Anti-bacterial polypeptides

Abstract

The present disclosure relates to mutated polypeptides comprising a fragment crystallisable (Fc) region with reduced affinity for Staphylococcus aureus protein A (SpA). The present disclosure further relates to the treatment of Staphylococcus aureus infections.

Description

[0001] ANTI-BACTERIAL POLYPEPTIDES

[0002] Technical Field

[0003] The present disclosure relates to mutated polypeptides comprising a fragment crystallisable (Fc) region with reduced affinity for Staphylococcus aureus protein A (SpA). The present disclosure further relates to the treatment of Staphylococcus aureus infections.

[0004] Background

[0005] Staphylococcus aureus (S. aureus) is a bacterium commonly found on the skin and mucous membranes of humans, which is a significant concern in healthcare settings. While it can exist harmlessly as a part of the normal microbiota, it is also capable of causing a range of infections and health issues. S. aureus infections can take various forms, including skin and soft tissue infections (such as boils, abscesses, and cellulitis), respiratory infections, bloodstream infections, pneumonia, endocarditis (infection of the heart valves) and sepsis. S. aureus produces a wide array of virulence factors, which contribute to its ability to evade the immune system and cause damage to host tissues. Some of these factors can also reduce the effectiveness of therapeutic antibodies (and other associated immunotherapies).

[0006] In particular, S. aureus expresses Staphylococcal Protein A (SpA), an IgG-binding protein that binds human IgG antibodies via their Fc-domain. S. aureus further expresses some additional IgG-binding proteins which also bind human IgG-antibodies via their Fc-domain: staphylococcal binder of immunoglobulins (Sbi). An equivalent protein in Streptococcus spp. is Streptococcal Protein G (SpG). Due to the abundant surface expression of SpA (and to a lesser extent Sbi), S. aureus essentially coats itself in human IgG-antibodies, thereby not only obscuring its own antigens but also effectively inhibiting Fc-mediated effector functions of the antibodies. This ability of S. aureus affects proximal human IgG antibodies and other Fc- containing proteins in general. Thus, chiefly through the expression of SpA, S. aureus reduces the effectiveness of therapeutic antibodies and other related Fc-containing immunotherapies. For this reason, for an effective use of a therapeutic antibody (or related immunotherapy) in S. aureus infections, the Fc-mediated capture of the therapeutic antibody etc. should be inhibited.

[0007] Summary

[0008] There remains a need in the art for Fc mutations and combinations thereof to provide Fc region polypeptides with reduced affinity to SpA, for use against Staphylococcus aureus infections.

[0009] The present disclosure provides the recognition that identification of Fc mutations and Fc region polypeptides that have reduced affinity for SpA is far from straightforward, as there is a need to balance many different factors such as production capability, stability and reduced SpA affinity. For example, Chen et al., PNAS 119(4) e2114478119 (2022) reported some preliminary alleged results into the possibility of mutating an antibody Fc region to reduce SpA affinity, but the investigators encountered difficulties in maintaining the properties of the Fc regions with the mutations that were introduced. Two mutated Fc regions for this purpose are disclosed in Chen et al. “AESP” with the mutations S254A, Q311 E, L432S and N434P, and “R” with the mutation H435R. However, the corresponding patent publication US 2023 / 0041644 A1 by the same group indicates that AESP had unfavourable properties, which precluded further development. Chen et al. further discloses the mutations T307R and A378V in combination with the H435R mutation. US 2020 / 0291099 A1 discloses several Fc mutations for allegedly reducing SpA affinity, but only discloses that a single antibody “Mab2”, with the mutations K274Q, H435R and Y436F, was considered for being carried through to preliminary testing. Thus, despite exploration in the field, the need for Fc mutations and Fc region polypeptides that have reduced affinity for SpA remains.

[0010] The present disclosure further recognizes that other characteristics of Fc regions used in therapeutics against S. aureus would be beneficial. For instance, there is also a need in the art for the Fc mutants to have advantageous FcRn binding characteristics for antibody halflife (e.g., binding at pH 6 and weak binding at pH 7). Further, it would be advantageous for Fc mutants to maintain Fc effector functions through FcyR interactions.

[0011] The present disclosure relates to mutations in the Fc region of polypeptides, e.g., for therapeutic or diagnostic use in Staphylococcus aureus infections. The provided mutations address certain needs in the field for Fc mutations and Fc region polypeptides that have reduced affinity for SpA and / or achieve certain objectives beneficial to uses (e.g., therapeutic uses) of such Fc mutations and Fc region polypeptides. In particular, the present disclosure provides polypeptides comprising substitution mutations and combinations thereof in an Fc region, which substitution mutations comprise T307I, Q311 R, M428L, N434L, Y436K, or any combination thereof. In some embodiments, polypeptides provided herein have reduced affinity for Staphylococcus aureus protein A (SpA). In some embodiments, polypeptides provided herein have other advantageous properties, such as maintained stability and expressibility. In some embodiments, polypeptides provided herein have favourable properties regarding Fc function, such as a maintained immune response, retained C1q binding and even improved complement component 1q (C1q) recruitment. Moreover, polypeptides of the present disclosure can have favourable properties regarding FcRn binding and antibody halflife. Furthermore, the polypeptides of the present disclosure can be useful in treating and / or diagnosing Staphylococcus aureus infections. Accordingly, in an aspect, the present disclosure provides a polypeptide comprising a fragment crystallisable (Fc) region having reduced affinity for Staphylococcus aureus protein A (SpA), wherein the polypeptide sequence of the Fc region comprises one or more of the following substitution mutations: T307I, Q311 R, M428L, N434L and Y436K.

[0012] In an aspect, the present disclosure provides one or more nucleic acid sequences capable of expressing a polypeptide provided herein.

[0013] In an aspect, the present disclosure provides a cell comprising a polypeptide or one or more nucleic acid sequences provided herein.

[0014] In an aspect, the present disclosure provides a composition comprising a polypeptide, one or more nucleic acid sequences or a cell provided herein.

[0015] In an aspect, the present disclosure provides an in vitro method comprising contacting a cell with a polypeptide, one or more nucleic acid sequences, a cell or a composition provided herein.

[0016] In an aspect, the present disclosure provides a polypeptide, one or more nucleic acid sequences, a cell or a composition provided herein for use in a method of therapy or a diagnostic method.

[0017] In an aspect, the present disclosure provides a polypeptide, one or more nucleic acid sequences, a cell or a composition provided herein for use in the manufacture of a medicament for a therapy or a diagnostic agent.

[0018] In an aspect, the present disclosure provides a method of therapy or a diagnostic method comprising administering a polypeptide, one or more nucleic acid sequences, a cell or a composition provided herein to a subject.

[0019] In an aspect, the present disclosure provides a method for making a polypeptide provided herein, comprising expressing one or more nucleic acids capable of expressing the polypeptide.

[0020] Brief description of the Figures

[0021] Figure 1: FcRn binding of select Fc mutants at pH 7, tested via ELISA. For efficient antibody recycling within the cell, it is advantageous for FcRn binding to be weak at pH 7. Fc185 showed strong binding to FcRn at pH 7. Binding to FcRn at pH7 is acceptable for all other tested mutants.

[0022] Figure 2: FcyR binding of select Fc mutants, tested via ELISA in respect of various different receptors, including FcyRI (Figure 2A), FcyRlla (Figure 2B), FcyRllb / c (Figure 2C) and FcyRllla (Figure 2D). Binding to Fcy-Receptors for the screening candidate mutant Fc136 was comparable to the wildtype control.

[0023] Figure 3: Functional testing of select Fc mutants, including assays for ADCC (Figure 3A), ADCP (Figure 3B), cell surface binding (Figure 3C) and C1q recruitment (Figure 3D). Fc136 performed comparably to the wildtype protein in Figures 3A-3C, and surprisingly showed favourably strong signals in the C1q recruitment assay in Figure 3D.

[0024] Figure 4: Binding assay testing of select Fc mutants, including Sbi binding assays (Figure 4A), SpA binding assays (Figure 4B) and SpG binding assays (Figure 4C) for several Fc mutants as well as a wild-type control (Fc001 / WT). Sbi, SpA and SpG did not substantially bind to Fc136. To the contrary, SpG showed binding to the Fc185 mutant.

[0025] Figure 5: Binding assays for the mutants Fc136, Fc183 and Fc185, as well as a wild-type control (Fc001) in highly purified forms, to the target antigen (Figure 5A), FcyRlla (Figure 5B), FcyRI (Figure 50) and FcyRllla (Figure 5D).

[0026] Figure 6: Binding assays for the mutants Fc136, Fc183 and Fc185, as well as a wild-type control (Fc001 / WT) in highly purified forms, to SpA (Figure 6A), SpG (Figure 6B) and Sbi (Figure 60). SpA, SpG and Sbi did not substantially bind to Fc136.

[0027] Figure 7: Serum concentrations of select VHH-Fc-fusions in NSG hFcRn (32) Tg mice over time. Serum concentrations of the mutants Fc136, Fc183, Fc185, as well as the wild-type control (Fc001) and a negative control (DPBS) over time. Fc136 showed consistently higher serum concentration than all other samples, indicative of a prolonged in vivo half-life.

[0028] Detailed Description

[0029] Polypeptides and mutations

[0030] Polypeptides according to the present disclosure may be present in a monomeric form (e.g. a single polypeptide chain such as an immunoglobulin chain) or in the form of an antibody construct (e.g. a dimeric form such as an antibody comprising two immunoglobulin heavy chains). Thus, the term “polypeptide” as used herein encompasses monomeric, multimeric, dimeric, homodimeric and heterodimeric forms of polypeptides. In the context of the monomeric forms of the polypeptides of the present disclosure, the term “Fc region” may refer to the part of the polypeptide sequence that comprises the constant domains, e.g. the CH2 domain and CH3 domain. In the context of the antibody constructs of the present disclosure, the term “Fc region” may refer to the part of the construct that comprises the constant domains, e.g. the CH2 domains and CH3 domains, such as the region of the antibody dimer construct that comprises the CH2 and CH3 domains. In an aspect, the present disclosure provides a polypeptide comprising a fragment crystallisable (Fc) region that comprises one or more of the following substitution mutations: T307I, Q311 R, M428L, N434L and Y436K. In some embodiments, an Fc region comprising one or more of the following substitution mutations: T307I, Q311 R, M428L, N434L and Y436K has reduced affinity for Staphylococcus aureus protein A (SpA).

[0031] In some embodiments, the polypeptide sequence of the Fc region comprises one substitution mutation. In some embodiments, the substitution mutation is T307I, Q311 R, M428L, N434L or Y436K.

[0032] In some embodiments, the polypeptide sequence of the Fc region comprises two substitution mutations. In some embodiments, the polypeptide sequence of the Fc region comprises the substitution mutations T307I and Q311 R; T307I and M428L; T307I and N434L; T307I and Y436K; Q311R and M428L; Q311R and N434L; Q311 R and Y436K; M428L and N434L; M428L and Y436K, or N434L and Y436K.

[0033] In some embodiments, the polypeptide sequence of the Fc region comprises three substitution mutations. In some embodiments, the polypeptide sequence of the Fc region comprises the substitution mutations T307I, Q311 R, and M428L; T307I, Q311 R, and N434L; T307I, Q311R, and Y436K; T307I, M428L, and N434L; T307I, M428L, and Y436K; T307I, N434L, and Y436K; Q311R, M428L, and N434L; Q311R, M428L, and Y436K; or Q311 R, N434L, and Y436K.

[0034] In some embodiments, the polypeptide sequence of the Fc region comprises four substitution mutations. In some embodiments, the polypeptide sequence of the Fc region comprises the substitution mutations T307I, Q311R, M428L, and N434L; T307I, Q311R, M428L, and Y436K; T307I, Q311R, N434L, and Y436K; T307I, M428L, N434L, and Y436K; or Q311R, M428L, N434L, and Y436K.

[0035] In some embodiments, the polypeptide sequence of the Fc region comprises the substitution mutation M428L, and optionally comprises one or more of T307I, Q311 R, N434L and Y436K. In some embodiments, the substitution mutation M428L is of particular interest because it was identified in just a single variant during a screen. Thus, in a generally preferred embodiment herein, the polypeptide sequence of the Fc region comprises (at least) the substitution mutation M428L.

[0036] In some embodiments, the polypeptide sequence of the Fc region comprises five substitution mutations. In a most preferred embodiment, the polypeptide sequence of the Fc region comprises all of the substitution mutations T307I, Q311 R, M428L, N434L and Y436K.

[0037] In some embodiments, the polypeptide sequence of the Fc region comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3. In some embodiments, the % identity language does not apply to the substitution mutations that are specified in the Fc region of the polypeptide. In some embodiments, the polypeptide sequence of the Fc region comprises SEQ ID NO: 3.

[0038] In some embodiments, the polypeptide sequence of the Fc region does not comprise the substitution mutation H435R. In some embodiments, the polypeptide sequence of the Fc region does not comprise the substitution mutations S254A, Q311 E, L432S or N434P. In some embodiments, the polypeptide sequence of the Fc region does not comprise the substitution mutations T307R or A378V. In some embodiments, the polypeptide sequence of the Fc region does not comprise the substitution mutations T307Q, Q311V or A378V. In some embodiments, the polypeptide sequence of the Fc region does not comprise the substitution mutations T256D, N286D, T307R, Q311V or A378V.

[0039] In some embodiments, the polypeptide sequence of the Fc region comprises H435. In some embodiments, the polypeptide sequence of the Fc region comprises S254, Q311 , L432 or N434. In some embodiments, the polypeptide sequence of the Fc region comprises T307 or A378. In some embodiments, the polypeptide sequence of the Fc region comprises T307, Q311 or A378. In some embodiments, the polypeptide sequence of the Fc region comprises T256, N286, T307, Q311 or A378.

[0040] It will be understood that the antibody and Fc amino acid numbering herein corresponds to the Ell numbering scheme. Thus, in some embodiments, the amino acid position numbering corresponds to Ell numbering. The Ell numbering scheme, including how it compares to other antibody residue numbering schemes, is well-known in the art and can be viewed at e.g. https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0041] In some embodiments herein, the mutations are defined simply by the position of the mutation in the Fc region (according to the Ell numbering scheme) and the residue which is present. For example, in some embodiments herein the polypeptide having the substitution mutation M428L is simply defined as the polypeptide having the residue “L” at position 428.

[0042] In embodiments herein, the mutations T307I, Q311 R, M428L, N434L and Y436K may alternatively be defined as the polypeptide sequence of the Fc region comprising “I” at Ell position 307, “R” at EU position 311 , “L” at EU position 428, “L” at EU position 434, and / or “K” at EU position 436 respectively.

[0043] In embodiments herein, the mutations S254A, T256D, N286D, T307R, T307Q, Q311 E, Q311V, A378V, L432S, N434P and H435R may alternatively be defined as the polypeptide sequence of the Fc region which does not comprise “A” at EU position 254, “D” at EU position 256, “D” at EU position 286, “R” at EU position 307, “Q” at EU position 307, “E” at EU position 311 , “V” at EU position 311 , “V” at EU position 378, “S” at EU position 432, “P” at EU position 434 and / or “R” at EU position 435, respectively.

[0044] Polypeptide functions

[0045] In some embodiments, the function(s) of the Fc region of the polypeptides of the present disclosure are provided when the polypeptide is comprised within an antibody construct. In some embodiments, the polypeptide is comprised within an antibody construct. In some embodiments, the polypeptide is comprised within a dimer of immunoglobulin heavy chains, optionally further comprising one or more immunoglobulin light chains.

[0046] In some embodiments, the Fc region of the polypeptide has reduced affinity for SpA. In some embodiments, the Fc region of the polypeptide has reduced affinity for the second immunoglobulin-binding protein of Staphylococcus aureus (Sbi). In some embodiments, the Fc region of the polypeptide has reduced affinity for Streptococcal protein G (SpG).

[0047] Herein, in some embodiments, “reduced affinity” of a mutant Fc region for SpA, Sbi and / or SpG means that the affinity is reduced relative to the equivalent wild-type Fc region, i.e. the same Fc region polypeptide sequence which does not comprise any of the mutations that are specified in the claim. It will be understood that SpA, Sbi and SpG typically have affinity for and are able to capture the Fc regions of polypeptides which contain such regions (e.g. antibodies). Thus, an Fc region provided herein with substitution mutations having reduced affinity for SpA, Sbi and / or SpG means that the Fc region has reduced affinity relative to the same Fc region without the substitution mutations. In this context, for example, an Fc region “without the substitution mutation” M428L does not have the residue “L” at EU position 428, and instead has the residue “M” at EU position 428.

[0048] In some embodiments, an Fc region of a polypeptide provided herein has reduced affinity for SpA, Sbi and / or SpG relative to the Fc region of the polypeptide that does not comprise the substitution mutations.

[0049] In some embodiments, “reduced” means that SpA, Sbi and / or SpG substantially do not bind to the Fc region of the polypeptide of the present disclosure. In some embodiments, “reduced” means that SpA, Sbi and / or SpG do not bind to the Fc region of the polypeptide of the present disclosure. In some embodiments, the Fc region of the polypeptide of the present disclosure having “reduced affinity” means that the binding of SpA, Sbi and / or SpG to the Fc region is inhibited. In some embodiments, SpA-mediated antibody capture is inhibited. In some embodiments, the reduced affinity for SpA (and / or Sbi) means that Fc effector functions are maintained in the presence of S. aureus or SpA. It will be understood that the affinity for SpA, Sbi and / or SpG herein refers to the ability of these proteins to bind to and capture antibodies and other immunoglobulin domain-containing polypeptides by binding to the Fc region (and possibly the VH3 Fab region). This affinity does not include the specific antibody binding of the polypeptides of the present disclosure, e.g. to SpA, where present. Thus, this affinity does not include the polypeptides of the present disclosure specifically binding to SpA, Sbi and / or SpG via antibody-like binding, e.g. via the CDRs of one or more variable immunoglobulin domain.

[0050] It will also be understood that reduced affinity for SpA, Sbi and / or SpG can be determined by in silico modelling / screening and (if necessary) can be further verified by cellular assays.

[0051] In some embodiments, the Fc-mediated effector functions of the polypeptide are not inhibited in the presence of Staphylococcus aureus.

[0052] In some embodiments, the Fc region of the polypeptide provided herein binds to the neonatal Fc receptor (FcRn). In some embodiments, the Fc region of the polypeptide binds to FcRn with greater affinity at pH 6 than at pH 7. In some embodiments, the binding of the Fc region of the polypeptide to FcRn is equivalent, comparable or similar to that of the Fc region of the polypeptide that does not comprise the substitution mutations provided herein. In some embodiments, the binding affinity of the Fc region of the polypeptide to FcRn is equivalent, comparable or similar to that of the Fc region of the polypeptide that does not comprise the substitution mutations provided herein. In some embodiments, the binding affinity of the Fc region of the polypeptide to FcRn at pH 6 and at pH 7 is equivalent, comparable or similar to that of the Fc region of the polypeptide that does not comprise the substitution mutations provided herein. It will be understood that binding to the FcRn receptor can be readily determined and measured, e.g. by ELISA.

[0053] In some embodiments, the polypeptide has an in vivo half-life that is at least equivalent to the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations provided herein. In some embodiments, the polypeptide has an in vivo half-life that is similar to or greater than the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations provided herein. In some embodiments, the polypeptide has an in vivo half-life that is comparable to or greater than the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations provided herein. In some embodiments, the in vivo half-life is in a mouse. In some embodiments, the in vivo half-life is in a human.

[0054] In some embodiments, the Fc region of the polypeptide binds to the Fc-gamma receptor (FcyR). In some embodiments, the Fc region of the polypeptide binds to FcyRI. In some embodiments, the Fc region of the polypeptide binds to FcyRlla. In some embodiments, the Fc region of the polypeptide binds to FcyRHb / c. In some embodiments, the Fc region of the polypeptide binds to FcyRllla. In some embodiments, the binding of the Fc region of the polypeptide to FcyR is equivalent to that of the Fc region that does not comprise the substitution mutations of the invention. In some embodiments, the binding of the Fc region of the polypeptide to FcyRI, FcyRlla, FcyRllb / c and / or FcyRllla is equivalent to that of the Fc region that does not comprise the substitution mutations. It will be understood that binding to FcyR receptors can be readily determined and measured, e.g. by ELISA.

[0055] In some embodiments, the polypeptide induces antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the polypeptide induces antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the polypeptide induces complementdependent cytotoxicity (CDC). In some embodiments, the polypeptide induces ADCC, ADCP and CDC. In some embodiments, the polypeptide induces ADCC and / or ADCP that is equivalent to that of the polypeptide that does not comprise the substitution mutations provided herein.

[0056] In some embodiments, the polypeptide induces antibody-dependent cellular cytotoxicity (ADCC) against S. aureus. In some embodiments, the polypeptide induces antibodydependent cellular phagocytosis (ADCP) against S. aureus. In some embodiments, the polypeptide induces complement-dependent cytotoxicity (CDC) against S. aureus. In some embodiments, the polypeptide induces ADCC, ADCP and CDC against S. aureus. In some embodiments, the polypeptide induces ADCC and / or ADCP against S. aureus that is equivalent to that of the polypeptide that does not comprise the substitution mutations provided herein.

[0057] In some embodiments, the polypeptide induces complement component 1q (C1q) recruitment. In some embodiments, the polypeptide has improved C1q recruitment. In some embodiments, the polypeptide has improved C1q recruitment relative to the polypeptide that does not comprise the substitution mutations provided herein. C1q recruitment to target cells bound by IgG antibodies / VHH-Fc fusion proteins etc. initiates the classical pathway of complement, thereby promoting CDC. Accordingly, in some embodiments, the polypeptide induces CDC. In some embodiments, the polypeptide has enhanced CDC induction. In some embodiments, the polypeptide induces greater CDC than the polypeptide that does not comprise the substitution mutations provided herein.

[0058] In some embodiments, the polypeptide induces complement component 1q (C1q) recruitment against S. aureus. In some embodiments, the polypeptide has improved C1q recruitment against S. aureus. In some embodiments, the polypeptide has improved C1q recruitment against S. aureus relative to the polypeptide that does not comprise the substitution mutations provided herein. In some embodiments, the polypeptide induces CDC against S. aureus. In some embodiments, the polypeptide has enhanced CDC induction against S. aureus. In some embodiments, the polypeptide induces greater CDC against S. aureus than the polypeptide that does not comprise the substitution mutations provided herein.

[0059] In some embodiments, the polypeptides provided herein comprise one or more further mutations that enhance effector function. These further mutations that may also be included may be those that have previously been developed in the art.

[0060] In some embodiments, the polypeptides provided herein comprise the S239D / A330L / I332E "3M" mutation. In some embodiments the mutation increases ADCC. Further information on the mutation can be found in Lazar et al. (2006) Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10 (incorporated herein by reference).

[0061] In some embodiments, the polypeptides provided herein comprise the F243L Mutation. In some embodiments the mutation increases ADCC and ADCP. Further information on the mutation can be found in Stavenhagen et al. (2007) Cancer Res 2007 Sep 15;67(18):8882-9 (incorporated herein by reference).

[0062] In some embodiments, the polypeptides provided herein comprise the S267E / H268F / S324T Mutation. In some embodiments the mutation enhances CDC activity. Further information on the mutation can be found in Moore et al. (2010) MAbs. 2010 Mar-Apr;2(2):181-9 (incorporated herein by reference).

[0063] In some embodiments, the polypeptides provided herein comprise the E345R / E430G / S440Y Mutation. In some embodiments the mutation enhances C1q binding and CDC activity. Further information on the mutation can be found in Diebolder et al. (2014) Science, 14; 343(6176): 1260-3 (incorporated herein by reference).

[0064] Improved half-life

[0065] In preferred embodiments, polypeptides provided herein have been found to possess an in vivo half-life that is unexpectedly greater than, e.g. substantially greater than, the in vivo halflife of polypeptides comprising an Fc region that does not comprise the substitution mutations provided herein. This also presents a substantial improvement of the polypeptides provided herein over the polypeptides of the prior art. In some embodiments of this type, the improved in vivo half-life is provided by an antibody construct comprising the polypeptide(s) of the present disclosure, wherein the construct comprises a Fc region dimer. In some embodiments of this type, the improved in vivo half-life is provided by an immunoglobulin heavy chain dimer comprising the polypeptide(s) of the present disclosure. In some embodiments of this type, the improved in vivo half-life is provided by an antibody comprising the polypeptide(s) of the present disclosure.

[0066] In some embodiments, the polypeptides provided herein comprise one or more further mutations that enhance half-life. These further mutations that may also be included may be those that have previously been developed in the art.

[0067] In some embodiments, the polypeptides provided herein comprise the M252Y / S254T / T256E (YTE) mutation. In some embodiments the mutation increases FcRn binding affinity. Further information on the mutation can be found in Dall'Acqua et al. (2002) J Immunol 2002 Nov 1 ;169(9):5171-80 (incorporated herein by reference).

[0068] In some embodiments, the polypeptides provided herein comprise the M428L / N434S (LS) mutation. In some embodiments, the mutation enhances FcRn binding. Further information on the mutation can be found in Zalevsky et al. (2010) Nat Biotechnol. 2010 Feb;28(2): 157-9 (incorporated herein by reference).

[0069] In some embodiments, the polypeptides provided herein comprise the M252Y / S254T / T256E / M428L / N434S (YTE-LS) mutation. Further information on the mutation can be found in Ko et al. (2022) Ex. Mol Med 54(11):1850-1861 (incorporated herein by reference).

[0070] In some embodiments, the polypeptides provided herein comprise the M252Y / S254T / T256E / H433K / N434F (YTE-HN) mutation. In some embodiments, the mutation increases FcRn binding affinity. Further information on the mutation can be found in Monnet et al. 2019 Nat Commun. 2019 Nov 6;10:5031 (incorporated herein by reference). In further detail, as demonstrated in the Examples herein, the Fc183 control mutant comprises the H435R mutation, but does not possess a substantially greater in vivo half-life compared to the equivalent wild-type polypeptide (Fc001). Further, the Fc185 control mutant comprises the H435R, T307R and A378V mutations, but still possesses a lesser in vivo half-life to that of Fc136. To the contrary, the Fc136 mutant, a polypeptide according to present disclosure, comprises the mutations T307I, Q311 R, M428L, N434L and Y436K, and has been determined to possess an in vivo half-life that is substantially greater than all of: the equivalent wild-type polypeptide (Fc001), Fc183 and Fc185.

[0071] Thus, without wishing to be bound by theory, a further advantage of an improved in vivo halflife is provided by the mutations that are present in the polypeptides of the present disclosure (e.g. Fc136), relative to the equivalent wild-type polypeptide of Fc001. Moreover, this improvement of in vivo half-life is unexpectedly significantly greater than Fc183 (which shows no difference vis-a-vis Fc001) and Fc185. Accordingly, in some embodiments, a polypeptide provided herein has an in vivo half-life that is greater than, such as substantially greater than, the in vivo half-life of a ‘control’ or ‘reference’ polypeptide. Accordingly, in some embodiments, a polypeptide provided herein has an in vivo half-life that is greater than, such as substantially greater than, the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations provided herein. In some embodiments, a polypeptide has an in vivo half-life that is greater than, such as substantially greater than, the in vivo half-life of the polypeptide comprising an Fc region that comprises the H435R mutation. In some embodiments, a polypeptide has an in vivo half-life that is greater than, such as substantially greater than, the in vivo half-life of the polypeptide comprising an Fc region that comprises the H435R, T307R and A378V mutations. In embodiments, it will be understood that the appropriate ‘control’ or ‘reference’ polypeptide does not comprise (e.g. any of the) mutations provided herein. In embodiments, the appropriate ‘control’ or ‘reference’ polypeptide does not comprise any mutations that increase the half-life of the polypeptide relative to the wild-type Fc region, e.g. the equivalent wild-type Fc region to the polypeptide. In embodiments, the appropriate ‘control’ or ‘reference’ polypeptide does not comprise any mutations relative to the wild-type Fc region, e.g. the equivalent wild-type Fc region to the polypeptide.

[0072] In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is greater than a polypeptide comprising an Fc region having the sequence as set forth in SEQ ID NO: 4. In some embodiments, the polypeptide of the present disclosure has an in vivo halflife that is greater than that of Fc183. In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is greater than a polypeptide comprising an Fc region having the sequence as set forth in SEQ ID NO: 5. In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is greater than that of Fc185.

[0073] In some embodiments herein, an in vivo half-life “greater than” means “substantially greater than”. In some embodiments herein, it will be understood that an in vivo half-life “greater than” means that the polypeptide is capable of having a greater or more prolonged effect in vivo than the relevant ‘control’ or ‘reference’ polypeptide.

[0074] In some embodiments herein, a polypeptide having an in vivo half-life “greater than” a polypeptide comprising an Fc region that does not comprise the substitution mutations provided herein, e.g. an equivalent wild-type polypeptide (such as Fc001), means at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% greater half-life than a polypeptide comprising an Fc region that does not comprise the substitution mutations. In some embodiments herein, a polypeptide having an in vivo half-life that is “greater than” a polypeptide comprising an Fc region that comprises a H435R mutation but does not comprise the substitution mutations provided herein (such as Fc183) means at least 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26% or 27% greater half-life than a polypeptide comprising an Fc region that comprises a H435R mutation but does not comprise the substitution mutations(e.g. Fc183).

[0075] In some embodiments herein, an in vivo half-life that is “greater than” a polypeptide comprising an Fc region that comprises H435R, T307R and A378V mutations but does not comprise the substitution mutations provided herein (such as Fc185) means at least 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% greater than a polypeptide comprising an Fc region that comprises H435R, T307R and A378V mutations but does not comprise the substitution mutations provided herein (e.g. Fc185).

[0076] In some embodiments, the polypeptide of the present disclosure has an in vivo half-life of at least 71 hours, at least 72 hours, at least 73 hours, at least 74 hours, at least 75 hours, at least 76 hours, at least 77 hours, at least 78 hours, at least 79 hours, at least 80 hours, at least 81 hours, at least 82 hours, at least 83 hours, at least 84 hours, at least 85 hours, at least 86 hours, at least 87 hours, at least 88 hours, at least 89 hours, at least 90 hours, at least 91 hours or at least 92 hours.

[0077] In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is at least equivalent to, substantially the same as or the same as the in vivo half-life of a polypeptide comprising a Fc region that comprises the T307I, Q311 R, M428L, N434L and Y436K mutations. In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is at least equivalent to, substantially the same as or the same as the in vivo half-life of a polypeptide comprising a Fc region that comprises “I” at position 307, “R” at position 311 , “L” at position 428, “L” at position 434 and “K” at position 436. In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is at least equivalent to, substantially the same as or the same as the in vivo half-life of a polypeptide comprising an Fc region having the sequence set forth in SEQ ID NO: 3.

[0078] It is to be understood herein that half-life may be considered as a measure of longevity in vivo. Thus, in some embodiments, the reference to “in vivo half-life” is considered a reference to “in vivo longevity”. In some embodiments, a “greater” in vivo half-life is a more prolonged in vivo half-life. In some embodiments, a “greater” in vivo half-life means that the time taken for the in vivo amount of the polypeptide to drop to half of a pre-determined initial amount is longer. In some embodiments, the in vivo half-life is in a human. In some embodiments, the in vivo half-life is in a mouse. In some embodiments, the in vivo half-life is the serum half-life. In some embodiments, the in vivo half-life is the serum half-life in a human. In some embodiments, the in vivo half-life is the serum half-life in a mouse. In some embodiments, the in vivo half-life is the serum half-life in a mouse model expressing human FcRn instead of murine FcRn.

[0079] In some embodiments wherein a comparison is made between the in vivo half-life of a polypeptide of the present disclosure and another polypeptide, it is to be understood that - unless otherwise stated, e.g. except for any stated differences in mutations - the ‘reference’ polypeptide is the same as, substantially the same as or equivalent to the polypeptide of the present disclosure.

[0080] Accordingly, in some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is greater than the equivalent polypeptide that does not comprise T307I, Q311 R, M428L, N434L and / or Y436K mutations. In some such embodiments, the equivalent polypeptide comprises the H435R mutation, and optionally further comprises the T307R and A378V mutations. In some embodiments, the polypeptide of the present disclosure has an in vivo half-life that is greater than the equivalent polypeptide comprising an Fc region that does not comprise “I” at position 307, “R” at position 311 , “L” at position 428, “L” at position 434 and / or “K” at position 436. In some such embodiments, the equivalent polypeptide comprises “R” at position 435, and optionally further comprises “R” at position 307 and “V” at position 378.

[0081] Polypeptide forms

[0082] In some embodiments, the polypeptide or Fc region provided herein may be combined with various further domains, e.g. immunoglobulin variable domains. Additionally, the polypeptide provided herein may be combined with a second polypeptide, e.g. in the form of an antibody construct. It is understood that the polypeptide or Fc region provided herein may be incorporated into any conceivable antibody, antigen-binding fragment or other immunoglobulin domain-comprising construct.

[0083] In an embodiment, a polypeptide provided herein further comprises a binding moiety or fragment or part (e.g. one or more CDRs) thereof that specifically binds to Staphylococcus aureus.

[0084] In some embodiments, an Fc region of the polypeptide provided herein is an immunoglobulin G (IgG) Fc region. In some embodiments, the Fc region of the polypeptide provided herein is an lgG1 , lgG2, lgG3 or lgG4 Fc region. In some embodiments, the Fc region of the polypeptide provided herein is a VHH-Fc antibody Fc region.

[0085] In some embodiments, the polypeptide is an antibody, an antigen-binding fragment thereof, or a polypeptide comprising a single domain antibody (VHH) fused to an Fc region. In some embodiments, the polypeptide comprises an Fc region and an antigen-binding domain, such as a domain comprising one or more variable immunoglobulin domains. In some embodiments, the polypeptide specifically binds to an S. aureus antigen.

[0086] In some embodiments, the polypeptide provided herein comprises or is a heavy chain of an antibody construct, antibody, antigen-binding fragment thereof comprising an Fc region, or VHH-Fc antibody.

[0087] In some embodiments, if the polypeptide of the present disclosure comprises one or more VH3 family antibody fragment (Fab) sequences, SpA does not bind to the VH3 Fab sequences. In some embodiments, SpA does not bind to the variable region of the polypeptide. In some embodiments, SpA does not bind to the variable region of the antibody or construct etc. that comprises the polypeptide of the present disclosure. In some embodiments, SpA does not bind to the Fab of the polypeptide or antibody / construct comprising the polypeptide. In some embodiments, SpA does not bind to the VHH domain of the polypeptide or antibody / construct comprising the polypeptide. In embodiments, this has no effect on the antibody-like specific binding of the variable region, which may specifically bind to S. aureus. In some embodiments, SpA does not bind via unspecific binding to the variable domains, VHHs or VH3 sequences etc. In some embodiments, SpA does not bind via non-antigen-specific binding to the variable domains, VHHs or VH3 sequences etc. In some embodiments, the polypeptide or antibody construct specifically binds to SpA (e.g. via antigenspecific binding) and optionally does not have any other affinity / binding to SpA, e.g. outside of the specific binding to SpA.

[0088] In some embodiments, it is generally understood that the Fc region of the polypeptide of the present disclosure is a dimeric Fc region. In some embodiments, it is generally understood that the Fc region of the polypeptide comprises the mutations of the present disclosure, e.g. T307I, Q311 R, M428L, N434L, Y436K, or any combination thereof, on both of the Fc region polypeptide sequences in the dimeric Fc region.

[0089] Further Forms

[0090] In an aspect, the present disclosure provides one or more nucleic acid sequences capable of expressing the polypeptide according to the invention.

[0091] In some embodiments, the nucleic acid is RNA encoding a polypeptide provided herein. In some embodiments, the nucleic acid is mRNA encoding a polypeptide provided herein. In some embodiments, the nucleic acid is a vector encoding a polypeptide provided herein.

[0092] In some embodiments, the nucleic acid encodes an antibody. In some embodiments, the nucleic acid encodes a polypeptide comprising a VHH domain and an Fc region. In some embodiments, the one or more nucleic acid sequences encode an antibody. In some embodiments, the one or more nucleic acid sequences encode a polypeptide comprising a VHH domain and an Fc region.

[0093] As used herein, the term “antibody construct” may refer to any multimeric immunoglobulin construct comprising a polypeptide of the present disclosure, e.g. including full-length antibodies, antibody-related binding molecules and antigen-binding fragments thereof comprising an Fc region. As used herein, the term “antibody” may refer to a protein having an antigen-binding domain which comprises at least one complementarity determining region (CDR). In general, it is understood that an antibody comprises a dimer e.g. a homodimer of two immunoglobulin heavy chains and optionally two immunoglobulin light chains. In some embodiments, a polypeptide of the present disclosure may be referred to as an antibody or antibody-related construct, which will be understood as a reference to an antibody or construct comprising the polypeptide of the present disclosure. The term “antibody construct” includes VHH-Fc constructs or antibodies.

[0094] The term “complementarity determining region” or "CDR" generally refers to one of the 6 hypervariable regions within the variable domain of an antibody, and may also refer to one of the 3 hypervariable regions within the VHH domain of an sdAb.

[0095] “Complementarity determining region” or “CDR” with regard to antigen-binding domain or antibody refers to a hypervariable region or a highly variable loop in the variable region of the heavy chain and / or the light chain of an antibody, which contributes primarily to antigen binding. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0096] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0097] As used herein, “antigen binding site” or “antigen-binding domain” means a protein or polypeptide which comprises at least one complementarity determining region (CDR). The antigen binding site may comprise 3 CDRs, e.g. it may be equivalent to that of a single domain antibody (sdAb) domain such as a VHH domain.

[0098] As used herein, the term “constant immunoglobulin domain” may refer to a constant domain of an immunoglobulin, e.g. a CH3 domain. In conventional, full-length antibodies (e.g. IgG antibodies) that comprise four polypeptides - two light chains and two heavy chains - the Fc region comprises dimer of the CH2-CH3 domains of each heavy chain polypeptide.

[0099] As used herein, the term “fragment crystallisable (Fc) region” may refer to the Fc region or Fc regions of an immunoglobulin (e.g. an antibody or antibody construct), which comprises the CH2-CH3 domains, or any fragments, truncations, derivatives or variants thereof, including those encoded by pseudogenes, insofar as the Fc region(s) also comprise the mutation(s) of the present disclosure. Truncations may include as little as one immunoglobulin constant domain or a fragment thereof. Preferably, the Fc regions are full-length or essentially full- length Fc regions comprising the mutation(s) of the present disclosure. The Fc regions include those in which known / standard / routine modifications have been made. The Fc regions are not particularly limited by species, although human is preferred, Thus, Fc regions of all species are encompassed, including all animal, human, murine and other orthologues. Fc regions or Fc region-like sequences encoded by paralogues and pseudogenes are also encompassed. Suitably, in embodiments the Fc region may be an lgG1 , lgG2, lgG3, lgG4, immunoglobulin pseudogene-encoded, IgA, IgE, IgM or IgD Fc region. Suitably, in embodiments the Fc region may be an lgG1 , lgG2, lgG3 or lgG4 Fc region. Preferably, the Fc region is an lgG1 Fc region. In some embodiments, a first polypeptide and a second polypeptide form a homodimer and each comprise an Fc region of the same immunoglobulin isotype.

[0100] The terms VHH domain, VHH and single domain antibody (sdAb) are used interchangeably herein. sdAbs are found naturally in e.g. camelids and sharks. sdAbs are devoid of the light chain and lack the first constant domain of the heavy chain (CH1) of conventional IgGs. Consequently, the antigen-binding fragment of sdAbs solely comprises a single variable domain, often referred to as a Variable Heavy domain of Heavy chain (VHH domain). In some embodiments, a first and a second polypeptide of an Fc region polypeptide homodimer each comprise an Fc region lacking the CH1 domain.

[0101] As used herein, the term “variable immunoglobulin domain” or “variable domain” may refer to a variable domain of an immunoglobulin, e.g. a VHH domain.

[0102] In some embodiments, the polypeptide of the present disclosure may comprise a single-chain variable fragment (scFv); an Fab; an Fab’; an F(ab)’2; an Fv; a single domain antibody (sdAb); a VHH; a single chain variable domain; a designed ankyrin repeat protein (DARPin); or an aptamer.

[0103] In some embodiments, the antibody construct of the present disclosure is an antibody, full- length immunoglobulin (full-length antibody), a scFv-Fc, a Fab-Fc, a Fab’-Fc, a F(ab)’2-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc. In some embodiments, the antibody of the present disclosure is a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, or a polyclonal antibody.

[0104] The term "chimeric antibody" generally refers to an antibody obtained by fusing a variable region of a non-human antibody and a constant region of a human antibody, which can reduce an immune response induced by the non-human antibody. The non-human antibody may be, for example, a murine, camelid, rabbit, sheep, goat or chicken antibody. By way of example, for establishment of a chimeric antibody, a hybridoma secreting a specific monoclonal antibody can be established, and a variable region gene is cloned from the mouse hybridoma cells; then a constant region gene of human antibody can be cloned as required, and the mouse variable region gene and the human constant region gene are connected to form a chimeric gene; then the chimeric gene is inserted into an expression vector, wherein chimeric antibody molecules can be expressed in a eukaryotic system or a prokaryotic system.

[0105] The term "humanized antibody", also referred to as CDR-grafted antibody, generally refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e. , an antibody produced in a different type of human germline antibody framework sequence. Therefore, the heterogeneous reaction induced by the presence of a large number of mouse protein components in the chimeric antibody can be overcome. Such framework sequences can be obtained from public DNA databases or disclosed references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database.

[0106] The term "fully humanized antibody", "fully human antibody" or "completely human antibody", which may also be known as "fully humanized monoclonal antibody", may have both humanized variable region and constant region so as to eliminate immunogenicity and toxic side effects. The development of monoclonal antibodies has four stages, namely murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully humanized monoclonal antibodies. The antibodies or ligands described herein can be fully humanized monoclonal antibodies. Relevant technologies for the preparation of fully human antibodies may be: human hybridoma technology, EBV-transformed B-lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, single B-cell antibody preparation technology, and the like.

[0107] A “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. A monoclonal antibody is highly specific and targets a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies targeting (or specific for) different epitopes. The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as producing the antibody by any particular method.

[0108] Antibodies may be obtained by techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum. Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al. , J. Mol. Biol. 222:581-597 (1991), for example.

[0109] In some embodiments, according to the number of antigen-binding domains, e.g. VH, VL or VHH domains, that are present, the antibody for use according to the present disclosure may be monovalent, bivalent, trivalent, tetravalent or pentavalent. Similarly, In some embodiments, according to the number of different antigen-binding domains, e.g. VH, VL or VHH domains, that are present, the antibody for use according to the disclosure may be monospecific or multispecific, e.g., bispecific, trispecific, tetraspecific etc.

[0110] Any suitable multispecific or multivalent antibody format which is known in the art may be used in the practice of the present disclosure. Suitably, the bispecific antibody may be an IgG-scFv, IgG-sdAb, IgG-VHH, scFv-Fc-scFv, KiH-IgG, KA-body, KiH-Fc-Fab / scFv.

[0111] The variable domains (e.g. VH, VL or VHH domains) used are not particularly limiting, and may comprise any antigen binding site that is specific for a selected target protein. Methods for determining binding specificity of an antibody to a particular antigen include, but are not limited to, by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (e.g. using a BIAcore instrument), ELISA, western blot, in situ hybridisation, immunohistochemistry, flow cytometry, Forster resonance energy transfer (FRET), phage display libraries, yeast two- hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification. Binding affinity can also be determined using methods such as BLI, SPR analysis (e.g. using a BIAcore instrument), flow cytometry, fluorescence quenching, isothermal titration calorimetry.

[0112] Methods for providing variable domains, such as sdAbs (which comprise VHH domains), against a specific target can be practiced (see Caussinus et al , Nat Struct Mol Biol; 2011 ; 19(1); 117-121 & Fulcher et al , Open Biol; 2016; 6(10); pii 160255). Further, methods to isolate antigen-specific VHHs from immune or semisynthetic libraries using phage, yeast, or ribosome display can be carried out (see Muyldermans J Biotechnol. 2001 Jun; 74(4):277- 302. & Dufner et a / . Trends Biotechnol. 2006 Nov; 24(11):523-9).

[0113] By way of example, a VHH can be obtained by immunisation of e.g. dromedaries, camels, llamas or alpacas with the desired antigen and subsequent isolation of the mRNA coding for VHHs. Single domain shark variable domain of new antigen receptor (VNAR) antibodies are also known and suitable for use according to the present disclosure as an alternative sdAb to a VHH domain. Hence, by way of further example, a VNAR can be obtained by immunisation of sharks with the desired antigen and subsequent isolation of the mRNA coding for VNARs. Reverse transcription and PCR can then be used to generate a library of VHHs or VNARs. Standard screening techniques such as phage display and ribosome display may be used to identify the suitable clones binding the antigen of interest.

[0114] Once the most potent clones have been identified, their DNA sequence may be optimized, for example to improve their stability towards enzymes. Humanisation may also be performed.

[0115] VHHs and VNARs may be expressed in a cell using conventional vectors, such as those described herein.

[0116] The ability of the antibody to specifically bind its target may be determined by the skilled person. For example, determination of binding may be performed e.g. by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), western blot, flow cytometry, in situ hybridisation and / or microscopy. Suitably, determination of binding affinity may be performed by e.g. by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument) and / or flow cytometry.

[0117] In a further aspect, the present disclosure provides a nucleic acid particle comprising the construct or nucleic acid sequences as described herein. In one embodiment, the nucleic acid particle is a lipid nanoparticle (LNP).

[0118] In an aspect, the present disclosure provides a cell comprising the polypeptide or the one or more nucleic acid sequences as described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a human cell.

[0119] Methods for engineering such cells include but are not limited to genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection - DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell. A cell of the present disclosure may be generated by introducing DNA or RNA coding for the polypeptide provided herein by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0120] A cell described herein may be made by introducing to a cell (e.g. by transduction or transfection) the one or more nucleic acids sequence according to the present disclosure.

[0121] A cell described herein may comprise and / or secrete a polypeptide described herein.

[0122] In an aspect, the present disclosure provides a composition comprising the polypeptide, the one or more nucleic acid sequences, the LNP or the cell described herein. The composition may be a pharmaceutical composition.

[0123] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0124] Methods of use and applications

[0125] In an aspect, the present disclosure provides an in vitro method comprising contacting a cell with the polypeptide, the one or more nucleic acid sequences, the LNP, the cell or the composition described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a CHO cell. In some embodiments, the cell is a human cell.

[0126] In an aspect, the present disclosure provides the polypeptide, the one or more nucleic acid sequences, the LNP, the cell or the composition described herein for use in a method of therapy or a diagnostic method.

[0127] In an aspect, the present disclosure provides the polypeptide, the one or more nucleic acid sequences, the LNP, the cell or the composition described herein for use in the manufacture of a medicament for a therapy or a diagnostic agent.

[0128] In an aspect, the present disclosure provides a method of therapy or a diagnostic method comprising administering the polypeptide, the one or more nucleic acid sequences, the LNP, the cell or the composition described herein to a subject.

[0129] In some embodiments, the subject is an animal or human. In some embodiments, the subject is a mammal. Preferably, the subject is human. In some embodiments, the subject is suffering from or susceptible to S. aureus infection.

[0130] In some embodiments, the therapy comprises treating or preventing a Staphylococcus aureus infection. In some embodiments, the therapy comprises treating or preventing a Streptococcal infection, particularly wherein the polypeptide has reduced affinity for SpG. In some embodiments, the S. aureus is resistant to one or more treatments. In some embodiments, the S. aureus is methicillin resistant. In some embodiments, the therapy is treating or preventing infection in a human. In some embodiments, treating or preventing comprises one or more of promoting S. aureus decolonization, preventing invasive disease caused by S. aureus, and improving the outcome of S. aureus bloodstream infections in the subject.

[0131] In some embodiments, the antibody is administered intravenously. In other embodiments, one or more nucleic acids encoding the antibody is / are administered. In some embodiments, the nucleic acid(s) may be introduced by transduction. In some embodiments, the nucleic acid(s) may be introduced by transfection.

[0132] Method of manufacture

[0133] In an aspect, the present disclosure provides a method for making the polypeptide provided herein, comprising expressing one or more nucleic acids capable of expressing the polypeptide. In some embodiments, the method comprises i) providing one or more nucleic acids capable of expressing the polypeptide and ii) expressing the nucleic acid(s) capable of expressing the polypeptide.

[0134] Other general definitions

[0135] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino (N) to carboxy (C) orientation, respectively.

[0136] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0137] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids. The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”. The term “residues” is used to refer to amino acids in an amino acid sequence.

[0138] The term “variant” in relation to a polypeptide refers to a polypeptide that has an equivalent function to the amino acid sequences described herein, but which includes one or more amino acid substitutions, insertions or deletions.

[0139] The term "antibody" refers to an intact immunoglobulin of any isotype that can compete with the intact antibody for specific binding to the target antigen, and includes chimeric, humanized, fully human, and bispecific antibodies as well as fragments of all of these. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity. “Fc region”, “Fc domain” and “fragment crystallisable” region / domain are used interchangeably herein. The Fc region is a well-known part of an antibody, although the same Fc region may be present in other, antibody-like or antibody-derived polypeptides such as VHH-Fc fusion polypeptides. Herein, antibody residue numbering is according to the Ell residue numbering scheme.

[0140] An amino acid residue in an antibody “corresponds” to a given residue when it occupies the same essential structural position within the antibody as the given residue. For example, a selected residue in a comparison antibody corresponds to position 428 (according to the Ell numbering system as described herein) in an antibody provided herein when the selected residue occupies the same essential spatial or structural relationship to Ell position 428 as assessed using applicable methods in the art. For example, a comparison antibody may be aligned for maximum sequence homology with the antibody provided herein and the position in the aligned comparison antibody that aligns with Ell position 428 may be determined to correspond to it. Alternatively, instead of (or in addition to) a primary sequence alignment as described above, a three dimensional structural alignment can also be used, e.g., where the structure of the comparison antibody is aligned for maximum correspondence with an antibody provided herein and the overall structures compared. In this case, an amino acid that occupies the same essential position as Ell position 428 in the structural model may be said to correspond.

[0141] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen may possess one or more epitopes that are capable of interacting with different antibodies. The term “epitope” includes any region or portion of molecule capable eliciting an immune response by binding to an immunoglobulin or to a T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0142] As used herein, the terms “polynucleotide”, “nucleotide”, “nucleic acid sequence” and “nucleic acid” are intended to be synonymous with each other.

[0143] The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

[0144] “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms “% identical” and “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981 , Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK _LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1 , -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1 ; and (vi) conditional compositional score matrix adjustment.

[0145] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0146] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0147] In some embodiments, “isolated” means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0148] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence depending on the context. Typically, translation is required.

[0149] In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.

[0150] With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0151] The term “nucleic acid” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0152] The term “nucleoside” (abbreviated herein as “N”) relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0153] The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes II, A, T, C and G, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2’-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.

[0154] A modified purine (A or G) or pyrimidine (C, T, or II) base moiety is preferably modified by one or more alkyl groups, more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)- alkyl-uracil, such as N7-C1-4 alkyl-guanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5- C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl- adenine, 5-methyl-cytosine, 5-methyl-uracil, N1-methyl-pseudouridine, and N(1)-methyl- uracil.

[0155] Herein, the term “DNA” relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, “deoxyribonucleotide” refers to a nucleotide which lacks a hydroxyl group at the 2’-position of a p-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains “a majority of deoxyribonucleotide residues” if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0156] DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.

[0157] The term “RNA” relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2’-position of a -D- ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0158] “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA), immunostimulatory RNA (isRNA) and viral RNA. In some embodiments, “RNA” refers to mRNA. In some embodiments, mRNA comprises a coding sequence, 5’ cap (e.g. an m7G cap) and / or 3’ tail (e.g. a polyA tail).

[0159] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0160] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”.

[0161] The terms “equivalent to”, “similar to” or “comparable to” as used herein are interchangeable with “not substantially different from” and, in a stricter embodiment, “not different from” or “the same as”. It will be understood herein that the margin allowable within e.g. “substantially” is that which would be understood by the skilled person as not having a significant effect on the function in question. E.g., the polypeptide of the present disclosure which has an in vivo halflife that is equivalent to that of a wild-type polypeptide can be considered to have an in vivo half-life that is not substantially different from that of the equivalent wild-type polypeptide. In this embodiment, any minor difference between the in vivo half-life of the mutant and the wildtype polypeptide is not significant and does not alone make any substantial difference to the in vivo function of the polypeptides.

[0162] References herein to any function or effect in an immune system, in vivo, in a cell or in an organism are preferably in a human or human cell, as applicable.

[0163] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0164] The technologies provided herein will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the technologies provided herein and are not intended in any way to limit the scope of a claimed invention.

[0165] Numbered Embodiments

[0166] 1 . A polypeptide comprising a fragment crystallisable (Fc) region having reduced affinity for Staphylococcus aureus protein A (SpA). 2. The polypeptide of embodiment 1 , wherein the polypeptide sequence of the Fc region comprises one or more of the following substitution mutations: T307I, Q311 R, M428L, N434L and Y436K.

[0167] 3. A polypeptide comprising an Fc region, wherein the polypeptide sequence of the Fc region comprises T307I, Q311 R, M428L, N434L, Y436K, or any combination thereof.

[0168] 4. The polypeptide of embodiment 3, wherein the polypeptide has reduced affinity for SpA.

[0169] 5. The polypeptide of any one of embodiments 1 to 4, wherein the polypeptide sequence of the Fc region comprises the substitution mutation M428L, and optionally comprises one or more of T307l, Q311 R, N434L and Y436K.

[0170] 6. The polypeptide of any one of embodiments 1 to 5, wherein the polypeptide sequence of the Fc region comprises all of the substitution mutations T307I, Q311 R, M428L, N434L and Y436K.

[0171] 7. The polypeptide of any one of embodiments 1 to 6, wherein the polypeptide does not comprise the substitution mutation H435R.

[0172] 8. The polypeptide of any one of embodiments 1 to 7, wherein the polypeptide comprises H435.

[0173] 9. The polypeptide of any one of embodiments 1 to 8, wherein the amino acid position numbering corresponds to Ell numbering.

[0174] 10. The polypeptide of any one of embodiments 1 to 9, wherein the Fc region of the polypeptide has reduced affinity for the second immunoglobulin-binding protein of Staphylococcus aureus (Sbi).

[0175] 11. The polypeptide of any one of embodiments 1 to 10, wherein the Fc region of the polypeptide has reduced affinity for Streptococcal protein G (SpG).

[0176] 12. The polypeptide of any one of embodiments 1 to 11 , wherein the Fc region of the polypeptide has reduced affinity for SpA, Sbi and / or SpG relative to the Fc region of the polypeptide that does not comprise the substitution mutations.

[0177] 13. The polypeptide of any one of embodiments 1 to 12, wherein the Fc-mediated effector functions of the polypeptide are not inhibited in the presence of Staphylococcus aureus.

[0178] 14. The polypeptide of any one of embodiments 1 to 13, wherein the Fc region of the polypeptide binds to the neonatal Fc receptor (FcRn). 15. The polypeptide of any one of embodiments 1 to 14, wherein the polypeptide has an in vivo half-life that is equivalent to the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations.

[0179] 16. The polypeptide of any one of embodiments 1 to 15, wherein the Fc region of the polypeptide binds to the Fc-gamma receptor (FcyR).

[0180] 17. The polypeptide of any one of embodiments 1 to 16, wherein the polypeptide has improved complement component 1q (C1q) recruitment.

[0181] 18. The polypeptide of any one of embodiments 1 to 17, wherein the polypeptide further comprises a binding moiety that specifically binds to Staphylococcus aureus.

[0182] 19. The polypeptide of any one of embodiments 1 to 18, wherein the Fc region of the polypeptide is an immunoglobulin G (IgG) Fc region.

[0183] 20. The polypeptide of any one of embodiments 1 to 19, wherein the polypeptide is an antibody construct, an antibody, an antigen-binding fragment thereof comprising an Fc region, or a polypeptide comprising a single domain antibody (VHH) fused to an Fc region.

[0184] 21 . An antibody construct comprising the polypeptide of any one of embodiments 1 to 20.

[0185] 22. The polypeptide or antibody construct of embodiment 20 or 21 , wherein the antibody construct is a full-length immunoglobulin, a construct comprising an Fc region dimer, a scFv- Fc, a Fab-Fc, a Fab’-Fc, a F(ab)’2-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

[0186] 23. One or more nucleic acid sequences capable of expressing the polypeptide or the antibody construct according to any one of embodiments 1 to 22.

[0187] 24. The one or more nucleic acid sequences of embodiment 23, wherein the nucleic acid comprises DNA, comprises RNA, comprises mRNA, is RNA, is DNA or is mRNA encoding the polypeptide or the antibody construct according to any one of embodiments 1 to 22.

[0188] 25. A cell comprising the polypeptide, the antibody construct or the one or more nucleic acid sequences according to any one of embodiments 1 to 24.

[0189] 26. A composition comprising the polypeptide, the antibody construct or the one or more nucleic acid sequences or the cell according to any one of embodiments 1 to 25.

[0190] 27. The composition of embodiment 26, wherein the composition is a pharmaceutical composition comprising at least one pharmaceutically acceptable carriers, diluents, excipients, or any combination thereof. 28. An in vitro method comprising contacting a cell with the polypeptide, the antibody construct, the one or more nucleic acid sequences, the cell or the composition of any one of embodiments 1 to 27.

[0191] 29. The polypeptide, the antibody construct, the one or more nucleic acid sequences, the cell or the composition of any one of embodiments 1 to 27 for use in a method of therapy or a diagnostic method.

[0192] 30. The polypeptide, the antibody construct, the one or more nucleic acid sequences, the cell or the composition of any one of embodiments 1 to 27 for use in the manufacture of a medicament for a therapy or a diagnostic agent.

[0193] 31. A method of therapy or a diagnostic method comprising administering the polypeptide, the antibody construct, the one or more nucleic acid sequences, the cell or the composition of any one of embodiments 1 to 27 to a subject.

[0194] 32. The method of any one of embodiments 29 to 31, wherein the therapy is treating or preventing a Staphylococcus aureus infection.

[0195] 33. A method comprising administering a polypeptide, antibody construct, one or more nucleic acid sequences, cell or composition of any one of embodiments 1 to 27 to a subject.

[0196] 34. The method of embodiment 33, wherein the method is a method of treating or preventing an S. aureus infection in the subject, optionally wherein the subject is a human.

[0197] 35. The method of embodiment 33, wherein the method is a method of inducing an immune response to an S. aureus infection in the subject, optionally wherein the subject is a human.

[0198] 36. A method for making the polypeptide or the antibody construct according to any one of embodiments 1 to 21 , comprising expressing one or more nucleic acids capable of expressing the polypeptide.

[0199] 37. A method of making a polynucleotide encoding a polypeptide of any one of embodiments 1 to 20 comprising recombinantly linking a first nucleotide sequence encoding an Fc region having reduced affinity for SpA with a second nucleotide sequence encoding a binding moiety that specifically binds to S. aureus.

[0200] 38. A method of making a polynucleotide encoding a polypeptide of any one of embodiments 1 to 20 comprising recombinantly linking a first nucleotide sequence encoding an Fc region with a second nucleotide sequence encoding a binding moiety that specifically binds to S. aureus, wherein the polypeptide sequence of the Fc region comprises T307I, Q311 R, M428L, N434L, Y436K, or any combination thereof. Examples

[0201] Example 1 - Screening for Fc variants

[0202] For an effective use of a therapeutic antibody in S. aureus infections, the Fc-mediated capture of the antibody must be inhibited. To achieve this, mutations in the Fc-domain that inhibit SpA- mediated binding were identified.

[0203] In silico screening for FcRn affinity, SpA affinity and stability

[0204] A considerable challenge presented in the screening process was the highly overlapping binding sites of SpA / Sbi and the neonatal Fc receptor (FcRn). FcRn mediates antibody recycling via pH-dependent rescue from endosomal degradation, and is thus useful for the long serum half-life of antibodies. Both SpA / Sbi and FcRn bind to IgG-Fc-domains at the interface of CH2 and CH3 regions and partly involve the same amino acids. Using advanced in silico Al-driven screening techniques, the protein complexes Fc:FcRn and Fc:SpA were modelled in silico for a large number of Fc variants, and mutagenesis and stability predictions led to the identification of 180 candidate Fc mutants.

[0205] Expression of screening candidates as VHH-Fc-fusions

[0206] The 180 identified candidate Fc mutants, along with controls from literature (Chen etal., PNAS 2022) were subsequently expressed and characterized in the format of VHH-Fc-fusions with a VHH targeting a placeholder target antigen (e.g. for later replacement with S. at / ret / s-specific VHHs). The characterisation included testing for both SpA- and FcRn-binding. Of the 180 candidate proteins and the controls from the literature, 32 proteins were further characterized with respect to their functional capacities. The other candidates did not pass initial testing of SpA- and / or FcRn-binding. Through several rounds of testing, 3 candidate mutants of further interest were identified: Fc049, Fc067 and Fc136, as well as two control mutants from literature: Fc183 and Fc185. Details of the Fc domains of these proteins are provided in Table 1.

[0207] TABLE 1 : Candidate mutants and literature controls after initial screening

[0208] Notably, the candidate mutants identified in Table 1 did not comprise any of the same substitution mutations as the control mutants from the literature (although some of the amino acid positions are the same, the specific substitution mutations are not the same). Notably, none of the candidate mutants identified in Table 1 possessed the H435R mutation of the control mutants. Notably, Fc136 was the sole mutant identified (out of any of the 180 screened mutants) to possess the M428L mutation.

[0209] Expression and stability tests

[0210] The expression and stability of the screening candidate mutants and control mutants were tested, and results were analysed from a western blot.

[0211] All the tested screening candidate mutants could be well expressed. The selected lead screening candidate mutants listed in Table 1 showed no aberrant fragmentation in the western blot. However, Fc183 showed a smear in the western blot, indicating possible liabilities with the expression and stability of this protein.

[0212] FcRn binding tests

[0213] FcRn binding of the Fc mutants listed in Table 1 was tested via ELISA at pH 6 and at pH 7. Some additional variants from the literature were also tested. For efficient antibody recycling within the cell, it is advantageous for FcRn binding to occur at pH 6, but for binding to be weaker at pH 7.

[0214] At pH 6, the Fc mutants listed in Table 1 displayed binding to FcRn which was similar to the WT control (the unmodified VHH-Fc fusion). Binding to FcRn at pH 6 was considered to be acceptable for all tested mutants of Table 1 . However, two additional Fc mutants from the literature were tested and failed in this screening stage, as they exhibited weak binding to FcRn at pH 6. Fc182 (“AESP” from Chen etal. (2022), comprising the mutations S254A, Q311 E, L432S and N434P) and Fc184 (“AESP-RV” from Chen et al. (2022), comprising the mutations S254A, Q311 E, L432S, N434P, T307R and A378V) both exhibited unacceptably weak binding to FcRn at pH 6.

[0215] At pH 7, binding to FcRn was considered to be acceptable for all of the Fc mutants listed in Table 1 , with the notable exception of Fc185. Fc185 showed strong binding to FcRn at pH 7, to a degree which may negatively impact the antibody half-life of Fc185. This result is shown in Figure 1.

[0216] Further, two additional Fc mutants from the literature were tested and failed in this screening stage, as they exhibited strong binding to FcRn at pH 7. Fc186 (“R-QW" from Chen et al. (2022), comprising the mutations H435R, T307Q, Q311V and A378V) and Fc187 (“R-DDRVV” from Chen et al. (2022), comprising the mutations H435R, T256D, N286D, T307R, Q311V and A378V) both exhibited unacceptably strong binding to FcRn at pH 7, indicative of a shorter serum half-life.

[0217] Example 2 - Testing antibody effector functions

[0218] FcyR receptor binding tests

[0219] Antibody effector functions are mostly mediated by the interaction of the Fc-domain with relevant Fc-receptors. In the case of IgG antibodies, the Fc-domain of which was used in these experiments, Fcy-Receptors (FcyRs) are particularly relevant. As an approximation of Fc- effector functionality, binding to Fcy-Receptors was tested via ELISA (see Figure 2).

[0220] Binding to Fcy-Receptors for the screening candidate mutant Fc136 was comparable to the wildtype control, indicating that the inserted mutations did not negatively affect Fcy-Receptor binding. Binding was conserved for activating receptors, such as FcyRI (Figure 2A), FcyRlla (Figure 2B) and FcyRI I la (Figure 2D), as well as inhibitory receptors, such as FcyRllb / c (Figure 20). High affinity polymorphisms of FcyRlla and FcyRI I la were used in these tests.

[0221] The literature mutant Fc185 exhibited the lowest maximum effect for both FcyRI and FcyRlla. The literature mutant Fc183 exhibited the lowest maximum effect for FcyRllb / c.

[0222] These tests confirmed that the mutations in Fc136 did not significantly reduce FcyR binding.

[0223] Functional testing of Fc mutants

[0224] The Fc mutants listed in Table 1 were subjected to functional testing, including cell binding testing, an ADCC assay, an ADCP assay and a C1 g recruitment assay. Cell binding was assessed with a target antigen over-expressing Flpln CHO cell line, an experimentally validated cell line based on the commercially available Flpln CHO system (https: / / www.thermofisher.com / order / catalog / product / de / en / R75807). The ADCC assay was carried out with a kit obtained from Promega GmbH (https: / / www.promega.de / en / products / reporter-bioassays / fc-effector-activity-bioassays / adcc- bioassays / ?catNum=G7010). The ADCP assay was also carried out with a kit obtained from Promega GmbH (https: / / www.promega.de / en / products / reporter-bioassays / fc-effector-activity- bioassays / adcp-bioassays / ?catNum=G9991). The C1q recruitment assay was designed based on available literature (Pawluczkowycz et al., J Immunol 1 ;183(1):749-58 (2009) [Pubmed ID: 19535640]).

[0225] The results of the functional tests are shown in Figure 3.

[0226] It was determined that cell surface binding to the target antigen over-expressing cell line was unaffected by the inserted mutations (Figure 3C).

[0227] However, some differences in Fc-effector functions were observed. Of note, Fc136 performed at least comparably to the wildtype protein in ADCC and ADCP reporter assays. Moreover, Fc136 showed favourably strong signals in the C1q recruitment assay. It was also noted that Fc049 showed ADCC signals but the signals were weaker than those of Fc136.

[0228] These tests confirmed that the mutations did not significantly reduce cell binding. The tests further confirmed that Fc136 performed well in all assays, and that Fc136 showed favourably strong signals in the C1q recruitment assay, compared to the other tested mutants. C1q recruitment to target cells bound by IgG antibodies / VHH-Fc fusion proteins etc. initiates the classical pathway of complement, thereby promoting complement-dependent cytotoxicity (CDC). Thus, these tests indicate that Fc136 may induce enhanced CDC.

[0229] Example 3 - Testing for inhibited Sbi, SpA and SpG binding

[0230] The Fc mutants including Fc136 were further tested via ELISA for inhibition of binding to the second immunoglobulin-binding protein of Staphylococcus aureus (Sbi), as well as SpA and SpG, in comparison with a wild-type control (Fc001 / WT).

[0231] The ELISA results are shown in Figure 4. Fc136 showed inhibited Sbi, SpA and SpG binding, indicating that Fc136 has reduced affinity for Sbi, SpA and SpG. In particular, Fc136 has substantially reduced affinity for Sbi, SpA and SpG relative to the wild-type equivalent protein.

[0232] Example 4 - Further confirmation with purified polypeptides

[0233] The VHH-Fc fusion mutants Fc136, Fc183 and Fc185 and a wild-type control (WT) were further tested in purified forms. This was done to further confirm that the Fc136 mutant is no longer bound to by Staphylococcal Protein A and Streptococcal Protein G, whilst maintaining other properties that are similar to that of the wild-type control (and are therefore desirable).

[0234] Protein production

[0235] VHH-Fc-fusions were produced via transient transfection of ExpiCHO cells. The cell culture supernatant was harvested 8 days post-transfection and subsequently purified via affinity chromatography (FLAG-tag) and preparative size exclusion chromatography. This yielded protein solutions of high purity (> 95%, confirmed via SDS-PAGE), with the target protein running at a molecular weight of around 95 kDa under non-reducing conditions and around 45 kDa under reducing conditions. Reducing conditions led to reduction of the disulfide bonds in the hinge-region of the antibody constructs and thus to single protein chains. Proteins were normalized to 2 mg / mL in sterile PBS.

[0236] ELISA testing interactions with target antigen, Fc-receptors, SpA, SpG and Sbi

[0237] Uniform binding of the VHH-Fc-fusions was detected on the target antigen, with EC50 values ranging from 0.39 to 0.51 nM (Figure 5A).

[0238] Binding to FcyRI (CD64), FcyRlla (CD32a) and FcyRllla (CD16) was comparable between Fc136 and the WT (Fc001) (Figures 5B-D).

[0239] All candidate Fc-mutants (Fc136, Fc183, Fc185) displayed reduced binding to Protein A, with binding only being detected in very high concentrations - EC50 values were increased by > factor 1000 (Figure 6A).

[0240] In contrast, only Fc136 showed reduced binding to Streptococcal Protein G, whereas the H435R-comprising candidates - Fc183 and Fc185 - showed comparable binding to Protein G as the WT (Figure 6B).

[0241] Further, all candidate Fc-mutants (Fc136, Fc183, Fc185) displayed reduced binding to Sbi, again with binding only being detected in very high concentrations (Figure 6A).

[0242] In short, further studies with purified polypeptides confirmed that the Fc136 mutant VHH-Fc fusion displayed reduced affinity for SpA, SpG and Sbi, whilst maintaining the same desired properties of target antigen and Fc-receptor binding as that of the wild-type VHH-Fc fusion. In particular, Fc136 has substantially reduced affinity for SpA, SpG and Sbi relative to the equivalent wildtype polypeptide.

[0243] Example 5 - Improved half-life relative to wild-type and literature polypeptides

[0244] To assess binding of the identified Fc-mutants to human FcRn in vivo, a pharmacokinetics experiment was conducted. For this, NOD. Cg-Fcgritm1DcrPrkdc5 1H2rgtm1WJITg(FCGRT)32Dcr / J (NSG FcRn- / - hFcRn (32) Tg) mice expressing human instead of murine FcRn were selected as a model organism, since binding of the Fc mutants to human versus murine FcRn showed different patterns.

[0245] 5 groups, each including 4 mice, were injected with one of the following samples:

[0246] • Fc136: 250 pg in 150 pL PBS

[0247] • Fc183: 250 pg in 150 pL PBS

[0248] • Fc185: 250 pg in 150 pL PBS

[0249] • Fc001 : 250 pg in 150 pL PBS

[0250] • PBS: 150 pL

[0251] Blood was drawn at different time points across a timeframe of 18 days. VHH-Fc concentrations in serum were subsequently quantified via ELISA, leading to the results shown in Figure 7 and Table 1 below.

[0252] Fc136 consistently showed the highest serum concentrations in comparison to all other samples tested. Serum concentrations of Fc136 were significantly higher than those observed for the respective WT protein (Fc001). Fc136 also showed significantly higher concentrations than Fc185. Fc183 did not differ substantially in half-life and serum concentrations from the wildtype.

[0253] Table 2: Serum half-lives of Fc mutants as determined via Two-Phase Decay Model

Claims

Claims1 . A polypeptide comprising a fragment crystallisable (Fc) region having reduced affinity for Staphylococcus aureus protein A (SpA), wherein the polypeptide sequence of the Fc region comprises T307I, Q311 R, M428L, N434L, Y436K, or any combination thereof.

2. The polypeptide of claim 1 , wherein the polypeptide sequence of the Fc region comprises the substitution mutation M428L, and optionally comprises T307I, Q311 R, N434L, Y436K or any combination thereof.

3. The polypeptide of claim 1 or 2, wherein the polypeptide sequence of the Fc region comprises all of the substitution mutations T307I, Q311 R, M428L, N434L and Y436K.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide does not comprise the substitution mutation H435R.

5. The polypeptide of any one of claims 1 to 4, wherein the Fc region of the polypeptide has reduced affinity for the second immunoglobulin-binding protein of Staphylococcus aureus (Sbi), optionally wherein the Fc region of the polypeptide has reduced affinity for Streptococcal protein G (SpG).

6. The polypeptide of any one of claims 1 to 5, wherein the Fc region of the polypeptide has reduced affinity for SpA, Sbi and / or SpG relative to the Fc region of the polypeptide that does not comprise the substitution mutations, optionally wherein the Fc-mediated effector functions of the polypeptide are not inhibited in the presence of Staphylococcus aureus.

7. The polypeptide of any one of claims 1 to 6, wherein the Fc region of the polypeptide binds to the neonatal Fc receptor (FcRn), optionally wherein the polypeptide has an in vivo half-life that is equivalent to the in vivo half-life of the polypeptide comprising an Fc region that does not comprise the substitution mutations.

8. The polypeptide of any one of claims 1 to 7, wherein the Fc region of the polypeptide binds to the Fc-gamma receptor (FcyR), optionally wherein the polypeptide has improved complement component 1q (C1q) recruitment.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptide further comprises a binding moiety that specifically binds to Staphylococcus aureus, optionally wherein the Fc region of the polypeptide is an immunoglobulin G (IgG) Fc region.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is an antibody or a polypeptide comprising a single domain antibody (VHH) fused to the Fc region.

11. An antibody construct comprising the polypeptide of any one of claims 1 to 10, optionally wherein the antibody construct is a full-length immunoglobulin, a constructcomprising an Fc region dimer, a scFv-Fc, a Fab-Fc, a Fab’-Fc, a F(ab)’2-Fc, an Fv-Fc, a sdAb-Fc, or a VHH-Fc.

12. One or more nucleic acid sequences capable of expressing the polypeptide or the antibody according to any one of claims 1 to 11 , wherein the nucleic acid is optionally mRNA encoding the polypeptide or the antibody according to any one of claims 1 to 11.

13. A cell comprising the polypeptide, the antibody or the one or more nucleic acid sequences according to any one of claims 1 to 12.

14. A composition comprising the polypeptide, the antibody, the one or more nucleic acid sequences or the cell according to any one of claims 1 to 13, optionally wherein the composition is a pharmaceutical composition comprising at least one pharmaceutically acceptable carriers, diluents, excipients, or any combination thereof.

15. An in vitro method comprising contacting a cell with the polypeptide, the antibody, the one or more nucleic acid sequences, the cell or the composition of any one of claims 1 to 14.

16. The polypeptide, the antibody, the one or more nucleic acid sequences, the cell or the composition of any one of claims 1 to 15 for use in a method of therapy or a diagnostic method, optionally wherein the therapy is treating or preventing a Staphylococcus aureus infection.

Images