilibrated in binding buffer (20 mM NaH2PO4 pH 7.0) and loaded with cell culture supernatant, the column was washed with binding buffer to a baseline OD 280 nm, and the bound antibody was eluted with 0.1 M sodium citrate tribasic dihydrate (pH 3.5), and 1 ml fractions were collected and immediately neutralized with 1 M Tris-HCl pH 9.0, and the antibody containing fractions were pooled.

[0261] Size Exclusion Chromatography (SEC) of mAbs

[0262] After protein A affinity purification, size exclusion chromatography (SEC) is used to further purify and characterize antibody. Using 30kDa molecular weight cut-off centrifugal concentrator device (Merck Millipore) the antibody of protein A purification is concentrated to OD280nm is 6-8. In PBS pH 7.2, Superose 6 10 / 300GL posts (GE Healthcare Life Sciences) are balanced, and then the antibody of concentrated protein A affinity purification is loaded and separated with a flow rate of 0.5 ml / min in PBS, and 0.5ml fraction is collected. For some mAb, SEC is carried out at pH 5.0, so the mAb of concentrated protein A purification is dialyzed overnight in buffer (100mM sodium citrate, 100mM NaCl, pH 5.0), then applied to the Superose 610 / 300 post pre-equilibrated in the same buffer. The antibody through dialyzation is applied to Superose post SEC with a flow rate of 0.5 ml / min, and 0.5ml fraction is collected from post.

[0263] Assessment of antigen binding by monoclonal antibodies

[0264] Prior to functional analysis, purified antibodies were tested for antigen binding. 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. Anti-CD20 mAbs based on rituximab or 11B8 and anti-CD38 mAbs based on daratumumab were tested for binding to Ramos lymphoma cells expressing CD20 and CD38. Anti-HER2 mAbs based on trastuzumab and pertuzumab mAbs were tested on the HER2-expressing ovarian cancer cell line SK-OV-3. Briefly, mAbs were titrated by serial two-fold dilutions in 25 μl of FACS buffer (PBS containing 0.5% (w / v) BSA). Twenty-five microliters of a concentration of 5x10 6Target cells at 400 μL / ml were added to the titrated mAb and incubated on ice for 30 minutes. The cells were then washed twice in FACS buffer, resuspended with 50 μL of anti-human IgG (Fab')2-Alexa647 conjugate and incubated on ice for 30 minutes, washed twice in cold FACS buffer, and then resuspended in 200 μL FACS buffer. BD FACSCanto TM II flow cytometer to analyze cells by flow cytometry.

[0265] Flow cytometric detection of C1q binding to cells opsonized with monoclonal antibodies

[0266] Binding of C1q to antibody-opsonized cells was assessed by flow cytometry. Ramos lymphoma cells or SK-OV-3 adenocarcinoma cells (5x10 6 / ml in FACS buffer) were incubated on ice for 30 minutes with serially diluted antibodies based on anti-CD20 rituximab or based on anti-HER2 trastuzumab. The 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 minutes. The cells were then washed twice in cold FACS buffer, and bound C1q was detected by resuspending the cells in rabbit antiserum (1:500 dilution) for detecting human C1q and incubating on ice for another 30 minutes. The cells were then washed twice in cold FACS buffer, resuspended in phycoerythrin-conjugated donkey antiserum for detecting rabbit antibodies for 30 minutes, washed twice more and resuspended in 200 μL ice-cold FACS buffer, and then incubated on a BD FACSCanto TM The cells were analyzed on a flow cytometer II (Bedi Medical).

[0267] Complement-dependent lysis of cells

[0268] After conditioning the target cells with mAb, CDC was measured by flow cytometry using a Zombie Green fixable viability kit (Baijin Bio). 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). Therefore, for each WT mAb or modified mAb (i.e., H429-mutant mAb) or combination thereof, 25 μl was then diluted to a concentration of 5x10 in 25 μl of PBS / BSA / G. 6 / ml of target cells were added to the titrated mAb and incubated on ice for 30 minutes. The cells were then washed twice in buffer and resuspended in 50 μl of complement (normal human serum (NHS) was thawed immediately before use and diluted 1:3 in buffer) and incubated at 37°C for 30 minutes. After incubation, the cells were washed twice in cold buffer without BSA and then resuspended in 50 μl Zombie Green diluted 1 / 500 in PBS (prepared in DMSO according to the manufacturer's instructions; Baijin Bio) and incubated on ice in the dark for another 30 minutes. The cells were then washed once in buffer, resuspended in 50 μl of 2% paraformaldehyde / BSA / PBS / G, and incubated on ice for 30 minutes, then washed once in PBS / BSA and resuspended in 200 μl PBS / BSA / G. BD FACSCanto TM Cells were analyzed by flow cytometry using a Flow Cytometer II (Bedi Medical).

[0269] mAbs collaborate to enhance C1q binding and CDC

[0270] Ramos cells were cultured at 5×10 in 25 μl of PBS / BSA / G in the presence of 0.025 μg / ml daratumumab-WT or rituximab-H429F. 5 Cells / ml were incubated with two-fold dilutions of rituximab-WT, two-fold dilutions of 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 minutes, washed twice with PBS, incubated with 1 / 500 Zombie Green on ice for 30 minutes, washed again with PBS / BSA / G and fixed with 2% paraformaldehyde. Cells were washed, resuspended in PBS / BSA / G and fixed on a BD FACSCanto TM The cells were analyzed on a Flow Cytometer II (Bedi Medical).

[0271] Results and discussion

[0272] pass Antigen binding assay by monoclonal antibodies

[0273] Prior to functional analysis, the purified antibodies used in this example were tested and confirmed for their antigen binding ability.

[0274] Purified mAbs detecting cell surface antigens were also tested by flow cytometry on antigen-positive Ramos cells (CD20 + CD38 +) or SK-OV-3 cells (HER2 + ) binding, and all mAbs showed readily detectable levels of antigen binding ( Fig.14 In addition, within each group, modified mAbs carrying mutations in the IgG heavy chain showed binding activity similar to that of the unmodified (WT) form (e.g., anti-CD20 mAb Rituximab-WT and 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-CD2011B8-WT and its CH3-modified mutants all gave binding curves equivalent to their corresponding wild-type forms ( Fig.14 Likewise, the anti-HER2 mAb Trastuzumab-WT and its modified CH3 mutant forms Trastuzumab-H429F and Trastuzumab-H429Y showed readily detectable and equivalent binding to SK-OV-3 adenocarcinoma cells as Pertuzumab-WT and its modified CH3 mutants ( Fig.14 ).

[0275] The effect of modification at position 429 on antibody properties was further studied using mAbs that bind to cell surface molecules. Specifically, it was found that the substitution of H429 changed the physical properties of the antibody and selectively promoted antibody oligomerization. For example, mutants of the rituximab antibody were used; that is, rituximab-H429F mAb and rituximab-H429Y mAb, in which the incorporated tyrosine and phenylalanine amino acids were structurally similar, but it was found that although both rituximab-H429F mAb and rituximab H429Y mAb were eluted from the protein A affinity column as a single homogenous peak, their elution characteristics were similar to those of the unmodified rituximab-WT IgG ( Fig.15 A), but purified by size exclusion chromatography (SEC) at pH 7.2 ( Fig.16 A) shows that Rituximab-H429Y mAb IgG exhibits a surprisingly different SEC profile (i.e., compared to the profiles of Rituximab-WT and Rituximab-H429F antibodies). That is, the Rituximab-H429Y mAb SEC profile reveals two distinct peaks, one IgG peak ( Fig.16 to the right of the vertical line in A), which is consistent with a single IgG peak in SEC for both rituximab-WT and rituximab-H429F mAbs, and a second peak believed to contain preformed oligomers (IgG(oli)) of rituximab-H429Y (see Fig.16A, left side of vertical line), and indicates the balance between the formation of oligomeric and non-oligomeric forms. Although the presence of both oligomeric IgG 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 that observed in Rituximab-H429F mAb. Therefore, the different biophysical properties of Rituximab-H429Y and Rituximab-H429F mAbs are determined solely by the presence of a hydroxyl group in the tyrosine at position 429 of Rituximab-H429Y mAb.

[0276] SDS-PAGE revealed that oligomerization (IgG oli ) and non-oligomeric (IgG) forms behaved identically to each other and to rituximab-WT IgG and rituximab-H429F mAb IgG. That is, although rituximab-H429Y IgG oli It is oligomeric in nature, but under non-reducing conditions it migrates as a single IgG species of approximately 150 kDa identical to its non-oligomeric rituximab-H429Y IgG (H2L2), importantly indicating that the oligomerization of the mAb promoted by the H429Y mutation is non-covalent in nature ( Fig.17 A). In addition, this clearly demonstrates that rituximab-H429Y can be synthesized as preformed oligomers of IgG (IgG, H2L2) and IgG (IgG oli ) are present, which is caused by 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 and non-oligomeric rituximab-H429Y IgG resolved into the expected approximately 50 kDa heavy (H) chain and approximately 25 kDa light (L) chain species with the same SDS-PAGE characteristics as rituximab-WT and rituximab-H429F mutants.

[0277] Thus, the H429Y modification imparts a new characteristic to rituximab, in which the mAb exists as a preformed IgG non-covalent oligomer in equilibrium with a single IgG molecule in solution at pH 7.2. In contrast, rituximab-H429F and rituximab-WT mAbs exist only as a single IgG species in solution. Thus, the choice of amino acid at position H429 unpredictably affects the physical properties of rituximab IgG.

[0278] These effects of mutations at position H429 were also found to be independent of the V domain. That is, an equivalent evaluation of H429 substitutions in the unrelated mAb trastuzumab yielded similar results for detection of a cell surface molecule structurally distinct from CD20 (i.e., HER2) (see Fig.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 ( Fig.15 B), as was the case for the elution of trastuzumab-H429F and trastuzumab-H429Y mutant mAbs (the single IgG peak was consistent with the peak obtained for the unmodified trastuzumab-WT IgG mAb). Then, the elution was performed by size exclusion chromatography (SEC) at pH 7.2 ( Fig.16 B), it was found that the unmodified trastuzumab-WT and trastuzumab-H429F mAb each contained the expected single major IgG species, while in contrast, trastuzumab-H429Y mAb showed two distinct peaks ( Fig.16 B). The first trastuzumab-H429Y IgG peak is consistent with the single IgG peak observed for trastuzumab-WT mAb and trastuzumab-H429F mAb ( Fig.16 B, right side of vertical line), and the second trastuzumab-H429Y IgG peak contains oligomeric IgG (IgG oli )( Fig.16 B, left side of vertical line). In addition, SDS-PAGE ( Fig.17 B) shows that under non-reducing conditions (i.e., without disulfide bond reduction), the IgG peaks of both trastuzumab-WT and trastuzumab-H429F mAb each migrated at the expected mass of 150 kDa. Also, in the case of trastuzumab-H429Y mAb, SDS-PAGE ( Fig.17 B) shows that under non-reducing conditions, oligomeric trastuzumab-H429Y (IgG oli ) and non-oligomeric (IgG H2L2) species of trastuzumab-H429Y IgG migrated identically as a single 150 kDa species. This clearly demonstrated 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 associations between IgG heavy chains carrying the H429Y mutation.

[0279] Thus, the amino acid substitution at position 429 in the trastuzumab heavy chain confers the same properties as observed for the equivalent substitution in the rituximab-based mAb, and thus, the effects promoted by the H249Y mutation on the physical properties of the mutated mAb, particularly oligomerization, are independent of antibody specificity, molecular target, epitope, and V H and V L Structural domain.

[0280] Preformed oligomeric and non-oligomeric H429Y IgG antibodies exhibit enhanced CDC 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 cytotoxicity (CDC) phagocytosis or killing of target cells). Such oligomers / hexamers can be formed in solution or on the target (i.e., "on-target" oligomerization or assembly).

[0281] The effect of the H429Y modification of the IgG heavy chain CH3 domain on antibody effector function was assessed by complement-dependent cytotoxicity assay (CDC) (see Fig.18 ). For example, using CD20-positive Ramos lymphoma cells as target cells, the effect of H429 mutation of oligomeric and non-oligomeric forms of rituximab-H429Y on CDC potency was determined. Therefore, both oligomeric rituximab-H429Y (IgG(oli)) and non-oligomeric IgG (H2L2) forms separated by SEC at pH 7.2 ( Fig.18 The CDC potency of the cells was evaluated and compared with that of unmodified rituximab-WT IgG. Fig.18 A) or oligomeric rituximab-H429Y IgG H2L2 species ( Fig.18 Ramos cells were incubated with serial dilutions of p2 fractions of A. Normal human serum (diluted 1 / 3) 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 ( Fig.18 B). Non-oligomeric rituximab-H429Y IgG (H2L2) (P1 fraction) showed a surprising enhancement in CDC potency compared to unmodified rituximab-WT. oli) also showed enhancement of CDC. Interestingly, both the oligomeric p2 form and the non-oligomeric p1 form showed similar enhanced CDC efficacy, which is consistent with the H429Y oligomer forming hexamers in solution at pH 7.2 before binding to target cells, or in the case of the non-oligomeric form of IgG (H2L2), forming hexamers on the target cell surface after antigen binding and thereby providing the optimal Fc configuration for C1q binding and thus enhancing complement activation, as observed in the mAb-H429Y form of IgG.

[0282] Therefore, substitution of H429 with tyrosine not only alters the physical properties of the mAb allowing oligomerization in solution, but also enhances complement-dependent killing of target cells, regardless of whether the IgG oligomerizes in solution or on the target.

[0283] Oligomerization of mAbs containing the H429Y mutation is controlled by changing the pH

[0284] The nature of the non-covalent oligomerization of mAbs containing the H429Y modification in the CH3 domain was further investigated by varying the pH of the buffer (environment). Fig.18 C). Specifically, the trastuzumab-H429Y mutant mAb (as purified by protein A affinity chromatography) was dialyzed into a pH 7.2 buffer and then also subjected to SEC at pH 7.2, or dialyzed into a pH 5.0 citrate buffer and then subjected to SEC at pH 5.0. At pH 7.2, both oligomeric and non-oligomeric IgG were present, but lowering the pH of the buffer completely reversed the formation of IgG hexameric oligomers, and only the IgG (H2L2) species was evident ( Fig.18 C). Thus, the H429Y modification of the CH3 domain promotes pH-sensitive non-covalent oligomerization of the mAb and thereby promotes oligomerization / hexamerization in solution or on target that enhances the effector efficacy of complement. In addition, the ability to control oligomerization by changing pH may be useful for preparing immunoglobulins carrying this modification.

[0285] The H429F mutation enhances activation of the serum complement cascade

[0286] The extent to which modification of the CH3 domain at position 429 of the H chain affects C1q binding was investigated by flow cytometry on cells treated with an irrelevant mAb that recognizes CD20 or HER2 and carries the H429F mutation in the CH3 of its heavy chain. Fig.19 AD. C1q binding was assessed on CD20 Ramos cells treated with rituximab-WT mAb or rituximab-H429F mAb ( Fig.19A, B). Similarly, C1q binding was measured on HER2-positive SK-OV-3 cells treated with trastuzumab-WT mAb or trastuzumab-H429F mAb ( Fig.19 C, D). C1q and Ramos cells treated with rituximab-WT mAb ( Fig.19 A) and SK-OV-3 cells treated with trastuzumab-WT had binding detectably above background control ( Fig.19 C). Importantly, C1q was significantly upregulated in Ramos cells treated with rituximab-H429F compared with rituximab-WT and trastuzumab-WT ( Fig.19 B) and SK-OV-3 cells treated with trastuzumab-H429F ( Fig.19 D) Both binding was enhanced. Thus, substitution of H429 in the CH3 domain of the heavy chain enhanced C1q binding in these unrelated anti-CD20 and anti-HER2 mAbs. Thus, it is also clear that this functional enhancement of the H429 modification is independent of the antibody variable region and the molecular target and epitope detected. Furthermore, enhanced C1q binding is consistent with enhanced antibody-dependent complement activation in the classical complement pathway, which leads to the development of MAC and thus cell lysis via CDC.

[0287] Therefore, since 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 potency was also determined using rituximab-H429F mAb and compared with the killing potency of unmodified rituximab-WT ( Fig.19 E). Specifically, at each of the indicated mAb concentrations, the proportion of Ramos cells killed by CDC was assessed by flow cytometry ( Fig.19 E). As with the surprising enhancement of C1q binding conferred by the H429F modification of the CH3 domain in rituximab-H429F mAb ( Fig.19 A compared with 19B), CDC killing efficacy (EC 50 ) also significantly increased (>10 times) ( Fig.19 E) The striking improvement in CIq binding and CDC killing potency caused by the H429F mutation is consistent with oligomerization (particularly hexamerization) of the antibody on the target surface and appears to provide an optimal mAb Fc configuration for binding of CIq, which is itself a hexamer, as exemplified by analysis of the rituximab-H429F mAb.

[0288] Enhanced complement activation by mAbs with H429 mutation is epitope-independent

[0289] As discussed above, the H429 substitution in the anti-CD20 rituximab mAb was found to strongly increase 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 studied using a second anti-CD20 mAb. Figure 20-22 shown.

[0290] The type II anti-CD20 mAb 11B8 exhibits naturally poor CDC activity, but importantly also detects an epitope distinct from that of the type I anti-CD20 mAb rituximab (Meyer S et al., Br J Haematol 180(6):808-820, 2018). 11B8-WT mAb and its mutant containing the H429F substitution were generated as described above and purified by protein A affinity chromatography ( Fig. 20 ). A single homogenous IgG peak was obtained for 11B8-WT and 11B8-H429F mAb ( Fig. 20 ), indicating that the modifications failed to alter the purification properties of the modified mAb compared to the unmodified 11B8-WT mAb. Similarly, size exclusion chromatography demonstrated that the 11B8-WT mAb contained a single non-oligomeric IgG (H2L2) species ( Fig.21 A), and the same is true for 11B8-H429F mAb. SDS-PAGE analysis confirmed this ( Fig.21 B), the SDS-PAGE analysis revealed the expected 150 kDa IgG species before disulfide bond reduction of wild-type and mutant mAbs that resolved into approximately 50 kDa heavy chain and 25 kD light chain species after reduction in DTT ( Fig.21 B).

[0291] The CDC potency of 11B8 WT was also compared with that of the H429-modified antibody 11B8-H429F ( Fig. 22 11B8-WT and 11B8-H429F were titrated in the presence of normal human serum as a complement source, and the percentage of killing of Ramos cells by each mAb concentration was assessed. It was found that 11B8-WT mAb failed to induce significant CDC, but in contrast, Fc-modified 11B8-H429F mAb mediated efficient CDC ( Fig. 22 ). Thus, CDC potency was enhanced in two different and unrelated mAbs (rituximab-H429F and 11B8-H429F) (compare Fig.19E and 22), and showed that the increase in CDC produced by modifying the H429 position can be achieved in different mAbs targeting different epitopes within the same target molecule. This also shows that the enhanced CDC obtained by CH3 modification is also independent of the variable domains of the modified antibody.

[0292] The enhancing effect of H429F is independent of the antibody, molecular target, and cell type

[0293] The enhanced complement activation potency conferred by H429 substitution of other mAbs was evaluated in the antibody daratumumab, which recognizes a fourth and unrelated molecular target, CD38 (a cell surface molecule structurally unrelated to CD20 or HER2) (de Weers M et al., J. Immunol. 2011; 186: 1840-1848, 2011; Overdijk mB et al., MAb 7: 311-321, 2015). As described above, daratumumab-WT mAb was formatted as a human IgG1 and kappa light chain mAb. A mutant of this antibody was engineered in which the H429 residue was substituted with phenylalanine to generate daratumumab-H429F mAb. In each case, the daratumumab-based IgG mAb showed equivalent characteristics and eluted from the protein A affinity column as a single homogenous peak ( Fig.23 Further purification by SEC yielded a single IgG (H2L2) peak for daratumumab-WT mAb and an equivalent peak for daratumumab-H429F mutant mAb ( Fig.24 In addition, SDS-PAGE confirmed that the SEC-purified mAb included the expected 150 kDa IgG species (before disulfide bond reduction), as well as approximately 50 kDa heavy chain and 25 kDa light chain species after reduction in DTT ( Fig.24 B).

[0294] The CDC potency of daratumumab-H429F mAb was compared with that of daratumumab-WT mAb ( Fig.25 Each mAb was titrated individually by serial two-fold dilution in the presence of normal human serum as a complement source. For each mAb concentration, the percent killing of Ramos cells expressing CD38 was assessed and the percentage of killing was calculated as follows: Fig.25 As shown. Both daratumumab-WT and daratumumab-H429F mAb achieved about 80% killing of Ramos lymphoma cells, however, daratumumab-H429F mAb showed stronger CDC efficacy, with an EC50 of about 15 times higher. The improved CDC efficacy of daratumumab-H429F mAb was then investigated using CD38-positive KMS-12-PE myeloma cells, which are resistant to CDC ( Fig.26A). Daratumumab-WT mAb showed barely detectable CDC of KMS-12-PE cells above background control lysis (approximately 20%) in the presence of complement alone (no mAb only C′), even at a concentration (5 μg / ml) 5-fold higher than that required for maximal killing of Ramos lymphoma cells (1 μg / ml) (approximately 80% lysis; see Fig.25 ). In contrast, efficient CDC of KMS-12-PE cells was mediated by daratumumab-H429F mAb ( Fig.26 A). In further experiments, CDC efficacy was evaluated on SUP-15 acute lymphoblastic leukemia (ALL) cells expressing CD38. Fig.26 As can be seen 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, substitution at position 429 of the CH3 domain not only enhances CDC against certain targets, but also rescues effective CDC against lysis-resistant targets. In addition, the enhanced CDC efficacy against the CD38 target (which is structurally different from CD20 detected by rituximab and 11B8, and HER2 detected by trastuzumab) also indicates that the improved efficacy is independent of the target and the detected epitope, and therefore also independent of the V domain.

[0295] H429 substitution promotes functional synergy between mAbs targeting different epitopes

[0296] Functional synergy between mAbs and mediated by H429 modification was investigated by measuring the extent of C1q binding in a mixture of mAbs (trastuzumab and pertuzumab) targeting different epitopes in HER2.

[0297] ( Fig. 27 and 28 Purification profiles of Pertuzumab-WT mAb and a mutant in which H429 has been substituted with phenylalanine (i.e., Pertuzumab-H429F mAb) are shown in A). For each mAb, a single equivalent IgG peak was obtained from Protein A affinity chromatography ( Fig. 27 ), and when further purified by size exclusion chromatography, a single homogeneous non-oligomeric IgG species was observed ( Fig.28 A, right side of vertical line). SDS-PAGE analysis ( Fig.28 B) Demonstrates that these peaks contain a 150 kDa IgG (H2L2) species that resolves upon reduction into a heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa ( Fig.28 B).

[0298] Collaboration between the unrelated anti-HER2 mAbs trastuzumab-WT and pertuzumab-WT, which detect different epitopes in HER2, was determined by assessing the extent of C1q binding in the mAb mixture quantified by flow cytometry ( Fig.29 A). Ovarian cancer cells SK-OV-3 treated with equal concentrations of a mixture of HER2 mAbs containing the H429F modification, Trastuzumab-H429F or Pertuzumab-H429F, show binding comparable to that observed with the mAbs alone at the same concentrations ( Fig.29 A) Enhanced CIq binding (MFI=10,877) compared to Pertuzumab-H429F (MFI=798) or Trastuzumab-H429F (MFI=1739).

[0299] The cooperative enhancement of C1q binding was further assessed by ( Fig.29 B): Anti-HER2 mAbs alone or paired mixtures of said mAbs were titrated in two-fold serial dilutions starting at 5 μg / ml (for each mAb alone) or 2.5 μg / ml:2.5 μg / ml (for each mAb in the mixture in a 1:1 ratio mixture of mAbs) Fig.29 B). Trastuzumab-H429F and pertuzumab-H429F act synergistically to further enhance complement activation, especially when the concentration of mAb is limiting (e.g., 1.25 μg / ml; Fig.29 B is indicated by an arrow). The C1q binding of a mixture of 1.25 μg / ml Trastuzumab-H429F and 1.25 μg / ml Pertuzumab-H429F (MFI=6327) was greater than the C1q binding 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 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 the wild-type mAb used alone (i.e., Trastuzumab-WT 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). MFI = 258, or 405 at 2.5 μg / ml, and Pertuzumab-WT MFI = 424 at 1.25 μg / ml, or MFI = 483 at 2.5 μg / ml). This increased complement activation potency further suggests that greater functional potency can be achieved through cooperative synergy of mixtures of CH3 domain-modified mAbs recognizing different epitopes.

[0300] Modification of the CH3 domain enables mAbs with different specificities to synergize in CDC independently of the target

[0301] 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, the effects of enhanced CDC cleavage of the target, such as enhanced C1q binding ( Fig.29 ) reflects the synergistic effect of mAbs with greater functional efficacy. Fig.30 shown.

[0302] The cooperation in CDC between antibodies detecting different molecular structures and different epitopes was determined using a mixture of pairwise combinations of rituximab-WT or rituximab-H429F mAb targeting CD20 and daratumumab-WT or daratumumab-H429F mAb targeting an unrelated surface molecule CD38. When rituximab-WT mAb was titrated in the presence of 0.25 μg / ml daratumumab-WT mAb, no cooperation was evident in CDC ( Fig.30 A); that is, the CDC observed at any concentration of rituximab-WT was no greater than that observed in the presence of 0.25 μg / ml daratumumab-WT alone (0 μg / ml rituximab, Fig.30 A) or baseline CDC in the presence of any concentration of rituximab-WT alone (i.e., titrated in the absence of daratumumab-WT). However, in contrast to the unmodified wild-type mAb, rituximab-H429F and daratumumab-H429F mAbs acted to enhance CDC cell killing ( Fig.30 B). Specifically, when used with daratumumab-H429F mAb at 0.025 μg / ml, cooperation between mAbs in mediating CDC was readily observed over a concentration range of rituximab-H429F from 0.5 μg / ml to 0.125 μg / ml (note: enhancement by Fig.30 Indicated by the upward arrow in B). The CDC mediated by rituximab-H429F mAb and daratumumab-H429F mAb was significantly greater than that of H429F mAb alone (rituximab-H429F or 0.025 μg / ml daratumumab; enhancement by Fig.30 Indicated by the upward arrow in B).

[0303] Functional synergy between mAbs detecting different epitopes but within the same target molecule was investigated in a mixture of different CD20 mAbs. Fig.30C. Titration of rituximab-H429F mAb in the presence (0.5 μg / ml) or absence of 11B8-H429F mAb revealed that the mAbs had synergistic effects. When the concentrations of both mAbs were limiting (0.25 μg / ml-0.031 μg / ml rituximab-H429F and 0.05 μg / ml 11B8-H429F), cooperation between the mAbs was easily detected, with CDC greater than that of the individual mAbs ( Fig.30 enhancement indicated by the upward arrow in C).

[0304] Thus, mAb cooperation and synergy by modifying the antibody H chain at position 429 (specifically H429F) has a broad effect. Regardless of the epitope detected by the individual mAbs (i.e., whether the epitope is present on the same or different molecular targets), it promotes greater functional efficacy through cooperation and functional synergy in the mixture of mAbs.

[0305] Example 4 Killing of B lymphocytes by anti-CD20 therapeutic antibodies with H429 mutation

[0306] Monoclonal antibodies are used to treat inflammatory diseases, such as autoimmune diseases, by targeting normal (i.e., non-malignant) cells. For example, the anti-CD20 mAb rituximab is used to treat inflammatory diseases by targeting normal B lymphocytes known to express CD20 (Lee DSW et al., Nature Reviews: Drug Discovery 20:179-199, 2021).

[0307] Methods and Materials

[0308] Isolation of leukocytes from human peripheral blood

[0309] Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated venous blood (Vacutainer ACD-A Bedi Medical) by centrifugation on a Ficoll gradient. 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 5×10 6 / ml concentration.

[0310] Complement-dependent lysis of cells

[0311] After opsonization of 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 Fixable Viability Kit (Bio-Gene).

[0312] CDC was performed in 96-well plates. Cells (25 μl, 5×10 6 / ml) was reacted with an equal volume of mAb in L15-BSA on ice for 30 minutes, then washed in L15-BSA (diluted in 100-200 μl of buffer) and centrifuged (200xg, 5 minutes, 4°C) twice. The IgG-opsonized 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 minutes. The treated cells were then washed once in L15-BSA, resuspended in 50 μl of anti-CD19-APC antibody (Baijin Bio) in L15-BSA, and incubated on ice for another 30 minutes. After washing twice in L-15 lacking BSA, the cells were resuspended in 50 μl of Zombie Green (1 / 500 dilution in protein-free L-15 medium or PBS according to the manufacturer's instructions) and incubated on ice for 30 minutes. Cells were washed once in L15-BSA and fixed by resuspending in 2% paraformaldehyde buffer on ice for 30 min and washed once more and then resuspended in 200 μl L15-BSA for flow cytometric analysis. B lymphocytes were identified by staining for CD19 and were positive for CD19 in PBMC samples treated with negative control mAb trastuzumab-WT or trastuzumab-H429F antibody. + Zombie Green + Compared with the background control of cells, the proportion (%) of dead B lymphocytes that were specifically killed by CDC was counted as CD19 + Zombie Green + The percentage of cells.

[0313] Results and discussion

[0314] CDC killing of normal B lymphocytes by rituximab-H429F was studied by flow cytometry and compared with that of unmodified rituximab-WT ( Fig.31 ). Compared with killing only 30% of peripheral blood B lymphocytes ( Fig.31 A) The killing of unmodified rituximab-WT was much lower compared to that of rituximab-H429F, which showed a stronger potency, killing more than 85% of B cells ( Fig.31 B). Since HER2 is not expressed on B lymphocytes, the negative control anti-HER2 mAbs trastuzumab-WT or trastuzumab-H429F had no CDC on B lymphocytes ( Fig.31 C, D).

[0315] Example 5 H429F modification improves the function of other types of immunoglobulins

[0316] Histidine 429 is conserved among the equivalent positions in all human Ig classes and subclasses ( Figure 3 and Figure 4 ); that is, in the CH3 domains of all IgG subclasses 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 modification of H429 on the functions of other immunoglobulins were evaluated using human IgG3 and human IgG4 as examples.

[0317] Methods and Materials

[0318] Antibodies and Antibody Constructs

[0319] The mAb used in this example includes a heavy chain of the IgG3 or IgG4 subclass. The IgG3 heavy chain used herein contains three amino acid substitutions N392K, M397V and R435H, which are introduced to avoid aggregation of purified antibodies (Saito S et al., Prot Sci 28(5): doi: 10.1002 / pro, 2019).

[0320] The rituximab-IgG3 CD20-specific heavy chain polypeptide (SEQ ID NO: 38) includes, in order from N-terminus to C-terminus, the CD20-specific VH domain of rituximab fused to the CH1-hinge-CH2-CH3 domain of aggregation-resistant human IgG3 (https: / / go.drugbank.com / drugs / DB00073) and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 39. Similarly, the polypeptide of the anti-CD20 mAb rituximab light chain (SEQ ID NO: 30) as previously described includes the CD20-specific VL domain of rituximab fused to a human kappa constant domain (https: / / go.drugbank.com / drugs / DB00073) and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0321] The CD20-specific heavy chain polypeptide formatted with rituximab-IgG4-WT (SEQ ID NO: 40) includes, in order from N-terminus to C-terminus, the CD20-specific VH domain of rituximab fused to the CH1-hinge-CH2-CH3 domain of human IgG4, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 41. Similarly, the polypeptide of the anti-CD20 mAb rituximab light chain (SEQ ID NO: 30) as previously described by reference includes the CD20-specific VL domain of rituximab fused to the human kappa constant domain, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 31.

[0322] Modifications to mAb heavy chains are produced by synthesizing codon-optimized polynucleotide sequences of complete antibody chains, comprising specific modifications (GeneArt; Thermo Fisher Scientific) encoding, for example, H429F, and antibody expression vectors are produced by standard methods known to those skilled in the art. In brief, antibody expression vectors consist of synthetic polynucleotide sequences encoding unmodified antibody heavy chains or with modifications / mutations or light chains, appropriately placed in expression plasmids, for example, pcDNA3.4 (Thermo Fisher Scientific).

[0323] Expression of antibody constructs

[0324] The expression of antibodies was carried out using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions as rituximab and therapeutic mAbs described elsewhere in this specification. For all mAbs, cell cultures were harvested after transfection and centrifuged at 2500 rpm for 20-30 minutes, and the supernatant was filtered with a 0.2 μm high flow filter (Sartorius) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE electrophoresis by the method of Laemmli.

[0325] Protein A affinity purification of Mab

[0326] mAbs were purified from supernatants of transfected Expi293F cells by protein A affinity chromatography as described herein.

[0327] Size Exclusion Chromatography (SEC) Purification of mAbs

[0328] Following protein A affinity purification, size exclusion chromatography (SEC) was used to further purify and characterize the antibodies as described herein.

[0329] Assessment of antigen binding by monoclonal antibodies (mAbs)

[0330] Prior to functional analysis, purified antibodies were tested for antigen binding by flow cytometry on CD20-positive Ramos lymphoma cells.

[0331] Complement-dependent lysis of cells

[0332] After opsonization of Ramos cells with mAb, CDC mediated by mAb was measured by flow cytometry as described herein using the Zombie Green Fixable Viability Kit (Bio-Gene).

[0333] Results and discussion

[0334] The anti-CD20 mAb rituximab was formatted with unmodified aggregation-resistant IgG3 heavy chain (rituximab-IgG3), aggregation-resistant IgG3 heavy chain including the H429F mutation (rituximab-IgG3-H429F), wild-type IgG4 heavy chain (rituximab-IgG4-WT), or IgG4 heavy chain including the H429F modification (rituximab-IgG4-H429F).

[0335] Rituximab-IgG3 mAb is formatted as aggregation-resistant human IgG3 and κ light chain mAb, and the mutant of this antibody is engineered, wherein H429 residue is replaced by phenylalanine to produce rituximab-IgG3 (KVH)-H429F mAb. Rituximab-IgG4-WT mAb is also produced, and it is formatted as human IgG4 and κ light chain mAb. In each case, rituximab IgG3 and IgG4 mAb show equivalent characteristics, and are eluted from protein A affinity column as a single homogeneous peak. The further purification by SEC produces each single IgG (H2L2) peak in mAb. In addition, SDS-PAGE confirms that the mAb purified by SEC includes expected complete IgG species (before disulfide bond reduction), and about 50kDa heavy chain and 25kDa light chain species after reduction in dithiothreitol.

[0336] The CDC potency of rituximab-IgG3-H429F was evaluated by flow cytometry on Ramos cells and compared with that of rituximab-IgG3-WT ( Fig.32 A) Each mAb was titrated by serial two-fold dilutions in the presence of normal human serum as a complement source. Fig.32Under each mAb concentration shown in A, the killing percentage of the Ramos cells expressing CD20 was evaluated. Rituximab-IgG3-H429F mAb shows a stronger CDC effect than rituximab-IgG3 mAb, particularly when mAb concentration is restrictive. These results are important because the results prove that the effect of H429 modification can be successfully incorporated into antibodies containing mutations elsewhere located in the heavy chain (e.g., N392K, M397V and R435H mutations that overcome the aggregation of IgG). Further, it is known that R435H mutations can enhance the in vivo half-life of IgG3, and therefore the enhanced CDC ( Fig.32 A) Demonstration that the H429 modification can be successfully incorporated into an antibody with other mutations that affect its in vivo half-life.

[0337] The CDC potency of rituximab-IgG4-H429F was also evaluated on Ramos cells and compared with that of rituximab-IgG4-WT ( Fig.32 B). Unmodified Rituximab-IgG4-WT failed to kill Ramos cells, but surprisingly, H429-modified Rituximab-IgG4-H429F mAb mediated readily detectable CDC.

[0338] Clearly, the functional effects of the H429F modification are not limited to human IgG1 and may be more broadly applicable to other immunoglobulin classes. This is important because histidine 429 is present in all human immunoglobulins (IgG, IgA, IgD, IgE, and IgM; see Figure 3 , Figure 4 ) and are conserved at equivalent positions in immunoglobulins of other mammals (e.g., primates).

[0339] Example 6 Antibodies with H429 mutations also rely on complement to kill CDC

[0340] CDC produced by mutant antibodies depends on both antibodies and complement

[0341] Ramos cells were opsonized with mAbs targeting different molecular targets, CD20 and CD38, or targeting epitopes within the same molecular target (CD20). CDC of Ramos cells using daratumumab-H429F or 11B8-H429F was evaluated in the presence of complement (mAb and human complement), in the presence of mAb but not complement (mAb alone), or in the absence of mAb but in the presence of complement (complement alone or C′ alone) ( Fig.33. Only when both mAb and complement are present, CDC lysis of Ramos cells is evident. Cells opsonized with mAb alone (i.e., in the absence of complement) show little lysis under CDC assay conditions. Similarly, unopsonized cells show little lysis when incubated with complement alone. Therefore, lysis of opsonized cells in CDC assays depends on both antibody and complement.

[0342] Example 7 H429 modification confers enhanced CDC to monoclonal antibodies

[0343] Isaatuximab 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). Isaatuximab inhibits the ADP-ribosyl cyclase activity of the CD38 molecule and detects an epitope different from daratumumab that inhibits the enzyme activity poorly (Deckert J et al., 2014 supra). The effect of H429 modification on the CDC activity of functionally different isatuximab on cells resistant to CDC killing was evaluated.

[0344] Methods and Materials

[0345] Antibodies and Antibody Constructs

[0346] The isatuximab-WT mAb used in this example includes a CD38-specific heavy chain polypeptide having an amino acid sequence shown as SEQ ID NO: 42, which includes, in order from N-terminal to C-terminal, the CD38-specific VH domain of the isatuximab mAb fused to the CH1-hinge-CH2-CH3 domain of human IgG1, and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 43. The polypeptide of the anti-CD38 mAb isatuximab light chain includes an amino acid sequence shown as SEQ ID NO: 44 (including the CD38-specific VL domain of the isatuximab mAb fused to a human kappa constant domain), and is encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 45.

[0347] Synthesis of unmodified and mutated heavy chains

[0348] Antibody expression vectors are produced by standard methods known to those skilled in the art. Briefly, antibody expression vectors consist of synthetic polynucleotide sequences encoding antibody heavy or light chains, appropriately placed in plasmids, such as pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors with antibodies of different specificities are generated by adding a variable domain (V H or V L ) borders. Newly synthesized DNA encoding the new V domain and flanked by sequences homologous to the cleaved vector (e.g., 25 nucleotides) is then incorporated by reaction with NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0349] Fc variants were generated by synthesizing synthetic polynucleotide sequences encoding the variants or by cleaving the Fc coding sequence of an antibody expression plasmid with appropriate restriction enzymes and incorporating the newly mutagenized synthetic DNA by performing a reaction using NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0350] Expression of antibody constructs

[0351] The expression of antibodies was carried out using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions as described above for the therapeutic mAbs described elsewhere in this specification. For all mAbs, cell cultures were harvested after transfection and centrifuged at 2500 rpm for 20-30 minutes, and the supernatant was filtered with a 0.2 μm high flow filter (Sartorius) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0352] Purification of mAbs and assessment of antigen binding

[0353] As described herein, mAbs were purified from the supernatant of transfected Expi293F cells by protein A affinity chromatography. After protein A affinity purification, the antibodies were further purified and characterized as described herein using size exclusion chromatography (SEC). The purified antibodies were then tested for antigen binding on CD38-positive SUP-15 acute lymphoblastic leukemia (ALL) cells by flow cytometry prior to functional analysis.

[0354] Complement-dependent lysis of cells

[0355] After opsonization of SUP-15 acute lymphoblastic leukemia (ALL) cells with mAbs, CDC mediated by mAbs was measured by flow cytometry as described herein using the Zombie Green Fixable Viability Kit (Bio-Gene).

[0356] Results and discussion

[0357] As described above, aisatuximab-WT mAb is formatted as human IgG1 and κ light chain mAb.The mutant of this antibody is engineered, and wherein H429 residue is replaced by phenylalanine, to produce aisatuximab-H429F mAb.In each case, the mAb based on aisatuximab shows equivalent features, and is eluted from protein A affinity column as a single homogeneous peak.The further purification by SEC also produces each single IgG (H2L2) peak in mAb.In addition, SDS-PAGE confirms that the mAb purified by SEC includes expected complete IgG species (before disulfide bond reduction), and about 50kDa heavy chain and 25kDa light chain species after reduction in dithiothreitol.

[0358] The CDC potency of isatuximab-H429F mAb was compared with that of isatuximab-WT mAb ( Fig.34 ). Each mAb was titrated individually by serial two-fold dilution in the presence of normal human serum as a complement source. The CDC efficacy percentage (% killing) was assessed on SUP-15 acute lymphoblastic leukemia (ALL) cells expressing CD38. Fig.34 As can be seen in the figure, SUP-15 cells resist CDC killing by isatuximab-WT mAb, but are easily killed by isatuximab-H429F mutant mAb. Therefore, substitution at position 429 of the CH3 domain not only enhances CDC against certain cellular targets, but also confers effective CDC against targets resistant to cleavage. Importantly, the results reveal that cleavage of resistant targets can be achieved by H429 modification of an unrelated mAb that also detects a different epitope - isatuximab-H429F ( Fig.34 ) and daratumumab-H429F ( Fig.26 Additionally, the results also suggest that mAbs with other functional attributes, such as the ability to inhibit the function of a target molecule, in this case the ability of isatuximab to inhibit the ADP-ribosyl cyclase action of CD38, may be endowed with improved CDC efficacy.

[0359] Example 8 Generation of anti-death receptor antibodies with H429 mutation

[0360] Methods and Materials

[0361] The mAbs used in this example are listed below in Table 3. The mAbs were designated BDR5 and TDR5 and detect two different epitopes of human death receptor 5 (DR5; Overdijk MB et al., Mol Cancer Therapy 19:2126-2138, 2020) and were produced in H2L2 format with heavy chains of human IgG1, IgG2, or IgA2 subclasses and their appropriate light chains of human κ class.

[0362] Table 3 DR5-specific mAbs detecting different epitopes

[0363]

[0364]

[0365] Generation of DR5-specific antibody constructs with IgG heavy chains

[0366] Immunoglobulin variable region sequences and constant region sequences were assembled into complete synthetic DNA molecules by standard molecular biology techniques to encode complete immunoglobulin H and L chains. These molecules were used in the expression vector pcDNA 3.4 (Thermo Fisher Scientific).

[0367] 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 DR5-specific mAbs described in U.S. Pat. No. 10,882,913; the entire disclosure of which is incorporated herein by reference). The mAb was produced in the H2L2 format as a monoclonal IgG antibody.

[0368] The unmodified DR5-specific mAbs produced comprise the specific VH and VL domains of the mAbs indicated in Table 3 and are referred to as wild-type (WT) forms; for example, the "wild-type" BDR5-1 WT mAb antibody used in this example comprises a DR5-specific heavy chain and a light chain, wherein the DR5-specific heavy chain of the IgG1 subclass comprises the previously described HC-hDR5-01-G56T sequence, but lacks the C-terminal lysine (see SEQ ID NO: 36 of U.S. Pat. No. 10,882,913). The DR5-specific kappa light chain comprises the previously described polypeptide sequence of LC-hDR5-01 (see SEQ ID NO: 39 of U.S. Pat. No. 10,882,913). In other cases, an unmodified antibody is produced by fusing the VH domain of a DR5-specific antibody to a wild-type heavy (H) chain constant domain of a particular immunoglobulin subclass or class; for example, the unmodified H chain of the BDR5-2WT mAb includes a previously described DR5-specific VH domain polypeptide sequence of the HC-hDR5-01-G56T sequence fused to a constant domain of a wild-type IgG2 H chain lacking a C-terminal lysine (i.e., the VH domain of SEQ ID NO: 36 from U.S. Pat. No. 10,882,913). The DR5-specific kappa light chain of the BDR5-2WT mAb includes a previously described polypeptide sequence of LC-hDR5-01 (i.e., the LC of SEQ ID NO: 39 from U.S. Pat. No. 10,882,913). More specifically, the BDR5-1WT antibody of the IgG1 subclass used in this example includes the previously described DR5-specific heavy chain polypeptide sequence of HC-hDR5-01-G56T (i.e., SEQ ID NO: 36 from U.S. Pat. No. 10,882,913), but lacks the lysine residue at the C-terminus of the H chain, the polypeptide sequence of which is provided herein as SEQ ID NO: 46, and is encoded by a codon-optimized DNA sequence (SEQ ID NO: 47). The DR5-specific kappa light chain of the BDR5-1WT mAb was previously described as LC-hDR5-01 in SEQ ID NO: 39 of U.S. Pat. No. 10,882,913, and the polypeptide sequence herein is provided as SEQ ID NO: 48, and is encoded by a codon-optimized DNA having a nucleotide sequence shown as SEQ ID NO: 49.

[0369] Separately, the DR5-specific BDR5-2WT antibody was formatted as a human IgG2 antibody subclass (which included, in order from N-terminus to C-terminus, a DR5-specific VH domain from the previously described HC-hDR5-01-G56T sequence (i.e., SEQ ID NO: 36 from U.S. Pat. No. 10,882,913) fused to a CH1-hinge-CH2-CH3 domain of a human IgG2 subclass H chain lacking a lysine residue at the C-terminus of the H chain (from the polypeptide sequence SEQ ID NO: 50 as provided herein) and encoded by a codon-optimized DNA sequence (SEQ ID NO: 51). The polypeptide of the DR5-specific kappa light chain of mAb BDR5-2WT was previously described as LC-hDR5-01 (i.e., SEQ ID NO: 39 of U.S. Pat. No. 10,882,913) and included the polypeptide sequence SEQ ID NO: 48 as provided herein and encoded by a codon-optimized DNA sequence of SEQ ID NO: 49. Additionally, a DR5-specific antibody called TDR5 was generated that detects an epitope different from that of the BDR5 mAb, specifically a TDR5 mAb based on the VH domain of the DR5-specific VH domain of the previously described HC-hDR5-05 polypeptide sequence (see SEQ ID NO: 40 of U.S. Patent No. 10,882,913) and was generated with a DR5-specific kappa light chain having a polypeptide sequence as previously described, such as LC-hDR5-05 (see SEQ ID NO: 43 of U.S. Patent No. 10,882,913). More specifically, the TDR5-1 WT antibody of the IgG1 subclass used in this example includes the previously described DR5-specific heavy chain polypeptide sequence of HC-hDR5-05 (i.e., SEQ ID NO: 40 from U.S. Pat. No. 10,882,913), but lacks a lysine residue at the C-terminus of the H chain, the polypeptide sequence of which is provided herein as SEQ ID NO: 52, and is encoded by a codon-optimized DNA sequence having a sequence provided as SEQ ID NO: 53. The polypeptide sequence of the DR5-specific kappa light chain of the TDR5-1 WT antibody is as previously described as LC-hDR5-05 (i.e., SEQ ID NO: 43 of U.S. Pat. No. 10,882,913), and is provided as SEQ ID NO: 54, and is encoded by a codon-optimized DNA sequence (SEQ ID NO: 55).

[0370] Separately, the TDR5-2WT antibody was formatted as a human IgG2 antibody (which included, in order from N-terminus to C-terminus, the DR5-specific VH domain from the previously described HC-hDR5-05 sequence (i.e., SEQ ID NO: 40 from U.S. Pat. No. 10,882,913) fused to the CH1-hinge-CH2-CH3 domain of the human IgG2 subclass lacking a lysine residue at the C-terminus of the H chain (see the polypeptide sequence provided as SEQ ID NO: 56)), and was encoded by a codon-optimized DNA sequence (SEQ ID NO: 57). The DR5-specific kappa light chain polypeptide of mAb TDR5-2WT was as previously described in (i.e., SEQ ID NO: 43 of U.S. Pat. No. 10,882,913) as LC-hDR5-05, and included the DR5-specific kappa light chain of the TDR5-1WT antibody (SEQ ID NO: 54), and was encoded by a codon-optimized DNA sequence (SEQ ID NO: 55).

[0371] Generation of DR5-specific antibody constructs with IgA H chain

[0372] DNA sequences corresponding to the immunoglobulin variable region sequences and constant region sequences of DR5 mAb were assembled into complete synthetic nucleotide sequences by standard methods well known to those skilled in the art to encode complete immunoglobulin H chain and immunoglobulin L chain. These sequences were used in expression vectors such as pcDNA 3.4 (Thermo Fisher Scientific).

[0373] The unmodified mAb used in this example was based on the VH and VL chain domains of a DR5-specific mAb described in US Pat. No. 10,882,913 and formatted on a human IgA heavy chain and a human kappa light chain (Table 3).

[0374] Unmodified IgA mAbs comprised the specific VH and VL domains of the indicated mAbs (Table 3) and were referred to as wild-type (WT) forms; for example, the "wild-type" BDR5-A2WT mAb used in this example included a DR5-specific VH domain polypeptide sequence from within the previously described HC-hDR5-01-G56T sequence (SEQ ID NO: 36 of U.S. Pat. No. 10,882,913), and a DR5-specific kappa light chain comprising the polypeptide sequence of the previously described LC-hDR5-01 sequence (i.e., SEQ ID NO: 39 of U.S. Pat. No. 10,882,913). More specifically, the BDR5-A2WT mAb used in this example includes a DR5-specific heavy chain polypeptide (whose amino acid sequence is provided as SEQ ID NO: 58) (which includes, in order from N-terminus to C-terminus, a previously described DR5-specific VH domain of the HC-hDR5-01-G56T sequence fused to the CH1-hinge-CH2-CH3 domain of the human IgA2 subclass (i.e., the VH portion of SEQ ID NO: 36 of U.S. Patent No. 10,882,913)) and is encoded by a codon-optimized DNA (SEQ ID NO: 59). The polypeptide of the DR5-specific kappa light chain of the BDR5-A2WT mAb is as previously described as LC-hDR5-01 (i.e., SEQ ID NO: 39 of U.S. Pat. No. 10,882,913), and includes the DR5-specific VL domain of LC-hDR5-01 fused to a human kappa constant domain light chain polypeptide sequence (SEQ ID NO: 48), and is encoded by a codon-optimized DNA sequence (SEQ ID NO: 49). Additional DR5-specific antibodies formatted as IgA mAbs were generated. Specifically, the IgA WT mAb, TDR5-A2WT, was generated based on the VH and VL domains of the DR5-specific HC-hDR5-05 sequence described in U.S. Pat. No. 10,882,913. More specifically, the TDR5-A2WT antibody used in this example includes a DR5-specific heavy chain polypeptide (SEQ ID NO: 60) (which includes, in order from N-terminus to C-terminus, a DR5-specific VH domain within the previously described polypeptide sequence of HC-hDR5-05 fused to the CH1-hinge-CH2-CH3 domain of the human IgGA2 subclass (SEQ ID NO: 40 of U.S. Patent No. 10882913) and is encoded by a codon-optimized DNA sequence (SEQ ID NO: 61).The DR5-specific light chain polypeptide of the DR5-specific mAb TDR5-A2WT mAb includes the previously described polypeptide sequence of LC-hDR5-05 (SEQ ID NO:43 of U.S. Pat. No. 10,882,913), including the polypeptide sequence provided herein as SEQ ID NO:54, and is encoded by a codon-optimized DNA sequence (SEQ ID NO:55).

[0375] Generation of DR5-specific antibody constructs encoding mutated IgG and IgA heavy chains

[0376] mAbs comprising H chains carrying modifications to the sequence at position 429, wherein, for example, histidine 429 in the Fc portion of IgG1 is substituted with phenylalanine 429 (H429F) to generate BDR5-1HF mAb, are generated from synthetic nucleotide sequences encoding variants by standard methods known to those skilled in the art, and the mutations are verified by DNA sequence analysis. In the case of human IgA H chains, H436 defined according to the Bur numbering convention is equivalent to the H429 position in human IgG1 (EU numbering) and is referred to herein as H436 defined according to the Bur numbering convention. Figure 3 and Figure 4 The sequence alignment shown defines H429. The antibodies generated are as follows:

[0377] (i) BDR5-1HF, which comprises an H chain of the IgG1 subclass carrying the H429F modification and comprises the polypeptide sequence shown as SEQ ID NO: 62 encoded by a codon-optimized DNA sequence (SEQ ID NO: 63).

[0378] (ii) BDR5-2HF, which comprises an H chain of the IgG2 subclass carrying the H429F modification and comprises the polypeptide sequence as shown in SEQ ID NO: 64 encoded by a codon-optimized DNA sequence (SEQ ID NO: 65).

[0379] (iii) TDR5-1HF, which comprises an H chain of the IgG1 subclass carrying the H429F modification and comprises the polypeptide sequence as shown in SEQ ID NO: 66 encoded by a codon-optimized DNA sequence (SEQ ID NO: 67).

[0380] (iv) TDR5-2HF, which comprises an H chain of the IgG2 subclass carrying the H429F modification and comprises the polypeptide sequence as shown in SEQ ID NO: 68 encoded by a codon-optimized DNA sequence (SEQ ID NO: 69).

[0381] (v) BDR5-A2HF, which comprises an H chain of the IgA2 subclass carrying the H429F modification and comprises the polypeptide sequence as shown in SEQ ID NO: 70 encoded by a codon-optimized DNA sequence (SEQ ID NO: 71).

[0382] (vi) TDR5-A2 HF, which comprises an H chain of the IgA2 subclass carrying the H429F modification and comprises the polypeptide sequence as shown in SEQ ID NO: 72 encoded by a codon-optimized DNA sequence (SEQ ID NO: 73).

[0383] All H chain Fc variants were generated with their appropriately matched human κL chains as follows: BDR5 H chain variants were generated with human κL chains comprising the polypeptide sequence of LC-hDR5-01 as previously described in (i.e., SEQ ID NO: 39 of U.S. Pat. No. 10,882,913), including the polypeptide sequence provided as SEQ ID NO: 48, and encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 49. TDR5 H chain variants were generated with human κL chains comprising the polypeptide sequence of LC-hDR5-05 as previously described in (i.e., SEQ ID NO: 43 of U.S. Pat. No. 10,882,913), including the polypeptide sequence of SEQ ID NO: 54, and encoded by a codon-optimized DNA having the sequence shown as SEQ ID NO: 55.

[0384] Transient transfection of Expi293F cells (Thermo Fisher Scientific) was used for antibody expression. Expi293F cells were cultured in Expi293 expression medium (Life Technologies) and cells were split to 2×10 24 hours before transfection. 6 On the day of transfection, 7.5x10 7Live logarithmic phase cells were centrifuged and resuspended in 25 ml of preheated, antibiotic-free Expi293 expression medium and maintained at 37°C until transfection. The cells were then transfected at room temperature using the Expifectamine transfection kit (Life Technologies), as described below. Eighty microliters of ExpiFectamine 293 reagent were diluted with 1.5 ml Opti-MEM-I reduced serum medium (Gibco) and incubated at room temperature for 5 minutes. Thirty μg of DNA (15 μg of H-chain DNA and 15 μg of L-chain DNA) were diluted in 1.5 ml of Opti-MEM-I reduced serum medium and then added to the diluted ExpiFectamine reagent, incubated at room temperature for 20-30 minutes, and then added dropwise to the Expi293F cell suspension, which was then cultured at 37°C for 16 to 18 hours, at which time 150 μL of the manufacturer's enhancer 1 and 1.5 ml of enhancer 2 were added, and the cells were cultured for another four days at 34°C or 37°C.

[0385] The cell culture was harvested and centrifuged at 2500 rpm for 20-30 minutes and the supernatant was filtered using a 0.2 μm high flow filter (Sartorius) prior to purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0386] IgG mAb Protein A affinity purification

[0387] mAbs were purified from supernatants of transfected Expi293F cells by protein A affinity chromatography. TM Protein A high performance column (GE Healthcare Life Sciences) was washed and balanced in binding buffer (20 mM NaH2PO4, pH 7.0). Cell culture supernatant was loaded, the column was washed with binding buffer to a baseline OD280 nm, and the bound antibody was eluted with 0.1 M sodium citrate tribasic dihydrate (pH 3.5), and 1 ml fractions were collected and immediately neutralized with 1 M Tris-HCl pH 9.0. The fractions containing the antibody were combined.

[0388] Size Exclusion Chromatography (SEC) for IgG mAb

[0389] After protein A affinity purification, size exclusion chromatography (SEC) is used to further purify and characterize antibody. Using 30kDa molecular weight cut-off centrifugal concentrator device (Merck Millipore) the antibody of protein A purification is concentrated to OD280nm is 6-8. In PBS, pH 7.2, balance Superose 6 10 / 300GL post (GE Healthcare Life Sciences), then the antibody of concentrated protein A affinity purification is loaded and separated with the flow velocity of 0.5 ml / min in PBS, and 0.5ml fraction is collected. For some mAb, SEC is carried out under pH 5.0, therefore the mAb of concentrated protein A purification is dialyzed overnight in buffer (100mM sodium citrate, 100mM NaCl, pH 5.0), then applied to the Superose 610 / 300 post pre-equilibrated in the same buffer. The antibody through dialyzation is applied to Superose post SEC with the flow velocity of 0.5 ml / min, and 0.5ml fraction is collected from post.

[0390] Assessment of antigen binding by IgG or IgA mAb

[0391] When using BD Biosciences Canto TM Purified antibodies were tested for DR5 antigen binding prior to functional analysis by flow cytometry as described in Trist et al., Journal of Immunology, 192(2):792-803, 2014. DR5-specific mAbs were tested for binding to Colo205 colorectal cells, Ramos lymphoma cells, and KMS-12-PE cells. Briefly, purified mAbs or culture supernatants from mAb-producing Expi293 cells were titrated by serial two-fold dilution in 25 μl of FACS buffer (phosphate-buffered saline containing 0.5% (w / v) bovine serum albumin). Note that in the case of binding to KMS-12-PE cells, mAbs were used at a single concentration. Twenty-five microliters of a concentration of 5x10 6Target cells of 100 μg / ml were added to mAb and incubated on ice for 30 minutes. The cells were then washed twice in FACS buffer and resuspended with 50 μl of anti-human IgG (Fc)-FITC conjugate (1 / 500 dilution; Chemicon Merck, Burlington, MA, United States of America) to detect bound IgG mAb. After incubation on ice for 30 minutes, the cells were washed and resuspended in 200 μl FACS buffer for analysis. IgA mAbs were detected with 50 μl of biotin-conjugated anti-human IgA (0.5 μg / ml) (BD Pharmingen, San Diego, CA, United States of America), incubated on ice for 30 min, washed and resuspended in APC-conjugated streptavidin (1 / 500 dilution), washed twice in cold FACS buffer, and then resuspended in 200 μl FACS buffer for analysis.

[0392] Viability assay

[0393] The ability of the antibodies to induce cell death was assessed in a viability assay. The antibodies were tested alone as single agents or as a mixture of two antibodies. Viability assays were performed using cell lines routinely cultured in a medium consisting of RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum plus 4 mM glutamine and 100 U / ml penicillin and 0.1 mg / ml streptomycin.

[0394] Cells were harvested from cell culture, washed in culture medium by centrifugation at 200 x g, and resuspended in culture medium. The cells were washed again and resuspended in culture medium to 1.2 x 10 6 / ml concentration. One hundred microliters (100 μL) of cell suspension (10,000 cells) was added to the wells of a 96-well flat-bottom sterile plate. Purified mAbs were added to 100 μL of culture medium as a single mAb or mAb mixture at a single concentration, or alternatively diluted twice in a certain concentration range (10 μg / ml to 0.075 μg / ml) in culture medium, and 100 μL of each dilution was added to a 96-well plate pre-seeded with cells, while 75 μL of culture medium was added to bring the total final volume of each well to 200 μL. For mAb mixtures, the total concentration of mAb was the same as that in the equivalent wells treated with a single antibody. The plate was then incubated at 37°C for 48-72 hours in 5% CO2, and in some experiments, 10 μl of 1% SDS was added to determine maximum cell death in the last hour of incubation (i.e., at the end of the incubation period). After the incubation period, the viability of the cells was determined using a colorimetric assay using a cell counting kit 8 (CCK-8; Abcam, Cambridge, United Kingdom). This assay indicates cell viability by producing a formazan dye upon bioreduction of the tetrazolium salt WST-8 by living cells, which is proportional to the number of living cells. The WST-8 solution was diluted 1:2 with culture medium, and 40 μl was added to each well of a 96-well plate. The plates were incubated at 37°C for 2-5 hours. Color development was determined by measuring the absorbance at 450 nm (Abs450) on a Fluorostar OPTIMA (BMG LABTECH, Ortenberg, Germany).

[0395] The viability assay analysis of the IgA formatted DR5 mAb was performed as for IgG, except that the cell culture supernatant from the mAb-producing Expi293 cells was used as the source of mAb and serial dilution was performed as for the IgG mAb. In the absence of antibody, the maximum viability control value was determined from cells cultured in 200 μL of culture medium. The control for maximum death was determined from a well inoculated with only a cell suspension in 200 μL of culture medium but treated with 10 μL of 1% SDS at 37°C for 1 hour (i.e., in the last hour of the incubation period) at the end of the incubation period. After the incubation period, the viability of the cells was determined using a colorimetric assay using a cell counting kit 8 (CCK-8; Abcam). This assay indicates cell viability by producing a formazan dye when the living cells bioreduced the tetrazolium salt WST-8, which is proportional to the number of living cells. The WST-8 solution was diluted 1:2 with culture medium, and 40 μl was added to each well of a 96-well plate. The plate was incubated at 37°C for 2-5 hours. Color development was determined by measuring the absorbance at 450 nm (Abs450) on a Fluorostar OPTIMA (BMG).

[0396] result

[0397] mAb-induced apoptotic cell death

[0398] Taking DR5-specific antibody-induced apoptosis as an example, the effect of antibody Fc modification on the agonistic activity of monoclonal antibodies was evaluated. mAbs (Table 3) that recognize different epitopes on human death receptor 5 (DR5, TRAILR2) cell surface molecules were generated and their ability to induce cell death was studied, as determined in cell viability assays. mAbs BDR5 and TDR5 are directed against two different epitopes on human DR5 and are formatted as H2L2 antibodies, which include DR5-specific antibody light chains and also include DR5-specific H chains lacking C-terminal lysine, but are otherwise wild-type human IgG1 or human IgG2 or human IgA subclasses (Table 3), or otherwise include Fc-modified DR5-specific human IgG1, IgG2 and IgA H chains, wherein the histidine at position 429 of the CH3 domain of the H chain has been replaced by phenylalanine (H429F).

[0399] Quantification of cell binding by IgG-formatted DR5-specific mAb

[0400] Purified mAbs for DR5 were tested by flow cytometry with DR5-positive Colo205 colorectal cells ( Fig.35 ) or Ramos lymphoma cells ( Fig.36). All mAbs showed readily detectable levels of target antigen binding. In addition, Fc-modified mAbs carrying mutations in the IgG heavy chain showed the same binding activity as the wild-type (WT) form. For example, BDR5-1HF, an IgG1 formatted with the H429F modification, showed the same binding to Colo205 cells as the unmodified wild-type IgG1 BDR5-1WT ( Fig.35 A). DR5-specific mAbs BDR5-2HF and BDR5-2WT formatted with an IgG2 backbone ( Fig.35 B) also showed identical binding activity to each other, thus clearly demonstrating that modification of the Fc does not affect the interaction of the mAb with its target DR5 epitope. Analysis of the TDR5 mAb detecting a second epitope different from that detected by the BDR5 mAb confirmed that Fc modification at position 429 did not affect antigen binding of the modified mAb. Binding of TDR5-1WT and TDR5-1HF was identical ( Fig.35 C), the binding of IgG2 formatted forms TDR5-2WT and TDR5-2HF was also the same ( Fig.35 D). Further analysis of mAb binding to DR5 expressed on Ramos lymphoma cells showed that the mAb binding curve was not affected by the Fc mutation. IgG1 formatted mAbs BDR5-1WT and BDR5-1HF ( Fig.36 A) and IgG2 formatted BDR5-2WT and BDR5-2HF ( Fig.36 B) shows the same binding to Ramos lymphoma cells. TDR5-1WT, TDR5-1HF ( Fig.36 C) and TDR5-2WT, TDR5-2HF ( Fig.36 D) also binds equivalently. Thus, binding to DR5 is not affected by the H429F mutation. This was further confirmed by binding to KMS-12-PE myeloma cells, where binding of wild-type or H429F mAbs was identical for each mAb (e.g., binding of BDR5-1WT and BDR5-1HF was identical to binding of IgG2 mAbs BDR5-2WT and BDR5-2HF; Fig.37 ). This again confirmed that binding to the target antigen was not affected by the H429F mutation and that binding of WT and mutant mAbs was similar to that of colorectal cells, B lymphoma cells, or myeloma cells.

[0401] Evaluation of the cell-killing potency of DR5-specific mAbs including IgG H chains

[0402] The ability of DR5 mAbs to induce cell death of different lineages was determined in a colorimetric assay that measures the viability of cells cultured in the presence of DR5-specific antibodies ( Fig.38 , Fig.39 The relative potency of mAbs comprising a heavy chain carrying the H429F modification or comprising a wild-type H chain was determined.

[0403] H429F enhances DR5 mAb-induced colorectal cell killing

[0404] Colo205 colorectal cells were incubated in the presence of 20 μg / ml of DR5 mAb. Cell viability was compared to that in the absence of antibody, Abs450 nm = 2.24 ( Fig.38 Compared with the wild-type counterpart TDR5-1WT (containing the wild-type IgG1 heavy chain and having almost no ability to induce cell death, Abs450 nm = 1.99), the TDR5-1HF mAb, which includes the IgG1 H chain containing the H429F modification, is a potent inducer of Colo205 cell death, Abs450 nm = 1.37. Fig.38 )) is much more effective.

[0405] The effect of the H429F modification on a DR5 mAb comprising an IgG2 heavy chain was also determined. Cell viability was compared to that in the absence of antibody, Abs450 nm = 2.24 ( Fig.38 Compared with the control group (no Ab), TDR5-2HF mAb (20 μg / ml) containing the IgG2 heavy chain with H429F modification was a more effective inducer of Colo205 cell death, with Abs450 nm = 1.32 ( Fig.38 ), and compared with its wild-type counterpart TDR5-2WT (including wild-type IgG2 heavy chain and almost no ability to induce cell death, Abs450 nm = 1.91 ( Fig.38 )) is significantly more effective.

[0406] Thus, the H429F modification of IgG Fc increases the ability of anti-death receptor mAbs to induce cell death, resulting in reduced cell viability. This increased potency caused by the H429F modification is independent of human IgG subclasses, as TDR5-1HF and TDR5-2HF mAbs comprising H chains of different IgG subclasses show equivalent increased potency compared to wild-type equivalents. Fig.38 ).

[0407] TDR5 and BDR5 mAbs detect different epitopes on DR5. Therefore, to further determine the impact of the H429F modification on the action of anti-death receptor mAbs, Colo205 cells were incubated in a 1:1 mixture of TDR5-2HF and BDR5-2HF mAbs (10 μg / ml of each mAb, total mAb concentration of 20 μg / ml), containing an IgG2 subclass H chain ( Fig.38 Although the TDR5-2HF mAb alone showed a clear improvement in killing potency (Abs450 nm = 1.32) compared to its wild-type counterpart (Abs450 nm = 1.91), the mixture of TDR5-2HF and BDR5-2HF showed an even greater improvement in killing and indeed demonstrated almost complete cell killing, Abs450 nm = 0.1 ( Fig.38 ).

[0408] H429F enhances DR5 mAb-induced killing of lymphoma cells

[0409] After determining the killing effect of DR5 mAb on colorectal Colo205 cells, we then studied the killing effect of mAb on cells of different lineages, Ramos lymphoma cells ( Fig.39 ).

[0410] Both the TDR5-1HF mAb comprising an IgG1 H chain modified with H429F and the TDR5-2HF mAb comprising an IgG2 H chain modified with H429F were potent inducers of Ramos cell death, with cell viability in the absence of antibody, Abs450 nm = 0.65 (no mAb), TDR5-1HF-Abs450 nm = 0.39, TDR5-2HF-Abs450 nm = 0.42, and significantly more potent than their wild-type counterparts (comprising wild-type IgG1 or IgG2 H chains, and having almost no ability to induce cell death, TDR5-1WT Abs450 nm = 0.59, TDR5-2WT Abs450 nm = 0.51 ( Fig.39 )). Therefore, as observed in the killing of colorectal cells ( Fig.38), H429F modification of IgG Fc increased the ability of anti-death receptor mAb to induce cell death of lymphoma cells, which resulted in decreased cell survival, indicating that the enhanced killing efficacy was not cell type dependent. Further, the improvement in killing of lymphoma cells caused by H429F modification was independent of human IgG subclass, as TDR5-1HF and TDR5-2HF mAbs comprising H chains of different IgG subclasses (i.e., IgG1 or IgG2) showed almost equivalent enhancement of efficacy compared to their IgG1 or IgG2 wild-type equivalents ( Fig.39 ).

[0411] Since the dual epitope targeted by the cocktail including the mAb carrying the H429F modified H chain showed the greatest killing of Colo205 colorectal cells compared to killing by either mAb alone or wild-type mAb ( Fig.38 ), so the same mixture was tested for its ability to kill lymphoma cells ( Fig.39 Therefore, Ramos cells were incubated in a 1:1 mixture of TDR5-2HF and BDR5-2HF mAbs (10 μg / ml for each mAb, 20 μg / ml total mAb concentration) containing an IgG2 subclass H chain carrying the H429F modification ( Fig.39 Although the TDR5-2HF mAb alone showed a clear improvement in killing potency (Abs450 nm = 0.42) compared to its wild-type counterpart (Abs450 nm = 0.51), the mixture of TDR5-2HF and BDR5-2HF showed an even greater improvement in killing and indeed demonstrated almost complete cell killing, Abs450 nm = 0.03 ( Fig.39 ).

[0412] Effectiveness of dual epitope targeting: killing by a cocktail of DR5 mAbs matched to IgG subclasses

[0413] Paired combinations of mAbs incubated with Colo205 cells ( Fig.40) studied the effect of H429 modification on the killing potency of a mixture of mAbs directed against different epitopes. In each paired mixture, BDR5 and TDR5 mAbs were present in a 1:1 ratio, and both mAbs included the same IgG heavy chain subclass with H429F modification (both BDR5-1HF and TDR5-1HF were IgG1, or both BDR5-2HF and TDR5-2HF were IgG2), and their killing potency was compared with that of the equivalent paired mAb of the wild-type H chain (both BDR5-1WT and TDR5-1WT were IgG1, or both BDR5-2WT and TDR5-2WT were IgG2). The mixture was titrated two-fold starting from 1 μg / ml mAb (BDR5 mAb 0.5 μg / ml + TDR5 mAb 0.5 μg / ml), and the effect on Colo205 cell viability was determined after 48 hours. A mixture of BDR5-1WT and TDR5-1WT containing IgG1 wild-type chains failed to induce significant killing of Colo205 cells. In fact, cell survival in this mixture, even at the highest concentration of 1 μg / ml, was equivalent to that in the absence of antibody (no ab, Fig.40 ). Substituting the IgG subclass failed to improve efficacy, as a mixture of IgG2 BDR5-2WT and TDR5-2WT lacked killing efficacy even at 1 μg / ml. Therefore, the lack of efficacy of the WT mAb mixture was not affected by the IgG subclass, as mixtures of both IgG1 and IgG2 were equally poor. However, mixtures containing mAbs with heavy chains containing the H429F modification were very effective inducers of killing. The killing level achieved by a mixture of BDR5-1HF and TDR5-1HF (including an IgG1 H chain with the H429F modification) was equivalent to the maximum cell death control determined by cell viability in the presence of SDS detergent (SDS, Fig.40 ). Further, its mAb mixture including BDR5-2HF and TDR5-2HF with H429F modified IgG2 H chain has similar potency to its equivalent mAb mixture based on IgG1 (BDR5-1HF and TDR5-1HF). Therefore, the improvement in potency depends on the H429F modification, but not on the IgG subclass, because whether formatted as human IgG1 or human IgG2 subclass H chain, these all show almost equivalent improvement in killing.

[0414] Effectiveness of dual epitope targeting: killing by a cocktail of DR5 mAbs with different specificities and IgG subclasses

[0415] Pairwise comparisons of mAbs detecting different epitopes of DR5 and including IgG heavy chains of different IgG subclasses were performed to determine whether the superior killing potency of the mAb mixture was determined by the properties of the individual mAbs of the combined IgG subclasses and / or epitopes in the mixture. Specifically, a pairwise mixture of mAbs in a 1:1 ratio was prepared by mixing two mAbs of equal concentration (0.5 μg / ml + 0.5 μg / ml) to produce a total anti-DR5 mAb concentration of 1 μg / ml in the mixture ( Fig.41 The potency of the DR5 mAb cocktail was then determined by incubating Colo205 cells with serial 2-fold titrations of the mAb cocktail, where each cocktail included a different human IgG H chain subclass ( Fig.41 ).

[0416] A mixture of BDR5-1WT mAb including a wild-type IgG1 heavy chain and TDR5-2WT mAb including a wild-type IgG2 heavy chain (BDR5-1WT+TDR5-2WT) showed only limited killing of Colo205 cells, and only at the highest concentration (0.5 μg / ml for each mAb; i.e., 1 μg / ml for total mAb). In contrast, a combination of BDR5-1HF mAb including an IgG1 heavy chain modified with H429F and TDR5-2HF including an IgG2 heavy chain modified with H429F (BDR5-1HF+TDR5-2HF) mediated a maximum level of killing comparable to that of the SDS killing control ( Fig.41 ), and importantly, showed a greater killing efficacy than the mixture of its wild-type mAb equivalents (BDR5-1WT + TDR5-2WT). Further, by titration, the killing efficacy of the mixture of H429F modified mAbs also showed an efficacy that was 10 times that of the WT equivalent.

[0417] The paired mixture (BDR5-2HF + TDR5-1HF) also showed a similar maximum killing level and titer to the BDR5-1HF + TDR5-2HF mixture, wherein BDR5-2HF includes an IgG2 heavy chain containing a H429F modification and is paired with a TDR5-1HF including an IgG1 heavy chain containing a H429F modification. An equivalent mixture of mAbs containing wild-type H chains (BDR5-2WT + TDR5-1WT) failed to show significant killing of Colo205 cells. It is also worth noting that the killing level achieved by the IgG including the H chain carrying the H429F modification is equivalent to the "homogeneous" mixture of mAbs of the same H chain subclass carrying the H429F modification, such as the killing level of IgG2 (BDR5-2HF with TDR5-2HF) ( Fig.40 , Fig.41 ).

[0418] Clearly, mixtures of DR5 mAb and wild-type H chain showed little efficacy in any pairwise combination, but inclusion of H429F in the mAb greatly increased cell killing.

[0419] Effect of changing the ratio of mAbs in the mixture on killing

[0420] When used alone, individual mAbs containing H429F showed higher potency than their wild-type counterparts (see Fig.38 , Fig.39 , Fig.42 and Fig.43 ), but the greatest potency was achieved in the mixture including the mAb carrying the H429F-modified H chain ( Fig.40 , Fig.41 ). However, the mixture used in the above experiment ( Figure 38-41 ) included the same concentration of mAb (ie, 1:1 ratio). Therefore, the effect of varying the ratio of the individual mAbs in the mixture on cell viability was determined.

[0421] By Fig.42 ) or Ramos cells ( Fig.43 ) were incubated with a mixture of BDR5-1HF mAb and TDR5-1HF mAb at a final concentration of 1 μg / ml of total DR5-specific mAb to determine cell viability as follows: 100% BDR5-1HF with 0% TDR5-1HF, 90% BDR5-1HF with 10% TDR5-1HF, 75% BDR5-1HF with 25% TDR5-1HF, 50% BDR5-1HF with 50% TDR5-1HF, 25% BDR5-1HF with 75% TDR5-1HF, 10% BDR5-1HF with 90% TDR5-1HF, and 0% BDR5-1HF with 100% TDR5-1HF. In the presence of the individual mAbs comprising the wild-type H chain, the viability of Colo205 cells was not affected, or barely affected in the case of Ramos cells. In fact, Colo205 cells treated with mAb alone had equivalent survival in the absence of mAb (no-ab control, Fig.42 , Fig.43). In the presence of only BDR5-1HF mAb alone and TDR5-1HF mAb alone (both of which included an H429F modified IgG1 H chain), survival was modestly reduced compared to wild type. However, surprisingly, all mixtures showed near maximal killing of Colo205 and Ramos cells, equivalent to the SDS maximum death control. Also, surprisingly, this effective killing was evident even when as little as 10% of one mAb was present in the mixture, e.g., BDR5-1HF 90:10 TDR5-1HF ( Fig.42 , Fig.43 ). Further, the relative abundance of mAbs with one epitope specificity in the mixture did not affect potency (i.e., BDR5-1HF:TDR5-1HF mixtures were equivalent at all ratios, e.g., BDR5-1HF 90:10 TDR5-1HF was as potent as BDR5-1HF 10:90 TDR5-1HF; Fig.42 , Fig.43 ).

[0422] Evaluation of mAbs carrying the H429-modified IgAH chain

[0423] The DR5-specific mAbs used in the above IgG experiments were also generated that included wild-type IgA H chains or included IgA chains containing the H429F modification and were evaluated for their ability to induce cell death.

[0424] DR5-specific mAbs BDR5 and TDR5 are directed against two different epitopes on human DR5 and were produced according to the above method as antibodies comprising H chains of human IgA, and specifically of human IgA2 subclass as an example (Table 3). Thus, BDR5-A2WT mAb and TDR5-A2WT mAb comprise wild-type IgA2 subclass H chains, and BDR5-A2HF mAb and the TDR5-A2HF mAb comprise IgA2 subclass H chains carrying the equivalent of the H429F modification of IgG1 ( Figure 3 , Table 3); and in all cases, the H chain was paired with a specific BDR5 or TDR5 human kappa light chain. Antibodies including its L chain and IgA H chain were produced in Expi293 cells, and supernatants from these cultures were used as a source of DR5 mAbs.

[0425] Binding of IgA-formatted DR5-specific antibodies to target cells was assessed using indirect immunofluorescence and quantified by flow cytometry ( Fig.44All mAbs detected DR5 on Colo205 cells at levels of binding significantly above background control levels of nonspecific binding of the fluorescent conjugate to cells in the absence of antibody (no ab) or background levels of cellular fluorescence (cells) ( Fig.44 Turning to the ability of DR5-specific mAbs (including IgAH chains) to induce cell death, this was assessed by culturing Colo205 cells in the presence of serial dilutions of the mAbs ( Fig.45 ). The titration curves revealed that inclusion of H429F in the IgA heavy chain conferred potent cell death, as mAbs comprising an IgA heavy chain containing the H429F modification were many times more potent inducers of cell death than equivalent mAbs comprising a wild-type IgA heavy chain. Thus, for example, BDR5-A2HF (EC50>1 / 256 dilution) and TDR5-A2HF (EC50>1 / 512) were potent inducers of killing compared to IgABDR5-A2WT (which showed little killing at any dilution) and TDR5-A2WT (which showed killing only at high concentrations (low dilutions) (EC50~1 / 6 dilution).

[0426] Clearly, the conversion of killing potency brought about by the inclusion of the H429F modification in the immunoglobulin heavy chain is evident in the IgA heavy chain as well as the IgG heavy chain.

[0427] Detection of cell specificity and killing efficacy of different molecular targets by a mixture of H429F-modified mAbs

[0428] As described above, a mixture of H429F-modified mAbs, each of which detects a different epitope on the same molecular target, was compared in terms of C1q binding levels (see Fig.29 ), CDC killing efficacy (see Fig.30 C) and DR5-induced cell death levels ( Figure 38-43 ) showed functional improvement over its individual components. Further, improved CDC ( Fig.30 B). Here, mAb-mediated cell death experiments were performed to evaluate the efficacy of a mixture of H429F-modified mAbs against different molecular targets (e.g., CD38 and DR5). The isatuximab mAbs comprising WT or H429F-mutated IgG H chains used herein were as described in Example 7. Specifically, Ramos lymphoma cells expressing CD38 and DR5 ( Fig.46) were treated with anti-CD38 mAb isatuximab including WT H chain, wild-type isatuximab (Isa-WT) or anti-DR5 mAb BDR5-1 including WT H chain (BDR5-1WT). Compared with untreated cells, the viability of cells treated with these individual mAbs was almost unchanged (A450nm=0.720). Similarly, a 1:1 mixture of Isa-WT and BDR5-1WT mAbs (Isa-WT+BDR5-1WT; Fig.46 ) treatment resulted in only a small decrease in cell viability (A450 nm = 0.576). Similar decreases in viability were obtained by treatment with either the H429F-modified mAb alone, Isa-HF (A450 nm = 0.550) or BDR5-1HF (A450 nm = 0.564) mAb. However, in contrast, a mixture of Isa-HF and BDR5-1HF mAbs (both of which include an H chain carrying the H429F modification) (Isa-HF + BDR5-1HF, Fig.46 ) was significantly more effective in reducing cell viability by inducing cell death (A450 nm = 0.372).

[0429] discuss

[0430] The results obtained in this example clearly demonstrate that the replacement of histidine 429 in the IgG heavy chain gives mAb the ability to induce increased cell death in target cells expressing the target antigen. Specifically, compared with mAb with wild-type H chain (i.e., lacking any modification at position 429), it was found that the H429F modification of the CH3 domain significantly improved the killing efficacy of anti-death receptor mAb. Importantly, it was found that the improvement in killing efficacy was independent of the epitope on the target, independent of the IgG heavy chain subclass entered by the H429 modification, and independent of the cell target, and it is worth noting that the H429F modification overcomes the poor killing ability of the WTmAb for different epitopes. In addition, it was found that improved killing by BDR5 and TDR5 mAbs including the H429F modification was achieved when they were formatted into IgG1 and IgG2 backbones, and their killing was further increased by combining two BDR5 and TDR5 mAbs regardless of the subclass format, i.e., when two DR5 clones were formatted as H429F-modified mAbs of the same IgG subclass (IgG1 or IgG2), or when one clone was IgG1 and the second clone was IgG2. In summary, the effect of H429 modification of the Fc was robust, as measured by DR5-induced apoptosis in these examples, enhancing the receptor agonism of the mAbs alone or in combination, independent of the context of IgG subclass. Overall, these results are very meaningful because they demonstrate the broader utility of the H429 modification for other antibodies, antibody-like molecules, and antibody derivatives, because H429 is conserved in, for example, IgG subclasses of humans, non-human primates, and mice (see Figure 3 and Figure 4 In addition, H429 is also found in the equivalent position of the H chain of other human Ig classes (i.e., IgA, IgE and IgM), which are structurally different from IgG and are not expected to mimic the Fc-related properties attributed to the IgG Fc portion. Nevertheless, using IgA as an example, DR5-specific mAbs used in IgG studies were generated, which included wild-type IgA H chains or IgA H chains modified with H429F, and were also found to exhibit enhanced cell killing ability.

[0431] Interestingly, the improved killing achieved by individual mAbs carrying the H429F modification was significantly further improved by culturing target cells in a mixture of mAbs that bind to different epitopes of the target antigen (e.g., different epitopes of DR5). Specifically, it was found that combining the TDR5-2HF and BDR5-2HF antibodies achieved more effective levels of killing than the individual mAbs (containing the H429F modification), with the mixture of mAbs almost completely killing the cells even at a 90:10 ratio. Finally, the results of this example also show that the improved killing achieved using mAbs containing the H429 modification, whether used as individual mAbs or in a mixture, is not limited to target cell types, and in fact, DR5-specific antibodies, whether containing IgG1 or IgG2 H chains modified at H429, showed similar levels of enhanced killing of colorectal and lymphoma cell types.

[0432] The method of more effectively focusing the killing mechanism on the expected target cell is desirable for improving immunotherapy with mAb. The characteristics of mAb and Fc fusion protein modified by H429 in forming an oligomeric platform (e.g., for optimal CDC) provide a way for combining mAbs with different specificities to increase cell target specificity. Therefore, using a mAb targeting a molecule present on a variety of cell types, but mixed with the mAb targeting a molecule expressed by a limited cell, specific cell killing efficacy can be achieved. For example, DR5 and CD38 have different cell and tissue distributions, but they are co-expressed on certain cell types, particularly hematopoietic cells (Zola et al., the same 2007). Fig.46 The results shown in demonstrate this approach and demonstrate that a single H429F-modified DR5 mAb or a single H429F-modified CD38 mAb induced moderate apoptosis, but when their antigens were co-expressed on target cells, their combination enhanced lymphoma killing. The enhancement of this killing by a combination of H429F-modified mAbs that detect different molecular targets may be caused by multiple mechanisms of H429F modification that are dependent on Fc, such as the formation of hetero-oligomers, in which different cell surface targets are incorporated into signaling clusters through interactions between H429F-modified H chains of different mAbs (e.g., containing both CD38: anti-CD38 and DR5: anti-DR5 oligomeric complexes). Similarly, such enhancements may reflect the integration of signals propagated by DR5 and CD38 through the combination of these mAbs (whether homo-oligomers or hetero-oligomers) and cross-linking or through a combination of these effects. Therefore, the combined use of H429F-modified mAbs that detect separate molecular target molecules combines target cell specificity with killing efficacy. The combination of such effects is desirable in overcoming often existing limiting factors in therapy, including lack of specificity and deleterious toxicity.

[0433] Example 9 Another example of H429 modification conferring enhanced CDC to anti-CD38 monoclonal antibodies

[0434] Mazetuzumab is a monoclonal antibody that recognizes the human CD38 cell surface molecule and exhibits functional properties that differ from several other anti-CD38 mAbs such as daratumumab (Deckert J et al., 2014 supra). The effect of H429 modification on the CDC activity of mazetuzumab on cells resistant to CDC killing was evaluated.

[0435] Methods and Materials

[0436] Antibodies and Antibody Constructs

[0437] The Mazetuzumab-WT mAb used in this example includes a CD38-specific heavy chain polypeptide having an amino acid sequence shown as SEQ ID NO: 74, which includes, from N-terminal to C-terminal, the CD38-specific VH domain of the Mazetuzumab mAb fused to the CH1-hinge-CH2-CH3 domain of human IgG1, and is encoded by a codon-optimized polynucleotide sequence shown as SEQ ID NO: 75. The polypeptide of the anti-CD38 mAb Mazetuzumab light chain includes an amino acid sequence shown as SEQ ID NO: 76 (including the CD38-specific VL domain of the Mazetuzumab mAb fused to a human lambda constant domain), and is encoded by a codon-optimized polynucleotide sequence shown as SEQ ID NO: 77.

[0438] Synthesis of unmodified and mutated heavy chains

[0439] Antibody expression vectors and Fc variants comprising replacement of H429 with phenylalanine (H429F) in the H chain of the CD38 mAb were generated by standard methods known to those skilled in the art, as described elsewhere in this specification for therapeutic mAbs (eg, Example 3).

[0440] Expression of antibody constructs

[0441] The expression of antibodies was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions as described above for other therapeutic mAbs described in this specification. For mAbs, cell cultures were harvested after transfection and centrifuged at 2500 rpm for 20-30 minutes, and the supernatant was filtered with a 0.2 μm high flow filter (Sartorius) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE analysis.

[0442] Purification of mAbs and assessment of antigen binding

[0443] Mazetuzumab-H429F and Mazetuzumab-WT mAbs were purified from the supernatant of transfected Expi293F cells using protein A affinity chromatography followed by size exclusion chromatography. SEC purified mAbs showed the expected H2L2 format of monomeric IgG confirmed by SDS-PAGE analysis. After purification, binding to CD38 positive cells was confirmed by flow cytometry analysis (data not shown).

[0444] Complement-dependent lysis of cells

[0445] After opsonization of cells with mAb, CDC of target cells mediated by mAb was measured by flow cytometry as described elsewhere in this specification (eg, Example 3) using the Zombie Green Fixable Viability Kit (Bio-Gene).

[0446] Results and discussion

[0447] The CDC killing of cells mediated by Mazetuzumab-H429F mAb was compared with that of its wild-type counterpart Mazetuzumab-WT mAb ( Fig.47 ). Each mAb was titrated individually by serial two-fold dilutions in the presence of normal human serum as a complement source. CDC efficacy (% killing) was assessed on Ramos cells expressing the CD38 antigen. Fig.47 As can be seen in the figure, Ramos cells resist CDC killing by Mazetuzumab-WT mAb, which showed a maximum killing level of 27.61% only at the highest concentration (5 μg / ml), but were easily killed by Mazetuzumab-H429F mutant mAb (which showed a maximum killing percentage of 89.74%, and 29% CDC killing even at a high dilution (0.16 μg / ml), which is similar to the maximum killing rate (27.61%) achieved by the wild-type form). Therefore, substitution at position H429 of the CH3 domain of Mazetuzumab not only enhances CDC against certain cellular targets, but can also confer effective CDC against targets that are resistant to lysis. Importantly, the enhanced level of cell killing conferred by the H429F modification in mazetumab-H429F further demonstrates that the H429 modification can be effectively applied to unrelated mAbs to enhance cell killing, including cells that are poorly killed by the unmodified WT mAb (i.e., cells that show resistance to lysis), as in, for example, daratumumab-H429F ( Fig.25 , Fig.26 ) and isatuximab-H429F ( Fig.34 )

[0448] Example 10 H429F modification of the Fc region component can produce hetero-oligomers

[0449] The H429F modification of IgG Fc enhances complement-mediated cytotoxicity (CDC). The mutations K439E and S440K of known IgG are used to inhibit IgG hexamer formation and to inhibit CDC function, but when two mutant IgGs are used together as a mixture, full activity can be restored (Diebolder CA et al., 2014 supra). Therefore, the effects of K439E and S440K mutations on the properties of H429-modified IgGs were evaluated in this example using CD20-specific mAb rituximab.

[0450] Methods and Materials

[0451] Antibodies and Antibody Constructs

[0452] Used in this example is the rituximab IgG1 WT heavy chain and the H429F mutant heavy chain as described in Example 3 above. Two additional antibodies were synthesized containing H429F plus additional point mutations in the H chain as described in Table 4. The first additional mAb includes an H chain containing the H429F modification plus an additional mutation of K439E (rituximab-H429F / K439E). The second additional mAb includes an H chain containing the H429F modification plus an additional mutation of S440K (rituximab-H429F / S440K).

[0453] Synthesis of unmodified and mutated heavy chains

[0454] Antibody expression constructs and Fc variants comprising replacement of H429 with phenylalanine (H429F), K439 with glutamic acid (K439E), and S440 with lysine (S440K) in the H chain of rituximab mAb were generated by standard methods known to those skilled in the art.

[0455] Expression of antibody constructs

[0456] The expression of antibodies was performed using transient transfection of Expi293F cells (Thermo Fisher Scientific) under the same conditions as described above with other therapeutic mAbs. For mAbs, cell cultures were harvested after transfection and centrifuged at 2500 rpm for 20-30 minutes, and the supernatant was then filtered with a 0.2 μm high flow filter (Sartorius) before purification. The presence of the expected antibody in the supernatant was confirmed by SDS-PAGE.

[0457] Purification of mAbs and assessment of antigen binding

[0458] mAbs were purified from the supernatant of transfected Expi293F cells by protein A affinity chromatography as described elsewhere in Example 3. After protein A affinity purification, the antibodies were further purified and characterized as described herein using size exclusion chromatography (SEC). The purified antibodies were then tested for antigen binding on CD20 positive cells by flow cytometry prior to functional analysis.

[0459] Complement-dependent lysis of cells

[0460] After opsonization of cells with mAb, CDC of target cells mediated by mAb was measured by flow cytometry using Zombie Green Fixable Viability Kit (Bio-Bio).

[0461]

[0462] Results and discussion

[0463] Rituximab-WT, Rituximab-H429F and additional C-terminal mutants H429F / K439E and H429F / S440K showed equivalent elution from the protein A affinity column, and further purification by SEC also produced a single IgG peak of 150 kDa for each of the mAbs by SDS-PAGE, which included approximately 50 kDa heavy chain and 25 kDa light chain species after reduction with dithiothreitol as described in Example 3 for similar therapeutic mAbs.

[0464] The CDC potency of rituximab-H429F was compared with that of rituximab-WT ( Fig.48 A). Each mAb was titrated individually by serial two-fold dilutions in the presence of normal human serum as a complement source. CDC potency (% killing) was assessed on CD20 expressing Ramos cells. Fig.48As shown in A, Ramos cells are resistant to CDC killing by rituximab-WT, but are easily killed by rituximab-H429F mutant. However, compared with rituximab-H429F, both double mutants rituximab-H429F / K439E and rituximab-H429F / S440K showed significantly reduced killing. Therefore, substitutions at positions 439 or 440 strongly inhibit the CDC enhancing effect of mutations such as H429F. Others have shown that K439E and S440K mutations inhibit Fc: Fc interactions, oligomer formation (including hexamers) and subsequent CDC by introducing charge repulsion (Diebolder CA et al., 2014 supra). However, when rituximab-H429F / K439E and rituximab-H429F / S440K mAbs were used together as a mixture, fully enhanced CDC activity equivalent to rituximab-H429F was restored. It should be noted that all mAbs bound to CD20 almost identically ( Fig.48 B). Therefore, the difference in CDC potency conferred by mutations K439E and S440K is not related to differences in binding to target cells. Although not wishing to be bound by theory, it is believed that by using these two mutants together as a mixture, charge repulsion can be avoided by heterotypic interactions between the K439E and S440K mutant Fcs.

[0465] The full CDC activity of the combination of rituximab-H429F / K439E and rituximab-H429F / S440K as a mixture demonstrated the effective formation of hetero-oligomers (e.g., hexamers). This can be used to generate hetero-oligomers from two mAbs with different specificities to target two different epitopes on the same antigen, or two completely different antigens. Such hetero-oligomers comprising hetero-hexamers are expected to have specificity advantages, for example, in cases where effector functions are expected to be directed only or selectively directed to targets expressing two specificities that are recognized separately by each o...

Claims

1. An immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptides comprising an Fc region component, wherein the Fc region component comprises at least one constant heavy chain domain 3 (CH3) domain (or at least one constant heavy domain 4 (CH4) domain), wherein the one or more polypeptides comprise an amino acid substitution at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain (Eu numbering).

2. An immunotherapeutic protein according to claim 1, wherein the Fc region component comprises the constant heavy domain 3 (CH3) and the constant heavy domain 2 (CH2) of an immunoglobulin heavy chain, optionally in combination with a lower, core and / or upper hinge sequence.

3. The immunotherapeutic protein of claim 1, wherein the Fc region component comprises only the constant heavy domain 3 (CH3).

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

5. The immunotherapeutic protein according to any one of claims 1 to 4, wherein the immunotherapeutic 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 an Fc region component comprising a CH2 domain and a CH3 domain, such that the first polypeptide and the second polypeptide form an Fc fragment or an Fc-like fragment by dimerization. The immunotherapeutic protein of claim 4 , wherein the Fc region component is derived from an IgG1 heavy chain polypeptide.

7. The immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgG2 heavy chain polypeptide.

8. The immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgG3 heavy chain polypeptide.

9. The immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgG4 heavy chain polypeptide.

10. The immunotherapeutic protein of claim 4, wherein the Fc region component is derived from an IgA heavy chain polypeptide.

11. The immunotherapeutic protein of claim 1, wherein the immunotherapeutic 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 an Fc region component comprising a CH2 domain and a CH4 domain, such that the first polypeptide and the second polypeptide form an Fc fragment or an Fc-like fragment by dimerization.

12. The immunotherapeutic protein according to claim 5 or 11, wherein the immunoglobulin is an antibody, an antibody-like molecule or an antibody derivative.

13. The immunotherapeutic protein according to claim 12, wherein the immunoglobulin comprises at least one antigen recognition structure, and the at least one antigen recognition structure is capable of specifically binding to an antigen or epitope with therapeutic significance.

14. An immunotherapeutic protein according to claim 13, wherein the at least one antigen recognition structure specifically binds to: cancer-associated antigens, self-antigens, allergens, antigens associated with inflammatory diseases, antigens from transplanted tissues or organs, or antigens from infectious sources, such as antigens of bacterial or viral pathogens.

15. The immunotherapeutic protein of claim 14, wherein the at least one antigen recognition structure specifically binds to a cancer-associated antigen selected from CD20, CD38 and CD52.

16. An immunotherapeutic protein according to claim 14, wherein the at least one antigen recognition structure specifically binds to the following antigens: SARS-CoV-2 virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Respiratory Syncytial Virus (RSV) or Dengue Virus.

17. The immunotherapeutic protein of claim 14, wherein the at least one antigen recognition structure specifically binds to an antigen of a viral pathogen selected from an antigen of a coronavirus.

18. The immunotherapeutic protein of claim 13, wherein the at least one antigen recognition structure specifically binds to a cell surface molecule known to induce cell activation or cell proliferation and / or stimulate inhibitory pathways to inhibit or reduce cellular responses.

19. The immunotherapeutic protein of claim 13, wherein the at least one antigen recognition structure specifically binds to a cell surface molecule known to induce a cellular response selected from stimulating an inhibitory pathway to inhibit or reduce the response.

20. The immunotherapeutic protein of claim 13 or 15, wherein the at least one antigen recognition structure specifically binds to an immune checkpoint molecule.

21. The immunotherapeutic protein of claim 20, wherein the immune checkpoint molecule is selected from the group consisting of: 4-1BB (CD137), cluster of differentiation (CD40, CD154), OX40 receptor (TNFRSF4, CD134), tumor necrosis factor receptor type II (TNFR2, CD120b), glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18, CD357), cluster of differentiation 27 (CD27), T cell immunoglobulin and mucin domain-containing 3 (TIM -3), B and T lymphocyte attenuator (BTLA, CD272), lymphocyte activation gene-3 (LAG3, CD223), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), inducible T cell co-stimulator (ICOS, CD278), cluster of differentiation 28 (CD28), T cell immunoreceptor with Ig and ITIM domains (IGIT, Vstm3), programmed death ligand 1 (PDL-1, CD274) and programmed cell death protein 1 (PD-1, CD279).

22. The immunotherapeutic protein of claim 13, wherein the at least one antigen recognition structure specifically binds to a cell surface molecule, wherein the cell surface molecule induces cell death when engaged by a ligand or the immunotherapeutic protein.

23. The immunotherapeutic protein of claim 22, wherein the cell surface molecule is selected from the group consisting of tumor necrosis receptor 1 (TNFR1), Fas (CD95), death receptor 3, death receptor 4, death receptor 5, death receptor 6, and cluster of differentiation (CD38).

24. The immunotherapeutic protein of any one of claims 12 to 23, wherein the immunotherapeutic protein is an antibody that forms oligomers by self-association in solution at physiological pH.

25. The immunotherapeutic protein of claim 24, wherein the immunotherapeutic protein is a hexamer.

26. The immunotherapeutic protein of claim 24 or 25, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide is a H→Y substitution.

27. The immunotherapeutic protein of any one of claims 12 to 23, wherein the immunotherapeutic protein is an antibody that forms oligomers when bound to a relevant target by "on-target" oligomerization.

28. The immunotherapeutic protein of claim 28, wherein the immunotherapeutic protein forms a hexamer when bound to a relevant target via "on-target" oligomerization.

29. The immunotherapeutic protein of claim 28, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide is a H→F substitution.

30. The immunotherapeutic protein of claim 28, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG2 heavy chain polypeptide is a H→F substitution.

31. The immunotherapeutic protein of claim 28, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG3 heavy chain polypeptide is a H→F substitution.

32. An immunotherapeutic protein according to any one of claims 30 to 32, wherein the Fc region component comprises at least one constant heavy chain domain 3 (CH3) domain (or at least one constant heavy chain domain 4 (CH4) domain) and a constant heavy chain domain 2 (CH2) domain, and wherein the CH2 comprises L234A and / or L235A amino acid substitutions.

33. An immunotherapeutic protein according to any one of claims 1 to 4, wherein the immunotherapeutic protein comprises a fusion protein or protein conjugate comprising a partner polypeptide connected to an Fc region component, wherein the Fc region component comprises at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain).

34. The immunotherapeutic protein of claim 34, wherein the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof) or a co-receptor polypeptide (or a fragment thereof).

35. The immunotherapeutic protein of claim 34, wherein the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof), which is capable of binding to the structural protein of the virus, so that the immunotherapeutic protein can act as a "bait" to block viral interaction and viral entry into host cells.

36. An immunotherapeutic protein according to claim 34, wherein the partner polypeptide is angiotensin converting enzyme 2 (ACE2) extracellular domain, dipeptidyl peptidase 4 (DPP4, CD26), Hsp70, hepatitis A virus cellular receptor 1 (HAVCR1 / TIM-1), cluster of differentiation 155 (CD155), glucose transporter 1 (GLUT1), proto-oncogene tyrosine protein kinase MER (MERTK), TYRO3 protein tyrosine kinase (TYRO3), AXL or cluster of differentiation 4 receptor (CD4 receptor).

37. The immunotherapeutic protein of claim 34, wherein the partner polypeptide is a co-receptor polypeptide (or a fragment thereof).

38. The immunotherapeutic protein of claim 38, wherein the co-receptor polypeptide (or fragment thereof) is CXC chemokine receptor type 4 (CXCR4), CC chemokine receptor type 5 (CCR5), a tetraspanin or an occludin.

39. An immunotherapeutic protein according to claim 34, wherein the partner polypeptide is a receptor for cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (or a soluble extracellular fragment thereof), interleukin-1 receptor (IL-1R), interleukin-6 receptor (IL-6R), tumor necrosis factor receptor-2 (TNFR2, also known as CD120b) or a cytokine of the TGF-β superfamily.

40. The immunotherapeutic protein of claim 34, wherein the partner polypeptide is a ligand for a cell surface molecule known to induce cell activation and / or proliferation and / or stimulate inhibitory pathways to inhibit or reduce cellular responses.

41. The immunotherapeutic protein of claim 41, wherein the partner polypeptide is a ligand of an immune checkpoint molecule.

42. The immunotherapeutic protein of claim 42, wherein the immune checkpoint molecule is selected from the group consisting of 4-1BB (CD137), CD40 (CD154), OX40 (TNFRSF4, CD134), TNFR2 (CD120b), GITR (TNFRSF18, CD357), CD27, TIM-3, BTLA (CD272), LAG3 (CD223), CTLA4 (CD152), ICOS (CD278), CD28, TIGIT (Vstm3), PDL-1 (CD274), and PD-1 (CD279).

43. The immunotherapeutic protein of claim 34, wherein the partner polypeptide is the ligand for a cell surface molecule that induces cell death upon engagement by the ligand.

44. The immunotherapeutic protein of claim 44, wherein the partner polypeptide is a ligand of a cell surface molecule selected from the group consisting of: TNFR1, Fas, death receptor 3, death receptor 4, death receptor 5, death receptor 6, and CD38.

45. The immunotherapeutic protein of claim 45, wherein the partner polypeptide is TNF-related apoptosis-inducing ligand (TRAIL).

46. ​​The immunotherapeutic protein of any one of claims 34 to 46, wherein the immunotherapeutic protein is a fusion protein that forms oligomers by self-association in solution at physiological pH.

47. The immunotherapeutic protein of claim 47, wherein the immunotherapeutic protein is a hexamer.

48. The immunotherapeutic protein of claim 47 or 48, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain polypeptide is a H→Y substitution.

49. The immunotherapeutic protein of any one of claims 34 to 46, wherein the immunotherapeutic protein is a fusion protein that forms oligomers upon binding to a relevant target via "on-target" oligomerization.

50. The immunotherapeutic protein of claim 50, wherein the immunotherapeutic protein forms a hexamer when bound to a relevant target via "on-target" oligomerization.

51. The immunotherapeutic protein of claim 50 or 51, wherein the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide is a H→F substitution.

52. An immunotherapeutic protein according to any one of claims 34 to 52, wherein the immunotherapeutic protein is an antibody-like molecule in an H2 or H2L2 format, the antibody-like molecule comprising two fusion proteins, each of the two fusion proteins comprising an immunoglobulin heavy chain polypeptide and a partner polypeptide, the immunoglobulin heavy chain polypeptide comprising an Fc region component, the Fc region component comprising at least one constant heavy chain domain 3 (CH3) domain (or at least one constant heavy domain 4 (CH4) domain) and comprising an amino acid substitution (Eu numbering) located at a position corresponding to H429 of the amino acid sequence of a human IgG1 heavy chain, the partner polypeptide being connected to the Fc region component comprising at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain).

53. The immunotherapeutic protein of claim 53, wherein the partner polypeptides of the two fusion proteins are the same or different.

54. The immunotherapeutic protein of claim 53 or 54, wherein the immunotherapeutic protein is an antibody-like molecule in an H2L2 format and comprises two fusion proteins, each of which comprises an immunoglobulin light chain polypeptide and a partner polypeptide that may be the same or different and is linked thereto.

55. The immunotherapeutic protein of any one of claims 53 to 55, wherein each of the partner polypeptides is a target recognition structure.

56. Use of an immunotherapeutic protein according to any one of claims 1 to 56 for treating or preventing a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases and proliferative diseases.

57. Use of an immunotherapeutic protein according to any one of claims 1 to 56 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, other inflammatory diseases, infectious diseases and proliferative diseases.

58. A method for treating or preventing a disease or condition, the method comprising administering to a subject an effective amount of an immunotherapeutic protein according to any one of claims 1 to 56, wherein the disease or condition is selected from autoimmune diseases and conditions, other inflammatory diseases, infectious diseases, and proliferative diseases.

59. The method of claim 59, wherein the immunotherapeutic protein comprises a fusion protein comprising a partner polypeptide connected to an Fc region component, the Fc region component comprising at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain), and the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof), the cell surface receptor polypeptide (or a fragment thereof) being capable of binding to a structural protein of a virus, such that the immunotherapeutic protein can act as a "bait" to block viral interactions and viral entry into host cells, the method further comprising administering antibodies against the virus.

60. The method of claim 59, wherein the immunotherapeutic protein comprises a fusion protein comprising a partner polypeptide linked to an Fc region component, the Fc region component comprising at least one CH3 domain (or at least one constant heavy domain 4 (CH4) domain), and the partner polypeptide is directed against a target of therapeutic interest, the method further comprising administering an antibody directed against the target.

61. A pharmaceutical composition or medicament comprising an immunotherapeutic protein according to any one of claims 1 to 56 and a pharmaceutically acceptable carrier, diluent and / or excipient.

62. The pharmaceutical composition or medicament of claim 62, wherein the carrier is saline at physiological pH.

63. The pharmaceutical composition or medicament of claim 62, wherein the carrier is saline at a pH less than 6.

5.

64. A pharmaceutical composition or drug comprising the immunotherapeutic protein according to claim 51 or 52 and a pharmaceutically acceptable carrier, diluent and / or excipient, wherein the pH of the composition or drug is weakly acidic.

65. A pharmaceutical composition or medicament comprising a mixture of two or more different immunotherapeutic proteins according to any one of claims 1 to 56 and a pharmaceutically acceptable carrier, diluent and / or excipient.

66. A pharmaceutical composition or medicament according to claim 66, wherein the mixture of two or more immunotherapeutic proteins binds to different molecular targets.

67. A pharmaceutical composition or drug according to claim 67, wherein the immunotherapeutic protein is composed of a first immunoglobulin molecule having a first antigen recognition structure for a first antigen and a second immunoglobulin molecule having a second antigen recognition structure for a second antigen, wherein the first antigen is a cancer antigen present on the surface of cancer cells and the second antigen is a death receptor.

68. A pharmaceutical composition or medicament according to claim 67, wherein the immunotherapeutic protein binds to different epitopes on a single molecular target.

69. A kit of parts comprising at least one immunotherapeutic protein according to any one of claims 1 to 56.

70. A method of producing an immunotherapeutic protein according to claims 1 to 56, the method comprising culturing a host cell comprising 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 recovery of the immunotherapeutic protein in monomeric form; or (ii) Substantially neutral pH for recovery of the immunotherapeutic protein in oligomeric form.

71. A method for producing an immunotherapeutic protein according to any one of claims 1 to 56, the method comprising culturing a host cell comprising a construct encoding the protein under conditions suitable for expression of the protein, and recovering the protein from the culture supernatant using a method comprising affinity chromatography using an elution buffer comprising arginine at a concentration of less than 130 mM and a pH less than or equal to 5.

0.

72. The method of claim 71, wherein said recovery of the expressed immunotherapeutic protein comprises recovery by size exclusion chromatography (SEC).

Images