Immunotherapeutic proteins

Abstract

The present disclosure relates to immunotherapeutic protein and methods of use and production thereof are disclosed, comprising one or more immunoglobulin heavy chain polypeptide comprising one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) and a hinge region, wherein the one or more polypeptide comprises a hexamerising mutation, and wherein the one or more polypeptide comprises at least one C1q binding modification.

Description

IMMUNOTHERAPEUTIC PROTEINS TECHNICAL FIELD

[0001] The present disclosure relates to immunotherapeutic protein and methods of use and production thereof are disclosed, comprising one or more immunoglobulin heavy chain polypeptide comprising one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) and a hinge region, wherein the one or more polypeptide comprises a hexamerising mutation, and wherein the one or more polypeptide comprises at least one C1q binding modification. BACKGROUND

[0002] Human immunoglobulin is produced to recognise foreign antigens through their antigen combining regions. Such antibodies can be produced wherein the heavy chain of the immunoglobulin can be of distinct immunoglobulin classes for example human IgG, human IgA, human IgE human IgM, human IgG, human IgD.

[0003] In some cases the heavy chain of the immunoglobulin (Ig) may be composed of subclasses of an immunoglobulin class for example the antibody may comprise heavy chains of human IgG1 or human IgG2 or human IgG3 or human IgG4 which are subclasses of human IgG.

[0004] A number of effector systems can be harnessed by immunoglobulins in order to mediate an effective outcome. Such an outcome includes an immune or innate immune response to target antigens for example resistance to pathogens by antibodies induced either through infection or vaccination or monoclonal antibodies for example directed at the target for example a cancer cell or a pathogen. Another outcome is the modulation of inflammatory responses in autoimmune disease.

[0005] The heavy chains of immunoglobulins are responsible for the effector functions attributable to immunoglobulins. However not all classes or subclasses of immunoglobulins can mediate all effector functions. Indeed using the human IgG class as an example its subclasses vary in their capacity to initiate effector functions.

[0006] The two main effector systems that are harnessed by immunoglobulins are the serum complement system which is a multi-faceted cascade of serum protein activation that when activated can lead to death of targets by lysis via complement-dependent cytotoxicity (CDC) or by cellular mechanismsmeditated by inflammatory leukocytes attracted to area of complement activation or other leukocytes or tissue cells that can ingest or destroy circulating targets that have been opsonised with antibody and complement . Not all human Ig classes or subclasses are able to activate complement.

[0007] The second major system are the Fc receptors which are cell surface proteins expressed on the surface of leukocytes and other cells. These are cell surface receptors that specifically bind immunoglobulins often in the form of an immune complex (a complex of antigen with its cognate antibody). The receptors are characterised by the immunoglobulin class of the immunoglobulin they bind i.e. IgG is bound by the IgG Fc receptors – FcγR; IgA is bound by IgA Fc receptors – FcαR and so on. The interaction of the immunoglobulin with the Fc receptors can initiate a range of cellular effector responses.

[0008] Not all immunoglobulins are capable of utilising one or both effector systems. Human IgG1 or human IgG3 are universal activators of effector responses and as such are potent activators of both serum complement and cellular FcγR receptors i.e. human IgG1 or human IgG3 activate serum complement and bind to all activating type FcγR and inhibitory type FcγR.

[0009] In contrast, human IgG2 or human IgG4 subclasses are widely known to fail to activate serum complement. Furthermore they are not universal ligands of FcγR and demonstrate specific binding to limited types of FcγR i.e. IgG2 fails to bind to all but one activating-type FcγR, binding only to one allelic form of human FcγRIIa (FcγRIIa-H131) and fails to bind the inhibitory FcγRIIb and other activating-type FcγR. Human IgG4 binds only to the high affinity activating receptor FcγRI and to the inhibitory FcγRIIb.

[0010] Given the above, it is perhaps unsurprising that considerable research effort has been applied to elucidate the details of Fc interactions (e.g. with FcγRs and complement proteins such as C1q and the membrane attack complex (MAC) proteins C4-C9) and identify modifications (e.g. mutations) that may be made to the Fc in order to potentially enhance the potency of therapeutic mAbs. However, very few such Fc mutations have been included to date in therapeutic mAbs; the best known being antibodies wherein the heavy chain glycan has been engineered to lack fucose and thereby result in enhanced FcγRIII interaction, for example obinutuzumab and margetuximab (which comprises six point mutations of the heavy chain of an IgG1 antibody), and others that are undergoing clinical trials but yet to be approved such as various HexaBody™ mAbs (GenMab BV; Copenhagen, Denmark) which comprise a triple mutation of the Fc fragment of IgG1, namely amino acid substitutions at positions 345 (i.e. E345R), 430 (i.e. E430G) and 440 (i.e. S440Y) ((known 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 etal., PLoS Biol 14(1):e1002344, 2016). This mutation has been shown to enhance the ability of IgG1 antibodies to form hexamers on target molecules, and IgG1 mutants including the E430G mutation have been reported as displaying strongly enhanced CDC conditional on antigen binding at the target cell (de Jong et al., 2016 supra). It will therefore be apparent to those skilled in the art that there is a need in the art for modifications which enhance the therapeutic potency of immunotherapeutic proteins (e.g., mAbs). SUMMARY

[0011] In work leading to the present disclosure, the inventors produced a series of immunotherapeutic proteins (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 polypeptides. Some molecules including a mutation at position 429, considered to be a "buried" or inaccessible site within the IgG1 structure (see Figure 1) and occupied by a histidine (His / H) residue, were found to show, for example, enhanced activation of the complement system by the classical complement pathway including binding of C1q, formation of the membrane attack complex and complement-dependent cytotoxicity in assays of complement function. In addition, the inventors obtained evidence showing that some molecules comprising an Fc region component with a mutation at position 429 are capable of forming oligomers, either in solution or upon binding to a relevant target. Further, the inventors have identified a series of C1q binding modifications that modulate binding of the immunotherapeutic protein to C1q.

[0012] Thus, in an aspect, the present invention provides an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide comprising one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) and a hinge region, wherein the one or more polypeptide comprises a hexamerising mutation, and wherein the one or more polypeptide comprises at least one C1q binding modification.

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

[0014] In an aspect, the present invention an oligomer comprising the immunotherapeutic protein as described herein.

[0015] In an aspect, the present invention provides a nucleic acid encoding the immunotherapeutic protein as described herein.

[0016] In an aspect, the present invention provides for the use of the immunotherapeutic protein, oligomer, or nucleic acid as described herein for treating or preventing a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

[0017] In an aspect, the present invention provides for the use of the immunotherapeutic protein, oligomer, or nucleic acid as described herein in the manufacture of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

[0018] In an aspect, the present invention provides a method for treating or preventing a disease or condition, comprising administering to the subject an effective amount of the immunotherapeutic protein, oligomer, or nucleic acid as described herein, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

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

[0020] In an aspect, the present invention provides a kit comprising at least one immunotherapeutic protein, oligomer or nucleic acid as described herein.

[0021] In an aspect, the present invention provides a method of producing an immunotherapeutic protein as described herein, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant under conditions of:(i) mildly acidic pH to recover immunotherapeutic in a monomeric form; or (ii) substantially neutral pH to recover immunotherapeutic protein in an oligomeric form.

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

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

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

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

[0026] In an aspect, the present invention provides a method of producing an immunotherapeutic protein as described herein, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant, e.g., using a method comprising affinity chromatography which may comprise using an elution buffer comprising a concentration of arginine of less than 130 mM and at less than or equal to pH 5.0.

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

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

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

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

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

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

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

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

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

[0036] In an aspect, the present invention provides a method of enhancing complement-based lysis mediated by an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide, the method comprising: substituting an amino acid at a position corresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering) with F, Q, E, S, A, Y, L, V, G, W, R or P. BRIEF DESCRIPTION OF FIGURES

[0037] Figure 1 provides diagrammatic representations of the structure of the Fc of human IgG1 showing that the H429 residue is located in an inaccessible site within the Fc fragment. The representations are of the Human IgG1-Fc (PDB: 1Fc1) reproduced from Deisenhofer J., Biochemistry 20:2361-2370, 1981 with space filled rendering. (A) Side view of the Fc with the A-chain shown in black and B-chain in light grey with N-linked glycan shown in dark grey. The boxed area of the A-chain indicates the region enlarged in panels B and C; (B) the enlarged boxed area of panel A indicating in grey, solvent accessible amino acids of chain-B labelled according to the Eu numbering of human IgG1: methionine at position 428, (M428), glutamic acid 430 (E430), alanine 431 (A431), leucine 432 (L432), histidine H435 which sit above and obscure histidine 429 (H429) which is buried in the Fc; (C) The view of panel B after the solvent accessible residues shown in panel B are not rendered i.e. removed from view. Only now does the buried H429 shown in grey CPK, become apparent beneath these overlying neighbouring solvent accessible residues shown in panel B. Accessible surface area (ASA) for residues calculated using PISA (i.e. "Protein interfaces, surfaces and assemblies" service PISA; http: / / www.ebi. ac.uk / pdbe / prot_int / pistart.htm), and Krissinel E and K Henrick. J Mol Biol 372:774-797, 2007) indicates that H429 is inaccessible, recording a value of 0.0;

[0038] Figure 2 provides representations of immunoglobulin (antibodies) and antibody-like (Ab-like) molecules showing the modular nature of the antibody: Left panel: Provides representations of the prototypical immunoglobulin structure - Immunoglobulins are comprised of chains of variable (V) and constant (C) domains arranged in heavy (H) chains and, optionally, light (L) chains that self-assemble. Domains are further identified by chain (e.g. CH3 is the third constant domain of the heavy chain). Heavy chains (H) normally further include a distinct linking,or hinge sequence, between the Fab and Fc Some H chains comprise three constant H domains, as shown in the panel (with residue H429 indicated in the CH3 domain), and others comprise four constant H domains (e.g. IgM and IgE) where CH4 is the equivalent domain to the CH3 of other antibodies (e.g. IgG and IgA). Antibodies from many species include a light (L) chain. In antibodies that lack a light chain, two H chains dimerise (H2) and normally comprise the antibody. On the other hand, in antibodies that include a light (L) chain, the two antibody H chains will normally dimerise and a L chain is associated with each H chain (i.e. H2L2). Some antibodies lacking strong H chain interactions (e.g. such as human IgG4) may at times 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 parts of each H chain (i.e. two Fc regions of the H chain, which each comprise a hinge, CH2 and CH3 domain of a heavy chain). Right panel: Provides representations showing that the modular nature of immunoglobulins allows flexibility in the production of Ab-like molecules and fusion proteins (including immunoglobulin parts). The molecules shown all include an "H-like" chain, comprising at least a CH3 domain. In particular, the Ab-like molecules depicted each consist of a V domain for target antigen recognition joined by a linking sequence to a CH3 domain of an Fc region from, for example, an IgG (which, in variations of the molecules shown, could be an equivalent CH4 domain from an IgE or IgM). The CH3 domain can carry mutations such as those described herein at position 429 (Eu numbering). The V and CH3 domains may be linked by a sequence that may comprise heterologous linking sequences, synthetic linking sequences and / or comprise other homologous or heterologous sequences or domains linking the V and CH3 domains (e.g. as depicted in the examples shown in the figure, the V and CH3 domains can be linked by CH1-CH2 or CH2 with or without a further short polypeptide sequence such as a heterologous or synthetic linking sequence or an immunoglobulin hinge sequence), and the CH1, CH2 and hinge sequence modules may be provided in varied combinations and orders, not limited to the examples shown. The molecules shown are each in a monomeric form, which may dimerise to form homodimeric or heterodimeric forms. Similarly for the fusion proteins shown, these each consist of a target recognition moiety (depicted as "X") joined by a linking sequence to at least a CH3 domain of an Fc region from, for example, an IgG (or an equivalent CH4 domain of an IgE or IgM). Again, the CH3 domain may include mutations such as those at position 429 (Eu numbering), and the target recognition moiety and H chain modules (e.g. the CH3 domain, CH2-CH3 or CH3-CH2 components) are linked by a sequence that may comprise heterologous or synthetic linking sequences and / or comprise other homologous or heterologous sequences or domains linking the V and CH3 domains (e.g. CH1-CH2 or CH2 with or without a further short polypeptide sequence such as a heterologous or synthetic linking sequence or an immunoglobulin hinge sequence);

[0039] Figure 3 illustrates the conservation of the domain structure and sequence homology in immunoglobulin G (IgG) and immunoglobulin A (IgA) molecules: The sequence comparison of thehinges and constant domains of human heavy (H) chain sequences are provided depicting the domain-based structure of the H chain (i.e. CH1–Hinge–CH2–CH3 defined by IgG1; Eu numbering indicated (inverted triangle) for the first amino acid of each IgG1 domain and hinge is shown thus: CH1 domain, amino acids 118 to 215; hinge amino acids 216 to 230; CH2 domain amino acids 231 to 340; and CH3 domain amino acids 341 to 447). The other IgG subclasses IgG2, IgG3, IgG4, as well as IgA subclasses, IgA1 and IgA2, have a corresponding domain structure with amino acid sequence homology. Also shown is the conservation of the histidine residue at position 429 of human IgG1 across the other IgG subclasses and IgA subclasses (indicated by the arrow). Amino acid sequences were derived from translation of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). Accession numbers of 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. Alignments were performed using Clustal except for the hinges which were aligned manually;

[0040] Figure 4 shows that the histidine of position 429 in IgG1 is conserved in all immunoglobulin classes. Comparison of the amino acid sequence of the IgG1 CH3 domain and the corresponding domains of other human immunoglobulin classes shows that Histidine 429, indicated by the arrow, is conserved in the CH3 domains of the other IgG subclasses, IgG2, IgG3 and IgG4 and the IgA subclasses IgA1 and IgA2, IgD and in the equivalent domain (CH4) of IgE and IgM. The IgG1 CH3 domain is defined and numbered according to the Eu numbering and the first amino acid in the CH3 domain is indicated by the inverted triangle above glycine. The amino acid sequences shown were derived from translation of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser / ). Accession numbers of 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;

[0041] Figure 5 shows the results of the purification of an ACE2-Fc H429Y fusion protein according to the present disclosure: (A) Anion exchange (IEX) chromatography of flACE2-Fc-WT (flow through (ft), eluted fractions, and wash) with the ACE2-Fc-containing peak highlighted by *; (B) SDS-PAGE of flow through (ft) and IEX peak fractions with the flACE2-Fc H429Y migrating above the 250 kDa marker and low molecular weight (mw) impurities marked †; (C) Size-exclusion chromatography (SEC) of IEX fractions 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 the high proportion of oligomeric species. The monomeric (mn) species areconsidered to be single molecules (i.e. monomer molecules) comprising two copies of the respective ACE2-Fc fusion polypeptide self-associated through the Fc region components;

[0042] Figure 6 provides graphical results showing SARS-CoV-2 RBD binding activities of ACE2-Fc fusion proteins according to the present disclosure. ACE2-Fc fusion protein binding to immobilised RBD- Ig was determined by ELISA for : (A) trACE2-Fc-WT, (B) flACE2-Fc-WT and (C) and EflACE2-Fc-WT and for the following variants thereof: F ACE2-Fc proteins including a mutated H429F Fc region component YoliSEC purified ACE2-Fc proteins including a mutated H429Y Fc region component (oligomeric proteins) Ymn SEC purified ACE2-Fc proteins including a mutated H429Y Fc region component (monomer proteins; comprising two copies of the ACE2-Fc fusion protein dimerised through the Fc region components) kif trACE2-Fc-WT proteins produced in the presence of the mannosidase inhibitor, kifunensine. (D) Summary of EC50binding constants for the trACE2-Fc-WT, flACE2-Fc-WT and variant proteins demonstrating a higher apparent RBD binding affinity for the flACE2-Fc-WT over trACE2-Fc-WT and a weaker binding affinity for the flACE2-Fc-H429Y monomer (Ymn; i.e. comprising two copies of the flACE2-Fc-H429Y fusion protein dimerised through the Fc region components). Welch's unpaired t test p = 0.0332 (*), <0.0001 (****);

[0043] Figure 7 provides results showing that the flACE2-Fc H429Y fusion protein according to the present disclosure forms pH-dependant oligomers: SEC of flACE2-Fc H429Y purified by IEX was dialysed against (A) PBS 7.4 or (B) 100 mM citrate, 100 mM NaCl pH 5 and then SEC was performed in the same buffers. SEC at pH 5 yielded a greater proportion of monomeric (mn) fusion protein, than separation at pH 7.4. (C) Native PAGE (1 µg) of ACE2-Fc H429Y. Lane 1, SEC pH 5 oligomers (oli); Lane 2, SEC pH 5 monomer (mn); Lane 3, SEC pH 7.4 oligomers; Lane 4, SEC pH 7.4 monomer; Lane 5, SEC pH 5 monomer re-dialysed against PBS pH 7.4 and purified by SEC and the mn fractions collected as in panel D. (D) SEC chromatogram of pH 5.0 oligomers analysed in lane 1 of panel C, re- dialysed against PBS pH 7.4 and SEC performed in PBS pH 7.4. (E) Improved RBD-Ig binding activity of flACE2-Fc-H429Y monomer (Ymn) prepared at pH 5. The monomers are considered to be single molecules (i.e. monomer molecules) comprising two copies of the respective ACE2-Fc fusion protein dimerised through the Fc region components;

[0044] Figure 8 provides results showing that the SARS-CoV-2 neutralisation 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 mutation. Neutralisation potencies of the ACE2 polypeptide and the three groups ofACE2-Fc-WT fusion and variant proteins were by titration to cytopathic effect (CPE) endpoint in a micro-neutralisation assay. The fusion proteins are trACE2-Fc, flACE2-Fc and EflACE2-Fc WT, and Fc variant, H429F, F; H429Y oligomers on SEC, Yoliand H429Y monomers on SEC, Ymn(comprising two copies of the fusion protein dimerised through the Fc region components). The trACE2- Fc fusion proteins include the glycan-modified trACE2-Fc-kif. Neutralisation endpoint mean ± SEM, ANOVA with Dunnett's multiple comparisons test compared to ACE2 and ACE2-Fc WT. p = 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****);

[0045] Figure 9 provides the results of assays to assess the interaction of ACE2-Fc fusion proteins according to the present disclosure with FcγR. Ramos-S cells were opsonised with the ACE2-Fc WT fusion protein and variant proteins (5 µg / ml). Biotinylated (A) dimeric rsFcγRIIa or (B) dimeric rsFcγRIIIa probes, followed by streptavidin-APC were bound to the opsonised cells and binding described as median fluorescence intensity (Median FI) determined by flow cytometry. FcR binding activity of the ACE2-Fc fusion proteins was readily detected with the exception of ACE2-Fc H429Y fusion proteins which was greatly diminished (3 replicates, mean ± SEM);

[0046] Figure 10 provides the results of assays to assess the capacity of ACE2-Fc fusion proteins according to the present disclosure to mediate cell activation via FcγRIIIa. The results demonstrate that flACE2-Fc proteins are potent activators of FcγRIIIa with the exception of the Fc H429Y mutants in any ACE2 format which fail to stimulate. The afucosylated trACE2-Fc-kif is also a potent activator of FcγRIIIa. Ramos-S target cells were opsonised with (A) trACE2-Fc, (B) flACE2-Fc and (C) EflACE2-Fc, WT and variant proteins, including H429F, F; H429Y, oligomers, Yoli; H429Y monomer, Ymn (comprising two copies of the fusion protein dimerised through the Fc region components); or trACE2-Fc kif produced from trACE2-Fc WT in Expi293 cells in the presence of the mannosidase inhibitor, kifunensine. In some experiments, Ramos-S target cells were separately opsonised with anti-CD20 mAb rituximab, RIT. These opsonised targets were incubated with FcγRIIIa-NF-κB-RE nanoluciferase reporter cells, and FcγRIIIa activation measured by the induction of nanoluciferase (RLU). Data was fitted to agonist response curves to estimate EC50. (D) EC50(nM) values from the curve fits are shown. Mean ± SEM, n ≥4, ANOVA with Dunnett's multiple comparisons test comparing to trACE2-Fc WT. p = 0.0021 (**), 0.0002 (***), <0.0001 (****);

[0047] Figure 11 provides results showing that ACE2-Fc fusion proteins according to the present disclosure, comprising Fc region components with the H429F and H429Y mutations, strongly fix complement and direct complement dependent cytotoxicity killing of Ramos-S target cells; as determined using ELISA analysis of the complement fixing 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: C1q binding (A, B) to ACE2-Fcfusion protein variants (2.0 μg / ml) at different of avidin-captured RBD on the plate; 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 opsonised Ramos- S cells was determined using the presence of a 1 / 3 dilution of normal human serum as a source of complement (EC50 (nM) values from the curve fits are shown);

[0048] Figure 12 provides the results of assays showing that IgG1 antibodies comprising Fc region components with the H429F mutation of the CH3 domain strongly fix complement and show identical antigen binding. (A,B) TNP-BSA antigen was adsorbed at different concentrations (20 μg / ml-0.625 μg / ml) to the wells of ELISA plates and were reacted with a single concentration (2 μg / ml) of chimeric anti-TNP human IgG1 and IgG2 mAbs comprising unmodified wildtype (WT) heavy chains or with 2 μg / ml anti-TNP mAbs comprising IgG1 heavy chains comprising the Fc component mutation H429F (TNP-IgG1-H429F). The antibody-opsonised TNP-BSA coated wells were treated with (A) purified human C1q and the fixation of C1q detected with anti-C1q rabbit polyclonal antibody and (B) with human serum as a source of complement and the formation of the membrane attack complex (C5b-C9) was detected with anti-C5b-C9 rabbit polyclonal antibody. (C) provides the results from an ELISA showing the antigen binding activity of the chimeric anti-TNP mAbs used in panels A and B including the unmodified (TNP-IgG1-WT and TNP-IgG2-WT mAbs) and H429 modified mAb, namely TNP-IgG1- H429F. ELISA plates were coated with TNP-BSA then anti-TNP mAbs titrated and antibody binding detected with HRP-conjugated anti-human IgG. All mAbs showed similar antigen binding activity;

[0049] Figure 13 provides results showing C1q fixation and MAC (C5b-9) formation by mAbs comprising IgG1 heavy chains with glutamic acid, glutamine or serine modifications at position 429 of the heavy chain. TNP-BSA (20 μg / ml) was adsorbed to the wells and reacted with chimeric human anti- TNP mAbs, titrated across the concentration range 4 μg / ml – 0.125 μg / ml. The mAbs tested comprised unmodified wild type (WT) heavy chains of IgG1 (TNP-IgG1-WT) (panels A-D) or IgG2 (TNP-IgG2- WT) (panels A-C) or IgG1 H chains comprising mutations TNP-IgG1-H429Q or TNP-IgG1-H429E (A,C) and TNP-IgG1-H429S (B,D). Antibody opsonised TNP-BSA coated wells were treated with human serum as a source of complement. The fixation of C1q (A,B) was detected with anti-C1q rabbit polyclonal antibody and (C,D) the formation of the membrane attack complex (C5b-C9) was detected with rabbit anti-C5b – C9 polyclonal antibody;

[0050] Figure 14 provides the results of flow cytometric quantitation of SEC purified mAb binding to target cell surface antigens. The binding activity of the unmodified (WT) rituximab, daratumumab-WT, 11B8-WT mAbs and their CH3-modified mutants carrying the mutations H429F or H429Y (SEC (IgGH2L2) peak fraction p1 and IgGoli peak fraction evaluated by flow cytometry on Ramos lymphoma cells expressing CD20 and CD38. The binding activity of the unmodified trastuzumab-WT, pertuzumab-WT and their CH3 heavy chain mutants carrying the mutation H429F was evaluated on SK- OV-3 cells expressing HER2;

[0051] Figure 15 provides elution chromotagrams of Protein A affinity purification characteristics of rituximab-WT, trastuzumab-WT and their mutants bearing modification in the CH3 of the heavy chain. The IgG was recovered from the column following elution with sodium citrate buffer pH 3.0: (A) Elution profiles of anti-CD20 rituximab-based mAbs. The rituximab-WT IgG and each mutant IgG eluted as a single homogenous peak; (B) Elution profiles of anti-HER2 trastuzumab-based mAbs. The trastuzumab- WT IgG and each mutant IgG eluted as a single homogenous peak;

[0052] Figure 16 provides size exclusion chromatography (SEC) profiles revealing that H429 mutation can alter the physical properties of IgG. Following Protein A affinity purification, the mAbs were further purified by SEC at pH 7.2: (A) The SEC profiles of the unmodified rituximab-WT (WT) and the rituximab-H429F (H429F) mutant consisted of a single homogenous IgG peak (fractions right of the vertical dashed line) with minimal oligomeric species, (fractions left of the vertical dashed line). Thus, for example, the SEC profile of unmodified rituximab-WT showed a single major species corresponding, as expected, to IgG (H2L2) as confirmed by SDS-PAGE analysis (Figure 17A) where it migrated at the expected ~150kDa mass of unreduced IgG and which resolved, after reduction in DTT, to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species. The rituximab-H429Y mAb on the other hand comprised both non-oligomeric IgG and oligomeric IgG; (B) The SEC profiles of Protein A purified anti- HER2 trastuzumab wild-type (WT) (that is, an unmodified form of trastuzumab) and its mutants including the amino acid substitution of H429F or H429Y. A single peak of non-oligomeric IgG, to the right of the dashed vertical line, was observed for the trastuzumab-WT, and the H429F mutant mAb, but the trastuzumab-H429Y mutant IgG (H429Y) contained both non-oligomeric IgG and oligomeric IgG. The non-oligomeric IgG peak of the trastuzumab-H429Y was coincident with that of the unmodified trastuzumab-WT, or its H429F variant and the equivalent rituximab-based mAbs in panel (A). The SEC profile of the oligomeric IgG in trastuzumab-H429Y was coincident with the oligomeric IgG in rituximab-H429Y in panel (A);

[0053] Figure 17 provides images obtained from SDS-PAGE of SEC purified mAbs. mAbs purified by SEC were analysed by SDS-PAGE in 5-15% gradient gels with or without reduction of disulphide bonds: (A) Under non-reducing conditions, the non-oligomeric IgG peak of rituximab-WT (WT IgG) and rituximab-H429F (HF IgG) migrated as a single species at the expected ~ 150kDa molecular size of IgG (i.e. H2L2). Following reduction in DTT, the 150kDa IgG resolved as expected into ~50kDa heavy (H)chain and ~25kDa light (L) chains. Prior to the non-oligomeric IgG (HY IgG (H2L2)) and oligomeric (HY IgG(oli)) forms of rituximab-H429Y mAb (see Figure 16), both migrated identically as a single 150kDa IgG i.e. H2L2species. Following reduction, both forms migrated as ~50kDa heavy (H) chain and ~25kDa light (L) chain; WT=wild-type, HF=H429F, HY=H429Y; M= molecular-weight markers, their masses (kD) are shown on the left side; (B) Under non-reducing conditions, the IgG peak of trastuzumab-WT (WT IgG), and trastuzumab-H429F (HF IgG) migrated as a single species at the expected ~ 150kD molecular size of IgG i.e. H2L2. Following reduction with dithiothreitol (DTT), the 150kDa IgG resolved as expected into ~50kDa heavy (H) chain and ~25kDa light (L) chains. Prior to reduction, the non-oligomeric IgG (HY IgG (H2L2)) and oligomeric (HY IgG(oli)) forms of trastuzumab- H429Y mAb both migrated identically as a single 150kDa IgG species under non-reducing conditions and following reduction, as the expected ~50kDa heavy (H) chain and ~25kDa light (L) chain; WT=wild- type, HF=H429F, HY=H429Y; M = molecular-weight markers, their masses (kD) are shown on the left side;

[0054] Figure 18 provides the results of experimentation 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 the rituximab-H429Y at pH7.2 showing the presence of two major IgG forms (see also Figure 16A). The non-oligomeric IgG (H2L2) (right of the vertical dashed line, indicated as p1) and the oligomeric form of IgG (IgG(oli)), (left of the vertical dashed line indicated as p2) were separately collected for further evaluation of complement-dependent cytotoxicity (CDC) potency; (B) The oligomeric (p2) and non-oligomeric (p1) IgG forms from panel A, and the SEC purified unmodified rituximab-WT IgG were titrated and CDC potency was determined by flow cytometry using Ramos lymphoma cells and normal human serum diluted 1 / 3 as the source of complement. The extent of killing of the Ramos cells opsonised with the non-oligomeric rituximab H429Y p1 (open squares), oligomeric rituximab-H429Y p2 (triangles) or unmodified rituximab-WT (filled circles) was determined. The control background CDC of complement in the absence of mAb (no mAb C' only) is shown as a filled diamond; and (C) The formation of IgG oligomers is sensitive to pH. Protein A purified trastuzumab-H429Y was analysed by size exclusion chromatography (SEC) either at pH 7.2 (left panel) or at pH 5.0 (right panel). At pH 7.2, both oligomeric (IgG (oli)) and non-oligomeric IgG were present, however at pH 5.0, only the single peak corresponding to non-oligomeric IgG was present;

[0055] Figure 19 provides results which show that an H429F mutation in the CH3 domain of the IgG H chain potently promotes C1q binding and complement dependent cell-mediated cytotoxicity. Flow cytometric detection of C1q binding (fluorescence intensity) to Ramos lymphoma cells opsonised with unmodified rituximab-WT (A) or with the modified rituximab-H429F mAb (B), or to SK-OV-3 ovarian cancer cells opsonised with trastuzumab-WT (C) or with the modified trastuzumab-H429F mAb (D) isdepicted. The cells opsonised with the mAbs 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 C1q binding to the mAb opsonised cells is shown in the un-shaded histograms and the C1q background binding control (i.e. cells treated with serum complement in the absence of mAbs), is shown in the grey-shaded histograms. The median fluorescence intensity (MFI) values for each histogram are indicated in parentheses. (E) The complement-dependent cytotoxicity (CDC; i.e. % killing detected using Zombie Green) of Ramos lymphoma cells opsonised with rituximab-WT (filled circles) or its mutated variant mAb rituximab-H429F (filled squares), was determined by flow cytometry using normal human serum as the source of complement. CDC is greatly enhanced by mutation at position 429 in the rituximab-H429F to compared rituximab-WT mAb;

[0056] Figure 20 shows the results of Protein A affinity chromatography of Type-II anti-CD2011B8- WT mAb and mutant mAb including an H429 substitution. The mAbs were 11B8-WT mAb (WT) produced with a wild-type human IgG1 heavy chain. The 11B8-H429F (H429F) was produced with a modified IgG1 heavy chain wherein amino acid histidine at position 429 in the CH3 domain of the Ig heavy chain was replaced with phenylalanine;

[0057] Figure 21 shows the results of size exclusion chromatography (SEC) purification and SDS PAGE analysis of the 11B8-WT mAb and the 11B8-H429F (H429F) mutant. (A) Chromatograms of the mAbs purified by SEC at pH 7.2; monomeric non-oligomeric IgG is shown to the right of the vertical dotted line and (B) SDS-PAGE analysis (5-15% gradient gel) of the SEC-purified monomeric IgG mAbs from panel A. Prior to reduction (Non-Reduced), all SEC purified antibodies migrated at the expected ~ 150kDa molecular size of IgG i.e H2L2. Following reduction with DTT (Reduced), all of the antibodies resolved as expected into ~50kDa heavy (H) chain and ~25kDa light (L) chain. WT=wild-type, HF=H429F, M = molecular-weight markers, their masses (kD) are shown on the left side;

[0058] Figure 22 provides results showing that H429 modification of the CH3 domain confers CDC potency on a type II anti-CD20 mAb. CDC by the 11B8-WT mAb (solid circles) or CH3 mutated 11B8- H429F mAb (solid squares). Complement-dependent lysis was determined using Ramos lymphoma cells opsonised with the mAbs at the indicated concentrations and normal human serum diluted 1 / 3 as the source of complement. CDC (% killing) was determined by flow cytometry using Zombie Green. The control background CDC of complement in the absence of mAb (no mAb C' only) is shown as a filled diamond. The 11B8-WT mAb failed to mediate CDC whereas the 11B8-H429F mutant mAb mediated potent complement-dependent lysis of lymphoma cells (WT = wild type);

[0059] Figure 23 shows the results of Protein A chromatography of the anti-CD38 daratumumab-WT mAb and the daratumumab-H429F mAb. Elution chromatograms showed that each of the antibodies eluted as a single coincident homogenous peak;

[0060] Figure 24 shows the results of size exclusion chromatography (SEC) purification and SDS- PAGE analysis of the daratumumab-WT mAb as well as the mutant mAb, daratumumab-H429F. (A) Chromatograms of the SEC purified mAbs. In each case, the mAbs contained a single non-oligomeric IgG peak (fractions right of the vertical dashed line) and the absence of oligomeric species (fractions left of the vertical dashed line); (B) SDS-PAGE analysis (5-15% gradient gel) of SEC purified IgG from panel A. Prior to reduction (Non-Reduced), all of the antibodies migrated at the expected ~ 150kDa molecular size of IgG (i.e. H2L2), and following reduction with DTT (Reduced), all of the antibodies resolved as expected into ~50kDa heavy (H) chain and ~25kDa light (L) chain. WT=wild-type, HF=H429F, M = molecular-weight markers, their masses (kD) are shown on the left side;

[0061] Figure 25 provides results which show that the H429F substitution profoundly enhances complement-dependent lysis of lymphoma cells by daratumumab-WT. The graph shows the level of CDC (% killing) by the daratumumab-WT mAb (solid circles) and daratumumab-H429F (solid squares). Background lysis (solid triangle) was determined in the absence of mAb, but in the presence of only complement. CDC was determined by flow cytometry using Zombie Green. The daratumumab-H429F mAb mediated more potent complement-dependent lysis of lymphoma cells than the unmodified daratumumab-WT;

[0062] Figure 26 provides results showing that the H429F substitution confers complement-dependent lysis of myeloma and leukaemia cells resistant to lysis by anti-CD38 mAb. CDC by complement- dependent lysis was determined using: (A) KMS-12-PE myeloma cells opsonised with the daratumumab- WT mAb (solid circles) or daratumumab-H429F mutant mAb (solid squares) at the indicated concentrations. Background lysis (solid triangle) was determined in the absence of mAb, but in the presence of only complement; and (B) SUP-15 acute lymphoblastic leukaemia (ALL) cells which were opsonised with the daratumumab-WT mAb (solid circles) and daratumumab-H429F mutant mAb (solid squares) at the indicated concentrations. Background lysis (solid triangle) was determined in the absence of mAb but in the presence of only complement. The CDC (% killing) was determined by flow cytometry using Zombie Green;

[0063] Figure 27 shows the results of Protein A affinity chromatography of the anti-HER2 mAb pertuzumab-WT and CH3 H429F variant. Elution chromatograms showed that each of the antibodies eluted as a single coincident homogenous peak;

[0064] Figure 28 shows the results of size chromatography (SEC) purification and SDS- PAGE analysis of the anti-HER2 mAb pertuzumab-WT and CH3 H429F variant. (A) Chromatograms of SEC purified mAbs. Following Protein A affinity chromatography, the mAbs were further purified by size exclusion chromatography (SEC) at pH 7.2. The chromatographic profiles are shown for unmodified pertuzumab-WT and for an Fc modified variant, pertuzumab-H429F (H429F). In each case, the Protein A-purified mAbs contained a single non-oligomeric IgG peak (fractions right of the vertical dashed line) and the absence of oligomeric species (fractions left of the vertical dashed line). (B) SDS-PAGE analysis (5-15% gradient gel) of SEC purified IgG from panel A. Prior to reduction (Non-Reduced), all of the SEC purified antibodies migrated at the expected ~ 150kDa molecular size of IgG (i.e. H2L2). Following reduction with DTT (Reduced), all of the antibodies resolved as expected into ~50kDa heavy (H) chain and ~25kDa light (L) chain. WT=wild-type, HF=H429F, M = molecular-weight markers shown in kilodaltons (kD);

[0065] Figure 29 provides results showing the co-operation and functional synergy in mixtures of mAbs including amino acid substitution at position H429; in particular, the results show that H429F-modified mAbs can combine to enhance C1q binding to target cells: (A) Flow cytometric histograms of C1q binding to HER2 expressing SK-OV-3 cells opsonised (unfilled histograms) with trastuzumab-H429F alone, pertuzumab-H429F alone or a 1:1 mixture of trastuzumab-H429F and pertuzumab-H429F. Filled histograms show the background binding of C1q to non-opsonised cells. The Median Fluorescence Intensity (MFI) values for each histogram is 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 opsonised with titrated individual anti-HER2 mAbs, trastuzumab-WT, pertuzumab-WT, trastuzumab-H429F, pertuzumab-H429F or with 1:1 mixtures of trastuzumab-WT with pertuzumab-WT or trastuzumab-H429F with pertuzumab-H429F. The double-headed vertical arrow exemplifies extensive cooperation in the mixture of trastuzumab-H429F with pertuzumab-H429F where antibody concentrations are limiting compared to C1q binding of either mAb alone;

[0066] Figure 30 provides graphical results showing that the co-operation and functional synergy of H429-modified mAbs enhances complement dependent killing (CDC) of target cells. The indicated rituximab-based mAbs were titrated alone (light grey columns) on Ramos cells or titrated in the presence of a fixed concentration of the indicated anti-CD38 or anti-CD20 mAbs (dark columns) : (A) rituximab- WT titrated in the presence of 0.025 µg / ml, daratumumab-WT, (B) rituximab-HF titrated in the presence 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 dotted horizontal line in (B, C) indicates the percentage of CDC killing obtained in the absence (0µg / ml) of rituximab-based mAbs, but in the presence only of (B) 0.025µg / ml daratumumab-H429F or (C) 0.5µg / ml 11B8-H429F.The black arrows in (B, C) indicate the enhanced of the mixtures of mAbs above that of the individual mAbs (i.e. the CDC of either the rituximab-H429F alone at the indicated concentrations (light columns) or of 0.025µg / ml daratumumab-H429F or 0.5µg / ml 11B8-H429F (dark column, 0µg / ml rituximab));

[0067] Figure 31 provides the results of flow cytometry analysis of CDC potency of a rituximab antibody comprising an H429F mutation on normal peripheral blood CD19+B lymphocytes. The results indicate that the rituximab-H429F mAb exhibits more potent CDC killing of normal peripheral blood B cells compared to unmodified rituximab-WT. Following treatment of peripheral blood mononuclear cells with the indicated mAbs and human complement, the proportion of live or dead B cells was determined by staining with anti-CD19 to identify B cells and also with Zombie Green (ZG) to identify dead cells. The cytograms show Zombie Green median fluorescence intensity (ZG MFI) of gated CD19 B cells (CD19 median fluorescence intensity (CD19 MFI). In each cytogram, the % of dead cells (ZG positive) is shown in the upper section, Q1, of each cytogram 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 % live B cells are shown in the lower section, Q2;

[0068] Figure 32 provides results showing that the effects of modification of H429 on the function of antibodies are not restricted to IgG1 and extend to other immunoglobulin types. Here, CDC potency on Ramos lymphoma cells of rituximab antibodies formatted with wild-type (WT) or H429F (HF) modified heavy chains of (A) IgG3 or (B) IgG4 subclasses were assessed;

[0069] Figure 33 provides results showing that potent CDC of target cells mediated by mAbs is dependent on the presence of both the monoclonal antibody and serum complement. CDC (% killing) of Ramos cells by rituximab-H429F, daratumumab-H429F or 11B8-H429F, or of SUP-15 cells by daratumumab-H429F is shown. A = percent killing in the presence of both the indicated mAb and human serum as a source of complement; B = lysis in the presence of mAb only at the same concentration as in A; and C= lysis in the presence of complement only;

[0070] Figure 34 provides results showing that the H429F substitution confers complement-dependent lysis of leukaemia cells resistant to lysis by the unmodified anti-CD38 mAb isatuximab. CDC by complement-dependent lysis was determined using SUP-15 acute lymphoblastic leukaemia (ALL) cells which were opsonised with the isatuximab-WT mAb (dashed line with filled circles) and isatuximab- H429F mutant mAb (solid line with filled squares) at the indicated concentrations. Background lysis (unfilled circle) was determined in the absence of mAb but in the presence of only complement (C' only,no mAb). Background lysis induced by the mAbs 5 μg / ml in the absence of complement was determined for isatuximab-WT mAb (inverted unfilled triangle) and isatuximab-H429F mutant mAb (open unfilled square). The CDC (% killing) was determined by flow cytometry using Zombie Green;

[0071] Figure 35 provides the results of flow cytometric quantitation of the binding to Colo205 colorectal cells of purified DR5-specific mAbs that comprised unmodified wild-type (WT) H chains of the human IgG1 or IgG2 subclasses or comprised human IgG1 or human IgG2 subclass heavy chains that had been modified by replacement of histidine 429 with phenylalanine (H429F). The panels show: (A) binding of BDR5-1WT which comprised wild-type H chains of the human IgG1 subclass or binding of the BDR5-1HF mAb which comprised IgG1 heavy chains containing the H429F modification, (B) binding of BDR5-2WT which comprised unmodified heavy chains of the human IgG2 subclass or binding of the BDR5-2HF mAb which comprised IgG2 heavy chains containing the H429F modification, (C) the binding of the TDR5-1WT mAb which comprised unmodified heavy chains of the human IgG1 subclass or the TDR5-1HF mAb which comprised IgG1 heavy chains containing the H429F modification, and (D) binding of TDR5-2WT which comprised unmodified heavy chains of the human IgG2 subclass or binding the TDR5-2HF mAb which comprised IgG2 heavy chains containing the H429F modification. The mAbs were serially 2-fold titrated and the binding activity on Colo205 cells was quantified by flow cytometry using an anti-IgG secondary reagent labelled with goat anti-hIgG Fc FITC. In all panels, the level of non-specific binding of fluorescent conjugate to cells is shown (▲ conj); MFI = Median Fluorescence Intensity;

[0072] Figure 36 provides the results of flow cytometric quantitation of the binding to target Ramos lymphoma cells of purified DR5-specific mAbs that comprised unmodified wild-type (WT) H chains of the human IgG1 or human IgG2 subclasses or comprised human IgG1 or human IgG2 heavy chains that had been modified by replacement of histidine 429 with phenylalanine (H429F). The panels show: (A) binding of BDR5-1WT which comprised unmodified heavy chains of the human IgG1 subclass or binding of the BDR5-1HF mAb which comprised IgG1 heavy chains containing the H429F modification; (B) binding of BDR5-2WT which comprised unmodified heavy chains of the human IgG2 subclass or binding of the BDR5-2HF mAb which comprised IgG2 heavy chains containing the H429F modification; (C) binding of TDR5-1WT which comprised unmodified heavy chains of the human IgG1 subclass or binding of the TDR5-1HF mAb which comprised IgG1 heavy chains containing the H429F modification; and (D) binding of TDR5-2WT which comprised unmodified heavy chains of the human IgG2 subclass or binding of the TDR5-2HF mAb which comprised IgG2 heavy chains containing the H429F modification. The mAbs were serially 2-fold titrated and the binding to Ramos cells was quantified by flow cytometry using an anti-IgG secondary reagent labelled with goat anti-hIgG Fc FITC. In all panels, the level of non- specific binding to cells was determined using an irrelevant IgG antibody (negative IgG) and thebackground binding of the fluorescence anti-IgG to cells is shown (▲ conj); MFI = Median Fluorescence Intensity;

[0073] Figure 37 provides the results of flow cytometric quantitation of the binding to target KMS-12- PE myeloma cells of purified DR5-specific mAbs that comprised unmodified wild-type (WT) H chains of the human IgG1 or human IgG2 subclasses or comprised human IgG1 or human IgG2 heavy chains that had been modified by replacement of histidine 429 with phenylalanine (H429F). The binding activity is shown for the DR5-specific mAbs, BDR5 and TDR5, which comprised unmodified, wild-type, H chains of the human IgG1 subclass (BDR5-1WT, TDR5-1WT) or human IgG2 subclass (BDR5-2WT, TDR5- 2WT) or comprised Fc mutated H chains of the IgG1 subclass (BDR5-1HF, TDR5-1HF) or IgG2 subclass (BDR5-2HF, TDR5-2HF) containing the H249F mutation. KMS12-PE cells were incubated with mAbs at 5μg / ml. Binding activity was quantified by flow cytometry using an anti-IgG secondary reagent labelled with FITC goat anti-hIgG Fc. The background control of anti-IgG conjugate only binding is shown (conj.) along with the background fluorescence of cells only (cells);

[0074] Figure 38 provides results showing the survival of Colo205 colorectal cells in the presence of purified DR5-specific mAbs comprised of unmodified wild-type H chains of the human IgG1 or human IgG2 subclasses, or comprised of Fc mutated H chains of the human IgG1 or human IgG2 subclasses containing the H429F mutation. 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 which comprised unmodified wild-type H chains of the human IgG1 subclass, or TDR5-1HF mAb which comprised H chains of the human IgG1 subclass containing the H429F mutation, or TDR5-2WT mAb which comprised unmodified wild-type H chains of the human IgG2 subclass, or TDR5-2HF mAb which comprised H chain of the human IgG2 subclass containing the H429F mutation. Additionally, Colo205 cells were separately cultured 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 comprised Fc mutated human IgG2 H chains carrying H429F mutations. Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by the culture of cells in the absence of antibody (no ab). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium], the mean Abs450 and the four replicate values are shown;

[0075] Figure 39 provides results showing the survival of Ramos lymphoma cells in the presence of purified DR5-specific mAbs comprised of unmodified wild-type or Fc mutated H chains of the human IgG1 or IgG2 subclasses containing the H429F mutation. 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 whichcomprised unmodified wild-type H chains of the IgG1 subclass, or TDR5-1HF mAb which comprised H chain of the human IgG1 subclass containing the H429F mutation, TDR5-2WT mAb which comprised unmodified wild-type H chains of the human IgG2 subclass, or TDR5-2HF mAb which comprised H chains of the human IgG2 subclass containing the H429F mutation. Additionally, Ramos cells were separately cultured 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 comprised Fc mutated human IgG2 H chains carrying H429F mutations. Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by culture of cells in the absence of antibody (no ab). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium], the mean Abs450 and the four replicate values are shown;

[0076] Figure 40 provides results showing the survival of Colo205 colorectal cells in the presence of mixtures of purified DR5-specific mAbs wherein the mAbs comprised wild-type or Fc modified H chains of the same IgG subclass. The BDR5 and TDR5 mAbs were comprised of unmodified wild-type H chains of the human IgG1 or human IgG2 subclasses or were comprised of heavy chains of the IgG1 subclass or IgG2 subclasses containing the H429F modification. Colo205 cells, 30,000 cells per well of 96-well plate, were cultured in the presence of serial 2-fold dilutions of 1:1 mixtures of mAbs comprised of identical H chains. The starting concentration of 1 μg / ml was comprised of 0.5 μg / ml of each mAb in the mixture. Thus, the mixtures used were: BDR5-1WT mAb comprising unmodified wild-type IgG1 H chain mAb mixed with TDR5-1WT comprising unmodified wild-type IgG1 H chain (BDR5-1WT + TDR5-1WT); BDR5-2WT mAb comprising unmodified wild-type IgG2 H chain mAb mixed with TDR5-2WT comprising unmodified wild-type IgG2 H chain (BDR5-2WT + TDR5-2WT); BDR5-1HF mAb comprising IgG1 H chains containing H429F modification mixed with TDR5-1HF mAb comprising IgG1 H chains containing H429F modification (BDR5-1HF + TDR5-1HF); BDR5-2HF mAb comprising IgG2 H chains containing H429F modification mixed with TDR5-2HF mAb comprising IgG2 H chains containing H429F modification (BDR5-2HF + TDR5-2HF). Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by the culture of cells in the absence of antibody (no ab) and maximum death determined by culture of cells with sodium dodecyl sulphate (SDS). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium];

[0077] Figure 41 provides results showing the survival of Colo205 colorectal cells in the presence of different pairwise combinations of purified DR5-specific mAbs wherein the mAbs comprised wild-type or Fc mutated H chains of different IgG subclasses. MAbs comprised H chains of human IgG1 subclassor human IgG2 subclass comprising wild-type chains or comprising heavy chains modified by H429F modification. Colo205 cells, 10,000 cells per well of 96-well tissue culture plate, were cultured in the presence of 1:1 mixtures of mAbs serially diluted 2-fold from 1μg / ml. The starting concentration of 1 μg / ml was comprised of 0.5 μg / ml of each mAb in the mixture. The mixtures of wild-type mAbs used were: BDR5-2WT comprising wild-type human IgG2 H chain mixed with TDR5-1WT comprising unmodified wild-type human IgG1 H chain (BDR5-2WT + TDR5-1WT); BDR5-1WT comprising wild- type human IgG1 H chain mixed with TDR5-2WT comprising unmodified wild-type human IgG2 H chain (BDR5-1WT + TDR5-2WT); and a control mixture of BDR5-2WT comprising unmodified wild- type human IgG2 H chain mixed with TDR5-2WT comprising unmodified wild-type human IgG2 H chain (BDR5-2WT + TDR5-2WT) as observed in Figure 40. The mixtures of DR5 mAbs comprising H429F Fc-mutated H chains were: BDR5-2HF comprising H429F Fc-mutated human IgG2 H chain mixed with TDR5-1HF comprising H429F Fc-mutated human IgG1 H chain (BDR5-2HF + TDR5-1HF); BDR5-1HF comprising H429F Fc-mutated human IgG1 H chain mixed with TDR5-2HF comprising H429F Fc-mutated human IgG2 H chain (BDR5-2HF + TDR5-2HF); positive killing control mixture of BDR5-2HF comprising H429F Fc-mutated human IgG2 H chain mixed with TDR5-2HF comprising H429F Fc-mutated IgG2 H chain (BDR52HF+TDR5-2HF) as observed in Figure 38 and Figure 40. Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by culture of cells in the absence of antibody (no ab) and maximum death determined by culture of cells with sodium dodecyl sulphate (SDS). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium];

[0078] Figure 42 provides results showing the survival of Colo205 cells in the presence of pairwise mixtures of BDR5 and TDR5 mAbs at different ratios. Ten thousand Colo205 cells were cultured for 48 hours in the presence of individual mAbs BDR5-1WT or TDR5-1WT comprising wild-type IgG1 H chains or individual mAbs BDR5-1HF or TDR5-1HF comprising H chains with the H429F modification or mixtures of BDR5-1HF with TDR5-1HF mAbs at the following ratios, 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixes shown in the plot, the BDR5-1HF mAb is designated as B-1HF and TDR5-1HF is designated as T-1HF. Controls include maximum cell survival in the absence of mAbs (no ab) or maximum death control in the presence of sodium dodecyl sulphate (SDS). Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium];

[0079] Figure 43 provides results showing the survival of Ramos lymphoma cells in the presence of pairwise mixtures of BDR5 and TDR5 mAbs at different ratios.10,000 Ramos cells were cultured for 48hours in the presence of individual mAbs BDR5- or TDR5-1WT comprising wild-type H chains or individual mAbs BDR5-1HF or TDR5-1HF comprising H chains with the H429F modification or mixtures of BDR5-1HF with TDR5-1HF mAbs at the following ratios, 90:10, 75:25, 50:50, 25:75, and 10:90. In the mixes shown in the plot, the BDR5-1HF mAb is designated as B-1HF and TDR5-1HF is designated as T-1HF. Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by culture of cells in the absence of antibody (no ab) and maximum death determined by culture of cells with sodium dodecyl sulphate (SDS). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally – background Abs450 of cell culture medium];

[0080] Figure 44 provides results quantitating the binding to Colo205 cells of DR5-specific mAbs that detect distinct epitopes and comprise wild-type or Fc mutated heavy chains of the human IgA2 subclass. The mAbs, used as tissue culture supernatants from appropriately transfected Expi293 cells, comprised unmodified wild-type IgA2 heavy chain (BDR5-A2WT) and (TDR5-A2WT) or Fc mutated IgA2 H chains carrying the H429F mutation (BDR5-A2HF) and (TDR5-A2HF) of the human IgA2 subclass. Background fluorescence determined by measuring binding of fluorescence conjugate only (no ab) is shown along with the non-specific fluorescence of cells only (cells);

[0081] Figure 45 provides results showing the Colo205 cell survival in the presence of two distinct DR5-specific mAbs comprising heavy chains of the human IgA2 subclass. Colo205 cells, 10,000 cells per well of 96-well plate, were cultured for 48 hours in the presence of serial 2-fold dilutions of tissue culture supernatants from Expi293 cells producing the DR5 IgA mAbs used in Figure 44. The mAbs used were: BDR5-A2WT which comprised wild-type heavy chain of the human IgA2 subclass; TDR5-A2WT which comprised wild-type heavy chain of human IgA2 subclass; BDR5-A2HF which comprised heavy chains of the human IgA2 subclass carrying the H429F modification; TDR5-A2HF which comprised heavy chains of the human IgA2 subclass carrying the H429F modification. Cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by culture of cells in the absence of antibody (no ab) and maximum death determined by culture of cells with sodium dodecyl sulphate (SDS). Survival is represented by: [experimentally determined Abs450nm – background Abs450 of cell culture medium];

[0082] Figure 46 provides results showing the enhanced killing of Ramos lymphoma cells by mixtures of H429F-modified mAbs detecting distinct molecular targets. Ramos cells at 10,000 per well were separately cultured in the presence of anti-CD38 mAb isatuximab comprising IgG1 WT H Chains (Isa-WT), or anti-DR5 mAb BDR5-1 comprising H chains (BDR5-1WT), or a 1:1 mixture of both mAbs (Isa-WT + BDR5-1WT) or cultured with their Fc modified counterparts, isatuximab comprising H chains carrying the H429F modification (Isa-HF) as described in Example 7 and BDR5-1 comprising H chains carrying the H429F modification (BDR5-1HF) or a 1:1 mixture of both (Isa-HF + BDR5-1HF). All mAbs were present in the cultures at 10 μg / ml and, for the mixtures, at 10µg / ml of each mAb for a total mAb concentration of 20µg / ml. After culture for 48 h, cell viability was quantitated using a CCK8 colorimetric cell viability assay where absorbance (Abs450nm) of the supernatant of the CCK8 treated cells is a measure of cell survival and viability. Maximum cell viability was determined by the culture of cells in the absence of antibody (no Ab) and maximum cell death by cell incubation with SDS before CCK8 treatment of the cells. Survival is represented by: [experimentally determined Abs450nm – background Abs450 of cell culture medium], the mean Abs450 and the four replicate values are shown;

[0083] Figure 47 provides results showing that H429F substitution confers complement-dependent lysis (CDC) of leukaemia cells resistant to lysis by the unmodified anti-CD38 mAb mezagitamab. CDC by complement-dependent lysis was determined using Ramos lymphoma cells which were opsonised especially with the mezagitamab-WT mAb or mezagitamab-H429F mutant mAb at the indicated concentrations. Background lysis was determined in the absence of mAb but in the presence of only complement (C' only). The CDC (% killing) was determined by flow cytometry using Zombie Green;

[0084] Figure 48 provides results showing that additional mutations of K439E or S440K in the Fc region component may suppress the enhanced CDC of H429F-modified rituximab and that the combination of rituximab-H429F / K439E with rituximab-H429F / S440K restores efficient CDC killing of Ramos cells. (A) Ramos lymphoma cells were opsonised with the individual rituximab-WT (WT) or rituximab-H429F (H429F) mAbs or with rituximab-H429F comprising the additional H chain mutation K439E (H429F / K439E) or with rituximab-H429F comprising the additional H chain mutation S440K (H429F / S440K), or with pairwise mixtures of rituximab-H429F / S440K with rituximab-H429F / K439E (H429F / K439E + H429F / S440K). Background lysis was determined in the absence of mAb but in the presence of only complement (no mAb C' only). The CDC (% killing) of the mAbs was measured in the presence of a 1 / 3 dilution of human serum by flow cytometry using Zombie green. The enhanced CDC mediated by H429F modification of rituximab was supressed by either Fc:Fc interactions inhibiting the K439E or S440K mutations, but was found to be fully recovered by the mixture of both IgG mutants. (B) For the binding analysis, the proteins were serially two-fold titrated and binding activity (on Ramos cells) quantified by flow cytometry using a FITC-conjugated anti-hIgG-Fc secondary reagent. The level of non- specific background binding of fluorescent conjugate to cells is shown (conj only); MFI = Median Fluorescence Intensity. All mAbs gave near identical binding to CD20;

[0085] Figure 49 provides the results of a flow analysis of binding (A, C) and CDC (B, D) of purified flACE2-Fc fusion protein (A, B) and SARS-CoV spike-specific mAbs (C, D) using Ramos-S cells. For the binding analysis, the proteins were serially two-fold titrated and binding activity (on Ramos-S cells) quantified by flow cytometry using a FITC-conjugated anti-hIgG-Fc secondary reagent. The level of non-specific background binding of fluorescent conjugate to cells is shown (conj); MFI = Median Fluorescence Intensity. For CDC analysis, the proteins were serially two-fold titrated and human serum used as a source of complement. The CDC lysis was quantitated using Zombie Green. Background lysis by complement in the absence antibodies was determined and is shown (no mAb C’ only);

[0086] Figure 50 provides graphical results from flow cytometric analysis showing that cooperative and functional synergy of the H429F-modified flACE2-Fc fusion protein and H429F-modified anti-SARS- CoV-2 mAbs further enhances complement dependent killing (CDC). (A) The mAbs S2P6-H429F (S2P6- HF) or S2P6-WT were titrated alone or titrated in the presence of a fixed concentration of the flACE2-Fc- H429 fusion protein (1 μg / ml final concentration) (S2P6-HF + flACE2-Fc-HF) that, when used alone, mediated CDC killing of 23.4% (indicated by open diamond symbol), or were titrated in the presence of fixed concentration of flACE2-Fc-WT (1 μg / ml final) (S2P6-WT + flACE2-Fc-WT) which, when used alone, mediated a % kill of 5.0% (filled diamond). The background lysis by complement in the absence of mAb or Fc fusion protein is shown (C’only); Arrow shows example of greatest synergy; (B) CDC killing potency of H429F- modified flACE2-Fc was evaluated on Ramos-S cells. The 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 concentrations of the mAbs was 2.5 μg / ml and of the flACE2-Fc-H429F was 1μg / ml). Killing potency was evaluated in the flow cytometric assay using Zombie Green. The % CDC killing mediated by flACE- 2-Fc-WT and background lysis by complement in the absence of mAb or Fc fusion protein (C'only) is also shown. Four replicate values and SEM are shown. Mean % kill value is shown above each column;

[0087] Figure 51 provides results showing that ACE2-Fc fusion proteins in three formats, namely trACE2-Fc, flACE2Fc and EflACE2-Fc, comprising Fc region components with the wild type sequence or the H429F mutation bind equivalently to Ramos-S target cells; as determined using flow cytometric analysis using an anti-IgG secondary reagent labelled with goat anti-hIgG Fc FITC: (A) trACE2-Fc (mean ± SEM, n = 3); (B) flACE2-Fc (mean ± SEM, n = 3); and (C) EflACE2-Fc (WT n = 3, H429F n = 1). In all panels, the level of non-specific binding of fluorescent conjugate to cells is shown (conj only); MFI = Median Fluorescence Intensity. The EC50 (nM) values from the curve fits are shown;

[0088] Figure 52 provides results showing that the ACE2-Fc fusion proteins in three formats (trACE2- Fc, flACE2-Fc and EflACE2-Fc) comprising Fc region components with the wild type sequence or theH429F mutation, strongly fix complement and complement dependent cytotoxicity (CDC) of Ramos-S target cells; as determined using flow cytometric analysis of CDC of opsonised Ramos-S cells using diluted normal human serum as a source of complement: (A) trACE2-Fc (mean ± SEM, n = 3); (B) flACE2-Fc (mean ± SEM, n = 3); (C) EflACE2-Fc (WT n = 3, H429F n = 1). In all panels, the level of non-specific complement killing in the absence of ACE2-Fc fusion proteins is shown complement only (C’ only). The EC50 (nM) values from the curve fits are shown for the H429F proteins. Fits for the WT proteins could not be determined (nd);

[0089] Figure 53 provides representations of immunoglobulin (antibodies) and immunoglobulin (antibody)-like molecules showing the modular nature of the antibody: (A) Left panel: The definition of the immunoglobulin molecule chains and components are as indicated and as also defined in the left panel of Figure 2. Middle panel: Provides one example of an Ab-like fusion protein, showing that the modular nature of immunoglobulins allows flexibility in the production of Ab-like molecules (as provided in Figure 2). In the particular depicted Ab-like fusion protein, the target recognition structure (shown as X1) is the same in all chains, as is the case of the Ab-like molecule described in Example 14 (where an EflACE2 polypeptide is separately linked to both the H chain at the CH1 domain and the L chain constant domain, enabling assembly into an H2L2 Ab-like fusion protein). Right panel: Depicts possible Ab-like fusion proteins comprising fusions to different target recognition structures (or enzymes and / or reporter molecules) in any combination of specificities (e.g. “X1 X1 X1 X1”, “X1 X1 X1 X2”, “X1 X1 X2 X2 , “X1 X1 X2 X3”, “X1 X2 X3 X4”; where “X1”, “X2”, “X3” and “X4” represent different target recognition structures (or enzymes or reporter molecules)); (B) SDS-PAGE analysis of EflACE2-Ab-like-WT fusion protein eluted from Protein A affinity matrix with 0.4 M arginine (pH 4) demonstrating that a fully disulfide-linked molecule consistent with an H2L2 Ab-like configuration was achieved (lane 1). This molecule comprises an EflACE2 polypeptide fusion to the immunoglobulin constant heavy chain (EflACE2-CH) which self assembles with an equivalent EflACE2 polypeptide fused to the immunoglobulin light chain constant domain (EflACE2-CL). Upon reduction with dithiothreitol, these two chains (i.e. EflACE2-CH (ACE2-CH) and EflACE2-CL (ACE2- CL); lane 2) are resolved separately. (C, D) Provide results which show that the EflACE2-Ab-like-H429F fusion protein (H429F) strongly directs CDC of Ramos-S target cells, whereas a corresponding fusion protein with H chains of wild type sequence (WT) was ineffective. CDC of the opsonised Ramos-S cells was determined using the presence of a 1 / 3 dilution of normal human serum as a source of complement. This potent CDC mediated by the EflACE2-Ab-like-H429F fusion protein was not attributable to different levels of opsonisation compared to the EflACE2-Ab-like-WT fusion protein, as the binding of these to Ramos cells expressing SARS- CoV-2 spike were comparable for both proteins. Binding was determined using flow cytometric analysisusing an anti-IgG secondary reagent labelled with anti-hIgG Fc FITC. MFI = median fluorescent intensity; background binding of the anti-Ig fluorescent conjugate shown as (conj only);

[0090] Figure 54 shows the results of purification of the EflACE2-Ab-like-Fc-H429F fusion protein using Protein A and elution with arginine. (A) Protein A chromatography using Hitrap™ Protein A column with gradient elution from 30 mM arginine (pH 4) to 35% of 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 oligomeric material indicated (HMW); and (C) SDS-PAGE analysis of the pooled Protein A eluate and the pooled SEC monomeric fractions under non-reducing (without DTT, dithiothreitol) and reducing (with DTT) conditions;

[0091] Figure 55 provides results showing the survival of Colo205 colorectal cells in the presence of purified DR5-specific mAbs comprising IgG1 H chains with the single H429F mutation (HF) or the L234A, L235A and H429F (LA / LA / HF) mutations. The 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 IgG1 H chains with only the H429F modification; Colo205 cells, 10,000 cells per well of 96-well plate, were cultured in the presence of serial two-fold dilutions of the indicated mAbs. The individual mAbs were titrated from 1μg / ml. For the titration of the mixtures, the starting concentration of 1 µg / ml was comprised of 0.5 µg / ml of each mAb in the mixture. Cell viability was quantitated using a CCK8 colorimetric cell viability assay, where absorbance (Abs450nm) of cell culture supernatant is a measure of cell survival and viability. Maximum cell viability was determined by the culture of cells in the absence of antibody (no Ab) and maximum death determined by culture of cells with sodium dodecyl sulphate (SDS). Survival is represented by: [Abs450nm of mAb or control treatment determined experimentally determined Abs450nm – background Abs450 of cell culture medium];

[0092] Figure 56 illustrates a sequence comparison of the hinges and constant domains of human immunoglobulin heavy (H) chain sequences and depicts the domain-based structure of the H chain (i.e. CH1–Hinge–CH2–CH3; Numbering of the amino acid positions is shown vertically and follows the Eu numbering and thus: CH1 domain, amino acids 118 to 215; hinge amino acids 216 to 230; CH2 domain amino acids 231 to 340 including the lower hinge positions; and CH3 domain amino acids 341 to 447). The other IgG subclasses IgG2, IgG4, have a corresponding domain structure with amino acid sequence homology. Amino acid sequences were derived from translation of open reading frames of sequences from the European Nucleotide Archive (https: / / www.ebi.ac.uk / ena / browser). Alignments were performed using Clustal except for the hinges which were aligned manually; gaps represented by a dot, may bepresent in the aligned sequences for the purpose sequence alignment; resides 231-237 inclusive, comprise the structural lower hinge of the IgG subclasses but are encoded by the CH2 exon and may also be known in the literature as the hinge proximal region of CH2. Accession numbers of the H chain sequences are: IgG1 H chain, J00228-IGHG1; IgG4 H chain, K01316-IGHG; IgG2 H chain, J00230-IGHG2;

[0093] Figure 57 provides the results of an ELISA showing the capacity of IgG antibodies comprising Fc region components to fix complement as measured by the binding of human C1q. Serial dilutions of mAbs, commencing at 4 µg / ml, were titrated on TNP-BSA antigen adsorbed to the wells of ELISA plates and the binding of human C1q determined and their C1q binding curves are shown. All mAbs showed similar antigen binding activity (not shown); the mAbs recognised TNP hapten and comprised unmodified wild-type (WT) heavy chains of the human IgG1 subclass (TNP-IgG1-WT) or comprised unmodified wild-type (WT) heavy chains of the human IgG2 subclass (TNP-IgG2-WT) or comprised heavy chains of the human IgG2 subclass which had been mutated and are shown as mAb TNP2x4, mAb TNP2x5, mAb TNP2x15 and mAb TNP2x17. O.D. = O.D. (450nm);

[0094] Figure 58 provides the results of an ELISA showing the capacity of IgG antibodies comprising Fc region components to fix complement as measured by the binding of human C1q. Serial dilutions of mAbs, commencing at 4 µg / ml, were titrated on TNP-BSA antigen adsorbed to the wells of ELISA plates and the binding of human C1q determined and their C1q binding curves are shown. All mAbs showed similar antigen binding activity (not shown); the mAbs tested recognised TNP and comprised unmodified wild-type (WT) heavy chains the human IgG1 subclass, mAb TNP-IgG1-WT, or comprised unmodified wild-type (WT) heavy chains of the human IgG2 subclass (TNP-IgG2-WT) or comprised mutated heavy chains of the human IgG1 subclass and are shown (A) as TNP1x32, and in (B) mAb TNP1x1, mAb TNP1x2, mAb TNP1x3, mAb TNP1x5, mAb TNP1x9, mAb TNP1x10, mAb TNP1x12, mAb TNP1x14 and mAb TNP1x15 O.D. = O.D. (450nm);

[0095] Figure 59 provides the results of an ELISA showing the capacity of IgG antibodies comprising Fc region components to fix complement is measured by the binding of human C1q. Serial dilutions of mAbs, commencing at 4 µg / ml, were titrated on TNP-BSA antigen adsorbed to the wells of ELISA plates and the binding of human C1q determined and their C1q binding curves are shown. All mAbs showed similar antigen binding activity (not shown); the mAbs tested recognised TNP hapten and comprised unmodified wild-type (WT) heavy chains of the human IgG1 subclass, (mAb TNP-IgG1-WT), or comprised unmodified wild-type (WT) heavy chains of the human IgG2 subclass (mAb TNP-IgG2-WT) or comprised mutated heavy chains of the human IgG1 subclass shown as mAb TNP1x16, mAb TNP1x22, mAb TNP1x23, mAb TNP1x24, mAb TNP1x25. O.D. = O.D.(450nm);

[0096] Figure 60 provides the results of flow quantitation of binding of SEC purified mAbs to target cell surface antigens. The mAbs detected CD20 and were based on rituximab variable domains and also comprised unmodified wild-type heavy chains of the IgG2 subclass or IgG1 subclass or comprising mutated IgG2 heavy chains carrying one or more mutations. Binding was evaluated by flow cytometry on Ramos lymphoma cells expressing CD20. The data shows that the mAbs comprising IgG2 based H chains bound similarly to one another and to Rit-IgG2-WT and in every case the level of binding of these IgG2 based mAbs was considerably lower than the binding of rituximab comprising unmodified wild-type heavy chains of the IgG1 subclass (Rit-IgG1-WT). HF is the mutation H429F, EG is the E430G mutation, LLGG is the PVA(233-236)ELLG mutation, FLGG is the PVA(233-236)EFLG mutation;

[0097] Figure 61 provides results showing that H429F modification of the mutants provides superior CDC to mAbs carrying H chain mutation without Stellabody. Complement-dependent cytotoxicity (CDC) of target cells was determined using Daudi lymphoma cells opsonised with the mAbs at the indicated concentrations and normal human serum diluted 1 / 3 as the source of complement. CDC (% killing) was determined by flow cytometry using Zombie Green. The control background CDC of complement in the absence of mAb (no mAb C' only) is shown. Whereas Rit-IgG2-WT comprising unmodified wild-type heavy chains failed to mediate detectable CDC, all mAbs that comprised specifically mutated IgG2 H chains mediated detectable CDC. (A) CDC mediated by Rit-IgG2-LLGG or Rit-IgG2-FLGG was superior in CDC compared to the unmodified Rit-IgG2-WT which failed to mediate CDC. (B) CDC mediated by mAb Rit-IgG2-FLGG HF, Rit-IgG2-LLGG HF, Rit-IgG2-FLGG-EG, Rit-IgG2-LLGG EG, Rit-IgG2-HF, Rit-IgG2-LLGG or Rit-IgG2-FLGG was superior compared to CDC by the Rit-IgG2-WT mAb which comprised unmodified H chains and which failed to mediate CDC. The background lysis control (no mAb C’ only) i.e. CDC in the presence of complement (serum) but without mAb is shown in both panels. HF is the mutation H429F, EG is the E430G mutation, LLGG is the PVA(233-236)ELLG mutation, FLGG is the PVA(233-236)EFLG mutation;

[0098] Figure 62 provides the results of a competitive binding assay for anti-CD20 rituximab mAbs on Daudi cells. The mAbs were serially 2-fold titrated from a starting concentration of 20 µg / mL and competition with biotinylated anti-CD20 IgG1-WT (RIT-IgG1-WT-biotin) for binding to the cell surface was measured by flow cytometry: (A) Comparison of RIT-IgG1-WT and RIT-IgG2-WT shows that IgG1 is a better competitor than IgG2 despite having the same VL and VH domains. A control for non-specific effects used each mAb titrated in the absence of the biotinylated anti-CD20 IgG1-WT antibody followed by incubation with the fluorescent conjugate (streptavidin-FITC) (mean ± SEM, n=4); (B) Comparison of the IgG1, IgG2, IgG3 and IgG4 human antibody subclasses showing that the IgG2 subclass uniquely demonstrates a reduced capacity to bind to the CD20 antigen (mean ± SEM, n=2). In all panels, the maximum binding signal of biotinylated anti-CD20 IgG1-WT in the absence of any competitor and thenon-specific binding of streptavidin-FITC alone only control) is shown. MFI = Median Fluorescence Intensity;

[0099] Figure 63 provides the binding profiles of anti-CD20 rituximab mAbs on target Ramos cells. The binding of mAbs to Ramos cells was detected using goat anti-human IgG Fc FITC and flow cytometry (A) RIT-IgG1 and RIT-IgG2 comprising a WT or a H429F mutant heavy chain, suggesting that rituximab formatted as an IgG2 displays reduced binding to target cells. MAbs were serially 2-fold titrated from 2 µg / mL (mean ± SEM, n=2); (B) RIT-IgG2 mAbs comprising the lower hinge residues of IgG4 (RIT- IgG2-EFLGG), the lower hinge residues of IgG1 (RIT-IgG2-ELLGG), the H429F mutation in the CH3 domain (RIT-IgG2-H429F), or a combination of lower hinge and H429F mutations (RIT-IgG2-EFLGG- H429F and RIT-IgG2-ELLGG-H429F). MAbs were serially 2-fold titrated from 20 µg / mL (mean ± SEM, n=2); The binding profile of IgG1-WT mAb is shown for comparison. The level of non-specific binding of anti-IgG Fc FITC to cells is shown (▲ conj. only); MFI = Median Fluorescence Intensity;

[0100] Figure 64 provides results showing anti-CD38 mAb variants’ ability to kill Ramos cells in the presence of serum. Purified mAbs were serially 2-fold titrated and CDC measured by flow cytometry. Normal human serum diluted 1 in 3 was used as the source of complement. (A) Comparison of the cytotoxic effect of RIT-IgG1 and RIT-IgG2 comprising a WT or a H429F mutant heavy chain. MAbs were serially 2-fold titrated from 2 µg / mL (n=1); (B) Comparison of the cytotoxic effect of RIT-IgG2 mAbs comprising the lower hinge residues of IgG4 (RIT-IgG2-EFLGG), the lower hinge residues of IgG1 (RIT-IgG2-ELLGG), the H429F mutation in the CH3 domain (RIT-IgG2-H429F), or a combination of lower hinge and H429F mutations (RIT-IgG2-EFLGG-H429F and RIT-IgG2-ELLGG-H429F). IgG2- WT and IgG1-WT mAbs were included for comparison. MAbs were serially 2-fold titrated from 40 µg / mL (mean ±SEM, n=3). Non-specific target cell killing in the absence of mAb (C' only) is shown as a filled triangle (▲);

[0101] Figure 65 shows representations of the light chain and the M1, M2 and M3 mutant IgG2 H- chains, inclusive of the VH, CH1, upper hinge, core hinge and lower hinge sequences only. Each indicate the disulfide linkages (depicted by a black line) expected in the upper hinge region of each IgG2 mutant mAb (M1, M2, and M3) between the light chain (LC) and heavy chain (HC). The core hinge, CPPC, forms inter-heavy chain disulfides which are not shown. Remaining cysteine residues capable of forming interchain disulfide bonds are shown as black circles, and cysteine residues that have been substituted with a serine are shown as black circles with a white cross. (A) The M1 mutant comprises the heavy chain upper hinge sequence of IgG2 and a cysteine-to-serine substitution at positions 131 (C131S) in the CH1 domain and 220 (C220S) and is thus expected to form a disulfide bond between the cysteine at position 214 in the light chain (C214) and the cysteine at position 219 (C219) in the upper hinge region of theheavy chain. (B) The M2 mutant comprises the chain upper hinge sequence of IgG2 and a cysteine- to-serine substitution at positions 131 (C131S) and 219 (C219S) and is thus expected to form a disulfide bond between the cysteine at position 214 in the light chain (C214) and the cysteine at position 220 (C220) in the upper hinge region of the heavy chain. (C) The M3 mutant comprises the heavy chain upper hinge sequence of IgG1 (EPKSCDKTHT) and a cysteine-to-serine substitution at positions 131 (C131S) and is thus expected to form a disulfide bond between the cysteine at position 214 in the light chain (C214) and the cysteine at position 220 (C220) in the upper hinge region of the heavy chain. As this mutant has the upper hinge sequence of IgG1, it has 2 additional amino acids;

[0102] Figure 66 provides size exclusion chromatography (SEC) elution profiles of the various isatuximab IgG1 and IgG2 mAbs as shown in each panel. Protein A affinity purified IgG was further purified by SEC at pH 7.2; (A) Isa-IgG1-WT, (B) Isa-IgG2-WT (C) Isa-IgG2-M1, (D) Isa-IgG2-M2, (E) Isa-IgG2-M3, (F) Isa-IgG2-C131S-C219S, and (G) Isa-IgG2-EFLGG are shown, each consisting of a single IgG peak with minimal oligomeric species;

[0103] Figure 67 shows the results of (A) non-reduced SDS-PAGE analysis of SEC purified mAbs, showing that each migrated at the expected ~150kDa mass of IgG (H2L2). (B) Purified mAbs were also reduced in dithiothreitol and examined by SDS-PAGE analysis, showing that each mAb resolved to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species. M = molecular weight marker;

[0104] Figure 68 provides the results of flow cytometric quantitation of the binding of anti-CD38 mAbs to Raji cells. The mAbs were serially 3-fold titrated from 5 µg / mL and the binding to Raji cells was detected by flow cytometry using a goat (Fab’)2 anti-human-IgG Fc FITC secondary reagent. (A) Isa- IgG1-WT and Isa-IgG2-WT, suggesting that isatuximab formatted as an IgG2 has reduced binding to target cells, (B) Isa-IgG2-WT and hinge-modified IgG2 mutants (Isa-IgG1-M1, Isa-IgG2-M2, and Isa- IgG2-M3), (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S and Isa-IgG2-EFLG, showing modest effects on binding by either the upper hinge or lower hinge mutations in isolation. In all panels, the level of non- specific binding of goat anti-human-IgG Fc FITC to cells is shown (▲ conj. only); MFI = Median Fluorescence Intensity. Mean ±SEM; n=4. P values were determined comparing to Isa-IgG2-WT by 2- way ANOVA for main column effect; and for B, C used Dunnett's multiple comparisons test;

[0105] Figure 69 provides the results of the binding of anti-CD38 isatuximab mAbs to Raji cells by a flow cytometric competitive binding assay. The mAbs were serially 3-fold titrated and used to compete with biotinylated Isa-IgG1-WT for binding. RIT-IgG1-WT was used as a negative control that does not compete with biotinylated Isa-IgG1-WT for binding. (A) Isa-IgG1-WT and Isa-IgG2-WT, shows that the IgG2-WT displays reduced binding to target cells (mean ± SEM, n=6); (B) Isa-IgG2-WT, Isa-IgG1-M1,Isa-IgG2-M2, and Isa-IgG2-M3, showing hinge- enhances binding and competition (mean ± SEM, n=6); (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S and Isa-IgG2-EFLGG, showing no effect on binding by either the upper hinge or lower hinge mutations in isolation. In all panels, the level of non- specific binding of fluorescent conjugate (streptavidin-FITC) to cells is shown (▲ conj only) and the maximum binding of biotinylated Isa-IgG1-WT (i.e. in the absence of a competitor mAb) is shown (Isa- IgG1-WT-biotin); MFI = Median Fluorescence Intensity. P values were determined comparing to Isa- IgG2-WT by 2-way ANOVA for main column effect and Dunnett's multiple comparisons test;

[0106] Figure 70 provides results showing anti-CD38 mAbs mediate the killing of Raji cells in the presence of serum. Purified mAbs were serially 3-fold titrated and CDC measured by flow cytometry. Normal human serum diluted 1 in 3 was used as the source of complement. (A) Isa-IgG1-WT and Isa- IgG2-WT (mean ±SEM, n=6), (B) Isa-IgG2-WT, Isa-IgG1-M1, Isa-IgG2-M2, and Isa-IgG2-M3 (mean ± SEM, n=6), (C) Isa-IgG2-WT, Isa-IgG2-C131S-C219S, Isa-IgG2-EFLGG (mean ± SEM, n=3). A, B, C) P values determined comparing to Isa-IgG2-WT by 2-way ANOVA by main column effect using B, C) Dunnett's multiple comparisons test; (D) The cell killing in the presence each mAb at the highest concentration tested (5 µg / mL) was measured with (+C’) and without (-C’) human serum as a source of complement (mean ±SEM, n=4);

[0107] Figure 71 provides results showing C1q binding, as the C1 complex, to opsonised Ramos cells. Ramos cells were opsonised with 5 µg / mL of anti-CD38 mAbs, IgG-WT and IgG2-mutants, followed by normal human serum (NHS; diluted 1 in 3 in DPBS+BSA) as a source of C1. The binding of C1 was detected with an anti-C1q rabbit polyclonal antibody followed by a fluorescently labelled anti-rabbit-Fc probe. Grey bars indicate C1 binding to mAb opsonized cells, and black bars indicate the level of background fluorescence from mAb opsonized cells in the absence of C1q. Background binding of C1 to cells without mAb is shown in the ‘no mAb’ control. Mean ± SEM, n=4; MFI = median fluorescence intensity;

[0108] Figure 72 provides size exclusion chromatography (SEC) profiles of the various isatuximab IgG1 and IgG2 mAbs generated with and without the H429F mutation. Following Protein A affinity purification, mAbs were further purified by SEC at pH 7.2. The SEC elution profiles of (A) Isa-IgG1-WT and Isa-IgG1-H429F, (B) Isa-IgG2-WT and Isa-IgG2-H429F, (C) Isa-IgG2-EFLGG and Isa-IgG2- EFLGG-H429F, (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F, (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F, and (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F are shown, each consisting of a single homogenous IgG peak with minimal oligomeric species. Fractions containing monomeric IgG are outlined in a box with a dotted line and were pooled for subsequent analyses of mAb function;

[0109] Figure 73 shows the results of (A) non- SDS-PAGE analysis of SEC purified mAbs as indicated, showing that each migrated predominately at the expected ~150kDa mass of IgG (H2L2). (B) Samples were also reduced in dithiothreitol and examined by SDS-PAGE , showing that each mAb resolved to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species. M = molecular weight marker;

[0110] Figure 74 provides the results of flow cytometric quantitation of the binding of anti-CD38 isatuximab mAb with or without the H429F mutation to Raji cells. MAbs were serially 3-fold titrated and the binding activity was quantified by flow cytometry using a goat anti-human IgG Fc-FITC: (A) Isa- IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5); (B) Isa-IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=6); (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=4); (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5); (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5); (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5). In all panels, the level of non- specific binding of the goat anti-human IgG Fc FITC conjugate to cells is shown (▲ conj); MFI = Median Fluorescence Intensity. P values of nonlinear fits were determined by two-way ANOVA by main column effect;

[0111] Figure 75 provides the results of a competitive binding assay comparing the binding of each anti-CD38 isatuximab mAb with its H429F equivalent. The mAbs were serially 3-fold titrated and competition with biotinylated anti-CD38 IgG1-WT (Isa-IgG1-WT-biotin) for binding to the cell surface was measured by flow cytometry: (A) Isa-IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5); (B) Isa- IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=6); (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=3); (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5); (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5); (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5). In all panels, the level of non-specific binding of fluorescent conjugate (streptavidin-FITC) to cells is shown (▲ conj only) and the maximum binding of biotinylated Isa-IgG1-WT is shown (Isa-IgG1-WT- biotin). MFI = Median Fluorescence Intensity. P values determined by two-way ANOVA;

[0112] Figure 76 provides results showing CDC mediated by isatuximab mAbs and their H429F- equivalents on Raji cells. SEC purified mAbs were serially 3-fold titrated and CDC potency was determined by flow cytometry using normal human serum diluted 1 in 3 as the source of complement: (A) Isa-IgG1-WT and Isa-IgG1-H429F (mean ± SEM, n=5); (B) Isa-IgG2-WT and Isa-IgG2-H429F (mean ± SEM, n=5); (C) Isa-IgG2-EFLGG and Isa-IgG2-EFLGG-H429F (mean ± SEM, n=3); (D) Isa-IgG2-M1 and Isa-IgG2-M1-H429F (mean ± SEM, n=5); (E) Isa-IgG2-M2 and Isa-IgG2-M2-H429F (mean ± SEM, n=5); (F) Isa-IgG2-M3 and Isa-IgG2-M3-H429F (mean ± SEM, n=5); In all panels, the level of non-specific target cell killing in the absence of mAb only) is shown as a filled triangle (▲). P values determined by two-way ANOVA testing for main column effect;

[0113] Figure 77 provides results showing the killing of Raji target cells in the presence of each mAb at the highest concentration tested (5 µg / mL) with (+C’) and without (-C’) human serum as a source of complement, revealing that the observed CDC activity was dependent on the presence of complement. Plots are shown for Isa-IgG variants: (A) IgG1-WT and IgG1-H429F; (B) IgG2-WT and IgG2-H429F; (C) IgG2-EFLGG and IgG2-EFLGG-H429F; (D) IgG2-M1 and IgG2-M1-H429F; (E) IgG2-M2 and IgG2-M2-H429F; (F) IgG2-M3 and IgG2-M3-H429F. Mean ± SEM, n=4. P values determined by pairwise t-test with Holm’s correction for multiple comparisons. *p<0.05; ns = not significant;

[0114] Figure 78 provides results showing that H429F mutation enhances C1 binding to Ramos cells opsonised with anti-CD38 IgG1-WT or IgG2-WT or the indicated modified IgG2 variants. Ramos cells were opsonised with 5 µg / mL of anti-CD38 mAb before normal human serum (diluted 1 in 3 in DPBS+BSA) was added as a source of C1. The binding of C1 was detected with an anti-C1q rabbit polyclonal antibody followed by a fluorescently labelled anti-rabbit-Fc donkey antiserum and is represented as median fluorescence intensity (MFI). Grey bars indicate C1 binding to mAb opsonized cells in the presence of serum, and black bars indicate the level of background fluorescence from mAb opsonized cells in the absence of serum C1 (mean ± SEM, n=4). Background binding of C1 to cells without mAb is shown in the ‘no mAb’ control;

[0115] Figure 79 provides size exclusion chromatography (SEC) profiles of the various isatuximab IgG1 and IgG2 mAbs with the indicated mutations. Following Protein A affinity purification, mAbs were further purified by SEC at pH 7.2. The SEC elution profiles of profiles are shown for Isa-IgG variants; (A) IgG1-H429F, (B) IgG1-LALA, (C) IgG1-LALA-H429F, (D) IgG1-K274Q (E) IgG1-K274Q-H429F, (F) IgG1-H268E-A327D-H429F, (G) IgG2-LALA-H429F, and (H) IgG2-Q274K-H429F, each consisting of a single homogenous IgG peak with minimal oligomeric species. Fractions containing monomeric IgG are outlined in a box with a dotted line and were pooled for subsequent analyses of mAb function;

[0116] Figure 80 shows the results of (A) non-reduced SDS-PAGE analysis of SEC purified mAbs, showing that each migrated predominately at the expected ~150kDa mass of IgG (H2L2). (B) Samples were also reduced in dithiothreitol and examined by SDS-PAGE, showing that each mAb resolved to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species;

[0117] Figure 81 provides the results of mAb binding assays with Raji cells, testing the effects of various mutations in the CH2 of anti-CD38 isatuximab IgG1 and IgG2 mAbs with, or without, H429Fmodification. The mAbs were serially 3-fold and the binding was quantified by flow cytometry using a goat anti-human IgG Fc-FITC secondary reagent. Binding profiles are shown for Isa-IgG variants: (A) IgG1-WT, IgG1-H429F, IgG1-LALA and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1- K274Q, IgG1-K274Q-H429F and IgG1-H268E-A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2- LALA-H429F and IgG2-Q274K-H429F. Note that in panel B the IgG1-WT and IgG1-H429F curves are as in panel A and are included for the sake of comparison. The level of non-specific binding of fluorescent conjugate to cells is shown (▲ conj); MFI = Median Fluorescence Intensity. Mean ± SEM, n=3. P values were determined by 2-way ANOVA testing for main column effect with comparison to IgG-WT by Dunnett's multiple comparisons test;

[0118] Figure 82 provides the results of competitive binding assays with Raji cells, testing the effects of various mutations in the CH2 of anti-CD38 isatuximab IgG1 and IgG2 mAbs with, or without, H429F modification. The mAbs were serially 3-fold titrated and binding activity of competing biotinylated isatuximab IgG1-WT was determined by flow cytometry. Isa-IgG competitors were: (A) IgG1-WT, IgG1- H429F, IgG1-LALA and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q- H429F and IgG1-H268E-A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F and IgG2- Q274K-H429F. Note that in panel B the IgG1-WT and IgG1-H429F binding curves are as in panel A and are included for the sake of comparison. P values were determined by 2-way ANOVA testing for main column effect with comparison to IgG-WT by Dunnett's multiple comparisons test. In all panels, the level of non-specific binding of fluorescent conjugate (streptavidin-FITC) to cells is shown (▲ conj only) and the maximum binding of biotinylated Isa-IgG1-WT is shown (Isa-IgG1-WT-biotin); MFI = Median Fluorescence Intensity. Mean ± SEM, n=2;

[0119] Figure 83 provides the results of cytotoxicity assays, testing the effects of various mutations in the CH2 of anti-CD38 Isa-IgG1 and IgG2 mAbs with, or without, H429F modification, on the killing of Raji cells in the presence of serum. Isa-IgG variants were: (A) IgG1-WT, IgG1-H429F, IgG1-LALA and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F and IgG1-H268E- A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F and IgG2-Q274K-H429F. Note that in panel B the same binding curves for IgG1-WT and IgG1-H429F are included as in panel A for the sake of comparison. P values were determined by 2-way ANOVA testing for main column effect with comparison to IgG-WT by Dunnett's multiple comparisons test. In all panels, mutant mAbs are compared to the relevant unmodified counterpart to determine the impact of the mutation, and the level of non- specific target cell killing in the absence of mAb (C' only) is shown as a filled triangle (▲). Mean ± SEM, n=3;

[0120] Figure 84 provides results showing the of Raji target cells in the presence of each mAb at the highest concentration tested (5 µg / mL) with (+C’) and without (-C’) human serum as a source of complement, revealing that the observed CDC activity was dependent on the presence of complement. The Isa-IgG variants were; (A) IgG1-WT, IgG1-H429F, IgG1-LALA and IgG1-LALA-H429F; (B) IgG1- WT, IgG1-H429F, IgG1-K274Q, IgG1-K274Q-H429F and IgG1-H268E-A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F and IgG2-Q274K-H429F and each experiment included a no mAb control for non-specific cytotoxicity. Note that in panel B the same data for IgG1-WT and IgG1-H429F are included as in panel A for the sake of comparison. Mean ± SEM, n=4;

[0121] Figure 85 provides results showing purified C1q binding on target Ramos cells opsonised with anti-CD38 mAbs. Ramos cells were opsonised with 5 µg / mL of anti-CD38 mAb before 10 µg / mL of purified human C1q was added. The binding of C1q was detected with an anti-C1q rabbit polyclonal antibody followed by a fluorescently labelled anti-rabbit-Fc probe. Isa-IgG variants were; (A) IgG1-WT, IgG1-H429F, IgG1-LALA and IgG1-LALA-H429F; (B) IgG1-WT, IgG1-H429F, IgG1-K274Q, IgG1- K274Q-H429F and IgG1-H268E-A327D-H429F; (C) IgG2-WT, IgG2-H429F, IgG2-LALA-H429F and IgG2-Q274K-H429F. Note that in panel B the same data for IgG1-WT and IgG1-H429F are included as in panel A for the sake of comparison. Grey bars indicate C1q binding to mAb opsonized cells (●+C1q), and white bars indicate the level of background fluorescence from mAb opsonized cells in the absence of C1q (o-C1q). Background binding of C1q to cells without mAb is shown in the ‘no mAb’ control. (Mean ± SD, n=2); MFI = median fluorescence intensity;

[0122] Figure 86 provides size exclusion chromatography (SEC) elution profiles of the S2P6 IgG1 mAbs. The mAbs were first purified from culture supernatants by Protein A affinity chromatography followed by SEC at pH 7.2. The SEC elution profiles of (A) S2P6-IgG1-WT, and (B) S2P6-IgG1-H429F are shown, each consisting of a single homogenous IgG peak of monomers with minimal oligomeric species;

[0123] Figure 87 provides the results SDS-PAGE analysis of SEC purified mAbs, S2P6-IgG1-WT and S2P6-IgG1-H429F. Prior to reduction with dithiothreitol (DTT-) both mAbs migrated predominately at the expected ~150kDa mass of IgG (H2L2). Following reduction in DTT (DTT+) each mAb resolved to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species;

[0124] Figure 88 provides size exclusion chromatography (SEC) elution profiles of the various S2P6 IgG2 mAbs generated. MAbs were purified initially by Protein A affinity purification followed by SEC at pH 7.2 as shown. The SEC elution profiles of (A) S2P6-IgG2-WT, (B) S2P6-IgG2-H429F (C) S2P6- IgG2-M1, (D) S2P6-IgG2-M1-H429F, (E) S2P6-IgG2-M2, (F) S2P6-IgG2-M2-H429F, (G) S2P6-IgG2-M3, and (H) S2P6-IgG2-M3-H429F are shown, consisting of a single monomeric IgG peak with minimal oligomeric species;

[0125] Figure 89 provides the results of (A) non-reduced SDS-PAGE analysis of SEC purified S2P6 IgG2 mAbs, showing that each predominately migrated at the expected ~150kDa mass of IgG (H2L2). (B) Samples were also reduced in dithiothreitol and examined by SDS-PAGE analysis, showing that each mAb resolved to its ~50kDa heavy (H) chain and ~25kDa light (L) chain species;

[0126] Figure 90 provides results showing antigen binding and cell cytotoxicity of rituximab-based mAb variants. (A) anti-CD20 variants RIT-IgG1-WT and RIT-IgG1-H429F were serially 3-fold titrated from 10 µg / ml and binding to Ramos-S cells was determined by flow cytometry using FITC conjugated F(ab’)2 fragments of goat anti-human IgG-Fc antiserum (n=4). Non-specific binding was determined by staining cells with FITC goat anti-human IgG-Fc antiserum in the absence of mAbs (Conj. only). Ramos- S cells are Ramos cells that express the Spike protein of SARS-CoV-2 (Wuhan). (B) anti-CD20 rituximab variants kill Ramos-S cell targets in the presence of serum. Purified mAbs were serially 3-fold titrated from 10 µg / mL and CDC measured by flow cytometry (n=3). Normal human serum diluted 1 in 3 was used as the source of complement. The concentration dependent cytotoxic effects of RIT-IgG1-WT and RIT-IgG1-H429F are shown. Non-specific target cell killing in the absence of mAb (C' only) is shown as a filled star (★);

[0127] Figure 91 provides graphical representation of binding of SARS-CoV-2 Spike-specific S2P6- based mAbs to Ramos-S cells. The S2P6 mAb comprised unmodified IgG1, IgG2 H chains or mutated H chain variants as indicated and were serially 3-fold titrated from 10 µg / ml. Their binding to Ramos-S cells was determined by flow cytometry using FITC conjugated F(ab’)2fragments of goat anti-human IgG-Fc antiserum (n=4). Non-specific binding was determined by staining cells with FITC goat anti- human IgG-Fc antiserum in the absence of mAbs (Conj. only). All mAbs displayed similar target cell binding;

[0128] Figure 92 provides results showing S2P6 mAb variants killing Ramos-S cell targets in the presence of serum. Ramos-S cells are Ramos cells that express the Spike protein of SARS-CoV-2 (Wuhan). Purified mAbs were serially 3-fold titrated from 10 µg / mL (n=3) and CDC measured by flow cytometry. Normal human serum diluted 1 in 3 was used as the source of complement. The concentration dependent cytotoxic effects of the S2P6 mAbs shown are (A) IgG1-WT, IgG1-H429F, IgG2-WT and IgG2-H429F; (B) IgG2-M1-H429F, IgG2-M2-H429F, IgG2-M3-H429F; (C) IgG2-M1, IgG2-M2, IgG2- M3 and for purposes of comparison the IgG2-M1-H429F, IgG2-M2-H429F, IgG2-M3-H429F curves from panel B are included. (D) Individual experiments from A-C were analysed as plots of CDC killingversus log10 mAb concentration by calculation under the curve (AUC) using the complement only control to set the baseline. Mean ± SD, n=3. P values determined by one-way ANOVA with Tukey’s multiple comparison test. *p<0.05, **p<0.01, ****p<0.0001; and

[0129] Figure 93 provides groupings of IgG and mutant variants by CDC in comparison to IgG2-WT. DETAILED DESCRIPTION

[0130] The present disclosure is directed to an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide comprising one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) domain and a hinge region, wherein the one or more polypeptide comprises an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering), and wherein the one or more polypeptide additionally comprises at least one C1q binding modification.

[0131] Amino acid numbering used herein is based on the so-called Eu numbering system which relates to the sequence numbering used in the description of the human IgG1 myeloma protein called Eu (Edelman GM et al., Proc Natl Acad Sci U S A 63(1):78-85, 1969). In accordance with this system, H429 of IgG1 for example (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 an antibody-like molecule, the amino acid in a given position number relates to, or corresponds to, the corresponding amino acid residue position number in the Eu sequence.

[0132] The heavy (H) chain of immunoglobulins such as IgG1 is a modular, multifunctional, but monomeric, polypeptide. The prototypical immunoglobulin or antibody structure (with an H429 mutation may be considered as consisting of a dimeric protein comprising two monomeric heavy (H) chain polypeptides each optionally associated with a light (L) chain and thus occurring in an H2L2 format (see Figure 2). However, other forms of immunoglobulin molecules may be formed, including where one heavy chain (H1) associates with one light (L1) chain in an H1L1 format, and other forms where two heavy (H) chains may form dimers in the absence of light (L) chains to produce an H2 formatted heavy chain complex (Figure 2). When a light (L) and heavy (H) chain is associated in the H1L1 format, the molecule can dimerise in this configuration to yield the typical immunoglobulin structure designated as H2L2. This is exemplified by the typical human IgG class of immunoglobulins wherein the two heavy (H) chains are covalently bonded to each other, and a light (L) chain is covalently bonded to each heavy (H) chain.

[0133] The dimeric form of H1L1 molecules (i.e. H2L2 proteins) is the fundamental structural "unit" of all human Ig classes (i.e. IgG, IgE, IgD, IgA and IgM) and indeed, is also the basis of most mammalian immunoglobulin classes noting that exceptions to this format are known (e.g. camelid immunoglobulins can form a heavy chain dimer without light chains (abbreviated as H2)). Thus, for example, human immunoglobulin G (IgG) molecules exist physiologically in solution as a single H2L2 unit. However, other covalently bonded, higher-order oligomers of the basic H2L2 unit do exist in nature and are common, particularly, for IgM and IgA.

[0134] For instance, IgM can form covalent pentameric or hexameric rings of the H2L2format with each H2L2unit having disulphide bonds to adjacent H2L2units to form the pentameric oligomer (H2L2)5or hexameric oligomer (H2L2)6 (Eskeland T and TB Christensen, Scand J Immunol 4(3):217-228, 1975), and notably, it is the hexameric form of IgM which is the most potent effector of the classical complement pathway (which is one of the two major effector systems of the innate immune system initiated by immunoglobulins), activated by the avid binding of the soluble hexameric protein C1q (Eskeland T and TB Christensen, 1975 supra; Randall TD et al., Proc Natl Acad Sci U S A 89:962-966, 1992; Hughey CT et al., J Immunol 161:4091-4097, 1998; and Randall TD et al., Eur J Immunol 20:1971-1979, 1990). IgM is also well recognised as the most potent agglutinin by virtue of its covalent oligomerisation which results in the presence of 10-12 specific, and identical, antigen binding variable domains in each IgM pentamer or hexamer. Since an individual antigen recognition structure (e.g. a Fab fragment) has a defined affinity for a target antigenic site or epitope with a strength defined by its monovalent binding to one target structure, the presence of multiple antigen recognition structures in the one molecule (e.g. the 12 antigen binding variable domains of each IgM hexamer) confers stronger or more avid binding of the oligomeric immunoglobulin. Thus, oligovalent binding arises from the combined strength of the individual antigen recognition interactions of the IgM oligomer and its target antigenic epitopes. By comparison, the avidity of IgG (which has two antigen recognition structures (Fabs) per H2L2 unit) arises only from the interaction of these two antigen recognition structures.

[0135] Immunoglobulins can also be regarded as modular, multifunctional proteins in which the target recognition structure is connected by a flexible linker to a function-activating structure (i.e. the antigen recognition structure provided by the V domain of the Fab is linked by a flexible hinge to the Fc region), and wherein, in the case of a typical immunoglobulin, each H chain and each L chain is comprised of different domains which can be considered structural or functional modules (Figure 2). An H chain is particularly comprised of an antigen target recognition domain (VH domain) of variable sequence followed by a series of constant domains that are unique to the heavy chain of an Ig class such as, for example, IgG or IgA, and sequence analysis shows that these constant domains are related between immunoglobulin classes (see Figures 3 and 4). The VHdomain is thus followed by a first constant domain(CH1). This first constant domain is connected flexible polypeptide that acts a linker known as the hinge region, to a second constant domain (CH2) which is followed, in turn, by a third constant domain (CH3), and thus a typical H chain consists of discrete structural molecules from the amino terminus (i.e. NH2-terminus VH–CH1–hinge–CH2–CH3 (Figure 2)) and is exemplified by the human IgG1 H chain sequence (SEQ ID NOS: 3) and is conserved across all human heavy chains (Figure 3, Figure 4) exemplified by human IgG3 (SEQ ID NOS: 4), IgG4 (SEQ ID NO: 5), IgG2 (SEQ ID NOS: 6), IgA1 (SEQ ID NOS: 7), IgA2 (SEQ ID NOS: 8) and mammalian immunoglobulins generally. Similarly, a light (L) chain is comprised of a variable antigen target recognition domain (VLdomain) followed by a constant domain unique to the light chain class.

[0136] It should be noted that the polypeptides that comprise the heavy chains of the IgG subclasses contain a structurally unique segment referred to as the hinge region. The hinge region of the IgG polypeptide is commonly considered to be composed of an upper hinge sequence, core hinge sequence, and a lower hinge sequence. The upper hinge is comprises residues 216-225 inclusive, the core hinge comprises residues 226-230 inclusive and the lower hinge comprises residues 231-237 according to Eu numbering. It should be noted that the amino acid residues which comprise the structural lower hinge of the IgG polypeptide are encoded by the CH2 exon and may also know in the literature as the hinge proximal region of CH2. In this application reference to CH2 includes reference to the lower hinge residues.

[0137] In some embodiments, the immunotherapeutic protein is a human immunotherapeutic protein. In some embodiments, the immunotherapeutic protein is a humanised or chimeric immunotherapeutic protein. In some embodiments, the immunotherapeutic protein is a non-human primate immunotherapeutic protein (e.g. monkey). In some embodiments, the immunotherapeutic protein is a rodent (e.g. a mouse immunotherapeutic protein).

[0138] In an embodiment, the immunotherapeutic protein comprises one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) domain and / or a hinge region. In an embodiment, the immunotherapeutic protein comprises a CH1 domain. In an embodiment, the immunotherapeutic protein comprises a CH2 domain. In an embodiment, the immunotherapeutic protein comprises a CH3 domain. In an embodiment, the immunotherapeutic protein comprises a hinge region. In an embodiment, the immunotherapeutic protein is derived from an IgG1 heavy chain polypeptide. In an embodiment, the immunotherapeutic protein is derived from an IgG2 heavy chain polypeptide. In an embodiment, the immunotherapeutic protein is derived from an IgG3 heavy chain polypeptide. In an embodiment, the immunotherapeutic protein is derived from an IgG4 heavy chain polypeptide.

[0139] Given their modular nature, provide a versatile platform for the creation of a diverse range of immunotherapeutic or diagnostic molecules, including bivalent antibodies such as a classical hybridoma-derived mAb, 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 a CH3 domain (or at least a CH4 domain)) and other immunoglobulin derivatives known to those skilled in the art as summarised in the "Periodic Table of Antibodies"; https: / / absoluteantibody.com / periodic-table-of-antibodies / #, the entire disclosure of which is herein incorporated by reference). Other specific examples include antibodies and antibody fragments such as single chain Fv (scFv) antibody fragments, asymmetric bi-specific antibodies (WO 2012 / 058768), strand-exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (WO2007110205); dual variable domain immunoglobulin (US 7,612,181), knobs-into-holes antibody formats (WO 1998 / 050431), duobody molecules (WO 2011 / 131746), IgG-like bispecific antibodies (Shen J et al., J Immunol Methods 318(1-2):65-74, 2007), and fusion proteins comprising an Fc or Fc region component such as scFv-fusions and dual scFv-fusions.

[0140] In this specification, a number of terms are used which are well known to those skilled in the art. Nevertheless, for the purposes of clarity, a number of these terms are hereinafter defined.

[0141] As used herein, the term "antibody" is to be understood as including (unless specifically indicated as otherwise) polyclonal antibodies (pAb), monoclonal antibodies (mAb), chimeric antibodies, humanised antibodies, antibody mixtures (e.g. recombinant polyclonal antibodies) such as those generated, for example, using methods well known to those skilled in the art for producing multiple antibodies with different specificities from a single host cell line (e.g. Oligoclonics® technology: Merus BV, Utrecht, The Netherlands) or a transgenic animal. Further, it is to be understood that an antibody may be of any immunoglobulin class (i.e. isotype) or allotype. Thus, for example, an antibody as disclosed herein may be of an isotype selected from the well-known immunoglobulin isotypes, or comprise components from more than one isotype (e.g. as may be the case with some chimeric antibody types). Also, an antibody may comprise "mixed" chains; for example, an antibody comprised of a H2 or H2L2 unit where the H chains are different (i.e. Hxand Hy(HxHyor LxHxHyLy).

[0142] The term "antibody-like molecule" (Ab-like molecule) is to be understood as referring to proteins which are not antibodies and minimally comprise a target recognition structure (e.g. a receptor or ligand such as an antigen recognition sequence (e.g. a variable (V) domain or complementarity- determining region (CDR) of an immunoglobulin) linked to at least a CH3 domain (or a CH4 domain) of an immunoglobulin heavy (H) chain (providing the molecule with an "H-like" chain), and includes, for example, receptor fusion proteins comprising at least a CH3 domain (or at least a CH4 domain) of an Hchain. Examples of Ab-like molecules include mentioned above (such as those depicted in Figure 2, Figure 53A and / or The Periodic Table of Antibodies). In some examples, an H-like chain comprising at least a CH3 domain is considered to comprise an Fc region component and may be, for example, a complete Fc region, or merely a CH3 domain, or a CH2-CH3 or CH3-CH2 component. In other examples, such an H-like chain may further comprise, for example, an immunoglobulin hinge sequence. The H-like chain may enable dimerisation, such that an antibody-like molecule may be provided, for example, in a homodimeric or heterodimeric form. As depicted in Figure 53, in some embodiments, an Ab-like molecule may comprise a H2L2format and may comprise, 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 X1 X2", "X1 X1 X2 X2" , "X1 X1 X2 X3" or "X1 X2 X3 X4", where X1, X2, X3 and X4 represent different target recognition structures (or enzymes or reporter molecules).

[0143] The term "fusion protein" is well known to those skilled in the art and refers to a protein expressed from a DNA construct comprising two or more open reading frames in a desired order, such that the protein may be regarded as a hybrid or chimeric protein. In some simple examples, the 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 as referring to certain types of an antibody-like molecule such as a fusion protein comprising a target recognition structure (e.g. a receptor or ligand such as an antigen recognition sequence (e.g. a variable (V) domain or complementarity-determining region (CDR) of an immunoglobulin)) linked to at least a CH3 domain (or at least a CH4 domain) of an immunoglobulin heavy (H) chain (see examples shown in Figure 2). Such fusion proteins may comprise, 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 comprises, 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 linked to different target recognition structures (e.g. ligand 1 and ligand 2). Those skilled in the art will understand that the target or antigen recognition structure may be linked to a polypeptide comprising at least a CH3 domain (or an equivalent CH4 domain of an IgE or IgM) that may, for example, comprise an entire H-chain or comprise an H-like chain which may comprise, for example, a complete Fc region, or merely a CH3 domain, or a CH2-CH3 or a CH3-CH2 component. In other examples, such an H-like chain may further comprise, for example, an immunoglobulin hinge sequence and / or a CH1 domain. A CH1 domain may, for example, provide a suitable site for linking the target or antigen recognition structure.

[0144] The term "Fc fragment" (Fc or Fc portion), as used herein, refers to a dimer formed by covalent and / or non-covalent interactions between parts of the immunoglobulin heavy (H) chain (i.e. a dimerformed between two Fc regions of the H chain each comprise CH2 and CH3 domains of a heavy chain and optionally a hinge sequence)), and which is responsible for much of the activation of the immune effector functions of immunoglobulins: particularly the cell-based effector responses initiated by antibodies and cell surface-located Fc receptors such as killing of target cells by antibody dependent cell- mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) or trogocytosis, and which also leads to the modulation and inhibition of the activity of innate and adaptive immune cells by inhibitory Fc receptors; and the effector responses of the innate immune system initiated by antibodies through activation of the the classical pathway of complement system, a cascade of proteins found in blood or biological fluids that is important in the destruction of pathogens and involves the killing of targets by direct lysis through complement-dependent cytotoxicity (CDC) and / or by killing via phagocytosis of targets through specific receptors of the components of complement (C'ADCP). The Fc fragment also provides a site of association between immunoglobulin molecules permitting the assembly (self-association) of immunoglobulin molecules into higher order oligomers (e.g. through covalent bonding between domains in the Fc region of the H chain of one immunoglobulin with an Fc region of an H chain of an adjacent immunoglobulin (such as seen in, for example, pentameric and hexameric forms of IgM) and non-covalent self-association such as that seen in IgG when bound to antigen and which, among other properties, leads to oligomerisation including hexamer formation (Diebolder CA et al., Science 343(6176):1260-1263, 2014) and to certain effector functions).

[0145] As used herein, the term "Fc region component" is to be understood as referring to a part of the Fc region of an immunoglobulin heavy (H) chain comprising at least a CH3 domain (or at least a CH4 domain), where H429 is located, but preferably comprising a CH2 and CH3 domain and optionally further comprising an immunoglobulin hinge sequence (which may, in turn, comprise all or a portion of the lower hinge, core hinge and upper hinge sequences), that is capable of forming (e.g. by dimerisation) an Fc fragment or Fc-like fragment. An "Fc-like fragment" is to be understood as referring to an Fc fragment-like structure, but which comprises fragments or components of the Fc region e.g. the CH3 domain (or CH4 domain) alone or a CH3 domain in combination with a CH2 domain and optionally further comprising an immunoglobulin hinge sequence which may, in turn, comprise all or a portion of the lower hinge, core hinge and upper hinge sequences.

[0146] As used herein, the term "treating" includes prophylaxis as well as the alleviation of established symptoms of a disease or condition. As such, the act of "treating" a disease or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the disease or condition developing in a subject suffering from, or predisposed to, the disease or condition; (2) inhibiting the disease or condition (i.e. arresting, reducing or delaying the development of the disease or condition or a relapse thereof, in case of a maintenance treatment, or at least one clinical or subclinical symptomthereof); and (3) relieving or attenuating the or condition (i.e. causing regression of the disease or condition or at least one of clinical or subclinical symptom thereof).

[0147] As used herein, the phrase "manufacture of a medicament" includes the use of one or more immunotherapeutic protein as defined herein directly as the medicament or in any stage of the manufacture of a medicament comprising one or more immunotherapeutic protein as defined herein.

[0148] The term "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. Typically, an effective amount is sufficient for treating a disease or condition or otherwise to palliate, ameliorate, stabilise, reverse, slow or delay the progression of a disease or condition. By way of example only, an effective amount of an immunotherapeutic protein such as a mutant IgG1 antibody may comprise between about 0.1 and about 250 mg / kg body weight per day, more preferably between about 0.1 and about 100 mg / kg body weight per day and, still more preferably between about 0.1 and about 25 mg / kg body weight per day. However, notwithstanding the above, it will be understood by those skilled in the art that an effective amount may vary and depend upon a variety of factors including the age, body weight, sex and / or health of the subject being treated, the activity of the particular immunotherapeutic protein, the metabolic stability and length of action of the particular immunotherapeutic protein, the route and time of administration of the particular immunotherapeutic protein, the rate of excretion of the particular immunotherapeutic protein and the severity of, for example, the disease or condition being treated.

[0149] As shown in the Examples provided hereinafter, the combination of a hexamerising mutation and a C1q binding modification have functional advantages. In an embodiment, the hexamerising mutation is a point mutation at the H429 position.

[0150] As shown in the Examples provided hereinafter, it has been found that a mutant antibody including a point mutation at the H429 position may confer significant functional changes. For example, an IgG mutant with an H429F substitution shows an enhanced ability to activate complement-dependent cytotoxicity (CDC) in complement assays (i.e. assays of complement function). Also, it has been found that a fusion protein comprising an Fc region component with a point mutation at position 429 (i.e. H429F) fused to an angiotensin-converting enzyme 2 (ACE2) ectodomain (which may act as a "decoy" to block viral interaction and cellular entry of coronaviruses to host cells) may provide an enhanced ability to provide an antiviral effect through CDC of infected cells, while a similar fusion protein with an H429Y substitution displays enhanced virus neutralisation with little or no complement activation (since immunotherapeutic proteins comprising an H429Y modified Fc component shows abrogated FcγR binding and activation, especially with FcγRIIIa). While not wishing to be bound by theory, it isconsidered that these effects may be due to of the proteins into oligomers through the self-association of the antibodies / fusion proteins either in solution or upon binding to a relevant target molecule (e.g. an antigen to which an antibody mutant is directed) through "on target" oligomerisation.

[0151] Further in this regard, it is to be noted that while monomeric IgG1 and IgG3 can deliver CDC, the level of CDC can be regarded as low or "poor" by comparison to that achieved with naturally pentameric or hexameric IgM. Moreover, IgG antibodies such as IgG1 and IgG3, unlike IgM, are also comparatively poor agglutinins by virtue of their bivalency. Thus, by enabling oligomerisation (by possibly enhancing the known weak inherent capacity of the Fc of an immunoglobulin such as IgG to self-associate, especially after the immunoglobulin has bound to an antigen (i.e. "on target" oligomerisation), an immunotherapeutic protein according to the present disclosure may, by forming oligomers, provide a more optimal platform for complement system activation and other functions enhanced by self-association. This oligomerisation may, particularly and / or optimally involve the formation of a hexamer; and such hexamerisation has been visualised in the crystallographic structure of anti-HIV antibody b12 (Saphire EO et al., Science 293:1155-1159, 2001), wherein CH3 residues form an interface between adjacent IgG:IgG molecules, so forming a hexamer that optimally presents binding sites for the six globular head domains of the C1q subunit which initiates the activation of the classical complement pathway.

[0152] Moreover, many cellular molecules, including cell surface molecules, require substantial clustering by a ligand (which may be soluble or another cell surface molecule) to induce a signal that consequently induces a cellular response, and thus the dimerisation that will typically be achieved with an antibody, may not be sufficient to induce a signal that leads to a meaningful cellular response. However, it is known that the strength of the signal can be increased by increasing clustering of the target molecule through, for example, approaches involving the addition of other entities that cross-link the ligand (Chenoweth et al., Immunol Cell Biol 98:287-304, 2020), and thus, in the case of an antibody (such as a mAb) bound to its target molecule, such "super cross-clustering" or "hyper-clustering" of the target molecule can be achieved by the use of additional anti-immunoglobulin antibodies that cross-link the mAb that is bound to the target molecule. While not wishing to be bound by theory, it is considered that proteins such as a mutant antibody including an H429F point mutation as mentioned above, through self- association upon binding to a target molecule, may similarly achieve super cross-clustering or hyper- clustering" of the target molecule. Depending upon the nature of the target molecule, such clustering might induce an enhanced signalling response that may lead, for example, to cell proliferation (e.g. where the target molecule is, for example, CD3 or CD28), the stimulation of an inhibitory pathway to inhibit or reduce cell responses (e.g. immune checkpoints; reviewed in Chenoweth et al., supra 2020), or the stimulation of a pathway that induces cell death such as, for example, programmed cell death includingapoptosis (see, for example, Ashkenazi A., Nat Discov 7(12):1001-1012, 2008). Stimulation of cellular pathways by mAbs for the development of therapeutic molecules is an approach being used to treat a range of diseases including cancers, inflammation and autoimmune diseases, infections, cardiovascular diseases and others (Ashkenazi, supra 2008).

[0153] Thus, in an aspect, the present disclosure provides an immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide comprising an Fc region component comprising at least a CH3 domain (or at least a CH4 domain), wherein said one or more polypeptide includes an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

[0154] In some embodiments, the immunotherapeutic protein as described herein comprises a dimeric immunotherapeutic protein comprising first and second immunoglobulin heavy chain polypeptides comprising an Fc region component comprising CH2 and CH3 domains (so that the first and second polypeptides may form (e.g. by dimerisation) an Fc fragment or Fc-like fragment), wherein the Fc region component of at least one of said first and second polypeptides comprises an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering). Accordingly, the immunotherapeutic protein of such embodiments may be an immunoglobulin molecule such as, for example, an antibody or an antibody-like molecule such as a dimeric polypeptide comprising a pair of single-chain Fv polypeptides linked via Fc fragments (i.e. scFv-Fc) or a mini-body (i.e. a protein comprising a pair of scFv polypeptides linked via CH3 domains (see the discussion of multivalent scFv- Fc and minibodies in, for example, Olafsen T et al., Generation of Antibody Fragments and Their Derivatives, Antibody Engineering Second edition, pp 69-84, 2010)).

[0155] In other embodiments, the immunotherapeutic protein as described herein comprises a partner polypeptide linked to an Fc region component comprising at least a CH3 domain (or at least a CH4 domain), wherein the Fc region component comprises an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering). Accordingly, the immunotherapeutic protein of such embodiments may be provided in the form of a fusion protein or protein conjugate. Those skilled in the art will understand 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- or C-terminus of the fusion partner (i.e. Fc component), whereas in a protein conjugate, the partner polypeptide will be covalently or non-covalently linked to the Fc component (i.e. as a conjugate partner) through a chemical linkage such as a disulphide bond (e.g. through one or more cysteine (C) residue) or cross-linkercompound such as a homobifunctional cross- as disuccinimidyl suberate (DSS) (e.g. bis(sulfosuccinimidyl)suberate (BS3); Thermo Fisher Scientific, Waltham, MA, United States of America) or disuccinimidyl tartrate (DST) to link amine groups or a heterobifunctional cross-linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MDS) and N-(ε-maleimidocaproloxy) succinimide ester (EMCS), or by other non-covalent bonding such as hydrogen bonding. Where the Fc component is a conjugate partner, the immunotherapeutic protein conjugate may be considered as a cross-linked protein, and the Fc component may be conjugated to the partner polypeptide at the N- or C- terminus, but otherwise at any other suitable site on the partner polypeptide. Alternatively, the Fc component (as a conjugate partner) may be conjugated to the partner polypeptide at the N- or C-terminus of the Fc component or otherwise at any other suitable site on the Fc component (e.g. within CH1 or the upper hinge sequence if these are included in the Fc region component).

[0156] The Fc region component (also referred to hereinafter as the "Fc component") of the immunotherapeutic protein may be derived from one or more immunoglobulin type (e.g. an IgG or IgA) and may comprise a full length (i.e. "complete") Fc region such as, for example, a heavy (H) chain polypeptide fragment corresponding to that generated by papain digestion (i.e. wherein the polypeptide is cleaved within the upper hinge sequence to generate an Fc region comprising the constant heavy domain 2 (CH2; amino acid A231 to K340 of the human IgG1 heavy chain polypeptide (Eu numbering)), constant heavy domain 3 (CH3; amino acid G341 to G446 or K447 of the human IgG1 heavy chain polypeptide (Eu numbering)) and lower hinge sequence (also known as hinge proximal sequence of CH2; amino acids P232 to P238 (Eu numbering)) and core hinge sequence (amino acids C226 to C229) and similar heavy chain polypeptide fragments that may be prepared through digestion of an immunoglobulin heavy chain polypeptide with plasmin and human neutrophil elastase (NHE). Further examples of suitable Fc region components may comprise fragments of the heavy chain polypeptide which comprise, in addition to the CH2 and CH3 domains and the lower and core hinge sequences, all or part of the upper hinge sequence and constant heavy domain 1 (CH1). On the other hand, other suitable Fc region components may comprise fragments of the heavy chain polypeptide which comprise only the CH3 domain (e.g. amino acid G341 to G446 or K447 of the human IgG1 heavy chain polypeptide (Eu numbering)) or a fragment thereof. In addition, suitable Fc region components may comprise heterogeneous ("hybrid") CH3 domains such as strand exchange engineered domain (SEED) forms of a CH3 domain comprising fragments derived from the IgG1 CH3 domain and other proteins such as IgA (Davies et al., Prot Eng Des Sel 23(4):195-202, 2009).

[0157] In some embodiments, the Fc region component is derived from a human immunoglobulin heavy chain polypeptide (e.g. such as those shown in Figures 3 and 4).

[0158] In other embodiments, the Fc region is derived from an IgG heavy chain polypeptide, preferably an IgG1 or IgG3 (e.g. human IgG1 or IgG3) heavy chain polypeptide, and more preferably, an IgG1. In some embodiments, the Fc region is glycosylated. In some embodiments, the Fc region does not comprise an E430G substitution.

[0159] The Fc region component comprises an amino acid substitution at a position corresponding to H429 of the amino acid sequence of the human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide (Eu numbering). Where the Fc region component is derived from another immunoglobulin type or isotype (or from an immunoglobulin from another species), it will be appreciated that those skilled in the art can readily determine a position corresponding to H429 of the human IgG1 heavy chain polypeptide IgG1 by, for example, routine sequence alignments (e.g. as shown in Figures 3 and 4). In some embodiments, the H429 amino acid substitution is an aromatic or cyclic amino acid. In some embodiments, the H429 amino acid substitution is an aromatic amino acid. In some embodiments, the H429 amino acid substitution is a cyclic amino acid. In some embodiments, the H429 amino acid substitution is hydrophobic amino acid. In some embodiments, the H429 amino acid substitution is a very hydrophobic amino acid (e.g. F or Y). In some embodiments, the H429 amino acid substitution has a hydrophobicity of T or greater. In some embodiments, the H429 substitution is not a neutral amino acid (e.g. not glycine). In some embodiments, the H429 substitution is not glycine or leucine. In some embodiments, the H429 substitution is not glycine. In some embodiments, the H429 substitution is not leucine.

[0160] Included among the suitable mutations at position 429 are: H→X, where X is selected from phenylalanine (H429F), glutamate (H429E), glutamine (H429Q), serine (H429S), alanine (H429A), threonine (H429T), tyrosine (H429Y), leucine (H429L), valine (H429V), glycine (H429G), tryptophan (H429W), arginine (H429R) and proline (H429P).

[0161] Included among the suitable mutations at position 429 are: H→X, where X is selected from tyrosine (H429Y), phenylalanine (H429F), tryptophan (H429W), glutamate (H429E), aspartate (H429D), glutamine (H429Q), serine (H429S), asparagine (H429N), and threonine (H429T).

[0162] Some preferred amino acid substitutions at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide include the H→Y (e.g. H429Y) substitution and the H→F (e.g. H429F) substitution.

[0163] In some embodiments, H429 is with phenylalanine (H429F). In some embodiments, H429 is substituted with glutamate (H429E). In some embodiments, H429 is substituted with glutamine (H429Q). In some embodiments, H429 is substituted with serine (H429S). In some embodiments, H429 is substituted with alanine (H429A). In some embodiments, H429 is substituted with threonine (H429T). In some embodiments, H429 is substituted with leucine (H429L). In some embodiments, H429 is substituted with valine (H429V). In some embodiments, H429 is substituted with glycine (H429G). In some embodiments, H429 is substituted with tryptophan (H429W). In some embodiments, H429 is substituted with arginine (H429R). In some embodiments, H429 is substituted with and proline (H429P).

[0164] As used herein “C1q binding modification” refers to a modification that increases or decreases C1q binding of an immunotherapeutic protein as described herein. In some embodiments, the modification directly decreases / increases binding of the immunotherapeutic protein to C1q compared to a control immunotherapeutic protein lacking the C1q binding modification. In some embodiments, the modification indirectly decreases / increases binding of the immunotherapeutic protein to C1q compared to a control immunotherapeutic protein lacking the C1q binding modification. For the sake of clarity, the term “C1q binding modification” does not require that the modification itself binds to C1q, however this is not excluded.

[0165] In some embodiments, the C1q binding modification is in the CH1 domain of the immunotherapeutic protein. In some embodiments, the C1q binding modification is in the CH2 domain of the immunotherapeutic protein. In some embodiments, the C1q binding modification is in the CH3 domain of the immunotherapeutic protein. In some embodiments, the C1q binding modification is in the hinge region of the immunotherapeutic protein. In some embodiments, the mutation is in the lower hinge. In some embodiments, the mutation is in the upper hinge.

[0166] In some embodiments, the C1q binding modification is selected from a modification in C1q group 1, C1q group 2, C1q group 3, C1q group 4 and C1q group 5 as described in Table 6. In some embodiments, the C1q binding modification is a C1q group 1 modification as described in Table 6. In some embodiments, the C1q binding modification is a C1q group 2 modification as described in Table 6. In some embodiments, the C1q binding modification is a C1q group 3 modification as described in Table 6. In some embodiments, the C1q binding modification is a C1q group 4 modification as described in Table 6. In some embodiments, the C1q binding modification is a C1q group 5 modification as described in Table 6.

[0167] In some embodiments, the C1q binding modification is selected from a modification in C1q Group 1, C1q Group 2 and C1q Group 3 and wherein the modification increases C1q binding comparedto a control IgG2 immunotherapeutic protein a control IgG1 immunotherapeutic protein lacking the C1q binding modification.

[0168] In some embodiments, the C1q binding modification is selected from a modification in C1q Group 4 and C1q Group 5 and wherein the modification decreases C1q binding compared to a control IgG2 immunotherapeutic protein and / or a control IgG1 immunotherapeutic protein lacking the C1q binding modification.

[0169] In some embodiments, the polypeptide is selected from an IgG1, IgG2, IgG3 or IgG4. In some embodiments, the polypeptide is an IgG1. In some embodiments, the polypeptide is an IgG2. In some embodiments, the polypeptide is an IgG3. In some embodiments, the polypeptide is an IgG4.

[0170] In an embodiment, the polypeptide is an IgG1, the C1q binding modification is in the CH1 domain and wherein the modification is a substitution at a position corresponding to S131 of the amino acid sequence of a human IgG1 heavy chain polypeptide substituted with a C (Eu numbering).

[0171] In an embodiment, the polypeptide is an IgG1, IgG2, IgG3 or IgG4, the modification is in the hinge region and wherein the modification is a substitution at a position corresponding to 216-225 or 217- 225 of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the hinge region is selected from: RKCCVE217-225 substituted with PKSCDKTHT, EPKSCDKTHT216-225 substituted with ERKCCVE and EPKSCDKTHT216-225 substituted with ESKYGPP (Eu numbering). In an embodiment, the substitution in the hinge region is RKCCVE217-225 substituted with PKSCDKTHT. In an embodiment, the substitution in the hinge region is EPKSCDKTHT216-225 substituted with ERKCCVE. In an embodiment, the substitution in the hinge region is EPKSCDKTHT216-225 substituted with ESKYGPP.

[0172] In an embodiment, the polypeptide is an IgG2, the modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to PVA233-236, L328 or S267 of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the CH2 domain is selected from: PVA233-236 substituted with EFLG, PVA233-236 substituted with ELLG, and PVA233-236 substituted with EFEG. In an embodiment, the substitution in the CH2 domain is PVA233-236 substituted with EFLG (Eu numbering). In an embodiment, the substitution in the CH2 domain is PVA233-236 substituted with ELLG (Eu numbering).

[0173] In an embodiment, the polypeptide is an IgG3 and IgG4, the modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to L235 of a human IgG1, IgG3 or IgG4 heavy chain polypeptide substituted with an E (Eu numbering).

[0174] In an embodiment, the polypeptide is an IgG1, IgG2, IgG3 or IgG4 and wherein the modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to QYN295-297, YNS296-298, ED269-270, L328 or S267 of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the CH2 domain is selected from: QYN295-297 substituted with NST, YNS296-298 substituted with NSS, ED269-270 substituted with AA, L328 substituted with F and S267 substituted with E (Eu numbering). In an embodiment, the substitution in the CH2 domain is QYN295-297 substituted with NST. In an embodiment, the substitution in the CH2 domain is YNS296-298 substituted with NSS. In an embodiment, the substitution in the CH2 domain is ED269-270 substituted with AA. In an embodiment, the substitution in the CH2 domain is L328 substituted with F and S267 substituted with E. In an embodiment, the substitution in the CH2 domain is L328 substituted with F. In an embodiment, the substitution in the CH2 domain is S267 substituted with E. In an embodiment, the polypeptide is an IgG2, IgG3 or IgG4 wherein modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to Q274 of a human IgG2, IgG3 or IgG4 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the CH2 domain is Q274 substituted with K. In an embodiment, the polypeptide is an IgG1, wherein the modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to K274 of a human IgG1 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the CH2 domain is K274 substituted with Q. In an embodiment, the polypeptide is an IgG1 or IgG3, and wherein the modification is in the lower hinge and wherein the modification is a substitution at a position corresponding to LL234- 235. In an embodiment, the substitution in the lower hinge is LL234-235 substituted with AA. In an embodiment, the polypeptide is an IgG4 and wherein the modification is in the lower hinge and wherein the modification is a substitution at a position corresponding to FL234-235. In an embodiment, the substitution in the lower hinge is FL234-235 substituted with AA.In an embodiment, the polypeptide is an IgG2, and wherein the modification is in the CH2 domain and wherein the modification is a substitution at a position corresponding to PVA234-236. In an embodiment, the substitution in the lower hinge is selected from: PVA234-235 substituted with AAG.

[0175] In an embodiment, the polypeptide is an IgG1, IgG2, IgG3 or IgG4 and wherein the modification is in the CH3 domain and wherein the modification is a substitution at a position corresponding to E430 or H435 of a human IgG1, IgG2, IgG3 or IgG4 heavy chain polypeptide (Eunumbering). In an embodiment, the substitution CH3 domain is selected from: E430 substituted with G, and H435 substituted with A.

[0176] In an embodiment, the polypeptide is an IgG1, IgG2, IgG3 or IgG4 and wherein the modification is the CH2 and the CH3 domain. In an embodiment, the polypeptide is an IgG1 and wherein the modification is the CH2 and the CH3 domain. In an embodiment, the polypeptide is an IgG2 and wherein the modification is the CH2 and the CH3 domain. In an embodiment, the mutation in the CH2 domain is PVA233-236 substituted with EFLG and the modification in the CH3 domain is E430G. In an embodiment, the mutation in the CH2 domain is PVA233-236 substituted with ELLG and the modification in the CH3 domain is E430G. In an embodiment, the mutation in the CH2 domain is PVA233-236 substituted with EFEG and the modification in the CH3 domain is E430G. In an embodiment, the polypeptide is an IgG2, mutation in the CH2 domain is PVA233-236 substituted with EFLG and the modification in the CH3 domain is E430G. In an embodiment, the polypeptide is an IgG2, the mutation in the CH2 domain is PVA233-236 substituted with ELLG and the modification in the CH3 domain is E430G. In an embodiment, the polypeptide is an IgG2, the mutation in the CH2 domain is PVA233-236 substituted with EFEG and the modification in the CH3 domain is E430G.

[0177] In an embodiment, the polypeptide is an IgG3, the modification is in the CH3 domain and wherein the modification is a substitution at a position corresponding to N392, M397 and R435 of a human IgG3 heavy chain polypeptide (Eu numbering). In an embodiment, the substitution in the CH3 domain comprises N392 substituted with K, M397 substituted with V and R435 substituted with H (Eu numbering).

[0178] In an embodiment, the polypeptide is an IgG2, the C1q binding modification is in the CH1 domain and wherein the modification is a substitution at a position corresponding to C131 of the amino acid sequence of a human IgG1 heavy chain polypeptide substituted with a S (Eu numbering).

[0179] In an embodiment, the polypeptide is an IgG2 and the C1q binding modification is PVA233-236 substituted with EFLG, C131 substituted with S, and C220 substituted with S.

[0180] In an embodiment, the polypeptide is an IgG2 and the C1q binding modification is PVA233-236 substituted with EFLG, C131 substituted with S, and C219 substituted with S.

[0181] In an embodiment, the polypeptide is an IgG2 and the C1q binding modification is PVA233-236 substituted with EFLG, C131 substituted with S, and ERKCCVE substituted with IgG1216- 225EPKSCDKTHT.

[0182] In an embodiment, wherein the is an IgG1 and the C1q binding modification is PVA233-236 substituted with EAAGG.

[0183] In an embodiment, the polypeptide is an IgG1 and the C1q binding modification is K274 substituted with a Q.

[0184] In an embodiment, the polypeptide is an IgG2, IgG3 or IgG4 and the C1q binding modification is Q274 substituted with K.

[0185] In some embodiments, the C1q binding modification is not E430 substituted with G. In some embodiments, the C1q binding modification is not L328 substituted with F or S267 substituted with E. In some embodiments, the C1q binding modification is not L328 substituted with F and S267 substituted with E.

[0186] In an embodiment, the immunotherapeutic protein is an anti-CD20 antibody. In an embodiment, the immunotherapeutic protein is an anti-trinitrophenyl antibody (anti-TNP antibody). In an embodiment, the immunotherapeutic protein is selected from: rituximab, ofatumumab, obinutuzumab, isatuximab, trastuzumab and pertuzumab. In an embodiment, the immunotherapeutic protein is selected from: rituximab, trastuzumab and pertuzumab. In an embodiment, the immunotherapeutic protein is rituximab. In an embodiment, the immunotherapeutic protein is trastuzumab. In an embodiment, the immunotherapeutic protein is pertuzumab. In an embodiment, the immunotherapeutic protein is selected from: rituximab (Rit), Rit-IgG2 FLGG HF, Rit-IgG2-FLGG EG, Rit-IgG2-LLGG HF, Isa-IgG2-M1- H429F, Isa-IgG2-M2-H429F, Isa-IgG2-M3-H429F, S2P6-IgG2-M1-H429F, S2P6-IgG2-M2-H429F and S2P6-IgG2-M3-H429F. In an embodiment, the immunotherapeutic protein comprises Rit-IgG2-FLGG HF. In an embodiment, the immunotherapeutic protein comprises Rit-IgG2 FLGG EG. In an embodiment, the immunotherapeutic protein comprises Rit-IgG2-LLGG HF. In an embodiment, the immunotherapeutic protein comprises Isa-IgG2-M1-H429F. In an embodiment, the immunotherapeutic protein comprises Isa-IgG2-M2-H429F. In an embodiment, the immunotherapeutic protein comprises Isa- IgG2-M3-H429F. In an embodiment, the immunotherapeutic protein comprises S2P6-IgG2-M1-H429F. In an embodiment, the immunotherapeutic protein comprises S2P6-IgG2-M2-H429F. In an embodiment, the immunotherapeutic protein comprises S2P6-IgG2-M3-H429F.

[0187] In an embodiment, the immunotherapeutic protein comprises: a) H429 substituted with an aromatic amino acid or cyclic amino acid wherein the C1q modification is PVA233-236 substituted with EFLG; b) H429 substituted with an aromatic amino acid or cyclic amino acid and wherein the C1q modification is PVA233-236 substituted with ELLG; c) H429 substituted with a hydrophobic amino acidand wherein the C1q modification is PVA233- with EFLG; and d) H429 substituted with a hydrophobic amino acid and wherein the C1q modification is PVA233-236 substituted with ELLG.

[0188] In an embodiment, the immunotherapeutic protein is an IgG2 and wherein the IgG2 comprises a further mutation producing a disulfide between the light chain and upper hinge region of the polypeptide. In an embodiment, the disulfide bond is formed between C214 and C219. In an embodiment, the disulfide bond is formed between C214 and C220. In an embodiment, the further mutation is M1, substitution of C131 with serine and substitution of C220 with serine. In an embodiment, the further mutation is M2, substation of C131 with serine and substitution of C219 with serine. In an embodiment, the further mutation is M3, substation of C131 with serine and substitution of ERKCCVE in the IgG2 upper hinge with EPKSCDKTHT.

[0189] In an embodiment, the immunotherapeutic protein comprises modifications selected from: a) H429 substituted with F and wherein the C1q modification comprises PVA233-236 substituted with EFLG; b) H429 substituted with F and wherein the C1q modification comprises PVA233-236 substituted with ELLG; c) H429 substituted with Q and wherein the C1q modification comprises PVA233-236 substituted with EFLG; d) H429 substituted with Q and wherein the C1q modification comprises PVA233-236 substituted with ELLG; e) H429 substituted with E and wherein the C1q modification comprises PVA233-236 substituted with EFLG; f) H429 substituted with E and wherein the C1q modification comprises PVA233-236 substituted with ELLG; g) H429 substituted with S and wherein the C1q modification comprises PVA233-236 substituted with EFLG; h) H429 substituted with S and wherein the C1q modification comprises PVA233-236 substituted with ELLG; i) H429 substituted with A and wherein the C1q modification comprises PVA233-236 substituted with EFLG; j) H429 substituted with A and wherein the C1q modification comprises PVA233-236 substituted with ELLG; k) H429 substituted with Y and wherein the C1q modification comprises PVA233-236 substituted with EFLG; l) H429 substituted with Y and wherein the C1q modification comprises PVA233-236 substituted with ELLG; m) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and C220 substituted with serine; n) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and C219 substituted with serine; o) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 substituted with serine, and ERKCCVE substituted with IgG1 EPKSCDKTHT; p) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with serine, and C220 substituted with serine; q) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLG, C131 substituted with serine, and C219 substituted with serine; and r) H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLG, C131 substituted and ERKCCVE in the IgG2 upper hinge substituted with EPKSCDKTHT.

[0190] In an embodiment, the immunotherapeutic protein comprises H429 substituted with F and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F and wherein the C1q modification comprises PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with Q and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with Q and wherein the C1q modification comprises PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with E and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with E and wherein the C1q modification comprises PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with S and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with S and wherein the C1q modification comprises PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with A and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with A and wherein the C1q modification comprises PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with Y and wherein the C1q modification comprises PVA233-236 substituted with EFLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with Y and wherein the C1q modification comprise PVA233-236 substituted with ELLG. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and C220 is substituted with serine. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and C219 is substituted with serine. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with EFLG, C131 is substituted with serine, and ERKCCVE in the IgG2 upper hinge is substituted with IgG1216225EPKSCDKTHT. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLG, C131 is substituted with serine, and C220 is substituted with serine. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification is PVA233-236 substituted with ELLG, C131 is substituted with serine, and C219 is substitute with serine. In an embodiment, the immunotherapeutic protein comprises H429 substituted with F, wherein the C1q modification isPVA233-236 substituted with ELLG, C131 is with serine, and ERKCCVE in the IgG2 upper hinge is substituted with IgG1216-225EPKSCDKTHT.

[0191] In an embodiment, the C1q binding modification increases complement-based lysis compared to a control immunotherapeutic protein lacking the C1q binding modification. In an embodiment, the C1q binding modification decreases complement-based lysis compared to a control immunotherapeutic protein lacking the C1q binding modification. In an embodiment, the control antibody is a wild type IgG2. In an embodiment, the control antibody is a wild type IgG1.

[0192] In an embodiment, the immunotherapeutic protein comprises at least two C1q binding modifications. In an embodiment, the immunotherapeutic protein comprises at least three C1q binding modifications.

[0193] In some embodiments, the immunotherapeutic protein as described herein may comprise one or more further mutation / s (e.g. amino acid substitution).For example, the Fc component may comprise one or more sequence mutation known to those skilled in the art (see, for example, the examples listed in Table 1 of Wang et al., Protein Cell 9(1):63-73, 2018; the entire disclosure of which is herein incorporated by reference) such as, for example: mutations that may modulate FcγR binding (e.g. S239D / I332E of IgG1 (Eu numbering) which increase FcγRIIIa binding); mutations to improve antibody- dependent cellular cytotoxicity (ADCC) such as S239D / I332E (Lazar GA et al., Proc Natl Acad Sci U S A 103(11):4005–4010, 2006), opsonic phagocytosis (e.g. G236A / S239D / I332E; Richards JO et al., Mol Cancer Ther 7:2517–2527, 2008) or complement activation (e.g. H268F / S324T; Moore GL et al., MAbs 2:181–189, 2010), or decrease effector function (e.g. IgG1: L234A / L235A; IgG4: F234A / L235A; Xu D et al., Cell Immunol 200(1):16–26, 2000); and mutations which confer enhanced binding to the neonatal Fc receptor (FcRn) such as M252Y / S254T / T256E (Dall’Acqua WF et al., J Immunol 169(9):5171-5180, 2002) to increase in vivo half-life and thereby improve pharmacokinetics (PK).

[0194] Based on the observations of the C1q binding by the mAbs as shown in Figure 57, Figure 58 and Figure 59, Figure 71, Figure 78, Figure 85 and grouping in Table 6 and the sequence alignment of Figure 56 and Tables 7, 8, 9 additional mutations could be incorporated onto an IgG2 backbone particularly mutation of the cysteine residues involved in disulphide bonding of light chain to heavy chain and or between heavy chains (at position 131 in CH1 or in the upper hinge at position 219 and 220 of IgG1, IgG2, IgG4 or IgG4 Eu numbering). Such mutations could be used alone or in combination for example with a C1q binding modification e.g. C131S in CH1 of IgG2 or S131C in CH1 of IgG1, IgG3 or IgG4. In some embodiments, the C1q binding modification is used in combination with C219S and / or C220S. In some embodiments, the C1q binding modification is used in combination with C219S. In someembodiments, the C1q binding modification is in combination with C220S. In some embodiments, the C1q binding modification is used in combination with C219S and / or C220S in an IgG2. Other point mutations might also be useful. For example at position 274 of IgG1 the mutation K274Q may result in recreased C1q binding (Figure 85) and is also inferred from the reduced CDC activity of this mutant compared with its IgG1-WT counterpart (Figure 84). This point mutation may be used in combination with other mutations such as those targeting the disulphide bonds in CH1 or the upper hinge.

[0195] In an embodiment, the further mutation is a substitution selected from K274 with Q, Q274 with K, C219 with S, S219 with C, and C220 with S.

[0196] In an embodiment, the polypeptide is an IgG2 and wherein the polypeptide comprises a further mutation that prevents formation of an intra-heavy chain disulfide bond in the upper hinge region of the polypeptide. In an embodiment, the intra-heavy chain disulfide bond is between C131 and C219, or between C131 and C220. In an embodiment, the polypeptide comprises a mutation at one or more of positions C131, C219 or C220 and wherein the mutation is a substitution to an amino acid other than cysteine. In one example, the substitution is to serine. In one example, the mutation is at positions C131 and C219 or C131 and C220.

[0197] In an embodiment, the polypeptide is an IgG2 and wherein the polypeptide comprises a further mutation that forms a disulfide bond between the light chain and upper hinge region of the polypeptide. In an embodiment, the disulfide bond is formed between C214 of the light chain and C219 of the heavy chain or between C214 of the light chain and C220 of the heavy chain. In an embodiment, the further mutation is M1, substitution of C131 with S and a substitution of C220 with S. In an embodiment, the further mutation is M2, substitution of C131 with S and substitution of C219 with S. In an embodiment, the further mutation is M3, a substation of C131 with S and substitution of ERKCCVE in the IgG2 upper hinge with EPKSCDKTHT.

[0198] In an embodiment, the immunotherapeutic protein is an anti-CD20 antibody. In an embodiment, the immunotherapeutic protein is an anti-trinitrophenyl antibody (anti-TNP antibody).

[0199] Other mutations that may be included in the immunotherapeutic protein component include mutations to enhance complement activation such as an amino acid mutation at a position(s) corresponding to K447 of the amino acid sequence of the human IgG1 heavy chain polypeptide (Eu numbering); in particular, K447X, where X is selected from null (i.e. K447del; an amino acid deletion or truncation of the Fc component), and glutamate (i.e. K447E) (see van der Bremer ETJ et al., mAbs 7(4):672-680, 2015). Further, the immunotherapeutic protein component may also include mutationswhich modulate glycosylation (e.g. a mutation at corresponding to Asn297 of the amino acid sequence of the 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. the lack of glycan at position 297) to abolish FcγR and complement C1 binding and / or activation.

[0200] Alternatively, the immunotherapeutic protein component may also be treated to achieve modified glycosylation by producing the immunotherapeutic protein in the presence of kifunensine (a mannosidase inhibitor which prevents normal maturation of the N-linked glycan including core fucosylation of the N-linked glycan); a modification that specifically enhances activity via FcγRIIIa. Further, an Fc component lacking core fucosylation of the Asn297 glycan can also be achieved by culturing host cells expressing the immunotherapeutic protein with inhibitors of fucosylation (e.g.2- fluoro peracetylated fucose, or similar) or by the expression of enzymes that modify glycosylation pathways (e.g. GDP-6-deoxy-D-lyxo-4-hexulose reductase; Neha M et al., J Biotech 5:100015, 2020) or by modification of these pathways by gene supression (e.g. siRNA silencing of the α-1,6- Fucosyltransferase fucosyl transferase gene FUT8; Imai-Nishiya H et al., BMC Biotechnol 7:84, 2007) or knock-out (Yamane-Ohnuki N et al., Biotechnol Bioeng 87:614-622, 2004.)

[0201] The immunotherapeutic protein as described herein may be monomeric, dimeric or oligomeric in solution (e.g. in physiological saline at neutral pH such as physiological pH of about 7.4).

[0202] For example, in some embodiments, the immunotherapeutic protein comprises a monomer in physiological saline, wherein each monomer comprises one copy of the Fc region component, while in other embodiments, the immunotherapeutic protein comprises a dimer in physiological saline wherein the Fc region components self-associate (i.e. to form the dimer) either by non-covalent bonding such as hydrogen bonding or through the formation of disulphide bonds through one or more cysteine (C) residue, particularly where situated within the hinge sequence, especially the core hinge sequence (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003). Thus, in some embodiments, the Fc region component comprises a core hinge sequence to enable the immunotherapeutic protein to self-associate (i.e. to form a dimer) through the formation of inter-chain disulphide bonds between one or more cysteine (C) residues of the core hinge sequence of two Fc region components, while in some other embodiments, the Fc region component comprises a CH3 domain to enable CH3:CH3 self-association by non-covalent interactions, or Fc region component comprises a CH3 domain and CH2 domain to enable self-association by CH2:CH2 and CH3:CH3 non-covalent interactions. It is to be noted that in the Examples and Figures hereinafter, dimeric forms of the immunotherapeutic protein are considered to be single molecules (i.e. each comprising two copies of the protein dimerised through the Fc region components) and are referred to as monomers / monomeric.

[0203] In other embodiments, the protein comprises an oligomer in physiological saline (at neutral pH such as physiological pH of about 7.4), wherein the immunotherapeutic protein comprises an amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide, which enables self-association of the Fc or Fc-like fragment comprising the mutated Fc components into soluble oligomeric forms in physiological saline (e.g. oligomeric forms comprising, for example, 3 copies, 4 copies, 5 copies, 6 copies or 12 copies assembled from, for example, dimeric forms of the immunotherapeutic protein, such that, in some particular embodiments, the oligomeric form of the immunotherapeutic protein may comprise six dimeric proteins (i.e. a hexameric form) comprising, in total, 12 copies of the immunotherapeutic protein). In such embodiments, the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide may be: H429X1, where X1is selected from tyrosine (i.e. H429Y), methionine, isoleucine, leucine, tryptophan and valine; but preferably, the immunotherapeutic protein comprises an H→Y (i.e. H429Y) substitution.

[0204] In yet other embodiments, the immunotherapeutic protein may form an oligomer upon binding to a relevant target through "on target" oligomerisation. Such oligomerisation may occur with an immunotherapeutic protein that is monomeric or dimeric in physiological saline (at neutral pH such as physiological pH of about 7.4), and comprises an amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide which enables on target oligomerisation into oligomeric forms (e.g. forms comprising 3 copies, 4 copies, 5 copies, 6 copies or 12 copies (e.g. a hexamer of dimeric forms) of the immunotherapeutic protein). In such embodiments, the amino acid substitution at the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide may be: H429X2, where X2is selected from phenylalanine (i.e. H429F), glutamate, glutamine and serine; but preferably, the immunotherapeutic protein comprises an H→F (i.e. H429F) substitution.

[0205] Alternatively, oligomeric forms of the immunotherapeutic protein as described herein may be produced by employing other techniques well known to those skilled in the art such as, for example, the use of Fc multimeric forms (stradomersTM) comprising linked multimerisation domain (MD) sequences from the hinge region of human IgG2 or the isoleucine zipper (ILZ) to the N- or C-terminus of murine IgG2a (Fitzpatrick EA et al., Front Immunol 11, article 496, 2020), and the use of multimerisationsequences from IgM (Melcheil et al., Sci Rep doi:10.1038 / srep0012, 2011), or docking and dimerisation sequences from unrelated proteins such as cyclic adenosine monophosphate-dependent protein kinase and A-kinase anchoring proteins (Rossi EA et al., Bioconjug Chem 23(3):309-323, 2012).

[0206] It has further been found that the immunotherapeutic protein as described herein may show enhanced binding of the neonatal Fc receptor (FcRn) by virtue of an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering). It is anticipated that this will mean that the immunotherapeutic protein will show a greater in vivo half-life (i.e. relative to an equivalent immunotherapeutic protein with no mutation at the H429 position) and thereby improved pharmacokinetics (PK), since FcRn is known to "recycle" antibodies so as to control how long they last in the body (Ward ES and RJ Orber, Trends Pharmacol Sci 39(10):892-904, 2018). Dimeric immunotherapeutic proteins (e.g. immunoglobulin molecules)

[0207] The immunotherapeutic protein as described herein may be a dimeric immunotherapeutic protein comprising an immunoglobulin molecule, wherein the immunoglobulin molecule comprises first and second immunoglobulin heavy chain polypeptides which each comprise an Fc region component comprising at least a CH3 domain (or at least a CH4 domain). The Fc region components of the first and second immunoglobulin heavy chain polypeptides may self-associate (i.e. to form the dimer) either by non-covalent bonding such as hydrogen bonding or through the formation of disulphide bonds through one or more cysteine (C) residue, particularly where situated within the hinge sequence, especially the core hinge sequence (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003), or otherwise be linked by, for example, a cross-linker compound such as those mentioned above (e.g. by cross-linking the CH3 domains of the Fc region components). As such, the immunotherapeutic protein may be an immunoglobulin molecule such as, for example, an antibody or antibody derivative such as an scFv-Fc (wherein a dimer of scFv polypeptides is formed through dimerisation of Fc region components), mini- body (wherein a dimer of scFv polypeptides is formed through linking CH3 domains / CH4 domains), or any of the other suitable Fc-containing antibodies or derivatives known to those skilled in the art (e.g. as summarised in the "Periodic Table of Antibodies" mentioned above).

[0208] Accordingly, the dimeric immunotherapeutic protein may further comprise at least one antigen recognition structure or, in other words, antigen binding region (e.g. an scFv or an antigen binding region comprising variable domains VL and VH such as a Fab fragment). The antigen binding region(s) may specifically bind to an antigen or epitope of therapeutic significance; for example, a cancer-associated antigen such as a cancer antigen present on the surface of a cancerous cell (e.g. a cell surface antigen differentially expressed and / or present in cancer cells such as the CD20, CD38 and CD52 antigens foundon the surface of CLL cells, and mucins (e.g. 1) or carbohydrate (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 of an infectious agent such as an antigen of a bacterial, yeast, parasite or viral pathogen (e.g. an antigen of the SARS-CoV-2 virus, Middle East respiratory syndrome coronavirus (MERS-CoV), respiratory syncytial virus (RSV) or dengue virus). In other examples, the antigen binding region(s) may specifically bind to a cell surface molecule known to induce cell proliferation (i.e. the cell surface molecule may be, for example, CD3 or CD28) and / or stimulate an inhibitory pathway to inhibit or reduce cell responses (i.e. the cell surface molecule may be, for example, an immune checkpoint molecule such as 4-1BB (CD137), cluster of differentiation (CD40, CD154), OX40 receptor (TNFRSF4, CD134), tumour 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 costimulatory (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); and wherein oligomerisation of the immunotherapeutic protein may bring about enhanced cross- linking (e.g. super-clustering) and, in turn, enhanced signalling to induce, for example, enhanced cell proliferation or inhibited / reduced cell responses (i.e. through enhanced stimulation of an inhibitory pathway). In yet other examples, the antigen binding region(s) may specifically bind to a cell surface molecule known to induce a cellular response selected from stimulation of an inhibitory pathway to inhibit or reduce responses. In still yet other examples, the antigen binding region(s) may specifically bind to a cell surface molecule which induces cell death when engaged by a ligand or other molecules such as agonistic mAbs. Such cell surface molecules include, for example, those of the TNF-receptor superfamily (also known as TNFRSF) including tumour necrosis factor receptor 1 (TNFR1, TNFRSF1A, CD120a), Fas (CD95, APO-1), Death Receptor 3 (DR3, TNFRSF25), Death Receptor 4 (DR4, CD261, TNFRSF10A, TRAILR1), Death Receptor 5 (DR5, CD262, TNFRSF10B, TRAILR2) and Death Receptor 6 (CD358, TNFRSF21). For example, under physiological conditions, the binding of TNF- related apoptosis-inducing ligand (TRAIL) to its receptor, DR5 (Carneiro BA et al., Nat Rev Clin Oncol 17(7):395-417, 2020) or the binding of the FAS ligand to its receptor, FAS (CD95) on the cell surface induces oligomerisation (Leukocyte and Stromal Cell molecules: The CD Markers, by Zola H et al., page 195, John Wiley & Sons, 2007) that initiates signalling leading to cell death. Other molecules (not related to the TNFRSF) which can induce apoptotic signals leading to cell death when cross-linked by, for example, mAbs or fragments thereof include CD38 (see Gambles MT et al., Molecules 26(15):4658,2021), CD20 (see Shan D et al., Cancer Immunol 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 a dimeric immunotherapeutic protein such that oligomerisation may bring about enhanced cross-linking and induction of apoptotic signals to bring about cell death as may be desired in a target cell.

[0209] In some embodiments, an immunoglobulin molecule according to the present disclosure may comprise two antigen binding regions, each of which specifically binds to different antigens or epitopes. As used herein, an “antigen” is any substance that causes the body to make an immune response (antibodies) against that substance. As used herein, an “epitope” also known as an “antigenic determinant” is the part of an antigen that is recognized by an antigen recognition structure.

[0210] In some embodiments, an immunoglobulin molecule according to the present disclosure that is an antibody, may be of, for example, an IgD, IgE or IgM isotype, but preferably, will be of an IgA or IgG isotype, such as an antibody of any of the human IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 sub-types.

[0211] The first and second immunoglobulin heavy chain polypeptides of a dimeric immunotherapeutic protein comprising an immunoglobulin molecule may be the same (i.e. a homodimer) or different (i.e. a heterodimer).

[0212] In some embodiments, an immunoglobulin molecule according to the present disclosure that is an antibody, may be an antibody that forms oligomers either through oligomerisation into oligomers (e.g. hexamers) through self-association of antibodies either in solution (e.g. in physiological saline at neutral pH such as physiological pH of about 7.4) or upon binding to a relevant target (e.g. an antigen to which an antibody mutant is directed) through "on target" oligomerisation.

[0213] For example, and while not wishing to be bound by theory, it is considered that an immunoglobulin molecule that is an antibody which includes H429Y amino acid substitution in the Fc region component, may form oligomers in solution (e.g. in physiological saline at physiological pH) which can lead to an overall increase in the strength of binding (for example, enhanced avidity) to a target binding partner (e.g. a cancer antigen, antigen of a bacterial or viral pathogen, or other soluble target molecule or molecular complex). Thus, in the context of an antibody directed against a cancer antigen present on the surface of a cancerous cell, an antibody according to the present disclosure (in the form of an oligomer in solution) may show enhanced ability to bind to the cancerous cells leading to their destruction by, for example, CDC by complement or by phagocytosis by macrophages and / or ADCC; and in the context of an antibody directed against a soluble target molecule or molecular complex, an antibodyaccording to the present disclosure (in the form of oligomer in solution) may show enhanced ability to bind to the soluble target molecule or molecular complex leading to their removal by phagocytosis by macrophages.

[0214] On the other hand, it is considered that an immunoglobulin molecule that is an antibody which includes an H429F amino acid substitution in the Fc region component, may form oligomers upon binding to a relevant target such that the Fc components assembled "on target" present a stabilised optimal platform for binding with, for example, enhanced avidity to the C1q complement protein complex, and thereby lead to complement activation and, in turn, complement-dependent cytotoxicity (CDC). Thus, in the context of an antibody directed against a cancer antigen present on the surface of a cancerous cell, an antibody according to the present disclosure may show enhanced capability to activate complement on the cancerous cells leading to their destruction.

[0215] In some embodiments, an immunoglobulin molecule according to the present disclosure may be provided or used as a first immunoglobulin molecule with a first antigen binding region directed to a first antigen, in combination with a second immunoglobulin molecule (according to the present disclosure) with a second antigen binding region directed to a second antigen. By way of example only, in such a combination, the first antigen may be cancer antigen present on the surface of a cancerous cell (e.g. CD38 found on the surface of CLL cells) and the second antigen may be a death receptor (e.g. DR5). Oligomerisation of the first and second immunoglobulin molecules may lead to the formation of hetero- oligomers such as hetero-hexamers wherein the different cell surface targets are incorporated into the cluster which may lead to, for example, the enhanced induction of apoptotic signals to bring about cell death (e.g. where a death receptor has been targeted) with target cell specificity achieved via binding of, for example, a cancer antigen present on a cancerous cell. Fusion / conjugate immunotherapeutic proteins

[0216] The immunotherapeutic protein as described herein may comprise a fusion protein or protein conjugate comprised of a partner polypeptide linked to an Fc region component comprising at least a CH3 domain (or at least a CH4 domain).

[0217] A fusion protein or protein conjugate according to the present disclosure may be monomeric, dimeric or oligomeric. For example, in some embodiments, the fusion protein or protein conjugate comprises a monomer, wherein each monomer comprises one copy of the partner polypeptide (or a fragment thereof) and one copy of the Fc region component, while in other embodiments, the immunotherapeutic protein comprises a dimer wherein the Fc region components self-associate (i.e. toform the dimer) either by non-covalent bonding as hydrogen bonding or through the formation of disulphide bonds through one or more cysteine (C) residue, particularly where situated within the hinge sequence, especially the core hinge sequence (if present) (Yoo EM et al., J Immunol 170:3134-3138, 2003). In such a dimer, the immunotherapeutic protein comprises two Fc region components (associated to one another to form, for example, an Fc fragment or Fc-like fragment) and two fused / conjugated partner polypeptides (or two fragments thereof), and as such may be considered as being bivalent in respect of the partner polypeptide (or fragment thereof). The two fused / conjugated partner polypeptides (or two fragments thereof) of a dimeric fusion protein or protein conjugate may be the same (i.e. a homodimer) or different (i.e. a heterodimer). In some embodiments of such a dimer, as depicted in Figure 53A, the fusion protein is antibody-like (Ab-like) and may have, for example a H2 format or H2L2 format (where the monomeric fusion proteins are produced with a light (L) chain, which in turn may be optionally fused / conjugated with a partner polypeptide (such as a target recognition structure)). As can be readily appreciated, each target recognition structure of an Ab-like molecule with a H2 or H2L2 format may be the same or different such as "X1 X1 X1 X1", "X1 X1 X1 X2", "X1 X1 X2 X2", "X1 X1 X2 X3" or "X1 X2 X3 X4", where X1, X2, X3 and X4 each represent a different target recognition structure. Also, one or more of the target recognition structures may be replaced with an alternative partner polypeptide type such as an enzyme(s) or reporter molecule(s).

[0218] The partner polypeptide may provide the immunotherapeutic protein with a beneficial function and / or characteristic.

[0219] In some embodiments, the partner polypeptide may be a cell surface receptor polypeptide (or a fragment thereof) or a co-receptor polypeptide (or fragment thereof).

[0220] For example, the partner polypeptide may be a cell surface molecule such as a cell surface receptor polypeptide (or a fragment thereof) that is capable of binding to a structural protein of a virus such that the immunotherapeutic protein may act as a "decoy" to block viral interaction and cellular entry of a virus to a host cell. Accordingly, some examples of such cell surface receptor polypeptides (or fragments thereof) that may comprise the partner polypeptide include the angiotensin-converting enzyme 2 (ACE2) ectodomain (ACE2 being the cellular entry receptor for SARS-CoV-2), nucleolin (the cellular entry receptor for RSV), dipeptidyl peptidase 4 (DPP4, CD26), Hsp70 (the cellular entry receptor for Japanese encephalitis virus), hepatitis A virus cellular receptor 1 (HAVCR1 / TIM-1; the cellular entry receptor for Hepatitis A virus and Ebola virus), cluster of differentiation 155 (CD155; the cellular entry receptor for poliovirus), Glucose transporter 1 (GLUT1; the cellular entry receptor for human T cell leukaemia virus 1), Proto-oncogene tyrosine-protein kinase MER (MERTK; a host factor that promotes swine fever virus entry), TYRO3 Protein Tyrosine Kinase (TYRO3; a cell surface protein associated withlymphocytic choriomeningitis (LMCV) infection) (a cell surface protein associated with lymphocytic choriomeningitis (LMCV) infection) and the cluster of differentiation 4 receptor (CD4 receptor; the cellular entry receptor for human immunodeficiency virus (HIV)), to name just 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 a virus, a point mutation at the position of the Fc region component corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering) may, for example, provide the immunotherapeutic protein with an enhanced ability to provide an antiviral effect through CDC of infected cells (e.g. where the mutation is H429F) or confer upon the immunotherapeutic protein enhanced virus neutralisation (e.g. where the mutation is H429Y). In other examples, the partner polypeptide may be a ligand for a cell surface molecule known to induce cell proliferation (i.e. the cell surface molecule may be, for example, CD3 or CD28) and / or stimulate an inhibitory pathway to inhibit or reduce cell responses (i.e. the cell surface molecule may 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 oligomerisation of the immunotherapeutic protein may bring about enhanced cross-linking (e.g. super cross-clustering) and, in turn, enhanced signalling to induce, for example, enhanced cell proliferation or inhibited / reduced cell responses (i.e. through enhanced stimulation of an inhibitory pathway). In yet other examples, the partner polypeptide may be a ligand for a cell surface molecule which induces cell death when engaged by a ligand or other molecules such as agonistic mAbs (e.g. the abovementioned cell surface molecules of the TNFRSF (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). Thus, in some embodiments, the immunotherapeutic protein as described herein may comprise a dimeric fusion protein or protein conjugate comprised of, for example, TRAIL (i.e. the ligand for DR4 or DR5) as the partner polypeptide linked to an Fc region component comprising at least a CH3 domain which includes an H429F amino acid substitution. Upon binding of the ligand to the cell surface molecule, oligomerisation of an immunotherapeutic protein of these embodiments may bring about enhanced cross-linking and induction of apoptotic signals to bring about cell death as may be desired in a target cell.

[0221] Where the partner polypeptide is a co-receptor polypeptide (or fragment thereof), the co- receptor polypeptide (or fragment thereof) may be, for example, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5) (co-receptors of the CD4 receptor which bind to the HIV viral glycoprotein gp120 and enable HIV to fuse with the host cell membrane), tetraspanin and occludin (which are co-receptors required to enable infection by Hepatitis C virus (HCV)), and Gas6 (which is a ligand of receptors (such as AXL and TYRO3) which together bind to phosphatidylserine displayed on viruses, including West Nile virus, Zika virus and Ebola virus among others, and facilitates host cell entry of such viruses), among many others.

[0222] In another example, the partner may be a cell surface receptor polypeptide (or a fragment thereof) that is capable of binding to a ligand for the cell surface receptor. Accordingly, some examples of such polypeptides (or fragments thereof) include the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (or a soluble extracellular fragment thereof). CTLA4 functions as an immune checkpoint and downregulates immune responses. An immunotherapeutic protein comprising a fusion protein or protein conjugate comprised of a CTLA4 partner polypeptide (e.g. a CTLA4-Fc component fusion protein) may, by forming a dimer or oligomer, show enhanced binding to the CTLA4 ligand and thereby act as a decoy to produce various therapeutic effects for the potential treatment of tumours (e.g. melanoma and colorectal cancer) and various autoimmune diseases such as SLE and rheumatoid arthritis (RA). Further examples of suitable cell surface receptor polypeptides (or a fragment thereof such as an ectodomain) that are capable of binding to a ligand for the cell surface receptor, include other immune checkpoints (e.g. PD1) and other cytokine receptors such as interleukin-1 receptor (IL-1R), interleukin-6 receptor (IL-6R), tumour necrosis factor receptor-2 (TNFR2, also known as CD120b) or a receptor for a cytokine of the TGF-β superfamily (see the review in Czajkowsky DM et al., Mol Med 4(10):1015-1028, 2012). An immunotherapeutic protein comprising a fusion protein or protein conjugate comprised of IL- 1R (or a fragment thereof) may potentially be used in an anti-IL-1 therapy for treatment of, for example, type 2 diabetes, and an immunotherapeutic protein comprising a fusion protein or protein conjugate comprised of IL-6R (or a fragment thereof) may potentially be used in an anti-IL-6 therapy for treatment of, for example, tumours and RA. An immunotherapeutic protein comprising a fusion protein or protein conjugate comprised of TNF-R2 or an ectodomain thereof may potentially be used in a treatment of RA or other inflammatory disease or condition.

[0223] While not wishing to be bound by theory, it is considered that a fusion protein or protein conjugate according to the present disclosure (whether in monomeric or dimeric form) which includes an H429Y amino acid substitution in the Fc region component may form oligomers in solution (e.g. in physiological saline at neutral pH such as physiological pH of about 7.4) that can lead to an overall increase in the strength of binding (for example, enhanced avidity) to a target binding partner (e.g. a structural protein of a virus or a ligand for a cell surface receptor, or other soluble target molecule or molecular complex). Thus, in the context of a fusion or conjugate protein which comprises a cell surface receptor polypeptide or a fragment thereof that is capable of binding to a structural protein of a virus, resulting in enhanced avidity of binding to the virus to provide enhanced virus neutralisation (possibly conferred by cross-linking and / or aggregating viral particles (virions)). However, it is considered that in at least some embodiments, such soluble oligomers are unable to substantially bind to Fc receptors and thus may be capable of virus neutralisation with little or no complement activation.

[0224] On the other hand, and again not be bound by theory, it is considered that a fusion protein or protein conjugate (whether in monomeric or dimeric form) which includes an H429F amino acid substitution in the Fc region component, may form oligomers upon binding to a relevant target (e.g. a structural protein of a virus or a ligand for a cell surface receptor) such that the Fc components assembled "on target" present a stabilised optimal arrangement for binding (with, for example, enhanced avidity) to the C1q complement protein complex, and thereby lead to complement activation and, in turn, enhanced complement-based effector functions such as complement-dependent cytotoxicity (CDC).

[0225] Immunotherapeutic proteins according to the present disclosure may be produced in accordance with any of the standard methodologies known to those skilled in the art. For instance, those skilled in the art can readily prepare an immunotherapeutic fusion protein by generating a construct, using standard molecular biology techniques, which comprises a polynucleotide sequence(s) encoding the fusion protein, introducing the construct into a suitable host cell (e.g. a human kidney (HEK) host cell or derivative thereof such as Expi293 cells (Thermo Fisher Scientific) for expression of the fusion protein or host animal (e.g. pig, monkey, rabbit or mouse), culturing the host cells according to standard culturing protocols and recovering the expressed fusion protein from the culture supernatant using, for example, any of the known suitable methodologies for purification (e.g. affinity chromatography (e.g. Protein A), ion exchange chromatography (IEX), size exclusion chromatography (SEC) and combinations thereof). In some embodiments, the immunotherapeutic protein is produced by a transgenic animal (the animal is modified to express the immunotherapeutic protein or nucleic acid as described herein). Similar methodologies can be used to prepare an immunotherapeutic protein which is an immunoglobulin molecule such as an antibody or an Ab-like molecule. That is, those skilled in the art can readily prepare a mutant antibody (i.e. an antibody including an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1 heavy chain (Eu numbering) by generating a construct(s) which comprises a polynucleotide sequence(s) encoding the variable heavy (VH) and light (VL) region sequence of a suitable antibody (e.g. one including an antigen binding region that binds to an antigen of interest) and a constant heavy (CH) region from, for example, an IgG1 antibody, and incorporate into the CH3 region-encoding polynucleotide sequence by standard molecular biology techniques such as site-directed mutagenesis, polynucleotide sequence changes to encode a mutation at H429 (e.g. H429F and H429Y). As with the preparation of an immunotherapeutic fusion protein, the construct(s) can be introduced into a suitable host cell (e.g. a human kidney (HEK) host cell or derivative thereof), cultured according to standard culturing protocols and the expressed mutant antibody purified from the culture supernatant using, for example, any of the known suitable methodologies for purification such as affinity chromatography. Further, it has been found that where affinity chromatography (e.g. Protein A) is used in the purification, especially where the immunotherapeutic protein is in the form of an Ab-like molecule, the use of mild elution conditions such as the use of an elution buffer comprising a lowconcentration of arginine (e.g. less than 130 mM) at less than or equal to pH 5, is advantageous so as to suppress the formation of aggregates. This can be achieved by, for example, standard liquid chromatography systems that can deliver a buffer gradient to the column, in this case, a linear gradient to, for example, 35% of 130 mM arginine (pH 4.0).

[0226] In some preferred embodiments, the recovery of an expressed immunotherapeutic protein according to the present disclosure is: (i) preferably conducted under conditions of mildly acidic pH (e.g. a pH of less than neutral pH such as pH 6.5, preferably pH 5.5, and more preferably, pH 5.0) where it is desired that the immunotherapeutic protein be provided in a monomeric form; or (ii) where it is desired that the immunotherapeutic protein be provided in a oligomeric form (e.g. as a hexamer), preferably conducted 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, at physiological pH of about 7.4); or (iii) preferably conducted using a method comprising affinity chromatography using an elution buffer comprising a low concentration of arginine (e.g. less than 130 mM) and at less than or equal to pH 5.0 (preferably about pH 4.0), especially where it is desired that the immunotherapeutic protein be provided as an antibody-like molecule.

[0227] In some particular embodiments, the recovery of an expressed immunotherapeutic protein according to the present disclosure comprises recovery by size exclusion chromatography (SEC), for example under conditions of mildly acidic pH for the production of monomeric forms of the immunotherapeutic protein, or under substantially neutral pH for the production of the immunotherapeutic protein in oligomeric forms. The SEC may, if desired, follow a recovery stage comprising ion exchange chromatography (IEX).

[0228] The US FDA grouped bsAbs into two main classes based on their mechanism of action, namely cell-bridging bsAbs and antigen-crosslinking bsAbs (non-cell-bridging molecules; Labrijn et al, 2019). Most cell-bridging bsAbs are designed for cancer treatment by linking immune cells to malignant cells. Through sequential binding, that is, by binding the cancer cell first owing to a higher affinity to tumor antigens, cell-bridging bsAbs can improve specificity and effectiveness with reduced non-specific side effects and lower dosage compared with mAbs. In contrast, antigen-crosslinking bsAbs target two antigens or two receptors simultaneously. Their main MoA is either blocking signals of cell growth / survival or activation of immune cells (Engelman et al, 2007). Antigen-crosslinking bsAbs basically act similar to mAbs except that they bind two different targets.

[0229] In an aspect, the present disclosure the use of an immunotherapeutic protein as described herein, for treating or preventing a disease or condition in a subject, wherein the disease or condition may be selected from, for example, autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

[0230] In an aspect, the present disclosure provides the use of an immunotherapeutic protein asdescribed herein, in the manufacture of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

[0231] In an aspect, the present disclosure provides a method for treating or preventing a disease or condition, comprising administering to the subject an effective amount of an immunotherapeutic protein as described herein, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

[0232] Among the diseases and conditions that may be treated or prevented by the method as described herein, are autoimmune diseases and conditions such as SLE and MS, other inflammatory diseases (e.g. immune complex vasculitis), infectious diseases and proliferative diseases (especially solid tumours such as breast cancers), blood cancers such as lymphoproliferative disorders (LPDs) including leukaemias (e.g. acute lymphoblastic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL)), adenocarcinomas and lymphomas, as well as multiple myeloma (MM) and X-linked proliferative disease.

[0233] The method as described herein will be typically applied to the treatment of a disease or condition in a human subject. However, the subject may also be selected from, for example, livestock animals (e.g. cows, horses, pigs, sheep and goats), companion animals (e.g. dogs and cats) and exotic animals (e.g. non-human primates, tigers, elephants etc).

[0234] In some embodiments, wherein the immunotherapeutic protein comprises a fusion protein comprising a partner polypeptide linked to an Fc region component comprising at least a CH3 domain (or at least a constant heavy domain 4 (CH4) domain) and said partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof) that is capable of binding to a structural protein of a virus such that the immunotherapeutic protein may act as a "decoy" to block viral interaction and cellular entry of a virus to a host cell, the method as described herein may further comprise administering an antibody directedagainst the said virus (i.e. target virus). For where the immunotherapeutic protein comprises an ACE2 polypeptide (or a fragment thereof) which binds with the RBD of the CoV-2 spike protein, the antibody may be selected from antibodies that are, for example, broadly neutralising coronavirus mAbs (ie bNmAbs which can neutralise multiple coronavirus types or strains), broadly reactive coronavirus mAbs (ie mAbs which may not be neutralising but can bind with multiple coronavirus types or strains), broadly neutralising SARS-CoV-2 mAbs (ie bNmAbs which can neutralise multiple SARS-CoV-2 strains) and broadly reactive SARS-CoV-2 mAbs, broadly neutralising coronavirus spike stem specific mAbs, broadly reactive coronavirus spike stem specific mAbs, are broadly neutralising SARS-CoV-2 spike stem specific mAbs, and broadly reactive SARS-CoV-2 spike stem specific mAbs. In some specific embodiments, the antibody may be, for example, targeted to 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 may be targeted to, for example, an epitope on the spike protein (S) but at a site distinct from the RBD. More generally, where the immunotherapeutic protein comprises a cell surface receptor polypeptide other than an ACE2 polypeptide, or a co-receptor polypeptide, or a fragment thereof, the antibody directed against the target virus may be selected from antibodies that are, for example, broadly neutralising against a class / family of viruses (e.g. human immunodeficiency viruses (HIV)), or which are broadly reactive against a class / family of viruses, broadly neutralising of strains of a specific virus type (e.g. bNmAbs which can neutralise multiple HIV-1 strains), and mAbs which are broadly reactive to a specific virus type. As shown hereinafter, it has been found that an immunotherapeutic protein of the present disclosure and an antibody directed against the target virus may synergistically cooperate to enhance CDC killing of cells (e.g. virus-infected cells).

[0235] Similarly, where the immunotherapeutic protein comprises a fusion protein comprising a partner polypeptide linked to an Fc region component comprising at least a CH3 domain (or at least a constant heavy domain 4 (CH4) domain) and said partner polypeptide is, for example, directed at a target of therapeutic significance (e.g. the partner polypeptide is a cell surface receptor polypeptide (or a fragment thereof) that is capable of binding to CTLA4 which functions as an immune checkpoint and downregulates immune responses), the method as described herein may further comprise administering an antibody directed against the said target (e.g. an antibody which binds to CTLA4) to, for example, provide an enhanced response such as enhanced CDC killing of cells (e.g. cancer cells).

[0236] Where the method as described herein does comprise administering an antibody in addition to 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 the position corresponding to H429 of the amino acid sequence of the human IgG1 heavy chain polypeptide (Eu numbering) such as, for example,an H429X2amino acid substitution, where X2is from phenylalanine (H429F), glutamate (H429E), glutamine (H429Q),serine (H429S), alanine (H429A), threonine (H429T), tyrosine (H429Y), leucine (H429L), valine (H429V), glycine (H429G), tryptophan (H429W), arginine (H429R) and proline (H429P).

[0237] In an aspect, the present disclosure provides a pharmaceutical composition or medicament comprising an immunotherapeutic protein as defined herein, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0238] The immunotherapeutic protein may be administered in combination with one or more additional agent(s) for the treatment of the particular disease or condition being treated. For example, in the context of treating proliferative diseases, the immunotherapeutic protein may be used in combination with other agents for treating cancer (including, for example, antineoplastic drugs such as cisplatin, gemcitabine, cytosine arabinoside, doxorubicin, epirubicin, taxoids including taxol, topoisomerase inhibitors such as etoposide, cytostatic agents such as tamoxifen, aromatase inhibitors (e.g. as anastrozole) and inhibitors of growth factor function (e.g. antibodies such as the antierbB2 antibody trastuzumab (Herceptin™)). In some embodiments, the immunotherapeutic protein may be administered with one or more additional agent(s) which may also be an immunotherapeutic protein(s) according to the present disclosure. For example, where a first immunotherapeutic protein according to the present disclosure comprises an ACE2 polypeptide (or a fragment thereof) which binds with the RBD of the CoV-2 spike protein, then the second immunotherapeutic protein of the present disclosure may be an antibody directed against the target CoV-2 virus, particularly one targeted to an epitope of a different structural protein (e.g. the envelope protein (E), the membrane protein (M) or the nucleocapsid protein (N)) or on the same structural protein (i.e. the spike protein (S)) but at a site distinct from the RBD. As shown hereinafter, it has been found that a combination of such immunotherapeutic proteins may synergistically cooperate to enhance CDC killing of cells (e.g. virus-infected cells).

[0239] Where used in combination with other agents, the immunotherapeutic protein can be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. If administered in separate pharmaceutical compositions, the immunotherapeutic protein and the other agent(s) may be administered simultaneously or sequentially in any order (e.g. within seconds or minutes or even hours (e.g.2 to 48 hours)).

[0240] The immunotherapeutic protein may be formulated into a pharmaceutical composition with a pharmaceutically acceptable carrier, diluent and / or excipient. Examples of suitable carriers and diluents are well known to those skilled in the art, and are described in, for example, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, PA 1995. of suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the Handbook of Pharmaceutical Excipients, 2ndEdition, (1994), Edited by A Wade and PJ Weller. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of carrier, diluent and / or excipient may be made with regard to the intended route of administration and standard pharmaceutical practice.

[0241] A pharmaceutical composition comprising an immunotherapeutic protein as defined herein may further comprise any suitable binders, lubricants, suspending agents, coating agents and solubilising agents. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, 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 and the like. Preservatives, stabilising agents, and even dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of phydroxybenzoic acid. Anti-oxidants and suspending agents may be also used.

[0242] A pharmaceutical composition comprising an immunotherapeutic protein as defined herein will typically be adapted for intravenous or subcutaneous administration. As such, a pharmaceutical composition may comprise solutions or emulsions which may be injected into the subject, and which are prepared from sterile or sterilisable solutions. A pharmaceutical composition may be formulated in unit dosage form (i.e. in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose).

[0243] The immunotherapeutic protein, uses and pharmaceutical composition of the present disclosure are hereinafter further described with reference to the following, non-limiting examples. EXAMPLES Example 1 ACE-2-Fc fusion proteins comprising an H429 mutation and activity analysis Methods and Materials

[0244] Constructs and fusion proteins The amino acid sequence of the human ACE2 polypeptide is available from the European Nucleotide Archive (ENA, European Molecular Biology Laboratory) at accession no. BAB40370. The ectodomain ofthe protein (amino acids 19 to 740; shown as NO: 1 in Table 1) is comprised of a catalytic domain and a collectrin domain. Different forms of the ACE2 ectodomain were produced and studied in this example; particularly, a truncated (tr) ACE2 ectodomain comprising amino acids 19 to 615 of the mature ACE2 polypeptide (named trACE2 shown as SEQ ID NO: 2 in Table 1) and excluding the collectrin domain, a full length (fl) ACE2 ectodomain to (flACE2; comprising amino acids 19 to 740 of the mature ACE2 polypeptide; SEQ ID NO: 1 in Table 1), and an enhanced flACE2 ectodomain (EflACE2) comprising a triple mutation within the ACE2 polypeptide that has been reported to improve its binding affinity to the S protein (Chan et al., 2020 supra). These proteins were produced as fusion proteins with an Fc region component derived from human IgG1 to generate trACE2-Fc, flACE2-Fc and EflACE2-Fc according to standard techniques (see Table 2).

[0245] For example, a construct encoding the trACE2 ectodomain in pcDNA3.4 (Thermo Fisher Scientific) was prepared by joining a polynucleotide sequence encoding the trACE2 ectodomain to a synthetic sequence encoding a linker and a sequence encoding human IgG1 Fc (particularly, IgG1 Fc having the amino acid sequence of accession no. AXN93652.1 (immunoglobulin gamma 1 constant region, partial [Homo sapiens]; National Center for Biotechnology Information (NCBI) database). For the generation of a flACE2-Fc expression construct, a KpnI digestion of the trACE2 construct was conducted followed by insertion of a codon-optimised polynucleotide sequence encoding the ACE2 collectrin domain (GeneArt, Thermo Fisher Scientific). For the generation of an EflACE2-Fc expression construct, a synthetic polynucleotide sequence equivalent to that encoding flACE2-Fc, but with three mutations (i.e. T27Y, L79T and N330Y; sACE2.v2.4 described by Chan et al., 2020 supra) was used. In addition, variants of the fusion proteins were produced that incorporated H429F and H429Y mutations in the Fc component introduced using cleavage at a unique AfeI site within the IgG Fc-encoding sequence, and the subsequent insertion of appropriate mutagenic oligonucleotides using NEBuilder (New England Biolabs, Ipswich, MA, United States of America) according to the manufacturer's instructions.

[0246] The H429 residue occupies a "buried" site within the IgG1 Fc structure (see Figure 1) and is occupied by a histidine (His / H) residue, which is also found in the corresponding position of the Fc fragment of all other human immunoglobulin classes (Figures 3, 4), and accordingly is found in the corresponding position of, for example, human IgG1, IgG2, IgG3 and IgG4, as well as primate IgG subclasses and some mouse IgG subclasses. For example, the structure published in 1981 of human IgG1- Fc (PDB:1Fc1; Deisenhofer, 1981 supra) indicates that H429 is not a surface accessible residue (Figure 1), and in a space-filled representation of the Fc, H429 is only "visible" when overlaying residues are rendered in a non-space filling manner, as shown in Figure 1C. Also, analysis of residues neighbouring H429 indicates that H429 lies beneath the side-chains of these residues and calculation of accessible surface area (ASA) for the Fc residues indicates that H429 is solvent inaccessible (0% ASA (Å2) Figure1C). H429 is also not a surface accessible residue other Fc structures including the Fc within the structure of the anti-HIV mAb (PDB:1HZH; Saphire et al., supra) which forms a hexameric ring structure, but H429 does not form part of the interface. Similarly, analysis of the Fc structure of the anti- Lewis Y mAb (Ramsland et al., J Immunol 187(6):3208-3217, 2011) showed that the H429 is also buried in this antibody. Table 1 SEQ ID NO: 1 ll e Q170 180 190 200 210 220 230 240 RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL 250 260 270 280 290 300 310 320 HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL o , o3 cells (Thermo Fisher Scientific). All expressed fusion proteins were first purified from the culture supernatant by ion exchange chromatography (IEX) followed by additional purification by size exclusion chromatography (SEC). Particularly, the supernatant of the Expi293 cells transiently transfected for the expression of the respective ACE2-Fc fusion protein (where the Fc region was according to the wild type (WT) hIgG1 Fc mentioned in the preceding paragraph) was extensively dialysed against 10 mM Tris-HCl pH 8.0 and applied to a High-Q column (BioRad Laboratories, Hercules, CA, United States of America). Bound proteins were eluted with a linear gradient to buffer A containing 0.4 M NaCl. Fractions were examined by SDS-PAGE, and those fractions containing the flACE2-Fc WT fusion protein were pooled and concentrated using a 30 kDa cut-off filtration device (Pall Corporation, Port Washington, NY, United States of America) and separated by size exclusion chromatography (SEC) using a Superose 6 column (GE Life Sciences, Chicago, IL, United States of America).

[0248] The SARS-CoV-2 RBD-Ig and RBD AviTag have been described previously. The RBD AviTag was biotinylated in situ using Expi293BirA cells (Wines BD et al., J Immunol 197(4):1507-1516, 2016).

[0249] Lamelli native PAGE (N-PAGE), h, 4oC, was according to Wines BD et al., J Immunol 162(4):2146-2153, 1999).

[0250] Virus neutralisation assays Antiviral titre was determined using SARS-CoV-2 (CoV / Australia / VIC01 / 2020) in a microneutralisation assay as described previously (Juno JA et al., Nat Med 26(9):1428-1434, 2020).

[0251] Bio-Layer interferometry Measurements of the affinity of the ACE2-Fc fusion proteins for the CoV-2 S RBD were performed on the 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 kinetics buffer (phosphate buffered saline (PBS) pH 7.4 supplemented with 0.1% (w / v) bovine serum albumin (BSA) and 0.05% (v / v) TWEEN-20). After a 60 second (60s) biosensor baseline step, the fusion proteins (20 mg / mL) were loaded onto anti-human IgG Fc capture (AHC) biosensors by submerging sensor tips for 200s and then washing in kinetics buffer for 60s. For most of the fusion proteins, association measurements were performed by dipping into a two-fold dilution series of SARS-CoV-2 RBD from 16–250 or 500 nM for 180s and then measuring dissociation in kinetics buffer for 180s. For EflACE2-Fc WT, a two-fold dilution series of 2–31 or 63 nM was used. The biosensor tips were regenerated five times using a cycle of 5s in 10 mM glycine pH 1.5 and 5s in kinetics buffer, and baseline drift was corrected by subtracting the average shift of a fusion protein-loaded sensor not incubated with SARS-CoV-2 RBD, and an unloaded sensor incubated with SARS-CoV-2 RBD. Curve fitting analysis was performed with Octet Data Analysis 10.0 software using a global fit 1:1 model to determine KD values and kinetic parameters. Curves that could not be fitted were excluded from the analyses. Kinetic constants reported were representative of two independent experiments.

[0252] ACE2-Fc fusion proteins and recombinant dimeric rsFcγR binding by flow cytometry The ACE2-Fc fusion proteins or rituximab (a chimeric mAb targeted to CD20), at 5µg / ml, or the indicated concentrations were incubated with Ramos cells expressing transfected spike protein (Ramos-S cells; Lee WS et al., medRxiv doi:10.1101 / 2020.12.13.20248143, 2020) at 5x106cells / ml in 25µl of PBS containing 0.5% (w / v) BSA and 1mM glucose (PBS / BSA / G), for 30 minutes on ice, and then washed twice with PBS / BSA / G, incubated with PE or FITC conjugated anti-human IgG-Fc for 30 minutes on ice, before being washed again and resuspended in 25 μl of PBS / BSA / G.

[0253] Evaluation of binding of dimeric recombinant soluble FcγR (rsFcγR) was performed as previously described (Wines et al., 2016 supra). The ACE2-Fc opsonised Ramos-S cells or rituximab- opsonised cells were resuspended in 0.5µg / ml of biotinylated dimeric rsFcγRIIa (H131 allelic form) ordimeric rsFcγRIIIa (V158 allelic form) or and incubated for 30 minutes on ice followed by 1 / 500 streptavidin-APC (or anti-hIgG-Fc FITC for confirmation of ACE2-Fc opsonisation) for 20 minutes on ice. The cells were washed, resuspended in PBS / BSA / G and analysed on a Canto™ II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, United States of America).

[0254] Complement dependent cytotoxicity (CDC) assay CDC was measured using Ramos-S cells opsonised with ACE2-Fc function proteins or the control mAb, rituximab, then incubation with human serum as a source of complement. Thus, Ramos-S cells were first incubated with a fusion protein or rituximab as above (5x106cells / ml in 25µl of PBS / BSA / G for 30 minutes on ice), then washed before resuspending in 1 / 3 diluted normal human serum for 30 minutes at 37°C. Cells were washed twice with PBS and the dead cells were then enumerated by staining with 1 / 500 Zombie Green (from a Zombie Green Fixable Viability kit according to the manufacturer's instruction, BioLegend, San Diego, CA, United States of America) before fixing with 2% paraformaldehyde in PBS and analysis on a Canto™ II flow cytometer (Becton Dickinson).

[0255] Complement fixation immunoassay for ACE2-Fc fusion proteins Ninety-six well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated with 5 μg / ml Avidin in PBS overnight, blocked, and then incubated with either a serial two-fold dilution or 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 for 1 hour at RT. The ACE2-Fc fusion proteins were then added over the indicated concentration range. To measure C1q fixation, the plates were incubated with 10 μg / ml purified human C1q (Merck Millipore, Burlington, MA, United States of America) for 30 minutes at RT followed by 1 / 2000 dilution of rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:452019) for 1 hour at RT. In experiments to measure C5b-C9 fixation, the plates were incubated with 10% fresh human serum for 30 minutes at RT followed by 1 / 2000 dilution of rabbit anti- C5b-C9 (Merck Millipore) for 1 hour at RT washed and then incubated with goat anti-rabbit IgG conjugated to HRP (Merck Millipore) at 1 / 2000 dilution for 1 hour at RT followed by TMB substrate (Life Technologies Corporation, Carlsbad, CA, United States of America) for 15-20 minutes at RT. Reactivity was stopped using 1 M 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 plates washed three times between each step using PBS containing 0.05% (v / v) TWEEN-20. Samples were tested in duplicate and corrected for background reactivity using negative control wells from which ACE2-Fc proteins were omitted. The mean and SEM from independent experiments are shown.

[0256] RBD variant multiplex assay A custom multiplex array was produced with the SARS-1 S1 subunit (ACROBiosystems, Newark, DE,United States of America), SARS-Cov-2 S1 and NL63 S1 and S2 subunits (Sino Biological Inc., Beijing, China), NL63 S trimer (BPS Bioscience, San Diego, CA, United States of America), and a hexahistidine-tagged RBD WT protein (amino acids 19-613) and 21 variants identified from the GISAID RBD surveillance repository that had been expressed from pcDNA3 in Expi293 cells and purified by affinity chromatography. Bead coupling, washing steps, and data acquisition on a FlexMap3D™ analyser (Luminex Corporation, Austin, TX, United States of America) was as previously described (Lee et al., 2020 supra). Briefly, direct binding of the ACE2-Fc fusion proteins trACE2-Fc, flACE2-Fc and EflACE2-Fc at 0.5 to 250 nM, were detected using 25µl of 1.3µg / ml anti-human IgG R-Phycoerythrin Conjugate (SouthernBiotech, Birmingham, AL, United States of America). Data were fitted as 3- parameter agonist versus response curves (r2> 0.85) to determine each EC50. In a competition assay, RBD- or S1-coupled beads were simultaneously incubated with 20µl of 25µg / ml of biotinylated, AviTagged ACE2 in the presence of the various unbiotinylated ACE2-Fc fusion protein "competitors", at 1 to 280 nM for 2 hour at RT. The binding of biotinylated ACE2 (aa 19 to 615) was performed using, first, Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Thermo Fisher Scientific) at 4µg / ml (1 hour), followed by 10µg / ml of R-Phycoerythrin, Biotin-XX Conjugate (Thermo Fisher Scientific) (1 hour).

[0257] FcγRIIIa-NF-κB-RE nanoluciferase reporter assay FcγRIIIa-NF-κB-RE nanoluciferase reporter assays were conducted using IIA1.6 / FcR-γ / FcγRIIIa V158 cells expressing a 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 were incubated with agonists and the FcγRIIIa / NF-κB- RE reporter cells for 5 hours before measurement of induced nanoluciferase with Nano-Glo substrate (Promega Corporation). Results and Discussion

[0258] ACE2-Fc fusion protein construction and production A series of ACE2-Fc fusion proteins (Table 2) were produced and analysed for their capacity to neutralise SARS-CoV-2 infection and mediate Fc-dependent effector functions normally attributed to the mechanisms of action of antibodies. Table 2 Protein name ACE2 ectodomain ACE2 IgG1 FctrACE2 Truncated ACE2 Not mutated N / A* (amino acids 19-615)

[0259] In an attempt to enhance the avidity of binding to the SARS-CoV-2 spike protein or to confer and improve Fc-dependent effector functions, three versions of the ACE2 ectodomain were fused to an Fc region of IgG1 either unmodified or altered by mutation (i.e. substitution of histidine 429 with phenylalanine (H429F) or tyrosine (H429Y)) or modified glycosylation (i.e. lacking core fucose; trACE2- Fc-kif).

[0260] ACE2-Fc fusion protein purification Proteins were produced in Expi293 cells and purified by (an)ion exchange (IEX) then by size exclusionchromatography (SEC) at pH 7.4 (Figure 5). All the fusion proteins showed a similar IEX purification profile to that of flACE2-Fc-WT (Figure 5A) which contained a major elution peak (peak *; Figure 5A), and SDS-PAGE analysis of the collected fractions showed a single major species of about 270 kDa (Figure 5B). Except for those fusion proteins comprising the H429Y mutation, SEC analysis and purification confirmed the presence of a major monomeric species (Figure 5C) (NB. The monomer species are considered to be single molecules (i.e. monomer molecules) comprising two (dimerised) copies of the respective ACE2-Fc fusion protein) which were collected for further analysis, and only trace quantities of higher molecular weight oligomers and other impurities were apparent for example for flACE2-Fc-WT (Figure 5). Indeed, the presence of a similar monomeric species was also evident following the IEX purification of all fusion proteins with unmodified Fc-WT or an Fc region component including an H429F modification (not shown). For the fusion proteins comprising the H429Y mutation, SEC analysis (Figure 5D) (i.e. of the major IEX peak from flACE2-Fc H429Y mutant, not shown), and also of the major IEX peaks from the trACE2-Fc H429Y and EflACE2-Fc H429Y proteins (not shown)), revealed the presence of significant quantities of oligomeric (Yoli) and monomeric (Ymn) species, which were subsequently evaluated separately for functional activity.

[0261] Binding of ACE2-Fc fusion proteins RBD of SARS-CoV-2 S by ELISA The binding of the trACE2-Fc WT, flACE2-Fc WT and EflACE2-Fc WT fusion proteins to SARS-CoV- 2 receptor binding domain (RBD) (Figure 6A-C) were similar overall (i.e. EC500.35nM, 0.27 nM and 0.25 nM respectively; Figure 6D) with the enhanced intrinsic affinity of the EflACE2-Fc being less apparent with binding to this bivalent form of RBD, which was presented in fusion with mouse IgG1 Fc (RBD-Ig). The binding activity of the various fusion proteins with mutated Fc components were also equivalent, excepting the monomeric flACE2-Fc-H429Y which was of slightly lower affinity (EC500.48 nM) (Figure 6D) and the other H429Y Fc variants likewise trending to a lower level of affinity (Figure 6).

[0262] Oligomerisation of the ACE2-Fc H429Y fusion protein is pH dependent Oligomerisation of fusion proteins including a mutated H429Y Fc component was examined by SEC separation at pH 5.0 of the flACE2-Fc-H429Y prepared by IEX. In contrast to SEC at pH 7.4 (Figure and Figure 7A), SEC at pH 5.0 (Figure 7B) revealed a greater proportion of monomer Ymnwhich N-PAGE showed was purified to homogeneity (Figure 7C: lane 1 cf lane 2). The purified monomeric flACE2-Fc- H429Ymn, pH 5.0, was re-analysed by dialysis at pH 7.4 followed by both N-PAGE (Figure 7C) and by SEC at pH 7.4 (Figure 7D). Re-exposure to pH 7.4 yielded a mix of oligomer Yoli and monomer Ymn species (Figure 7C; lane 2 pH 5.0 cf lane 5 pH 7.4) indicating some equilibrium between these forms occurs at neutral pH. The flACE2-Fc-H429Ymn prepared at pH 5.0 showed equivalent binding to RBD-Ig as the other flACE2-Fc WT fusion proteins and Fc variants (Figure 7E). It is also clear that theprevalence of oligomers is related to the tyrosine of histidine 429 as this was not observed in the fusion proteins with phenylalanine-substituted H429F Fc region component.

[0263] Evaluation of virus neutralisation potency The antiviral activities of the ACE2-Fc fusion proteins was determined in a micro-neutralisation assay of SARS-CoV-2 infection of Vero cells (Figure 8) where the EC50 endpoint corresponds to neutralisation of ~99% of the inoculum virions (Khoury DS et al., Nat Rev Immunol 20(12):727-738, 2020).

[0264] The SARS-CoV-2 neutralisation endpoint of the truncated ectodomain, trACE2 (2.70 µM), was improved ~10-fold by fusion to the wild type Fc region of IgG1 (trACE2-Fc WT, 283 nM) (Figure 8). The improved potency is consistent with improved avidity of binding to SARS-CoV-2 spike RBD because of ACE2-Fc bivalency resulting from fusion of ACE2 ectodomains to the IgG Fc region and was also similar to that of flACE2-Fc WT. The EflACE2-Fc-WT showed a further ~20-fold improvement (11 nM) over the flACE2-Fc WT and trACE2-Fc WT fusion proteins and ~200-fold more than un-fused trACE2.

[0265] Analysis of the Fc modifications revealed several interesting differences. First, the H429Y mutation in trACE2-Fc and flACE2-Fc improved their virus neutralisation potency (Figure 8); this was surprising as the neutralisation function of the ACE2-Fc fusion proteins occurs well away from the modifications at the H429 position (i.e. in the fusion proteins, the ACE2 polypeptide is expected to neutralise the virus, but the H429 residue is distal to the ACE2 being present in the CH3 domain of the Fc component). Secondly, it was found that the neutralisation activity (21.9 nM) of the oligomeric form of trACE2-Fc-H429Yoli isolated by SEC at pH 7.4 was enhanced 13-fold over the trACE2-Fc WT. Both the oligomer, Yoli, and the monomer, Ymn,,forms of flACE2-Fc-H429Y comprising two (dimerised) copies of the ACE2-Fc fusion protein (Figure 5B) (endpoints of 10.0 and 20.9 nM respectively; Figure 8) showed greater potency than the flACE2-Fc WT (124 nM) fusion protein and was similar to the viral neutralisation achieved with EflACE2-Fc WT (10.6 nM), but EflACE2-Fc-H429Yoli was seemingly more potent (4nM) which represents a neutralisation activity of the EflACE2-Fc WT (10.6 nM) that is over 200-fold greater than that of the unfused trACE2, or in the case of EflACE2-Fc-H429Y (4nM), over 600 fold greater (Figure 8).Thus, the Fc-H429Y mutation increased SARS-CoV-2 neutralisation in the trACE2-Fc and flACE2-Fc fusion protein formats, and also trended towards a greater level of potency when combined with the triple mutation of the ACE2 in the intrinsically higher affinity EflACE2-Fc.

[0266] The phenylalanine substitution of histidine 429 (H429F) of the ACE2-Fc fusion proteins did not enhance neutralisation.

[0267] Interaction of ACE2-Fc fusion proteins FcγR The interaction of FcγRIIa and FcγRIIIa with the ACE2-Fc fusion proteins was evaluated by flow cytometry using ACE2-Fc opsonised Ramos-S cells and dimeric recombinant soluble FcγR (Wines et al., 2016 supra). The trACE2-Fc, flACE2-Fc and EflACE2-Fc fusion proteins all bound to FcγRIIa and FcγRIIIa (Figure 9A, 9B), however, the variant proteins comprising a mutated H429Y Fc component largely ablated binding to both of these Fc receptor types.

[0268] FcγRIIIa activation by ACE2-Fc fusion proteins Antibody dependent cytotoxicity (ADCC) and Fc-dependent clearance of viruses are important antiviral effector mechanisms that may play a protective role during SARS-CoV-2 infection (Li D et al., bioRxiv doi: 10.1101 / 2020.12.31.424729, 2021; and Shafer A et al., J Exp Med 218(3):e20201993, 2021). Thus, in this experimentation, the ability of the trACE2-Fc, flACE2-Fc and EflACE2-Fc fusion proteins and variant proteins to activate FcγRIIIa was evaluated. It was found that Ramos-S cells opsonised with ACE2-Fc fusion proteins comprising the wild type (WT) Fc region all initiated FcγRIIIa activation (Figure 10). However, it was surprising that the flACE2-Fc fusion protein induced a greater level of activation than trACE2-Fc indicating that the inclusion of the collectrin domain in the ACE2 component of the fusion protein substantially, and surprisingly, improves the potency of FcγRIIIa activation by the ACE2-Fc fusion protein. Further, it was found that the increased affinity that the EflACE2-Fc fusion protein has for the SARS-CoV-2 spike protein, did not alter the level of FcγRIIIa activation which was equivalent to that of flACE2-Fc (Figure 10D).

[0269] Production of the trACE2-Fc fusion protein in the presence of kifunensine (van Berkel PHC et al., Biotechnol Bioeng 105(20:350-357, 2010), that is trACE2-Fc-kif, also improved a modest level of FcγRIIIa activation shown by trACE2-Fc to a level comparable to that of the flACE2-Fc and EflACE2-Fc fusion proteins (Figure 10D) and approaching that of the therapeutic anti-CD20 mAb, rituximab, on the CD20+Ramos-S cells (Figure 10A,10D). Kifunensine, a mannosidase inhibitor, 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., Proc Natl Acad Sci U S A 108(31):12669-12674, 2011). Thus, it was hypothesised that similar treatment of the flACE2-Fc and EflACE2-Fc fusion proteins, or amino acid residue substitution to increase affinity for FcγRIIIa (Wang et al., 2018 supra), would be likely to further improve their FcγRIII activating potency.

[0270] Modification of the trACE2-Fc, flACE2-Fc and EflACE2-Fc fusion proteins by including an H429F Fc component mutation did not affect FcγRIII activation by opsonised Ramos-S cells (Figure 10). In contrast, the inclusion of the mutated H429Y Fc component in all ACE2-Fc fusion proteins ablatedFcγRIIIa activation of cells, which was consistent the abovementioned failure to bind to Fc receptors, especially FcγRIIIa (Figure 9B). Thus, while enhancing virus neutralisation, the H429Y modified Fc component in the trACE2-Fc, flACE2-Fc and EflACE2-Fc fusion proteins were inactive in FcγR binding (Figure 9) and in activating cells through FcγRIIIa (Figure 10). The hierarchy of FcγRIIIa activation by the fusion proteins was flACE2-Fc WT = EflACE2-Fc > trACE2-Fc emphasising that the presence of the collectrin domain is a key component in optimal activation of FcγRIIIa by the ACE2-Fc fusion proteins.

[0271] Complement fixation and activation, and lysis of SARS-CoV-2 spike cells by ACE2 Fc proteins The ACE2-Fc fusion proteins comprising mutated Fc region components were examined for their capacity to fix complement components C1q and C5-C9 in an ELISA-based analysis using avidin- immobilised RBD-biotin (Figure 11A–F), and importantly to mediate complement-dependent killing of cells expressing SARS-CoV-2 spike protein (Figure 11G). When the RBD was limiting (Figure 11A, 11B), the activity of the fusion proteins comprising a mutated H429F Fc component was surprisingly enhanced over the counterparts comprising a wild type Fc region. Additionally, the binding of C5b-9 (Figure 11E, 11F) which forms the membrane attack complex on cells, was equivalent for the fusion proteins comprising a mutated H429F Fc region component. Further, despite their oligomerisation (Figures 6 and 7), a feature associated with superior CoV-2 neutralisation activities (Figure 8), both the trACE2-Fc-H429Y and flACE2-Fc-H429Y fusion proteins showed little difference in C1q or C5-9 fixation over the counterparts comprising a wild type Fc region (Figure 11A-F). Moreover, it was apparent that the trACE2-Fc fusion protein was more potent in fixing complement C1q than flACE2-Fc (cf Figure 11C, 11E with 11D, 11F); due to the presence of the collectrin dimerisation domain in flACE2-Fc possibly reducing the segmental flexibility of the fusion protein and thereby negatively affecting complement C1q binding. Notably however, this difference was much reduced by the inclusion of the H429F Fc mutation of the trACE2-Fc-H429F and flACE2-Fc-H429F fusion proteins; particularly evident in the fixation of the C5b-C9 complex (Figure 11E, 11F).

[0272] Surprisingly, the fusion proteins comprising a mutated H429F Fc region component, were the only highly active ACE2-Fc fusion proteins in serum complement-dependent cytotoxicity (CDC) of Ramos-S cells (Figure 11G). Despite the capacity of the trACE2-Fc WT, as well as the trACE2-Fc-kif fusion proteins and flACE2-Fc WT, to fix C1q and C5b-9 in the ELISA assay (Figure 11A, 11C, 11E), these could not induce complement mediated cell death nor could EflACE2-Fc WT despite higher affinity for the SARS-CoV-2 spike protein. The trACE2-Fc-H429Ymn fusion protein was also active in complement mediated killing albeit weakly.

[0273] Since it is well known that the of the complement cascade can also lead to, for example, the phagocytosis of cells, microbes or particles opsonised with C1q (or other complement fragments such as, for example C3b or C3bi; Ricklin D et al., Immunol Rev 274(1):33-58, 2016) which bind to specific cell surface receptors such as, for example, CR1 or CR3 (Vandendriessche S et al., Front Cell Dev Biol 9:624025, 2021) on phagocytic cells, it is considered that the observed enhancement of Fc- dependent complement lysis by the ACE2-Fc fusion proteins including an H429Y mutation, will also be reflected in similar enhancements in complement-dependent phagocytosis of ACE2-Fc-coated targets (e.g. virions) through the complement receptors. Conclusion

[0274] It has been found that immunotherapeutic proteins in the form of fusion proteins comprising an angiotensin converting enzyme 2 (ACE2) polypeptide (or a fragment thereof) fused to an Fc region component comprising an H429 mutation provides considerable potential for the treatment or prevention of coronavirus infection. Selection of, for example, a full length or truncated (i.e. fragment) of ACE2, and various modifications of the Fc component may also enable considerable "tuning" of the antiviral agent to modify the action(s) by which the antiviral effect is achieved. For example, by including an Fc component in the immunotherapeutic protein comprising an H429Y substitution, an oligomeric immunotherapeutic protein may be produced which shows increased virus neutralisation, and abrogated FcγR binding and activation. On the other hand, by including an Fc component in the immunotherapeutic protein comprising an H429F substitution, an antiviral effect including complement-dependent cytotoxicity (CDC) of spike protein-expressing cells (e.g. infected cells) may be achieved. Thus, these unique Fc mutations enable complementary approaches to tuning the function of the Fc region component that may aid in the development of ACE2-Fc fusion proteins and other fusion proteins of therapeutic significance by allowing the selection of desired functional profiles. Example 2 Chimeric anti-TNP IgG1 antibodies comprising an H429 mutation and activity analysis Methods and Materials

[0275] Production of anti-TNP human IgG and mutant anti-TNP human IgG plasmid constructs Chimeric anti-TNP human IgG antibody constructs consisting of the variable heavy (VH) and light (VL) region sequence of the mouse anti-trinitrophenyl (anti-TNP) antibody TIB142 and the sequence from the constant heavy (CH) region from human IgG subclasses have been described previously in detail - hIgG1 (Patel D et al., J Immunol 184(11):6283-6292, 2010), hIgG2 and hIgG4 (Wines et al., 2016 supra). Allchimeric antibody sequences were subcloned into pCR3vector. Mutations for H429F, H429Q, H429E, H429S, H433A and H435A substitutions were made into the cDNA sequence encoding the Fc region component using standard molecular biology techniques.

[0276] Expression and production of IgG antibodies by Expi293 cells The IgG antibodies were produced in Expi293 human embryonic kidney cells as described previously (Wines et al., 2016 supra). Briefly, Expi293 cells were maintained in Expi293 Expression Medium (Gibco, Waltham, MA, United States of America) for both cell growth and protein production. Cells were transfected simultaneously with the IgG heavy chain plasmid (15ug) and light chain plasmid (15μg) diluted in Opti-MEM I Reduced-Serum Medium (Gibco) using the Expifectamine transfection kit (Life Technologies) then cultured for four days. Culture supernatants were clarified by centrifugation and filtered through a 0.2μm filter after which the IgGs were purified by affinity chromatography using a Hi- Trap HP Protein A column (GE Healthcare Life Sciences, Marlborough, MA, United States of America) and eluted with 0.1 M citric acid, pH 3.5, followed by neutralisation with 1M Tris-HCl, pH 9.0 and dialysation against PBS pH 7.5. Aggregates were removed by subsequent gel filtration on a Superose 6 10 / 300GL column (GE Healthcare Life Sciences) and monomeric IgG peak fractions collected. The antigen binding activity of all antibody preparations was tested on BSA-TNP by ELISA as described (Wines et al, 2016 supra).

[0277] Evaluation of antigen binding activity by monoclonal anti-TNP mAbs The anti-TNP mAbs were evaluated by ELISA for antigen binding activity by binding to TNP haptenylated BSA (TNP-BSA) as previously described in Wines et al., 2016 supra.

[0278] ELISA immunoassay for detection of complement fixation by mAbs Ninety-six well flat-bottom MaxiSorp Nunc plates (Thermo Fisher Scientific) were coated with 20 μg / ml TNP-BSA in PBS overnight at 4°C. The following morning, plates were blocked with 0.1% (w / v) casein in PBS for 2 hours at 37°C and then incubated with anti-TNP mAbs (concentrations between 4 and 0.125 μg / ml) for 2 hours at RT. To measure C1q fixation, the TNP:anti-TNP plates were incubated with 10 μg / ml purified human C1q (Merck Millipore) for 30 minutes at RT followed by 1 / 2000 dilution of rabbit anti-C1q IgG (Kurtovic L et al., BMC Med 17:452019) for 1 hour at RT. In experiments to measure C5b- C9 fixation, the TNP:anti-TNP plates were incubated with 10% fresh human serum for 30 minutes at RT followed by a 1 / 2000 dilution of rabbit anti-C5b-C9 (Merck Millipore) for 1 hour at RT, before washing and then incubation with goat anti-rabbit IgG conjugated to HRP (Merck Millipore) at 1 / 2000 dilution for 1 hour at RT followed by TMB substrate (Life Technologies) for 15-20 minutes at RT. Reactivity was stopped using 1M sulfuric acid and absorbance was measured at OD450nm. Test samples and reagents were prepared in 0.1% (w / v) casein in PBS, and plates washed three times between each step using 0.05%(v / v) Tween20 in PBS. Samples were tested in and corrected for background reactivity using negative control wells from which antibodies were omitted. Results and Discussion

[0279] Anti-TNP antibody constructs The TNP-WT mAb used in this example comprised either of two human heavy chain isotypes; first, an IgG1 isotype polypeptide (SEQ ID NO: 32) comprising, in the order of the N-terminus to the C-terminus, the TNP-specific VH domain of mouse monoclonal antibody TIB142 fused to CH1-hinge-CH2-CH3 domains of human IgG1, encoded by the cDNA (SEQ ID NO: 33) (Patel D et al., J Immunol 184: 6283– 6292, 2010); and secondly, an IgG2 isotype polypeptide (SEQ ID NO: 34) comprised, in the order of N- terminus to the C-terminus, the TNP-specific VH domain of mouse monoclonal antibody TIB142 fused to CH1-hinge-CH2-CH3 domains of human IgG2, encoded by the codon-optimised cDNA with the sequence shown as SEQ ID NO: 35. The TNP-specific light chain polypeptide (SEQ ID NO: 36) comprised the TNP-specific VL domain of mAb TIB142 fused to the human kappa chain constant domain, encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 37.

[0280] Modification of the IgG enhances complement fixation When antibodies, such as mAbs, are bound to their target antigen the classical pathway of complement may be activated leading to physiological effects including target cell destruction by complement dependent lysis (CDC) or complement mediated antibody dependent phagocytosis (C’ADCP) by specialised phagocytic cells through specific receptors for complement proteins and fragments. Activation of this major amplifying effector system of innate immunity is critically dependent on, and initiated by, the binding of the hexameric complement protein C1q which is part of the C1 complex. This binding of C1q by the antibodies bound to their target antigens leads ultimately to the formation of the membrane attack complex (MAC) comprising the other complement components (C5b, C6, C7, C8, C9).

[0281] Since the H429F Fc region component mutations conferred potent CDC in the ACE2-Fc fusion proteins (see Example 1), the effects of these (Figure 12) and several other mutations (Figure 13) were evaluated in human IgG1 under varied conditions of antigen density or input mAb concentration. The Fc component mutations were examined in an ELISA for complement fixation in the context of an intact (chimeric) IgG1 antibody with mouse V domains recognising the TNP hapten and human IgG1 constant domains. Surprisingly, replacement of histidine at position 429 in the TNP-IgG1-H429F mAb resulted in enhanced C1q binding compared to the C1q binding of the unmodified TNP-IgG1-WT mAb (Figure 12). Next, the TNP-specific mAbs were evaluated for generation of the membrane attack complex proteins (Figure 12). Again, the TNP-IgG1-H429F mAb showed enhanced complement activation as seen by theincreased formation of the MAC proteins (C5b, C7, C9), and importantly, this is entirely consistent with its observed enhanced capacity to bind C1q conferred by the H429F mutation. Thus, modification at position 429 can unexpectedly alter the properties of antibodies. This enhanced function is surprising as amino acid 429 is buried within the CH3 domain of IgG1 and is distant from the complement C1q binding surface in CH2 of the H chain and from the binding site for the leukocyte Fc receptors (Hogarth PM and Pietersz GA. Nature Reviews Drug Discovery 11:311-331, 2012; Chenoweth et al., 2020 supra).

[0282] The H429F mutation in the IgG1 antibody enhanced both C1q (Figure 12A) and C5b-C9 fixation (Figure 12B). The complement enhanced activities of the mutant IgG1 antibody comprising an H429F mutation, particularly in fixing C1q, were most apparent when lower densities of TNP:BSA antigen were opsonised by the mutated IgG (Figure 12). Thus, in the context of both the ACE2-Fc fusion proteins and in intact IgG1 antibodies, the H429F Fc component mutation enhances complement activation.

[0283] The antigen binding activity of the anti-TNP mAbs, TNP-IgG1-WT, TNP-IgG2-WT and the modified (mutant) mAb, TNP-IgG1-H429F, were evaluated by ELISA for binding to TNP haptenylated BSA (TNP-BSA) as described (Wines et al., 2016 supra). All of the mAbs showed substantially equivalent binding activity to TNP-BSA (Figure 12C) indicating that the enhanced complement activation by the Fc modified anti-TNP antibodies was due to the Fc mutation and not to differences in antigen binding activity. Conclusion

[0284] The data obtained in this example showed that the IgG-H429F mutant was capable of enhanced C1q and C5b-C9 fixation to enable increased complement activation and can therefore be expected to be capable of inducing useful complement-based effector functions. Example 3 Production of modified forms of therapeutic antibodies comprising an H429 mutation and analysis

[0285] In this example, the effects of modifying position H429 (i.e. by amino acid substitution) on the properties of antibodies was evaluated using a number of unrelated chimeric mAbs containing distinct antigen binding variable domains which bind to unrelated epitopes in a range of different target molecules on different cell types. These antibodies were "based" upon various commercially used, and in some cases, clinically significant mAbs. In particular, the experimentation was directed at determining the possible functional effects of amino acid substitution at position 429 in antibodies bearing the V domainsof the anti-HER2 mAbs trastuzumab and anti-CD20 mAbs bearing the V domains of rituximab or 11B8; and the V domain of the anti-CD38 mAb daratumumab. Methods and Materials

[0286] Antibodies and antibody constructs DNA sequences obtained by RT-PCR or synthetic DNA corresponding to the 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 DNAs to encode entire immunoglobulin H and L chains. These sequences were utilised in expression vectors such as pcDNA3.1, pcDNA 3.4 (Thermo Fisher Scientific) and pCIneo (Promega Corporation).

[0287] The unmodified mAbs used in this example were formatted on human IgG heavy chains and produced with a human kappa light chain. The unmodified mAbs include the specific VH and VL domains of the indicated mAb and are referred to as the wild type (WT) forms; for instance, the "WT" trastuzumab antibody used in this example, comprised the wild type (WT) HER2-specific VH and VL domains of trastuzumab as previously described (https: / / go.drugbank.com / drugs / DB00072).

[0288] More particularly, the trastuzumab-WT mAb used in this example comprised the HER2-specific heavy chain polypeptide described at https: / / go.drugbank.com / drugs / DB00072 (the amino acid sequence of which is provided as SEQ ID NO: 12) comprising, in the order of N-terminus to C-terminus, the HER2-specific VH domain of trastuzumab fused to CH1-hinge-CH2-CH3 domains of human IgG1 and encoded by a codon-optimised DNA with the 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 comprises the HER2-specific VLdomain of trastuzumab fused to a human kappa constant domain and is encoded by a codon-optimised DNA with the sequence shown as SEQ ID NO: 15.

[0289] The 11B8-WT mAb used in this example comprised a previously described CD20 specific heavy chain polypeptide (US Patent No 8,529,902) (SEQ ID NO: 16) comprising, in the N-terminus to C- terminus order, the CD20-specific VH domain of 11B8 fused to CH1-hinge-CH2-CH3 domains of human IgG1, and is encoded by a codon-optimised DNA with the sequence shown as SEQ ID NO: 17. Similarly, the polypeptide of the anti-CD20 mAb 11B8 light chain was as previously described (SEQ ID NO: 18) and comprised the CD-20 specific VL domain of 11B8 fused to human kappa constant domain and is encoded by a codon-optimised DNA with the sequence shown as SEQ ID NO: 19.

[0290] The daratumumab-WT mAb used in comprised a previously described CD38- specific heavy chain polypeptide (https: / / go.drugbank.com / drugs / DB09331) (SEQ ID NO: 20) comprising, in the order N-terminus to C-terminus, the CD38-specific VHdomain of daratumumab mAb fused to CH1-hinge-CH2-CH3 domains of human IgG1, and is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 21. Similarly the polypeptide of the anti-CD38 mAb daratumumab light chain was as previously described at https: / / go.drugbank.com / drugs / DB09331 (SEQ ID NO: 22) comprising the CD38-specific VL domain of the daratumumab mAb fused to human kappa constant domain is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 23.

[0291] The pertuzumab-WT mAb used in this example comprised a HER2-specific heavy chain polypeptide as previously described at https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 24) comprising, in the order of the N-terminus to the C-terminus, the HER2-specific VH domain fused to CH1-hinge-CH2-CH3 domains of human IgG1, and is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 25. Similarly, the polypeptide of the anti-HER2 mAb trastuzumab light chain was as previously described at https: / / go.drugbank.com / drugs / DB06366 (SEQ ID NO: 26) comprising the HER2-specific VLdomain of the pertuzumab mAB fused to human kappa constant domain and is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 27.

[0292] The rituximab-WT mAb used in this example comprised a CD20-specific heavy chain polypeptide (as described at https: / / go.drugbank.com / drugs / DB00073) (SEQ ID NO: 28) comprising in the order N-terminus to C-terminus, the CD20-specific VH domain of rituximab fused to CH1-hinge- CH2-CH3 domains of human IgG1, and is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 29. Similarly, the polypeptide of the anti-CD20 mAb rituximab light chain as previously described at https: / / go.drugbank.com / drugs / DB000723 (SEQ ID NO: 30) comprising the CD20-specific VL domain of rituximab fused to human kappa constant domain, and is encoded by the codon-optimised DNA with the sequence shown as SEQ ID NO: 31.

[0293] Synthesis of unmodified and mutated heavy chains Antibody expression vectors were generated by standard methods that are known to those skilled in the art. Briefly, antibody expression vectors consisted of a synthetic polynucleotide sequence, encoding the antibody heavy chain or light chain, appropriately placed within plasmids such as, for example, pcDNA3 and pcDNA3.4 (Thermo Fisher Scientific). Expression vectors for antibodies of different specificities were produced by cleavage at restriction sites at the boundaries of the existing variable domain (VH or VL). A new synthetic DNA encoding the new V domain and flanked by sequences (e.g.25 nucleotides) that were homologous to the cleaved vector were then incorporated by reaction with NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0294] Fc variants were produced by synthesis synthetic polynucleotide sequences encoding the variant or by cleavage of the Fc encoding sequence of antibody expression plasmids with appropriate restriction enzymes and the incorporation of new mutagenic synthetic DNA by reaction using NEBuilder (New England Biolabs) according to the manufacturer's instructions.

[0295] Expression of antibody constructs Expression of the antibodies was conducted using transient transfection of Expi293F cells (Thermo Fisher Scientific). Expi293F cells were cultured in EXPI expression media (Life Technologies) and, 24 hours prior to transfection cells, were split to a concentration of 2 x106viable cells / ml. On the day of transfection, 7.5 x 107viable log phase cells were centrifuged and resuspended in 25 ml of pre-warmed, antibiotic-free Expi293 Expression Media and maintained at 37oC until transfection. Transfection of the cells was then performed at RT using the Life Technologies Expifectamine Transfection Kit as follows. Eighty microlitres of ExpiFectamine 293 reagent was diluted with 1.5 ml Opti-MEM-I Reduced Serum Medium (Gibco) and incubated for 5 minutes at RT. Thirty µg of DNA (15µg of H chain DNA and 15µg of L chain DNA) was diluted in 1.5 ml of Opti-MEM-I reduced serum medium then added to the diluted ExpiFectamine reagent, incubated for 20-30 minutes at RT 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 cultured for a further four days at 37°C.

[0296] Cell cultures were harvested and centrifuged at 2500 rpm for 20–30 minutes, and the supernatant filtered with a 0.2 µm high flow filter (Sartorius AG, Göttingen, Germany) prior to purification. The presence of expected antibody in the supernatant was confirmed by SDS-PAGE.

[0297] Protein A affinity purification of mAbs The mAbs were purified from the supernatant of the transfected Expi293F cells by Protein A affinity chromatography. Briefly, Hi-trap™ Protein A high-performance columns (GE Healthcare Life Sciences) were washed and equilibrated in binding buffer (20 mM NaH2PO4 pH 7.0) and the cell culture supernatant was loaded, the columns washed with binding buffer to baseline OD280nm and bound antibody eluted with 0.1M sodium citrate tribasic dihydrate (pH 3.5) and 1 ml fractions collected and neutralised immediately with 1M Tris-HCl pH 9.0 and fractions containing the antibody, pooled.

[0298] Size exclusion chromatography (SEC) of mAbs Following Protein A affinity purification, size exclusion chromatography (SEC) was used to further purify and characterise the antibodies. The Protein A purified antibodies were concentrated to an OD280 nm of 6-8 using a 30kDa molecular weight cut-off centrifuge concentrator device (Merck-Millipore).Superose 610 / 300GL columns (GE Healthcare Sciences) were equilibrated in PBS pH 7.2, then the concentrated Protein A affinity purified antibody was loaded and separated at a flow rate of 0.5 ml / min in PBS and 0.5 ml fractions collected. For some mAbs, SEC was performed at pH 5.0, thus the concentrated Protein A purified mAb was dialysed overnight against buffer (100 mM sodium citrate, 100 mM NaCl, pH 5.0), then applied to a Superose 610 / 300 column pre-equilibrated in the same buffer. The dialysed antibody was applied to the Superose column SEC at a flow rate of 0.5 ml / min and 0.5 ml fractions collected from the column.

[0299] Evaluation of antigen binding by monoclonal antibodies Purified antibodies were tested for antigen binding prior to functional analysis. The mAbs recognising cell surface antigens were tested for antigen recognition by flow cytometry as previously described in Trist et al., J Immunol 192(2):792-803, 2014. The anti-CD20 mAbs based on rituximab or 11B8 and the anti-CD38 mAb based on daratumumab were tested for binding on Ramos lymphoma cells expressing CD20 and CD38. The anti-HER2 mAbs based on the 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 dilution in 25 µl of FACS buffer (PBS with 0.5% (w / v) BSA). Twenty-five microlitres of target cells at the concentration of 5 x 106 / ml were then added to the titrated mAb and incubated for 30 minutes on ice. Cells were then washed twice in FACS buffer, resuspended with 50 μL of anti-human IgG(Fab')2–Alexa 647 conjugate and incubated for 30 minutes on ice, washed twice in cold FACS buffer and then resuspended in 200 µl FACS buffer. The cells were analysed by flow cytometry using a BD FACSCanto™ II flow cytometer .

[0300] Flow cytometric detection of C1q binding to cells opsonised with monoclonal antibody C1q binding to antibody-opsonised cells was evaluated by flow cytometry. Ramos lymphoma cells or SK-OV-3 adenocarcinoma cells (5 x 106 / ml in FACS buffer) were incubated with serial dilutions of anti- CD20 rituximab-based or anti-HER2 trastuzumab-based antibodies respectively on ice for 30 minutes. Cells were washed twice in FACS buffer, resuspended in normal human serum diluted 1 / 3 in FACS buffer and incubated on ice for a further 30 minutes. The cells were then washed twice in cold FACS buffer and bound C1q was detected by resuspending the cells in rabbit antiserum detecting human C1q (1:500 dilution) and incubated for a further 30 minutes on ice. The cells were then washed twice in cold FACS buffer, resuspended in phycoerythrin-conjugated donkey antisera detecting rabbit antibody for 30 minutes, washed a further two times and resuspended in 200 µL of ice-cold FACS buffer, before being analysed on a BD FACSCanto™ II flow cytometer (Becton Dickinson).

[0301] Complement Dependent Lysis of Cells CDC was measured by flow cytometry using the Zombie Green Fixable Viability kit (BioLegend)following opsonisation of target cells with mAb. mAb of interest was serially two-fold diluted starting at the initial concentration indicated on the figures, in 25 μl of PBS / BSA / G (PBS containing 0.5% (w / v) BSA and 1mM glucose) or Lebowitz-15 (L-15) medium (containing 0.5% (w / v) BSA and lacking phenol red). Thus, for each WT mAb or modified mAb (i.e. H429-mutant mAb), or combinations thereof, 25µl of target cells, at a concentration of 5 x 106 / ml in 25µl of PBS / BSA / G, were then added to the titrated mAb and incubated for 30 minutes on ice. Cells were then washed twice in buffer and resuspended in 50 µl of complement (normal human serum (NHS) thawed and diluted 1:3 in buffer immediately before use), and incubated for 30 minutes at 37°C. Following the incubation, the cells were washed twice in cold buffer without BSA then resuspended in 50 µl Zombie Green (prepared in DMSO according to the manufacturer's instructions; BioLegend) diluted 1 / 500 in PBS and incubated for a further 30 mins on ice, protected from light. These cells were then washed once in buffer, resuspended in 50µl of 2% paraformaldehyde / BSA / PBS / G and incubated for 30 minutes on ice, then washed once in PBS / BSA and re-suspended in 200µl PBS / BSA / G. The cells were analysed by flow cytometry using a BD FACSCanto™ II flow cytometer (Becton Dickinson).

[0302] MAb cooperation to enhance C1q binding and CDC Ramos cells, at 5 x 105cells / ml in 25µl of PBS / BSA / G, were incubated with two-fold diluted rituximab- WT in the presence of 0.025µg / ml daratumumab-WT or rituximab-H429F with two-fold diluted rituximab-H429F, in the presence of 0.025µg / ml daratumumab-H429F mutant, or 0.5µg / ml 11B8-H429F mutant, for 30 minutes at 37°C in the presence of 1 / 3 dilution of normal human serum, washed twice with PBS, incubated with 1 / 500 Zombie Green for 30 minutes on ice washed again with PBS / BSA / G and fixed with 2% paraformaldehyde. The cells were washed, resuspended in PBS / BSA / G and analysed on a BD FACSCanto™ II flow cytometer (Becton Dickinson). Results and Discussion

[0303] Antigen binding by monoclonal antibodies The antigen binding capacity of the purified antibodies used in this example were tested and confirmed prior to functional analysis.

[0304] The purified mAbs detecting cell surface antigens were also tested by flow cytometry for binding to either antigen positive Ramos cells (CD20+CD38+) or SK-OV-3 cells (HER2+) and all of the mAbs showed readily detectable levels of antigen binding (Figure 14). Moreover, within each group, the modified mAbs carrying a mutation in the IgG heavy chain, showed similar binding activity to that of the unmodified (WT) form (e.g. the anti-CD20 mAbs rituximab-WT and the rituximab-H429F and rituximab- H429Y mutants showed equivalent homogeneous binding to CD20 expressing Ramos cells). Similarly,the anti-CD38 daratumumab-WT mAb and its modified mutant, daratumumab-H429F, as well as the anti-CD2011B8-WT and its CH3-modified mutants all gave binding profiles equivalent to their respective wild type forms (Figure 14). Also, the anti-HER2 mAb trastuzumab-WT and its modified CH3-mutant form, trastuzumab-H429F and trastuzumab-H429Y, showed readily detectable and equivalent binding to SK-OV-3 adenocarcinoma cells as did pertuzumab-WT and its modified CH3 mutants (Figure 14).

[0305] The effects of modifications at position 429 on antibody properties were further investigated using mAbs that bind to cell surface molecules. It was found, in particular, that substitution of H429 alters the physical characteristics of antibodies and selectively promotes antibody oligomerisation. For example, using mutants of the rituximab antibody; that is, rituximab-H429F mAb and rituximab-H429Y mAb, wherein the introduced tyrosine and phenylalanine amino acids are structurally similar, it was found that while the rituximab-H429F mAb and rituximab-H429Y mAb both eluted from Protein A affinity columns as a single homogeneous peak with elution characteristics like those of the unmodified rituximab-WT IgG (Figure 15A), purification by size exclusion chromatography (SEC) at pH 7.2 (Figure 16A) showed that the rituximab-H429Y mAb IgG exhibited a surprisingly different SEC profile (i.e. as compared to the profile for rituximab-WT and rituximab-H429F antibodies. That is, the rituximab-H429Y mAb SEC profile revealed two distinct peaks, one IgG peak (right of the vertical line in Figure 16A) which was coincident with a single IgG peak in the SEC of rituximab-WT and rituximab-H429F mAbs, and a second peak considered to be containing preformed oligomers (IgG (oli)) of rituximab-H429Y (see Figure 16A, left of the vertical line) and indicating an equilibrium between the formation of oligomeric and non- oligomeric forms. Whilst the presence of both oligomeric IgG and non-oligomeric IgG in the rituximab- H429Y mAb was surprising and different from the rituximab-WT IgG, it was even more surprising that this differed from what was observed with the rituximab-H429F mAb. Thus, the different biophysical properties of the rituximab-H429Y and rituximab-H429F mAbs is determined only by the presence of a hydroxyl in the tyrosine at position 429 of the rituximab-H429Y mAb.

[0306] Using SDS-PAGE, it was revealed that the oligomeric (IgGoli) and non-oligomeric (IgG) forms observed in the SEC IgG of rituximab-H429Y mAb behaved identically to each other and to the rituximab-WT IgG and rituximab-H429F mAb IgG. That is, despite its oligomeric nature, the rituximab- H429Y IgGoli migrated, under non-reducing conditions as a single ~150kDa IgG species identical to its non-oligomeric rituximab-H429Y IgG (H2L2) form indicating, importantly, that the oligomerisation of the mAb, facilitated by the H429Y mutation was non-covalent in nature (Figure 17A). Moreover, this clearly demonstrated that the rituximab-H429Y can exist at pH 7.2 as both IgG (IgG, H2L2) and as preformed oligomers of IgG (IgGoli) which arise from non-covalent association between the IgG heavy chains carrying the H429Y mutation. Further analysis under reducing conditions, by reduction in dithiotheitol(DTT), showed that these non-covalent oligomers rituximab-H429Y as well as the non-oligomeric rituximab-H429Y IgG, resolved into the expected ~50kDa heavy (H) chain and light ~25kDa (L) chain species identical to the SDS-PAGE characteristics of rituximab-WT and the rituximab-H429F mutant.

[0307] Thus, the H429Y modification confers new characteristics on rituximab wherein, in solution at pH 7.2, the mAb exists as pre-formed, non-covalent oligomers of IgG which exist in equilibrium with single IgG molecules. In contrast, rituximab-H429F and rituximab-WT mAbs only exist in solution as the single IgG species. Thus, the choice of amino acid at position H429 unpredictably affects the physical properties of rituximab IgG.

[0308] It was also found that these effects of mutation at position H429 is independent of the V domains. That is, equivalent evaluation of H429 substitution in trastuzumab, a mAb unrelated to rituximab, and which detects a cell surface molecule (i.e. HER2) structurally distinct from CD20, achieved similar results (see Figures 15B, 16B and 17B). In particular, the elution of the trastuzumab-WT IgG from the Protein A affinity matrix with citrate buffer, pH 3.0, yielded a homogeneous single peak (Figure 15B), as did elution of the trastuzumab-H429F and trastuzumab-H429Y mutant mAbs (the single IgG peak being coincident with the peak obtained of the unmodified trastuzumab-WT IgG mAb). Then, by size exclusion chromatography (SEC) at pH 7.2 (Figure 16B), it was found that the unmodified trastuzumab-WT and the trastuzumab-H429F mAbs each contained, the expected, single major IgG species, while the trastuzumab-H429Y mAb, in contrast, showed two distinct peaks (Figure 16B). The first trastuzumab-H429Y IgG peak was coincident with the single IgG peaks observed for trastuzumab- WT mAb and trastuzumab-H429F mAb (Figure 16B, right of vertical line) and the second trastuzumab- H429Y IgG peak contained oligomeric IgG (IgGoli) (Figure 16B, left of vertical line). In addition, SDS- PAGE (Figure 17B) showed that under non-reducing conditions (i.e. without disulphide bond reduction), the IgG peak of the trastuzumab-WT and trastuzumab-H429F mAbs each migrated at the expected 150 kDa mass. And, in the case of the trastuzumab-H429Y mAb, SDS-PAGE (Figure 17B) showed that under non-reducing conditions both the oligomeric trastuzumab-H429Y (IgGoli) and the non-oligomeric (IgG H2L2) species of trastuzumab-H429Y IgG migrated identically as a single 150 kDa species. This clearly demonstrated that the trastuzumab-H429Y can exist at pH 7.2 as both single IgG and as preformed oligomers of IgG arising from non-covalent association between the IgG heavy chains carrying the H429Y mutation.

[0309] Thus, the amino acid substitution at position 429 in the trastuzumab heavy chain conferred the same properties as those observed for the equivalent substitution in rituximab-based mAbs, and accordingly, the effects on the physical characteristics of the mutated mAb (particularly ofoligomerisation) facilitated by H249Y mutation of antibody specificity, molecular target, epitope and the VHand VLdomains.

[0310] Preformed oligomeric and non-oligomeric H429Y IgG antibodies exhibit enhanced CDC While not wishing to be bound by theory, it is considered that oligomerisation, particularly hexamerisation, of mAbs provides the optimal basis for C1q binding and activation of the complement cascade that leads to complement-dependent effector responses (e.g. phagocytosis or killing of target cells by complement-dependent cytotoxicity (CDC)). Such oligomers / hexamers can either form in solution or on the target (ie "on target" oligomerisation or assembly).

[0311] The effect on antibody effector function arising from the H429Y modification of the CH3 domain of the IgG heavy chain was evaluated by complement dependent cytotoxicity assays (CDC) (see Figure 18). By way of example, the effect of the H429 mutation on CDC potency was determined for the oligomeric and non-oligomeric forms of rituximab-H429Y using CD20 positive Ramos lymphoma cells as target cells. Thus, both the oligomeric rituximab-H429Y (IgG (oli)) and the non-oligomeric, IgG (H2L2) forms, separated by SEC at pH 7.2 (Figure 18A) were evaluated for CDC potency and compared to the CDC of the unmodified rituximab-WT IgG. Ramos cells were incubated in the presence of serial dilutions of the rituximab-WT IgG or non-oligomeric rituximab-H429Y IgG (H2L2) (p1 fraction of Figure 18A) or oligomeric rituximab-H429Y IgG H2L2 species (p2 fraction of Figure 18A). Normal human serum (diluted 1 / 3) was then added as a source of complement and the proportion of Ramos cells killed by CDC at each mAb concentration was determined by flow cytometry (Figure 18B). The non- oligomeric rituximab-H429Y IgG (H2L2) (the p1 fraction), demonstrated a surprising enhancement in CDC potency compared to the unmodified rituximab-WT. The non-covalent oligomeric IgG form of rituximab-H429Y (IgGoli) (the p2 fraction) also showed enhancement of CDC compared to rituximab- WT. Interestingly, both the oligomeric p2 form and the non-oligomeric p1 form exhibited similarly enhanced CDC potency which is consistent with the H429Y oligomers forming hexamers in solution at pH 7.2 prior to binding the target cell or in the case of non-oligomeric forms IgG (H2L2) forming hexamers on the target cell surface after antigen binding and thereby providing the optimal Fc configuration for C1q binding and thus enhanced complement activation as observed in the mAb-H429Y forms of IgG.

[0312] Thus, the substitution of H429 with tyrosine not only alters the physical characteristics of mAbs permitting oligomerisation in solution but also enhances complement dependent killing of target cells whether the IgG is oligomerised in solution or on the target.

[0313] The oligomerisation of mAbs including H429Y mutation is controlled by altering the pH The nature of the non-covalent oligomerisation of the mAbs including an H429Y modification in the CH3 domain was investigated further by altering the pH of the buffer (environment) (Figure 18C). In particular, the trastuzumab-H429Y mutant mAb (as purified by Protein A affinity chromatography) was dialysed either into buffer at pH 7.2 and then subjected to SEC also at pH 7.2, or dialysed into citrate buffer at pH 5.0 and then subjected to SEC at pH 5.0. At pH 7.2, both oligomeric and non-oligomeric IgG was present but lowering the pH of the buffer completely reversed the formation of IgG hexameric oligomers and only the IgG (H2L2) species was apparent (Figure 18C). Accordingly, the H429Y modification of the CH3 domain facilitates pH-sensitive, non-covalent oligomerisation of mAbs and thereby the oligomerisation / hexamerisation in solution or on the target that enhances effector potency of complement. Moreover, the capacity to control oligomerisation by alteration of pH may be useful the manufacture of immunoglobulins carrying this modification.

[0314] The H429F mutation enhances the activation of the serum complement cascade The extent of the effect on C1q binding by modification of the CH3 domain at the H-chain position 429 was investigated by flow cytometry on cells treated with the unrelated mAbs recognising CD20 or HER2 and bearing the H429F mutation in the CH3 of their heavy chains. The results are shown in Figure 19A- D. C1q binding was evaluated on CD20 Ramos cells treated with either rituximab-WT mAb or with rituximab-H429F mAb (Figure 19A, 19B). Similarly, C1q binding was also measured on HER2 positive SK-OV-3 cells treated with trastuzumab-WT mAb or trastuzumab-H429F mAb (Figure 19C, 19D). C1q bound detectably above background controls to Ramos cells treated with rituximab-WT mAb (Figure 19A) and to SK-OV-3 cells treated with trastuzumab-WT (Figure 19C). Importantly, C1q binding was enhanced to both the rituximab-H429F treated Ramos cells (Figure 19B) and to trastuzumab-H429F treated SK-OV-3 cells (Figure 19D) compared to the rituximab-WT and trastuzumab-WT. Thus, the substitution of H429 in the CH3 domain of the heavy chain enhanced C1q binding in these unrelated anti- CD20 and anti-HER2 mAbs. It is also therefore clear that this functional enhancement by H429 modification is independent of the antibody variable region and of the molecular target and the epitope detected. Moreover, enhanced C1q binding is consistent with enhanced antibody dependent activation of complement in the classical complement pathway which leads to the development of the MAC that leads to cell lysis via CDC.

[0315] Accordingly, since the binding of C1q component of the C1 complex initiates the classical complement cascade that leads to the lysis of cells, the effect of the enhanced C1q binding on the CDC killing potency was also determined using the rituximab-H429F mAb and compared to the killing potency of unmodified rituximab-WT (Figure 19E). In particular, the proportion of Ramos cells killed by CDC was evaluated by flow cytometry at each of the indicated mAb concentrations (Figure 19E). Like thesurprising enhancement of C1q binding conferred the H429F modification of the CH3 domain in the rituximab-H429F mAb (Figure 19A compared to Figure 19B), CDC killing potency (EC50) was also dramatically improved (>10-fold) compared to CDC by the unmodified rituximab-WT (Figure 19E). This surprising improvement in C1q binding and in CDC killing potency by the H429F mutation is consistent with oligomerisation (particularly hexamerisation) of the antibody on the target surface, and appears to provide the optimal configuration of the mAb Fc for the binding of C1q which is itself a hexamer, as exemplified by the analysis of the rituximab-H429F mAb.

[0316] The enhanced complement activation of mAbs with H429 mutation is epitope independent As discussed above, it was found that an H429 substitution in the anti-CD20 rituximab mAb strongly increased its CDC activity; to evaluate whether the enhancement of antibody function can apply to a different epitope within a single molecular target, complement activation was investigated using a second anti-CD20 mAb. The results are shown in Figures 20-22.

[0317] The type-II anti-CD20 mAb, 11B8 exhibits naturally poor CDC activity, but importantly also detects an epitope distinct from type-I anti-CD20 mAb rituximab (Meyer S et al., Br J Haematol 180(6):808-820, 2018). The 11B8-WT mAb and a mutant thereof including H429F substitution were produced as described above and purified separately by Protein A affinity chromatography (Figure 20). A single homogeneous IgG peak was obtained for the 11B8-WT and 11B8-H429F mAbs (Figure 20) indicating that the modifications had not altered the purification characteristics of the modified mAbs compared to the unmodified 11B8-WT mAb. Also, size exclusion chromatography demonstrated that the 11B8-WT mAb contained a single non-oligomeric IgG (H2L2) species (Figure 21A) as did the 11B8- H429F mAb. This was confirmed by SDS-PAGE analysis (Figure 21B) that revealed the expected 150 kDa IgG species, prior to disulphide bond reduction for the wild type and mutant mAbs that resolved to the ~50 kDa heavy chain and 25 KD light chain species following reduction in DTT (Figure 21B).

[0318] The CDC potency of 11B8 WT was also compared to that of the H429 modified antibody 11B8-H429F (Figure 22). The 11B8-WT and 11B8-H429F were titrated in the presence of normal human serum as a source of complement and the percent killing of Ramos cells evaluated for each mAb concentration. It was found that the 11B8-WT mAb failed to induce appreciable CDC but in contrast, the Fc modified 11B8-H429F mAb mediated potent CDC (Figure 22). Thus, the CDC potency is enhanced in two distinct and unrelated mAbs, rituximab-H429F and 11B8-H429F and (compare Figures 19E and 22), which indicates that improvement of CDC by modification of the H429 position can be achieved in different mAbs targeting distinct epitopes within the same target molecule. This also indicates that the enhanced CDC achieved by the CH3 modification is also independent of the variable domain of the modified antibody.

[0319] The enhancing effect of H429F is of antibody, molecular target and cell type The enhanced complement activating potency conferred by H429 substitution to other mAbs was evaluated in an antibody, daratumumab, recognising a fourth and unrelated molecular target, namely CD38 (de Weers M et al., J Immunol 2011;186:1840-1848, 2011; Overdijk mB et al., MAbs 7:311-321, 2015), which is a cell surface molecule that is structurally unrelated to CD20 or HER2. The daratumumab-WT mAb was formatted as a human IgG1 and kappa light chain mAb as described above. A mutant of this antibody was engineered wherein the H429 residue was replaced with phenylalanine to create the daratumumab-H429F mAb. In each case, the daratumumab-based IgG mAbs showed equivalent characteristics and eluted from the Protein A affinity column as a single homogeneous peak (Figure). Further purification by SEC yielded a single IgG (H2L2) peak for the daratumumab-WT mAb and an equivalent peak also for the daratumumab-H429F mutant mAb (Figure 24A). Moreover, SDS- PAGE confirmed that the SEC purified mAbs comprised the expected 150 kDa IgG species (prior to disulphide bond reduction), and the ~50 kDa heavy chain and 25 kDa light chain species following reduction in DTT (Figure 24B).

[0320] The CDC potency of the daratumumab-H429F mAb was compared to daratumumab-WT mAb (Figure 25). Each mAb was titrated individually by serial two-fold dilution in the presence of normal human serum as a source of complement. The percent killing of CD38 expressing Ramos cells was evaluated for each mAb concentration and is shown in Figure 25. Both the daratumumab-WT and daratumumab-H429F mAbs achieved ~80% killing of Ramos lymphoma cells, however the daratumumab-H429F mAb exhibited greater CDC potency, ~15-fold higher EC50. The improved CDC potency of the daratumumab-H429F mAb was then investigated using CD38 positive cells KMS-12-PE myeloma cells that resist CDC (Figure 26A). The daratumumab-WT mAb showed barely detectable CDC of KMS-12-PE cells above the background control lysis (~20%) in the presence only of complement (no mAb C' only), even at concentrations 5-times greater (5 μg / ml) than that (1 μg / ml) required to maximally kill Ramos lymphoma cells (~80% lysis; see Figure 25). In contrast, potent CDC of the KMS-12-PE cells was mediated by the daratumumab-H429F mAb (Figure 26A). In a further experiment, CDC potency was evaluated on the CD38 expressing SUP-15 acute lymphoblastic leukaemia (ALL) cells. As can be seen from Figure 26B, the SUP-15 cells also resisted CDC killing by the daratumumab-WT mAb, but were readily killed by the daratumumab-H429F mutant mAb. Thus, substitution at position 429 of the CH3 domain not only enhances CDC against certain targets but can also rescue potent CDC against lysis-resistant targets. Moreover, the enhanced CDC potency against the CD38 target (which is structurally distinct from CD20 detected by rituximab and 11B8, and HER2 detected by trastuzumab), also indicates that the improved efficacy is independent of target and of the epitope detected and thus also of the V domain.

[0321] H429 substitution facilitates functional between mAbs directed at distinct epitopes Functional synergy between mAbs and mediated by the H429 modification was investigated by determining the extent of binding of C1q in mixtures of mAbs (trastuzumab and pertuzumab) targeting separate epitopes in HER2.

[0322] The purification characteristics of the pertuzumab-WT mAb and a mutant wherein H429 had been replaced with phenylalanine (i.e. pertuzumab-H429F mAb) are shown in (Figure 27 and 28A). For each mAb, a single equivalent IgG peak was obtained from Protein A affinity chromatography (Figure 27), and when further purified by size exclusion chromatography, single homogeneous non-oligomeric IgG species were observed (Figure 28A, right of vertical line). SDS-PAGE analysis (Figure 28B) confirmed that these peaks contained a 150kDa IgG (H2L2) species that resolved into ~50 kDa heavy chains and ~25 kDa light chains after reduction (Figure 28B).

[0323] Cooperation between the unrelated anti-HER2 mAbs, trastuzumab-WT and pertuzumab-WT, which detect distinct epitopes in HER2 was determined by evaluating the extent of C1q binding in mixtures of the mAbs which was quantitated by flow cytometry (Figure 29A). Ovarian cancer cells, SK- OV-3, treated with a mixture of equal concentrations of the HER2 mAbs, trastuzumab-H429F or pertuzumab-H429F containing the H429F modification showed enhanced C1q binding (MFI=10,877) over that observed when the mAbs were used individually at the same concentrations (Figure 29A) pertuzumab-H429F (MFI=798) or trastuzumab-H429F (MFI=1739).

[0324] The cooperative enhancement of C1q binding was further evaluated (Figure 29B) by titration of the individual anti-HER2 mAbs or pairwise mixtures of the mAbs at two-fold serial dilutions from a starting concentration of 5µg / ml for each mAb alone or in a mixture of the mAbs at a 1:1 ratio starting at 2.5µg / ml:2.5µg / ml of each mAb in the mixture (Figure 29B). The trastuzumab-H429F and pertuzumab- H429F acted synergistically to further enhance complement activation particularly when the concentration of mAbs was limiting (e.g. at 1.25 μg / ml; Figure 29B indicated by arrow). C1q binding by the mixture of 1.25 μg / ml trastuzumab-H429F and 1.25µg / ml Pertuzumab-H429F (MFI=6327) was greater than the C1q binding by the individual mAbs used alone at the same or twice the concentration (i.e. trastuzumab-H429F ΜFI= 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 the WT mAbs at the same 1:1 concentrations (e.g.1.25 μg / ml trastuzumab-WT and 1.25 μg / ml pertuzumab-WT MFI=522) and even greater than the wild type mAbs used alone (i.e. trastuzumab-WT MFI = 258 at 1.25 μg / ml, or 405 at 2.5 μg / ml) and pertuzumab-WT MFI= 424 at 1.25 μg / ml, or MFI=483 at 2.5 μg / ml). This improved complement activation potency further indicates that greater functional potency is achievable by cooperative synergy in mixtures of CH3 domain-modified mAbs recognising distinct epitopes.

[0325] Modification of the CH3 domain of distinct specificity to act synergistically in CDC independent of target The cooperative synergy of mAbs leading to greater functional potency as reflected in the enhanced C1q binding (Figure 29) was investigated for enhanced CDC lysis of targets using mixtures of mAbs detecting either two distinct molecular structures (i.e. CD20 and CD38) or two distinct epitopes within the same molecular structure, CD20. The results are shown in Figure 30.

[0326] Cooperation in CDC between antibodies detecting distinct molecular structures, and distinct epitopes, was determined using a mixture of rituximab-WT or rituximab-H429F mAbs that target CD20 in pairwise combination with daratumumab-WT or daratumumab-H429F mAbs which target the unrelated surface molecule, CD38. No cooperation in CDC was apparent when the rituximab-WT mAb was titrated in the presence of 0.25µg / ml daratumumab-WT mAb (Figure 30A); that is, the CDC observed at any concentration of rituximab-WT was no greater than the baseline CDC in the presence of 0.25µg / ml daratumumab-WT alone (0 µg / ml rituximab, Figure 30A) or at any concentration of the rituximab-WT alone (i.e. titrated in the absence of daratumumab-WT). However, in contrast to the unmodified wild type mAbs, the rituximab-H429F and daratumumab-H429F mAbs acted to enhance CDC cell killing (Figure 30B). In particular, the cooperation between the mAbs in mediating CDC was readily observed in the rituximab-H429F concentration range of 0.5 µg / ml – 0.125 µg / ml, when used with 0.025 µg / ml of the daratumumab-H429F mAb (NB. the enhancement is indicated by the up-arrows in Figure 30B). CDC mediated by the rituximab-H429F mAb with the daratumumab-H429F mAb was considerably greater than with either of the H429F mAbs alone (rituximab-H429F or 0.025 µg / ml daratumumab; enhancement is indicated by the up-arrows in Figure 30B).

[0327] Functional cooperativity between mAbs detecting distinct epitopes, but within the same target molecule, was investigated in mixtures of the different CD20 mAbs. The results are shown in Figure 30C. Titration of the rituximab-H429F mAb in the presence (0.5 μg / ml) or absence of 11B8-H429F mAb revealed that the mAbs acted synergistically. The cooperation between the mAbs was readily detected when the concentration of both mAbs was limiting (0.25 µg / ml – 0.031µg / ml rituximab-H429F and 0.05 µg / ml 11B8-H429F), where CDC was greater than with either mAb alone (enhancement indicated by the up-arrows in Figure 30C).

[0328] Thus, mAb cooperation and synergy by modification of the antibody H chain at position 429, particularly H429F, is broad in its effect. It facilitates greater functional potency through cooperation and functional synergy in mixtures of mAbs irrespective of the epitope detected by the individual mAbs (i.e. whether the epitopes are present on the same or distinct molecular targets).Example 4 B lymphocyte killing by an therapeutic antibody with an H429 mutation

[0329] Monoclonal antibodies are used in the treatment of inflammatory disease, such as autoimmune disease, by targeting normal (i.e. non-malignant) cells. For example, the anti-CD20 mAb Rituximab is used for the treatment of inflammatory disease (Lee DSW et al., Nat Rev Drug Discov 20:179-199, 2021) by targeting normal B lymphocytes known to express CD20. Methods and Materials

[0330] Isolation of leukocytes from human peripheral blood Peripheral blood mononuclear cells (PBMC) were isolated from anticoagulated venous blood (Vacutainer ACD-A Becton Dickinson) 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 a concentration of 5x106 / ml.

[0331] Complement dependent lysis of cells The CDC killing of normal peripheral blood B lymphocytes in PBMC, mediated by WT and mutated anti-CD20 mAbs was measured by flow cytometry as described above using the Zombie Green Fixable Viability kit (BioLegend) following opsonisation of cells with mAb.

[0332] CDC was performed in 96-well plates. Cells (25 μl, 5x106 / ml) were reacted with equal volume of mAb in L15-BSA for 30 minutes on ice, then washed (diluted in 100-200 μl of buffer and centrifuged (at 200xg for 5 minutes, 4°C) twice in L15-BSA. The IgG-opsonised cells were then resuspended in 50 μl of human serum diluted 1 / 3 in L15-BSA, as a source of complement, and incubated at 37°C for 30 minutes. The treated cells were then washed once in L15-BSA, resuspended in 50 μl anti-CD19-APC antibody (BioLegend) in L15-BSA and incubated for a further 30 minutes on ice. Following two washes in L-15 lacking BSA, cells were then 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. The cells were washed once in L15-BSA and the cells fixed by resuspension in 2% paraformaldehyde in buffer for 30 minutes on ice and finally washed once and then resuspended in 200 μl L15-BSA for flow cytometry analysis. B lymphocytes were identified by staining for CD19 and the proportion (%) of dead B lymphocytes specifically killed by CDC was enumerated as the percentage of CD19+Zombie green+cells compared to the background control of CD19+Zombie green+cells in PBMC samples treated with the negative control mAbs trastuzumab-WT or trastuzumab-H429F antibodies. Results and Discussion

[0333] The CDC killing of normal B by rituximab-H429F was investigated by flow cytometry and was compared to that of the unmodified rituximab-WT (Figure 31). The rituximab-H429F showed greater potency, killing over 85% of B cells (Figure 31B) compared to the much lower killing by the unmodified rituximab-WT which killed only 30% of the peripheral blood B lymphocytes (Figure 31A). As HER2 is not expressed on B lymphocytes there was no CDC of the B lymphocytes (Figure 31C, D) by the negative control anti-HER2 mAbs, trastuzumab-WT or trastuzumab-H429F. Example 5 H429F modification improves the function of immunoglobulins of other types

[0334] Histidine 429 is conserved in the equivalent position in all human Ig classes and subclasses (Figure 3, Figure 4); that is, in the CH3 domain of all IgG subclasses and IgA subclasses (Figure 3), in IgD (Figure 4), and also in IgE, and IgM where the CH4 domain is the equivalent of the CH3 domain of IgG (Figure 4). The effect of modification of H429 on the function of other immunoglobulins was evaluated using human IgG3 and human IgG4 as examples. Methods and Materials

[0335] Antibodies and antibody constructs The mAbs used in this example comprised heavy chains of the IgG3 or IgG4 subclasses as shown in Ta...

Claims

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

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

3. The immunotherapeutic protein of claim 1 or claim 2, wherein the substitution at H429 is selected from: H429 substituted with F, H429 substituted with Q, H429 substituted with E, H429 substituted with S, H429 substituted with A, H429 substituted with Y, H429 substituted with L, H429 substituted with V, H429 substituted with G, H429 substituted with H429W, H429 substituted with R and H429 substituted with P (Eu numbering).

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

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

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

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

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

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

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

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

12. The immunotherapeutic protein of claim 11, wherein the immunotherapeutic protein is a hexamer.

13. The immunotherapeutic protein of any one of claims 1 to 10, wherein the immunotherapeutic protein is an antibody that forms oligomers upon binding to a relevant target through "on target" oligomerisation.

14. The immunotherapeutic protein of claim 13, wherein the immunotherapeutic protein forms a hexamer upon binding to a relevant target through "on target" oligomerisation.

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

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

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

18. The immunotherapeutic protein of claim 15, wherein the C1q binding modification is selected from a modification in C1q Group 4 and C1q Group 5 and wherein the modification decreases C1q binding compared to a control IgG2 immunotherapeutic protein and / or a control IgG1 immunotherapeutic protein lacking the C1q binding modification.

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

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

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

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

23. The immunotherapeutic protein of claim 22, wherein the substitution in the hinge region is selected from: RKCCVE217-225 substituted with PKSCDKTHT, EPKSCDKTHT216-225 substituted with ERKCCVE, and EPKSCDKTHT216-225 substituted with ESKYGPP.

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

25. The immunotherapeutic protein of claim 24, wherein the substitution in the CH2 domain is selected from: PVA233-236 substituted with EFLG, PVA233-236 substituted with ELLG, and PVA233-236 substituted with EFEG.

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

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

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

29. The immunotherapeutic protein of claim 24 or claim 25, wherein the substitution in the CH2 domain is PVA233-236 substituted with EFLG (Eu numbering).

30. The immunotherapeutic protein of claim 24 or claim 25, wherein the substitution in the CH2 domain is PVA233-236 substituted with ELLG (Eu numbering).

31. The immunotherapeutic protein of claim 27 or claim 28, wherein the substitution in the CH2 domain is selected from: i) S267 substituted with E and L328 substituted with F (Eu numbering); ii) S267 substituted with E (Eu numbering); and iii) L328 substituted with L328 F (Eu numbering).

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

33. The immunotherapeutic protein of claim 32, wherein the substitution in the CH3 domain is selected from: E430 substituted with G, and H435 substituted with A.

34. The immunotherapeutic protein of claim 1 to 20, wherein the polypeptide is an IgG3, the modification is in the CH3 domain and wherein the modification is a substitution at a position corresponding to N392, M397 and R435 of the IgG3 heavy chain polypeptide (Eu numbering).

35. The immunotherapeutic protein of claim 34, wherein the substitution in the CH3 domain comprises N392 substituted with K, M397 substituted with V and R435 substituted with H (Eu numbering).

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

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

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

39. The immunotherapeutic protein of any one of claims 1 to 17 and 20, wherein the polypeptide is an IgG2 and the C1q binding modification is PVA233-236 substituted with EFLG, C131 substituted with S, and ERKCCVE substituted with IgG1216-225EPKSCDKTHT.

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

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

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

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

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

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

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

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

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

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

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

51. The immunotherapeutic protein of any one of claims 1 to 50, wherein the polypeptide comprises a further mutation selected from one or more of: i) an amino acid substitution at a position corresponding to 274, of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); ii) an amino acid substitution at a position corresponding to 219 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); iii) an amino acid substitution at a position corresponding to 220 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering); and iv) an amino acid substitution at a position corresponding to 219 and 220 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering).

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

53. The immunotherapeutic protein of claim 51, wherein the further mutation is a substitution selected from K274 with Q, Q274 with K, C219 with S, S219 with C, and C220 with S.

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

55. The immunotherapeutic of claim 54, wherein the intra-heavy chain disulfide bond is between C131 and C219, or between C131 and C220.

56. The immunotherapeutic protein of any one of claims 1 to 2022 to 25, 27 to 32, 36 to 39 and 43 and 53, wherein the polypeptide is an IgG2 and wherein the polypeptide comprises a further mutation that forms a disulfide bond between the light chain and upper hinge region of the polypeptide.

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

58. The immunotherapeutic protein of any one of claims 54 to 57, wherein the further mutation is M1, substitution of C131 with S and a substitution of C220 with S.

59. The immunotherapeutic protein of any one of claims 54 to 57, wherein the further mutation is M2, substitution of C131 with S and substitution of C219 with S.

60. The immunotherapeutic protein of claim 54 or 57, wherein the further mutation is M3, a substation of C131 with S and substitution of ERKCCVE in the IgG2 upper hinge with EPKSCDKTHT.

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

62. The immunotherapeutic protein of claim 17 or claim 19, wherein the control immunotherapeutic protein is IgG1.

63. The immunotherapeutic protein of claim 17 or claim 19, wherein the control immunotherapeutic protein is human IgG2.

64. An oligomer comprising the immunotherapeutic protein of any one of claims 1 to 63.

65. A nucleic acid encoding the immunotherapeutic protein of any one of claims 1 to 63.

66. The nucleic acid of claim 65, wherein the nucleic acid is selected from an RNA, DNA or combination thereof.

67. The use of the immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 66 for treating or preventing a disease or condition in a subject, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

68. The use of the immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 66 in the manufacture of a medicament for treating or preventing a disease or condition, wherein the disease or condition is selected from autoimmune diseases and conditions, cardiovascular disease, neurodegenerative disease, other inflammatory diseases, transplantation condition or rejection, infectious diseases and proliferative diseases.

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

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

71. The use of claim 67 or 68 or method of claim 69, wherein the immunotherapeutic protein enhances death receptor signalling responses.

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

73. A pharmaceutical composition or medicament comprising the immunotherapeutic protein, oligomer, or nucleic acid of any one of claims 1 to 66, and a pharmaceutically acceptable carrier, diluent and / or excipient.

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

75. A method of producing an immunotherapeutic protein of claim 1 to 66, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant under conditions of: (i) mildly acidic pH to recover immunotherapeutic protein in a monomeric form; or (ii) substantially neutral pH to recover immunotherapeutic protein in an oligomeric form.

76. A method of producing an immunotherapeutic protein of any one of claims 1 to 66, comprising culturing a host cell comprising a construct encoding said protein under conditions suitable for the expression of said protein, and recovering the protein from culture supernatant using a method comprising affinity chromatography using an elution buffer comprising a concentration of arginine of less than 130 mM and at less than or equal to pH 5.

0.

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

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

79. A method of producing / modifying a immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide comprising: substituting the amino acid at a positioncorresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering) with an aromatic, cyclic or hydrophobic amino acid, wherein the one or more polypeptide comprises one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) domain and a hinge region, and wherein the one or more polypeptide comprises at least one C1q binding modification.

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

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

82. A method of producing / modifying a immunotherapeutic protein comprising one or more immunoglobulin heavy chain polypeptide comprising: introducing a C1q modification as described in table 5 into the immunotherapeutic protein, wherein the one or more polypeptide comprises one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) domain and a hinge region, and wherein the one or more polypeptide comprises an amino acid substitution at a position corresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering) with F, Q, E, S, A, Y, L, V, G, W, R or P.

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

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

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

86. An immunotherapeutic protein comprising modified C1q binding wherein the immunotherapeutic protein comprises one or more immunoglobulin heavy chain polypeptide comprising one or more of: a constant heavy chain domain 1 (CH1), a constant heavy chain domain 2 (CH2), a constant heavy chain domain 3 (CH3) domain and a hinge region, wherein the one or more polypeptide comprises substitution of an amino acid at a position corresponding to H429 of the amino acid sequence of human IgG1, IgG2, IgG3 or IgG4 heavy chain (Eu numbering) with F, Q, E, S, A, Y, L, V, G, W, R or P.

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

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

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

90. The method of claim 89, wherein the substitution at H429 is H429 substituted with F (Eu numbering).

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