Antibacterial polypeptides

Abstract

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

Description

Technical Field

[0001] This disclosure relates to mutated polypeptides, said mutated polypeptides comprising antibodies against Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Protein A (SpA) has a crystallizable fragment (Fc) region with reduced affinity. This disclosure also relates to the treatment of Staphylococcus aureus infections. Background Technology

[0002] Staphylococcus aureus ( S. aureus Staphylococcus aureus (SAA) is a bacterium commonly found on human skin and mucous membranes, and it is a significant concern in the healthcare setting. While it can exist harmlessly as part of the normal microbiota, it can also cause a range of infections and health problems. SAA infections can take many forms, including skin and soft tissue infections (such as boils, abscesses, and cellulitis), respiratory infections, bloodstream infections, pneumonia, endocarditis (heart valve infection), and sepsis. SAA produces a variety of virulence factors that contribute to its ability to evade the immune system and damage host tissues. Some of these factors can also reduce the effectiveness of therapeutic antibodies (and other related immunotherapies).

[0003] Specifically, *Staphylococcus aureus* expresses staphylococcal protein A (SpA), an IgG-binding protein that binds to human IgG antibodies via its Fc domain. *Staphylococcus aureus* also expresses several other IgG-binding proteins (which also bind to human IgG antibodies via their Fc domains): staphylococcal conjugates of immunoglobulins (Sbi). *Streptococcus* spp. ( Streptococcus The equivalent protein in Staphylococcus aureus is Streptococcal protein G (SpG). Due to the abundant surface expression of SpA (and to a lesser extent Sbi), Staphylococcus aureus essentially coats itself with human IgG antibodies, thereby not only masking its own antigens but also effectively inhibiting the Fc-mediated effector function of the antibodies. This ability of Staphylococcus aureus generally affects proximal human IgG antibodies and other Fc-containing proteins. Therefore, primarily through SpA expression, Staphylococcus aureus reduces the effectiveness of therapeutic antibodies and other associated Fc-containing immunotherapies. Therefore, to effectively use therapeutic antibodies (or related immunotherapies) in Staphylococcus aureus infection, it is necessary to inhibit Fc-mediated capture of therapeutic antibodies, etc. Summary of the Invention

[0004] There remains a need in the art for Fc mutations and combinations thereof to provide Fc region peptides with reduced affinity for SpA for use against Staphylococcus aureus infections.

[0005] This disclosure provides the understanding that identifying Fc mutations and Fc region peptides with reduced affinity for SpA is not straightforward, as it requires balancing many different factors, such as productivity, stability, and reduced SpA affinity. For example, Chen et al., PNAS 119(4) e2114478119 (2022) reported some preliminary claims regarding the possibility of mutating the Fc region of an antibody to reduce SpA affinity, but researchers encountered difficulties in maintaining the properties of the Fc region with the introduced mutation. Chen et al. disclosed two mutated Fc regions for this purpose: one with the mutation S254. A Q311 E L432 S and N434 P "AESP", and H435 with mutation R The "R" in this context is missing. However, the corresponding patent publication US 2023 / 0041644 A1, published by the same group, indicates that AESP has unfavorable properties that hinder further development. Chen et al. also disclosed combinations of mutants T307R and A378V with mutant H435R. US 2020 / 0291099 A1 discloses several Fc mutations that allegedly reduce SpA affinity, but only discloses the consideration of using a single antibody "Mab2" with mutants K274Q, H435R, and Y436F to complete preliminary testing. Therefore, despite exploration in this field, there is still a need for Fc mutations and Fc region peptides with reduced affinity for SpA.

[0006] This disclosure further recognizes that other features of the Fc region used in therapeutic agents against Staphylococcus aureus would be beneficial. For example, there is a need in the art for Fc mutants to possess FcRn binding characteristics favorable to antibody half-life (e.g., binding at pH 6 and weak binding at pH 7). Furthermore, it would be advantageous for Fc mutants to maintain Fc effector function through FcγR interactions.

[0007] This disclosure relates to mutations in the Fc region of peptides, for example, for therapeutic or diagnostic use in Staphylococcus aureus infection. The provided mutations address certain needs in the art for Fc mutations and Fc region peptides with reduced affinity for SpA, and / or achieve certain objectives beneficial to the use (e.g., therapeutic use) of such Fc mutations and Fc region peptides. In particular, this disclosure provides peptides containing substitution mutations and combinations thereof in the Fc region, including T307I, Q311R, M428L, N434L, Y436K, or any combination thereof. In some embodiments, the peptides provided herein have reduced affinity for Staphylococcus aureus protein A (SpA). In some embodiments, the peptides provided herein have other advantageous properties, such as maintained stability and expressibility. In some embodiments, the peptides provided herein have advantageous properties regarding Fc function, such as maintained immune response, preserved C1q binding, and even improved recruitment of complement component 1q (C1q). Furthermore, the disclosed peptides may possess favorable properties regarding FcRn binding and antibody half-life. Additionally, the disclosed peptides may be used for the treatment and / or diagnosis of Staphylococcus aureus infections.

[0008] Therefore, in one aspect, this disclosure provides a polypeptide comprising a crystallizable fragment (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, Q311R, M428L, N434L, and Y436K.

[0009] In one aspect, this disclosure provides one or more nucleic acid sequences capable of expressing the polypeptides provided herein.

[0010] In one aspect, this disclosure provides a cell comprising a polypeptide or one or more nucleic acid sequences provided herein.

[0011] In one aspect, this disclosure provides a composition comprising a polypeptide, one or more nucleic acid sequences, or cells provided herein.

[0012] In one aspect, this disclosure provides an in vitro method comprising contacting cells with a polypeptide, one or more nucleic acid sequences, cells, or a composition provided herein.

[0013] In one aspect, this disclosure provides a polypeptide, one or more nucleic acid sequences, cells, or compositions provided herein for use in therapeutic or diagnostic methods.

[0014] In one aspect, this disclosure provides a polypeptide, one or more nucleic acid sequences, cells, or compositions provided herein for use in the manufacture of a medicament or diagnostic agent for therapeutic purposes.

[0015] In one aspect, this disclosure provides a treatment or diagnostic method comprising administering to a subject a polypeptide, one or more nucleic acid sequences, cells, or a composition provided herein.

[0016] In one aspect, this disclosure provides a method for preparing the polypeptide provided herein, the method comprising expressing one or more nucleic acids capable of expressing the polypeptide. Attached Figure Description

[0017] Figure 1 Selected Fc mutants were tested by ELISA for FcRn binding at pH 7. Weaker FcRn binding at pH 7 is advantageous for efficient antibody recycling within cells. Fc185 showed strong binding to FcRn at pH 7. For all other tested mutants, binding to FcRn at pH 7 was acceptable.

[0018] Figure 2: FcγR binding of selected Fc mutants to various receptors (including FcγRI (Figure 2A), FcγRIIa (Figure 2B), FcγRIIb / c (Figure 2C), and FcγRIIIa (Figure 2D)) by ELISA. The binding of the candidate mutant Fc136 to the Fcγ receptor was comparable to that of the wild-type control.

[0019] Figure 3: Functional assays of the selected Fc mutant, including measurements of ADCC (Figure 3A), ADCP (Figure 3B), cell surface binding (Figure 3C), and C1q recruitment (Figure 3D). Fc136 behaved comparably to the wild-type proteins in Figures 3A-3C and surprisingly showed a strong and favorable signal in the C1q recruitment assay in Figure 3D.

[0020] Figure 4: Binding assays for selected Fc mutants, including Sbi (Figure 4A), SpA (Figure 4B), and SpG (Figure 4C) binding assays for several Fc mutants and the wild-type control (Fc001 / WT). Sbi, SpA, and SpG showed virtually no binding to Fc136. Conversely, SpG showed binding to the Fc185 mutant.

[0021] Figure 5: Binding assays of highly purified mutants Fc136, Fc183, and Fc185, as well as the wild-type control (Fc001), with the target antigens (Figure 5A), FcγRIIa (Figure 5B), FcγRI (Figure 5C), and FcγRIIIa (Figure 5D).

[0022] Figure 6: Binding assays of highly purified mutants Fc136, Fc183, and Fc185, and the wild-type control (Fc001 / WT), with SpA (Figure 6A), SpG (Figure 6B), and Sbi (Figure 6C). SpA, SpG, and Sbi showed virtually no binding to Fc136.

[0023] Figure 7 Serum concentrations of the VHH-Fc- fusion in NSG hFcRn (32) Tg mice were selected over time. Serum concentrations of mutants Fc136, Fc183, Fc185, wild-type control (Fc001), and negative control (DPBS) over time were also shown. Fc136 consistently showed higher serum concentrations than all other samples, indicating an extended in vivo half-life. Detailed Implementation

[0024] Peptides and Mutations The polypeptides according to this disclosure can exist in monomeric form (e.g., a single polypeptide chain, such as an immunoglobulin chain) or in antibody construct form (e.g., a dimer form, such as an antibody containing two immunoglobulin heavy chains). Therefore, as used herein, the term "polypeptide" encompasses monomeric, multimeric, dimer, homodimer, and heterodimer forms of the polypeptide. In the context of the monomeric form of the polypeptides of this disclosure, the term "Fc region" can refer to a portion of a polypeptide sequence containing constant structural domains (e.g., CH2 and CH3 domains). In the context of antibody constructs of this disclosure, the term "Fc region" can refer to a portion of a construct containing constant structural domains (e.g., CH2 and CH3 domains), such as a region of an antibody dimer construct containing CH2 and CH3 domains.

[0025] In one aspect, this disclosure provides a polypeptide comprising a crystallizable fragment (Fc) region containing one or more of the following substitution mutations: T307I, Q311R, M428L, N434L, and Y436K. In some embodiments, the Fc region containing one or more of the following substitution mutations has a reduced affinity for Staphylococcus aureus protein A (SpA): T307I, Q311R, M428L, N434L, and Y436K.

[0026] In some embodiments, the polypeptide sequence in the Fc region contains a substitution mutation. In some embodiments, the substitution mutation is T307I, Q311R, M428L, N434L, or Y436K.

[0027] In some embodiments, the polypeptide sequence in the Fc region contains two substitution mutations. Specifically, in some embodiments, the polypeptide sequence in the Fc region contains substitution mutations T307I and Q311R; T307I and M428L; T307I and N434L; T307I and Y436K; Q311R and M428L; Q311R and N434L; Q311R and Y436K; M428L and N434L; M428L and Y436K; or N434L and Y436K.

[0028] In some embodiments, the polypeptide sequence in the Fc region contains three substitution mutations. Specifically, in some embodiments, the polypeptide sequence in the Fc region contains substitution mutations T307I, Q311R, and M428L; T307I, Q311R, 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 Q311R, N434L, and Y436K.

[0029] In some embodiments, the polypeptide sequence in the Fc region contains four substitution mutations. Specifically, in some embodiments, the polypeptide sequence in the Fc region contains substitution mutations T307I, Q311R, M428L, and N434L; T307I, Q311R, M428L, and Y436K; T307I, Q311R, N434L, and Y436K; or T307I, M428L, N434L, and Y436K; or Q311R, M428L, N434L, and Y436K.

[0030] In some embodiments, the polypeptide sequence in the Fc region contains the substitution mutation M428L, and optionally contains one or more of T307I, Q311R, N434L, and Y436K. In some embodiments, the substitution mutation M428L is of particular interest because it is identified in only a single variant during screening. Therefore, in the generally preferred embodiments herein, the polypeptide sequence in the Fc region contains (at least) the substitution mutation M428L.

[0031] In some embodiments, the polypeptide sequence in the Fc region contains five substitution mutations. In the most preferred embodiment, the polypeptide sequence in the Fc region contains all the substitution mutations T307I, Q311R, M428L, N434L, and Y436K.

[0032] In some embodiments, the polypeptide sequence in the Fc region comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 3. In some embodiments, the % identity does not apply to substitution mutations specified in the Fc region of the polypeptide. In some embodiments, the polypeptide sequence in the Fc region comprises SEQ ID NO: 3.

[0033] In some embodiments, the polypeptide sequence in the Fc region does not contain the substitution mutation H435R. In some embodiments, the polypeptide sequence in the Fc region does not contain the substitution mutations S254A, Q311E, L432S, or N434P. In some embodiments, the polypeptide sequence in the Fc region does not contain the substitution mutations T307R or A378V. In some embodiments, the polypeptide sequence in the Fc region does not contain the substitution mutations T307Q, Q311V, or A378V. In some embodiments, the polypeptide sequence in the Fc region does not contain the substitution mutations T256D, N286D, T307R, Q311V, or A378V.

[0034] In some embodiments, the polypeptide sequence of the Fc region includes H435. In some embodiments, the polypeptide sequence of the Fc region includes S254, Q311, L432, or N434. In some embodiments, the polypeptide sequence of the Fc region includes T307 or A378. In some embodiments, the polypeptide sequence of the Fc region includes T307, Q311, or A378. In some embodiments, the polypeptide sequence of the Fc region includes T256, N286, T307, Q311, or A378.

[0035] It should be understood that the antibody and Fc amino acid numbers in this document correspond to the EU numbering scheme. Therefore, in some embodiments, the amino acid position number corresponds to the EU number. The EU numbering scheme, including how it compares to other antibody residue numbering schemes, is well known in the art and can be viewed, for example, at https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0036] In some embodiments herein, mutations are defined solely by the location of the mutation in the Fc region (according to the EU numbering scheme) and the presence of the residues. For example, in some embodiments herein, a polypeptide with the substitution mutation M428L is simply defined as a polypeptide having the residue "L" at position 428.

[0037] In the embodiments described herein, the mutations T307I, Q311R, M428L, N434L, and Y436K may alternatively be defined as polypeptide sequences in the Fc region containing “I” at EU 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.

[0038] In the embodiments described herein, the mutations S254A, T256D, N286D, T307R, T307Q, Q311E, Q311V, A378V, L432S, N434P, and H435R may alternatively be defined as polypeptide sequences in the Fc region that do not contain “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.

[0039] peptide function In some embodiments, when the polypeptide of this disclosure is included in an antibody construct, it provides one or more functions of the Fc region of the polypeptide. In some embodiments, the polypeptide is included in an antibody construct. In some embodiments, the polypeptide is included in a dimer of immunoglobulin heavy chains, the dimer optionally also comprising one or more immunoglobulin light chains.

[0040] In some embodiments, the Fc region of the peptide has a reduced affinity for SpA. In some embodiments, the Fc region of the peptide has a reduced affinity for Staphylococcus aureus secondary immunoglobulin-binding protein (Sbi). In some embodiments, the Fc region of the peptide has a reduced affinity for streptococcal protein G (SpG).

[0041] In this document, in some embodiments, “reduced affinity” of the mutated Fc region for SpA, Sbi, and / or SpG means a reduced affinity relative to an equivalent wild-type Fc region (i.e., the same Fc region polypeptide sequence not containing any of the mutations specified in the claims). It should be understood that SpA, Sbi, and SpG generally have affinity for and are capable of capturing Fc regions of polypeptides (e.g., antibodies) containing such regions. Therefore, the Fc region with a substitution mutation having reduced affinity for SpA, Sbi, and / or SpG provided herein means that the Fc region has reduced affinity relative to the same Fc region without the substitution mutation. In this context, for example, the Fc region of “without substitution mutation” M428L does not have the residue “L” at EU position 428, but instead has the residue “M” at EU position 428.

[0042] In some embodiments, the Fc region of the peptides provided herein has reduced affinity for SpA, Sbi, and / or SpG compared to the Fc region of peptides that do not contain substitution mutations.

[0043] In some embodiments, "reduced" means that SpA, Sbi, and / or SpG substantially do not bind to the Fc region of the disclosed peptide. In some embodiments, "reduced" means that SpA, Sbi, and / or SpG do not bind to the Fc region of the disclosed peptide. In some embodiments, "reduced affinity" for the Fc region of the disclosed peptide means that 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, reduced affinity for SpA (and / or Sbi) means maintaining Fc effector function in the presence of Staphylococcus aureus or SpA.

[0044] It should be understood that the affinity for SpA, Sbi, and / or SpG in this article refers to the ability of these proteins to bind to and capture antibodies and other peptides containing immunoglobulin domains by binding to their Fc regions (and possibly VH3 Fab regions). This affinity... No This includes the binding of peptides of this disclosure to specific antibodies against SpA (when present). Therefore, this affinity does not include the specific binding of peptides of this disclosure to SpA, Sbi, and / or SpG via antibody-like binding (e.g., via CDRs of one or more variable immunoglobulin domains).

[0045] It should also be understood that reduced affinity for SpA, Sbi, and / or SpG can be achieved via computer ( in silico Modeling / screening can be used to determine this, and (if necessary) further validation can be achieved through cell assays.

[0046] In some implementations, the Fc-mediated effector function of the peptide is not inhibited in the presence of Staphylococcus aureus.

[0047] In some embodiments, the Fc region of the peptide provided herein binds to the neonatal Fc receptor (FcRn). In some embodiments, the Fc region of the peptide binds to FcRn with a greater affinity at pH 6 than at pH 7. In some embodiments, the binding of the Fc region of the peptide to FcRn is equivalent, comparable, or similar to the binding of the Fc region of a peptide without the substitution mutation provided herein to FcRn. In some embodiments, the binding affinity of the Fc region of the peptide to FcRn is equivalent, comparable, or similar to the binding affinity of the Fc region of a peptide without the substitution mutation provided herein to FcRn. In some embodiments, the binding affinity of the Fc region of the peptide to FcRn at pH 6 and pH 7 is equivalent, comparable, or similar to the binding affinity of the Fc region of a peptide without the substitution mutation provided herein to FcRn. It should be understood that binding to the FcRn receptor can be readily determined and measured, for example, by ELISA.

[0048] In some embodiments, the in vivo half-life of the peptide is at least equivalent to the in vivo half-life of a peptide containing an Fc region without the substitution mutations provided herein. In some embodiments, the in vivo half-life of the peptide is similar to or greater than the in vivo half-life of a peptide containing an Fc region without the substitution mutations provided herein. In some embodiments, the in vivo half-life of the peptide is equivalent to or greater than the in vivo half-life of a peptide containing an Fc region without the substitution mutations provided herein. In some embodiments, the in vivo half-life is measured in mice. In some embodiments, the in vivo half-life is measured in humans.

[0049] In some embodiments, the Fc region of the peptide binds to the Fcγ receptor (FcγR). In some embodiments, the Fc region of the peptide binds to FcγRI. In some embodiments, the Fc region of the peptide binds to FcγRIIa. In some embodiments, the Fc region of the peptide binds to FcγRIIb / c. In some embodiments, the Fc region of the peptide binds to FcγRIIIa. In some embodiments, the binding of the Fc region of the peptide to FcγR is equivalent to the binding of the Fc region without the substitution mutation of the present invention to FcγR. In some embodiments, the binding of the Fc region of the peptide to FcγRI, FcγRIIa, FcγRIIb / c and / or FcγRIIIa is equivalent to the binding of the Fc region without the substitution mutation to FcγRI, FcγRIIa, FcγRIIb / c and / or FcγRIIIa. It should be understood that binding to the FcγR receptor can be readily determined and measured, for example, by ELISA.

[0050] In some embodiments, the peptide induces antibody-dependent cytotoxicity (ADCC). In some embodiments, the peptide induces antibody-dependent phagocytosis (ADCP). In some embodiments, the peptide induces complement-dependent cytotoxicity (CDC). In some embodiments, the peptide induces ADCC, ADCP, and CDC. In some embodiments, the peptide induces ADCC and / or ADCP equivalent to peptides that do not contain the substitution mutations provided herein.

[0051] In some embodiments, the peptide induces antibody-dependent cytotoxicity (ADCC) against Staphylococcus aureus. In some embodiments, the peptide induces antibody-dependent phagocytosis (ADCP) against Staphylococcus aureus. In some embodiments, the peptide induces complement-dependent cytotoxicity (CDC) against Staphylococcus aureus. In some embodiments, the peptide induces ADCC, ADCP, and CDC against Staphylococcus aureus. In some embodiments, the peptide induces ADCC and / or ADCP against Staphylococcus aureus equivalent to peptides that do not contain the substitution mutations provided herein.

[0052] In some embodiments, the peptide induces the recruitment of complement component 1q (C1q). In some embodiments, the peptide exhibits improved C1q recruitment. In some embodiments, the peptide exhibits improved C1q recruitment compared to peptides that do not contain the substitution mutations provided herein. C1q recruitment to target cells bound by the like IgG antibody / VHH-Fc fusion protein initiates the classical complement pathway, thereby promoting CDC. Therefore, in some embodiments, the peptide induces CDC. In some embodiments, the peptide exhibits enhanced CDC induction. In some embodiments, the peptide induces a larger CDC than peptides that do not contain the substitution mutations provided herein.

[0053] In some embodiments, the peptide induces recruitment of complement component 1q (C1q) against Staphylococcus aureus. In some embodiments, the peptide has improved C1q recruitment against Staphylococcus aureus. In some embodiments, the peptide has improved C1q recruitment against Staphylococcus aureus compared to peptides that do not contain the substitution mutations provided herein. In some embodiments, the peptide induces CDC against Staphylococcus aureus. In some embodiments, the peptide has enhanced CDC induction against Staphylococcus aureus. In some embodiments, the peptide induces a larger CDC against Staphylococcus aureus than peptides that do not contain the substitution mutations provided herein.

[0054] In some embodiments, the peptides provided herein contain one or more additional mutations that enhance effector function. These additional mutations may also be those previously developed in the art.

[0055] In some embodiments, the peptides provided herein contain the S239D / A330L / I332E “3M” mutation. In some embodiments, the mutation increases ADCC. Further information about the mutation can be found in Lazar et al. (2006) Proc NatlAcad Sci US A. 2006 Mar 14;103(11):4005-10 (incorporated herein by reference).

[0056] In some embodiments, the peptides provided herein contain the F243L mutation. In some embodiments, the mutation increases ADCC and ADCP. Further information about the mutation can be found in Stavenhagen et al. (2007) Cancer Res 2007 Sep 15;67(18):8882-9 (incorporated hereby by reference).

[0057] In some embodiments, the peptides provided herein contain the S267E / H268F / S324T mutation. In some embodiments, the mutation enhances CDC activity. Further information about the mutation can be found in Moore et al. (2010) MAbs. Mar-April 2010;2(2):181-9 (incorporated herein by reference).

[0058] In some embodiments, the peptides provided herein contain the E345R / E430G / S440Y mutation. In some embodiments, the mutation enhances C1q binding and CDC activity. Further information about the mutation can be found in Diebolder et al. (2014) Science, 14;343(6176):1260-3 (incorporated herein by reference).

[0059] Improved half-life In preferred embodiments, it has been found that the peptides provided herein have an in vivo half-life that is unexpectedly longer (e.g., substantially longer) than that of peptides containing an Fc region without the substitutional mutations provided herein. This also presents a substantial improvement of the peptides provided herein compared to prior art peptides. In some embodiments of this type, the improved in vivo half-life is provided by an antibody construct comprising one or more peptides of this disclosure, wherein the construct comprises an Fc region dimer. In some embodiments of this type, the improved in vivo half-life is provided by an immunoglobulin heavy chain dimer comprising one or more peptides of this disclosure. In some embodiments of this type, the improved in vivo half-life is provided by an antibody comprising one or more peptides of this disclosure.

[0060] In some embodiments, the peptides provided herein include one or more additional mutations that enhance half-life. These additional mutations may also be those previously developed in the art.

[0061] In some embodiments, the peptides provided herein contain the M252Y / S254T / T256E (YTE) mutation. In some embodiments, the mutation increases FcRn binding affinity. Further information about the mutation can be found in Dall'Acqua et al. (2002) J Immunol 1 Nov 2002;169(9):5171-80 (incorporated herein by reference).

[0062] In some embodiments, the peptides provided herein contain the M428L / N434S (LS) mutation. In some embodiments, the mutation enhances FcRn binding. Further information about the mutation can be found in Zalevsky et al. (2010) Nat Biotechnol. Feb. 2010;28(2):157-9 (incorporated hereby by reference).

[0063] In some embodiments, the peptides provided herein contain the M252Y / S254T / T256E / M428L / N434S(YTE-LS) mutation. Further information about the mutation can be found in Ko et al. (2022) Ex. Mol Med 54(11):1850-1861 (incorporated hereby by reference).

[0064] In some embodiments, the peptides provided herein contain the M252Y / S254T / T256E / H433K / N434F (YTE-HN) mutation. In some embodiments, the mutation increases FcRn binding affinity. Further information about the mutations can be found in Monnet et al. 2019 Nat Commun. 2019 Nov 6;10:5031 (incorporated herein by reference). More specifically, as demonstrated in the examples herein, the Fc183 control mutant contains the H435R mutation but does not have a significantly longer in vivo half-life compared to the equivalent wild-type peptide (Fc001). Furthermore, the Fc185 control mutant contains the H435R, T307R, and A378V mutations but still has a shorter in vivo half-life than Fc136. In contrast, the Fc136 mutant (the polypeptide according to this disclosure) contains the mutations T307I, Q311R, M428L, N434L, and Y436K, and has been determined to have an in vivo half-life substantially greater than that of the equivalent wild-type polypeptides (Fc001), Fc183, and Fc185.

[0065] Therefore, not wanting to be bound by theory, the mutations present in the disclosed peptides (e.g., Fc136) provide another advantage of improved in vivo half-life relative to the wild-type equivalent peptide of Fc001. Furthermore, this improvement in in vivo half-life is unexpectedly significantly greater than that of Fc183 (which shows no difference relative to Fc001) and Fc185.

[0066] Therefore, in some embodiments, the in vivo half-life of the peptides provided herein is greater than (e.g., substantially greater than) the in vivo half-life of a 'control' or 'reference' peptide. Therefore, in some embodiments, the in vivo half-life of the peptides provided herein is greater than (e.g., substantially greater than) the in vivo half-life of peptides containing an Fc region that does not contain the substitution mutations provided herein. In some embodiments, the in vivo half-life of the peptide is greater than (e.g., substantially greater than) the in vivo half-life of peptides containing an Fc region containing an H435R mutation. In some embodiments, the in vivo half-life of the peptide is greater than (e.g., substantially greater than) the in vivo half-life of peptides containing an Fc region containing H435R, T307R, and A378V mutations. In embodiments, it should be understood that a suitable 'control' or 'reference' peptide does not contain any of the mutations provided herein. In embodiments, a suitable 'control' or 'reference' peptide does not contain any mutations that increase the half-life of the peptide relative to a wild-type Fc region (e.g., a wild-type Fc region equivalent to the peptide). In the implementation scheme, the appropriate 'control' or 'reference' peptide does not contain any mutations relative to the wild-type Fc region (e.g., the wild-type Fc region equivalent to the peptide).

[0067] In some embodiments, the in vivo half-life of the polypeptide of this disclosure is greater than the in vivo half-life of the polypeptide comprising an Fc region having the sequence shown in SEQ ID NO: 4. In some embodiments, the in vivo half-life of the polypeptide of this disclosure is greater than the in vivo half-life of Fc183. In some embodiments, the in vivo half-life of the polypeptide of this disclosure is greater than the in vivo half-life of the polypeptide comprising an Fc region having the sequence shown in SEQ ID NO: 5. In some embodiments, the in vivo half-life of the polypeptide of this disclosure is greater than the in vivo half-life of Fc185.

[0068] In some embodiments described herein, "greater than" in vivo half-life means "substantially greater than". In some embodiments described herein, it should be understood that "greater than" in vivo half-life means that the peptide can have a greater or longer effect in vivo than a related 'control' or 'reference' peptide.

[0069] In some embodiments herein, the in vivo half-life of a peptide “greater than” means a half-life that is 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% longer than that of a peptide containing an Fc region without a substitution mutation.

[0070] In some embodiments herein, the in vivo half-life of a peptide being “greater than” means a half-life that is 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% longer than that of a peptide containing an Fc region containing an H435R mutation but not such substitutional mutations (e.g., Fc183).

[0071] In some embodiments herein, an in vivo half-life “greater than” means that a polypeptide containing an Fc region with H435R, T307R, and A378V mutations but not the substitutional mutations provided herein (such as Fc185) is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12% longer than a polypeptide containing an Fc region with H435R, T307R, and A378V mutations but not the substitutional mutations provided herein (e.g., Fc185).

[0072] In some embodiments, the polypeptides of this disclosure have 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.

[0073] In some embodiments, the polypeptide of this disclosure has an in vivo half-life that is at least equivalent to, substantially the same as, or identical to that of a polypeptide comprising an Fc region containing mutations of T307I, Q311R, M428L, N434L, and Y436K. In some embodiments, the polypeptide of this disclosure has an in vivo half-life that is at least equivalent to, substantially the same as, or identical to that of a polypeptide comprising an Fc region containing "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 this disclosure has an in vivo half-life that is at least equivalent to, substantially the same as, or identical to that of a polypeptide comprising an Fc region having the sequence shown in SEQ ID NO: 3.

[0074] It should be understood herein that half-life can be considered a measure of in vivo lifespan. Therefore, in some embodiments, reference to "in vivo half-life" is considered a reference to "in vivo lifespan." 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 it takes longer for the amount of the peptide in vivo to decrease to half of a predetermined initial amount. In some embodiments, the in vivo half-life is in humans. In some embodiments, the in vivo half-life is in mice. In some embodiments, the in vivo half-life is a serum half-life. In some embodiments, the in vivo half-life is a serum half-life in humans. In some embodiments, the in vivo half-life is a serum half-life in mice. In some embodiments, the in vivo half-life is a serum half-life in a mouse model expressing human FcRn instead of mouse FcRn.

[0075] In some embodiments, where a comparison is made between the in vivo half-life of the polypeptide of this disclosure and another polypeptide, it should be understood—unless otherwise stated, such as in addition to any of the mutational differences described—that the 'reference' polypeptide is the same as, substantially the same as, or equivalent to the polypeptide of this disclosure.

[0076] Therefore, in some embodiments, the polypeptide of this disclosure has a longer in vivo half-life than equivalent polypeptides that do not contain the T307I, Q311R, M428L, N434L, and / or Y436K mutations. In some such embodiments, the equivalent polypeptide contains the H435R mutation and optionally also contains the T307R and A378V mutations. In some embodiments, the polypeptide of this disclosure has a longer in vivo half-life than equivalent polypeptides that contain an Fc region that does not contain "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 contains "R" at position 435 and optionally also contains "R" at position 307 and "V" at position 378.

[0077] polypeptide form In some embodiments, the peptide or Fc region provided herein can be combined with various other domains, such as immunoglobulin variable domains. Additionally, the peptide provided herein can be combined with a second peptide, for example, in the form of an antibody construct. It should be understood that the peptide or Fc region provided herein can be incorporated into any conceivable antibody, antigen-binding fragment, or other construct containing an immunoglobulin domain.

[0078] In one embodiment, the polypeptide provided herein also includes a binding portion or fragment or portion thereof that specifically binds to Staphylococcus aureus (e.g., one or more CDRs).

[0079] In some embodiments, the Fc region of the peptide provided herein is an immunoglobulin G (IgG) Fc region. In some embodiments, the Fc region of the peptide provided herein is an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region of the peptide provided herein is a VHH-Fc antibody Fc region.

[0080] In some embodiments, the polypeptide is an antibody, its antigen-binding fragment, 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 Staphylococcus aureus antigens.

[0081] In some embodiments, the polypeptides provided herein comprise or are antibody constructs, antibodies, antigen-binding fragments containing Fc regions, or heavy chains of VHH-Fc antibodies.

[0082] In some embodiments, if the polypeptide of this disclosure contains one or more VH3 family antibody fragment (Fab) sequences, then SpA does not bind to the VH3 Fab sequence. 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 an antibody or construct containing the polypeptide of this disclosure. In some embodiments, SpA does not bind to the Fab of the polypeptide or an antibody / construct containing the polypeptide. In some embodiments, SpA does not bind to the VHH domain of the polypeptide or an antibody / construct containing the polypeptide. In these embodiments, this has no effect on antibody-like specific binding to the variable region, which can specifically bind to Staphylococcus aureus. In some embodiments, SpA does not bind to the variable domain, VHH, or VH3 sequence, etc., by non-specific binding. In some embodiments, SpA does not bind to the variable domain, VHH, or VH3 sequence, etc., by non-antigen-specific binding. In some embodiments, the polypeptide or antibody construct (e.g., by antigen-specific binding) specifically binds to SpA and optionally does not have any other affinity / binding to SpA other than specific binding to SpA.

[0083] In some embodiments, it should be generally understood that the Fc region of the polypeptide of this disclosure is a dimer Fc region. In some embodiments, it should be generally understood that the Fc region of the polypeptide contains mutations of this disclosure on both Fc region polypeptide sequences in the dimer Fc region, such as T307I, Q311R, M428L, N434L, Y436K or any combination thereof.

[0084] Other forms In one aspect, this disclosure provides one or more nucleic acid sequences capable of expressing the polypeptides according to the invention.

[0085] In some embodiments, the nucleic acid is RNA encoding the polypeptide provided herein. In some embodiments, the nucleic acid is mRNA encoding the polypeptide provided herein. In some embodiments, the nucleic acid is a vector encoding the polypeptide provided herein.

[0086] 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.

[0087] In some embodiments, one or more nucleic acid sequences encode antibodies. In some embodiments, one or more nucleic acid sequences encode polypeptides comprising a VHH domain and an Fc region.

[0088] As used herein, the term "antibody construct" can refer to any multimeric immunoglobulin construct comprising the polypeptide of this disclosure, such as a full-length antibody comprising an Fc region, an antibody-associated binding molecule, and an antigen-binding fragment thereof. As used herein, the term "antibody" can refer to a protein having an antigen-binding domain comprising at least one complementarity-determining region (CDR). Generally, it should be understood that an antibody comprises a dimer, such as a homodimer of two immunoglobulin heavy chains and optionally two immunoglobulin light chains. In some embodiments, the polypeptide of this disclosure may be referred to as an antibody or antibody-associated construct, which will be understood as a reference to an antibody or construct comprising the polypeptide of this disclosure. The term "antibody construct" includes VHH-Fc constructs or antibodies.

[0089] The term "complementarity-determining region" or "CDR" typically refers to one of the six hypervariable regions within the variable domain of an antibody, and can also refer to one of the three hypervariable regions within the VHH domain of an sdAb.

[0090] The antigen-binding domain, or antibody's "complementarity-determining region" or "CDR," refers to a hypervariable region or highly variable loop within the variable regions of the antibody's heavy and / or light chains, which primarily facilitates 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). Both the heavy and light chain variable regions each contain three CDRs (heavy chain CDRs 1, 2, and 3, and light chain CDRs 1, 2, and 3, numbered from the amino terminus to the carboxyl terminus).

[0091] The techniques used to prepare and use various antibody-based constructs and fragments are well known in the art.

[0092] As used herein, "antigen binding site" or "antigen binding domain" refers to a protein or polypeptide containing at least one complementarity-determining region (CDR). An antigen binding site may contain three CDRs; for example, it may be equivalent to an antigen binding site of a single-domain antibody (sdAb) domain such as the VHH domain.

[0093] As used in this article, the term "constant immunoglobulin domain" can refer to the constant domain of an immunoglobulin, such as the CH3 domain.

[0094] In a conventional full-length antibody (such as an IgG antibody) containing four polypeptides (two light chains and two heavy chains), the Fc region contains a dimer of the CH2-CH3 domain of each heavy chain polypeptide.

[0095] As used herein, the term "crystallizable fragment (Fc) region" can refer to one or more Fc regions of an immunoglobulin (e.g., an antibody or antibody construct) containing a CH2-CH3 domain or any fragment, truncated, derivative, or variant thereof, including those encoded by pseudogenes, provided that the one or more Fc regions also contain one or more mutations of this disclosure. Truncation may include as few as one immunoglobulin constant domain or a fragment thereof. Preferably, the Fc region is a full-length or substantially full-length Fc region containing one or more mutations of this disclosure. Fc regions include those in which known / standard / routine modifications have been made. Fc regions are not particularly species-specific, although humans are preferred, and therefore encompass Fc regions of all species, including all animals, humans, rodents, and other orthologs. Fc regions or Fc-like sequences encoded by paralogs and pseudogenes are also included. Suitably, in embodiments, the Fc region may be an Fc region encoded by IgG1, IgG2, IgG3, IgG4, or immunoglobulin pseudogenes of IgA, IgE, IgM, or IgD. Suitably, in embodiments, the Fc region may be an IgG1, IgG2, IgG3, or IgG4 Fc region. Preferably, the Fc region is an IgG1 Fc region. In some embodiments, the first polypeptide and the second polypeptide form a homodimer and each contains an Fc region of the same immunoglobulin isotype.

[0096] The terms VHH domain, VHH, and single-domain antibody (sdAb) are used interchangeably herein. sdAbs are naturally found in animals such as camels and sharks. sdAbs lack the light chain and the first constant domain (CH1) of the heavy chain of conventional IgG. Therefore, the antigen-binding fragment of an sdAb contains only a single variable domain, commonly referred to as the variable heavy domain of the heavy chain (VHH domain). In some embodiments, the first and second polypeptides of the Fc region polypeptide homodimer each contain an Fc region lacking the CH1 domain.

[0097] As used herein, the term “variable immunoglobulin domain” or “variable domain” can refer to the variable domain of an immunoglobulin, such as the VHH domain.

[0098] In some embodiments, the polypeptide disclosed herein may comprise a single-chain variable fragment (scFv); Fab; Fab'; F(ab)'2; Fv; a single-domain antibody (sdAb); VHH; a single-chain variable domain; a designed ankyrin repeat protein (DARPin); or an aptamer.

[0099] In some embodiments, the antibody constructs disclosed herein are antibodies, full-length immunoglobulins (full-length antibodies), scFv-Fc, Fab-Fc, Fab'-Fc, F(ab)'2-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0100] In some embodiments, the antibodies disclosed herein are human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, or polyclonal antibodies.

[0101] The term "chimeric antibody" generally refers to an antibody obtained by fusing the variable region of a non-human antibody with the constant region of a human antibody, which can reduce the immune response induced by the non-human antibody. Non-human antibodies can be, for example, mouse, camel, rabbit, sheep, goat, or chicken antibodies. For instance, to create a chimeric antibody, a hybridoma that secretes a specific monoclonal antibody can be created, and the variable region gene can be cloned from mouse hybridoma cells; then, the constant region gene of the human antibody can be cloned as needed, and the mouse variable region gene and the human constant region gene can be linked to form a chimeric gene; the chimeric gene is then inserted into an expression vector, where the chimeric antibody molecule can be expressed in a eukaryotic or prokaryotic system.

[0102] The term "humanized antibody," also known as a CDR-transplanted antibody, generally refers to an antibody generated by transplanting a mouse CDR sequence into a human antibody variable region framework, i.e., an antibody generated within a different type of human germline antibody framework sequence. This overcomes the heterogeneity induced by the presence of numerous mouse protein components in the chimeric antibody. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database.

[0103] The terms "fully humanized antibody," "fully human antibody," or "fully human antibody," which can also be called "fully humanized monoclonal antibody," can contain both humanized variable and constant regions to eliminate immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages: mouse 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. Related technologies for preparing fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0104] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the antibodies constituting this population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically comprise a large number of antibodies that target (or are specific to) different epitopes. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as the antibody being produced by any particular method.

[0105] Antibodies can be obtained using techniques that include immunizing animals with target antigens and isolating antibodies from serum. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0106] In some embodiments, the antibodies used according to this disclosure may be monovalent, bivalent, trivalent, tetravalent, or pentavalent, depending on the number of antigen-binding domains (e.g., VH, VL, or VHH domains) present. Similarly, in some embodiments, the antibodies used according to this disclosure may be monospecific or multispecific, such as bispecific, trispecific, tetraspecific, etc., depending on the number of different antigen-binding domains (e.g., VH, VL, or VHH domains) present.

[0107] Any suitable form of multispecific or multivalent antibody known in the art may be used in the practice of this disclosure. Suitably, bispecific antibodies may be IgG-scFv, IgG-sdAb, IgG-VHH, scFv-Fc-scFv, KiH-IgG, κλ antibody, KiH-Fc-Fab / scFv.

[0108] There are no particular restrictions on the variable domains used (e.g., VH, VL, or VHH domains), and they can contain any antigen-binding site that is specific to the selected target protein. Methods for determining the binding specificity of an antibody to a specific antigen include, but are not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (e.g., using BIAcore instruments), ELISA, Western blotting, in situ hybridization, immunohistochemistry, flow cytometry, Förster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screening, 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 BIAcore instruments), flow cytometry, fluorescence quenching, and isothermal titration calorimetry.

[0109] Methods can be implemented to provide variable domains (such as sdAbs containing VHH domains) for specific targets (see Caussinus et al., Nat Struct Mol Biol, 2011, 19(1), 117-121 and Fulcher et al., Open Biol, 2016, 6(10), pii 160255). Furthermore, methods can be implemented to isolate antigen-specific VHHs from immune or semi-synthetic libraries using phage, yeast, or ribosome display (see Muyldermans J Biotechnol. 2001 Jun; 74(4):277-302; and Dufner et al. Trends Biotechnol. 2006 Nov; 24(11):523-9).

[0110] For example, VHH can be obtained by immunizing, for example, a dromedary camel, camel, llama, or alpaca with the desired antigen and then isolating the mRNA encoding VHH. The single-domain shark variable domain of neoantigen receptor (VNAR) antibodies is also known and suitable for use as an alternative sdAb to the VHH domain according to this disclosure. Therefore, as a further example, VNAR can be obtained by immunizing a shark with the desired antigen and then isolating the mRNA encoding VNAR. A library of VHH or VNAR can then be generated using reverse transcription and PCR. Standard screening techniques such as phage display and ribosome display can be used to identify suitable clones that bind to the target antigen.

[0111] Once the most effective clones are identified, their DNA sequences can be optimized, for example, to improve their stability against enzymes. Humanization can also be performed.

[0112] VHH and VNAR can be expressed in cells using conventional vectors (such as those described in this article).

[0113] The ability of an antibody to specifically bind to its target can be determined by a technician. For example, binding can be determined by biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), Western blotting, flow cytometry, in situ hybridization, and / or microscopy. Suitablely, binding affinity can be determined by, for example, biolayer interferometry (BLI), surface plasmon resonance (SPR) analysis (using a BIAcore instrument), and / or flow cytometry.

[0114] In another aspect, this disclosure provides a nucleic acid particle comprising a construct or nucleic acid sequence as described herein. In one embodiment, the nucleic acid particle is a lipid nanoparticle (LNP).

[0115] In one aspect, this disclosure provides a cell comprising a polypeptide or 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.

[0116] Methods for engineering such cells include, but are not limited to, genetic modifications of the cells, such as transduction (e.g., retroviral or lentiviral transduction), transfection (e.g., transient transfection—DNA or RNA based) (including lipid transfection), polyethylene glycol, calcium phosphate, and electroporation. Nucleic acid sequences can be introduced into the cells using any suitable method.

[0117] The cells disclosed herein can be generated by introducing DNA or RNA encoding the polypeptides provided herein via one of a variety of methods, including transduction with a viral vector, or transfection with DNA or RNA.

[0118] The cells described herein can be prepared by introducing one or more nucleic acid sequences according to this disclosure into cells (e.g., by transduction or transfection).

[0119] The cells described herein may contain and / or secrete the polypeptides described herein.

[0120] In one aspect, this disclosure provides a composition comprising the polypeptide, one or more nucleic acid sequences, LNPs, or cells described herein. The composition may be a pharmaceutical composition.

[0121] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active peptides and / or compounds. Such formulations may, for example, be in a form suitable for intravenous infusion.

[0122] Methods and applications used In one aspect, this disclosure provides an in vitro method comprising contacting cells with a polypeptide, one or more nucleic acid sequences, LNPs, cells, or compositions described herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are CHO cells. In some embodiments, the cells are human cells.

[0123] In one aspect, this disclosure provides the polypeptides, one or more nucleic acid sequences, LNPs, cells, or compositions described herein for use in therapeutic or diagnostic methods.

[0124] In one aspect, this disclosure provides polypeptides, one or more nucleic acid sequences, LNPs, cells, or compositions described herein for use in the manufacture of medicaments or diagnostic agents for therapeutic purposes.

[0125] In one aspect, this disclosure provides a treatment or diagnostic method comprising administering to a subject a polypeptide, one or more nucleic acid sequences, LNPs, cells, or a composition described herein.

[0126] In some embodiments, the subject is an animal or a human. In some embodiments, the subject is a mammal. Preferably, the subject is a human. In some embodiments, the subject has or is susceptible to Staphylococcus aureus infection.

[0127] In some embodiments, the treatment includes treating or preventing Staphylococcus aureus infection. In some embodiments, the treatment includes treating or preventing streptococcal infection, particularly wherein the peptide has a reduced affinity for SpG. In some embodiments, Staphylococcus aureus is resistant to one or more treatments. In some embodiments, Staphylococcus aureus is methicillin-resistant. In some embodiments, the treatment is treating or preventing infection in humans. In some embodiments, the treatment or prevention includes one or more of the following: promoting Staphylococcus aureus decolonization in a subject, preventing invasive diseases caused by Staphylococcus aureus, and improving outcomes of Staphylococcus aureus bloodstream infection.

[0128] In some embodiments, the antibody is administered intravenously. In other embodiments, one or more nucleic acids encoding the antibody are administered. In some embodiments, one or more nucleic acids can be introduced via transduction. In some embodiments, one or more nucleic acids can be introduced via transfection.

[0129] Manufacturing method In one aspect, this disclosure provides a method for preparing the polypeptide provided herein, the method 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 capable of expressing the polypeptide.

[0130] Other general definitions This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include numerical values ​​that define the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino (N) to carboxyl (C) direction.

[0131] Where a numerical range is provided, it should be understood that various intermediate values ​​between the upper and lower limits of that range are also explicitly disclosed, wherein the lower limit is accurate to the tenths place unless otherwise expressly specified by the context. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within that stated range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which any, or no, one or both limits are included is also covered in this disclosure, subject to any specific exclusions from the stated range. When a stated range includes one or two limits, ranges excluding any or both of those included limits are also included in this disclosure.

[0132] The term "polypeptide" is used in its conventional sense to refer to a series of amino acids (usually L-amino acids) typically linked together by peptide bonds between the α-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 "residue" is used to refer to an amino acid within an amino acid sequence.

[0133] The term “variant” in relation to peptides refers to a peptide that has the same function as the amino acid sequence described herein, but includes one or more amino acid substitutions, insertions, or deletions.

[0134] The term “antibody” refers to any isotype of intact immunoglobulin that can compete with intact antibodies for specific binding to a target antigen, and includes chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies, as well as fragments of all of these. As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to any of several classes of structure-associated proteins that function as part of an animal’s immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides containing antibody CDR domains that retain antigen-binding activity. The terms “Fc region,” “Fc domain,” and “crystallizable fragment” are used interchangeably herein. The Fc region is a well-known part of an antibody, but the same Fc region can be present in other antibody-like or antibody-derived polypeptides such as the VHH-Fc fusion polypeptide. In this document, antibody residue numbering follows the EU residue numbering scheme.

[0135] An amino acid residue in an antibody is "corresponding" to a given residue when the amino acid residue occupies the same basic structural position within the antibody. For example, when selected residues in a comparative antibody occupy the same basic spatial or structural relationship as EU position 428 in the antibody provided herein, the selected residues correspond to position 428 (according to the EU numbering system as described herein), as assessed using methods applicable in the art. For example, a maximum sequence homology alignment can be performed between the comparative antibody and the antibody provided herein, and the position in the compared comparative antibody that aligns to EU position 428 can be determined to correspond to it. Alternatively, instead of (or in addition to) primary sequence alignment as described above, three-dimensional structural alignment can also be used, for example, where the structure of the comparative antibody is aligned to maximize correspondence with the antibody provided herein and the overall structure being compared. In this case, the amino acid occupying the same basic position as EU position 428 in the structural model can be referred to as the correspondent.

[0136] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder, such as an antibody. An antigen may have one or more epitopes that can interact with different antibodies.

[0137] The term "epitope" includes any region or portion of a molecule capable of evoking an immune response by binding to immunoglobulins or T-cell receptors. Epitope determinants can include chemically active surface groups, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge features. Typically, antibodies specific to a particular target antigen will preferentially recognize epitopes on the target antigen within a complex mixture.

[0138] As used in this article, the terms “polynucleotide,” “nucleotide,” “nucleic acid sequence,” and “nucleic acid” are intended to be synonymous with each other.

[0139] The terms “variant,” “homologous,” or “derivative” related to nucleotide sequences include any substitution, variation, modification, replacement, deletion, or addition to one (or more) nucleic acids derived from the sequence or to the sequence.

[0140] The “sequence identity” between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences. The terms “%identity” and “%compatibility” or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the optimal alignment between the sequences being compared. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed across the entire length of the sequences being compared. The comparison of two sequences is typically performed after optimal alignment, relative to segments or a “comparison window”, to identify local regions of the corresponding sequences. The best alignment for comparison can be performed manually, or by means of the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm described by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search algorithm described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA88, 2444; or by means of computer programs using the algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some implementations, the BLASTN or BLASTP algorithm is used to determine the percentage of identity between two sequences. This algorithm is available on the website of the National Center for Biotechnology Information (NCBI) (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some implementations, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 28; (iii) a maximum match in the query range set to 0; (iv) match / non-match scores set to 1, -2; (v) a gap cost set to linear; and (vi) the use of filters for low-complexity regions. In some implementations, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) the expected threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum match in the query range is set to 0; (iv) the matrix is ​​set to BLOSUM62; (v) the space cost is set to exist: 11, extend: 1; and (vi) the conditional score matrix is ​​adjusted.

[0141] The identity percentage is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0142] In some embodiments, the degree of similarity or identity is given for a region comprising 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 (contiguous nucleotides in some embodiments). In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0143] In some implementations, "isolated" means removed (e.g., purified) from its natural state or from an artificial composition (such as a composition derived from a manufacturing process). For example, nucleic acids, peptides, or polypeptides naturally present in living animals are not "isolated," but the same nucleic acids, peptides, or polypeptides that are partially or completely separated from their natural counterparts are "isolated." Isolated nucleic acids, peptides, or polypeptides may exist in a substantially purified form or may exist in a non-natural environment (e.g., host cells).

[0144] As used in this article, the term "expression" is defined, depending on the context, as the transcription and / or translation of a specific nucleotide sequence. Typically, translation is required.

[0145] In the context of this disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). The RNA can then be translated into peptides or polypeptides.

[0146] For RNA, the terms “expression” or “translation” refer to the process by which the mRNA chain in the cell’s ribosomes directs the assembly of amino acid sequences to form peptides or polypeptides.

[0147] The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinant-produced, and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded molecules and as linear or covalently circularly closed molecules. Nucleic acids can be isolated. According to this disclosure, the term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example, by polymerase chain reaction (PCR) for DNA or by in vitro transcription (using, for example, RNA polymerase) for RNA, (ii) produced by clonal recombination, (iii) purified, for example, by cleavage and isolation via gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.

[0148] The term "nucleoside" (abbreviated as "N" in this document) refers to compounds that can be considered as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0149] The five standard nucleosides that typically constitute naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. These five nucleosides are usually abbreviated by their single-letter codes U, A, T, C, and G. However, thymidine is more often written as "dT" ("d" stands for "deoxy") because it contains a 2'-deoxyfuranose moiety instead of the furanose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) rather than ribonucleic acid (RNA). Conversely, uridine is found in RNA rather than DNA. The remaining three nucleosides can exist in both RNA and DNA. In RNA, they would be represented as A, C, and G; however, in DNA, they would be represented as dA, dC, and dG.

[0150] The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified with one or more alkyl groups (more preferably one or more C1-4 alkyl groups, even more preferably one or more methyl groups). Specific examples of the modified purine or pyrimidine base moiety 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-pseuuridine, and N(1)-methyl-uracil.

[0151] In this document, the term "DNA" refers to a nucleic acid molecule comprising deoxyribonucleotide residues. In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose group. DNA encompasses, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may target internal DNA nucleotides or add non-nucleotide material to one or both ends of the DNA. It is also contemplated herein that the nucleotides in the DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, these altered DNAs are considered analogs of naturally occurring DNA. If, based on the total number of nucleotide residues in a molecule, the content of deoxyribonucleotide residues is greater 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%), then the molecule contains "a majority of deoxyribonucleotide residues". The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0152] DNA can be recombinant DNA and can be obtained by cloning nucleic acids (especially cDNA). cDNA can be obtained through reverse transcription of RNA.

[0153] The term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group. RNA encompasses, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may target internal RNA nucleotides or add non-nucleotide material to the ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxyribonucleotides. For the purposes of this disclosure, these altered / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such altered / modified nucleotides (i.e., the altered / modified RNA) may be referred to as analogs of naturally occurring RNA. If, based on the total number of nucleotide residues in the molecule, the content of ribonucleotide residues is greater 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%), then the molecule contains "a majority of ribonucleotide residues". The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0154] “RNA” includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, repressive 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, a 5' cap (e.g., an m7G cap), and / or a 3' tail (e.g., a poly-A tail).

[0155] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” used herein and in the appended claims include plural references.

[0156] As used herein, the term “comprising / comprises / comprised of” is synonymous with “including / includes” or “containing / contains”, and is inclusive or open-ended, and does not exclude additional, unlisted members, elements, or method steps. The term “comprising / comprises / comprised of” also includes the term “composed of”.

[0157] As used herein, the terms “equivalent to,” “similar to,” or “equivalent to” are interchangeable with “substantially no different from,” and in more stringent embodiments, with “no different from” or “identical to.” It will be understood herein that the margin allowed within, for example, “substantially” is a margin that a person skilled in the art would understand to have no significant effect on the function in question. For example, a polypeptide of this disclosure having an in vivo half-life equivalent to that of a wild-type polypeptide can be considered to have an in vivo half-life substantially no different from that of an equivalent wild-type polypeptide. In this embodiment, any minute difference between the in vivo half-lives of mutant and wild-type polypeptides is not significant and, individually, does not produce any substantial difference in the in vivo function of the polypeptide.

[0158] If applicable, any function or role of the immune system, in the body, in cells or organisms mentioned herein is preferably in humans or human cells.

[0159] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art as claimed in the appended claims.

[0160] The techniques provided herein will now be further described by way of examples, which are intended to help those skilled in the art to implement the techniques provided herein and are not intended to limit the scope of the claimed invention in any way.

[0161] Numbering Implementation Plan 1. A polypeptide comprising a crystallizable fragment (Fc) region having reduced affinity for Staphylococcus aureus protein A (SpA).

[0162] 2. The polypeptide of embodiment 1, wherein the polypeptide sequence of the Fc region comprises one or more of the following substitution mutations: T307I, Q311R, M428L, N434L and Y436K.

[0163] 3. A polypeptide comprising an Fc region, wherein the polypeptide sequence of the Fc region comprises T307I, Q311R, M428L, N434L, Y436K or any combination thereof.

[0164] 4. The polypeptide as described in embodiment 3, wherein the polypeptide has a reduced affinity for SpA.

[0165] 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 T307I, Q311R, N434L and Y436K.

[0166] 6. The polypeptide according to any one of embodiments 1 to 5, wherein the polypeptide sequence of the Fc region comprises all of the substitution mutations T307I, Q311R, M428L, N434L and Y436K.

[0167] 7. The polypeptide as described in any one of embodiments 1 to 6, wherein the polypeptide does not contain the substitution mutation H435R.

[0168] 8. The polypeptide according to any one of embodiments 1 to 7, wherein the polypeptide comprises H435.

[0169] 9. The polypeptide as described in any one of embodiments 1 to 8, wherein the amino acid position number corresponds to the EU number.

[0170] 10. The polypeptide of any one of embodiments 1 to 9, wherein the Fc region of the polypeptide has a reduced affinity for Staphylococcus aureus secondary immunoglobulin-binding protein (Sbi).

[0171] 11. The polypeptide of any one of embodiments 1 to 10, wherein the Fc region of the polypeptide has a reduced affinity for streptococcal protein G (SpG).

[0172] 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 a polypeptide that does not contain the substitution mutation.

[0173] 13. The polypeptide of any one of embodiments 1 to 12, wherein the Fc-mediated effector function of the polypeptide is not inhibited in the presence of Staphylococcus aureus.

[0174] 14. The polypeptide of any one of embodiments 1 to 13, wherein the Fc region of the polypeptide binds to a neonatal Fc receptor (FcRn).

[0175] 15. The polypeptide of any one of embodiments 1 to 14, wherein the in vivo half-life of the polypeptide is equivalent to the in vivo half-life of the polypeptide comprising the Fc region without the substitution mutation.

[0176] 16. The polypeptide of any one of embodiments 1 to 15, wherein the Fc region of the polypeptide binds to an Fc-γ receptor (FcγR).

[0177] 17. The polypeptide of any one of embodiments 1 to 16, wherein the polypeptide has improved complement component 1q (C1q) recruitment.

[0178] 18. The polypeptide of any one of embodiments 1 to 17, wherein the polypeptide further comprises a binding portion that specifically binds to Staphylococcus aureus.

[0179] 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.

[0180] 20. The polypeptide of any one of embodiments 1 to 19, wherein the polypeptide is an antibody construct, an antibody, an antigen-binding fragment comprising an Fc region, or a polypeptide comprising a single-domain antibody (VHH) fused to an Fc region.

[0181] 21. An antibody construct comprising a polypeptide as described in any one of embodiments 1 to 20.

[0182] 22. The polypeptide or antibody construct as described in embodiment 20 or 21, wherein the antibody construct is a full-length immunoglobulin, a construct containing an Fc region dimer, scFv-Fc, Fab-Fc, Fab'-Fc, F(ab)'2-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

[0183] 23. One or more nucleic acid sequences, said one or more nucleic acid sequences being capable of expressing a polypeptide or antibody construct according to any one of embodiments 1 to 22.

[0184] 24. One or more nucleic acid sequences as described in embodiment 23, wherein the nucleic acid comprises DNA encoding a polypeptide or antibody construct according to any one of embodiments 1 to 22, comprises RNA encoding the polypeptide or antibody construct, comprises mRNA encoding the polypeptide or antibody construct, is RNA encoding the polypeptide or antibody construct, is DNA encoding the polypeptide or antibody construct, or is mRNA encoding the polypeptide or antibody construct.

[0185] 25. A cell comprising a polypeptide, antibody construct, or one or more nucleic acid sequences according to any one of embodiments 1 to 24.

[0186] 26. A composition comprising a polypeptide, antibody construct, or one or more nucleic acid sequences or cells according to any one of embodiments 1 to 25.

[0187] 27. The composition of embodiment 26, wherein the composition is a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier, diluent, excipient, or any combination thereof.

[0188] 28. An in vitro method comprising contacting cells with a polypeptide, antibody construct, one or more nucleic acid sequences, cells, or a composition as described in any one of embodiments 1 to 27.

[0189] 29. The polypeptide, antibody construct, one or more nucleic acid sequences, cells or compositions as described in any one of embodiments 1 to 27, for use in a treatment or diagnostic method.

[0190] 30. The polypeptide, antibody construct, one or more nucleic acid sequences, cells or compositions as described in any one of embodiments 1 to 27, for use in the manufacture of a therapeutic or diagnostic agent.

[0191] 31. A treatment or diagnostic method comprising administering to a subject a polypeptide, antibody construct, one or more nucleic acid sequences, cells, or a composition as described in any one of embodiments 1 to 27.

[0192] 32. The method of any one of embodiments 29 to 31, wherein the treatment is the treatment or prevention of Staphylococcus aureus infection.

[0193] 33. A method comprising administering to a subject a polypeptide, antibody construct, one or more nucleic acid sequences, cells, or a composition as described in any one of embodiments 1 to 27.

[0194] 34. The method of embodiment 33, wherein the method is a method for treating or preventing Staphylococcus aureus infection in the subject, optionally wherein the subject is a human.

[0195] 35. The method of embodiment 33, wherein the method is a means of inducing an immune response against Staphylococcus aureus infection in the subject, optionally wherein the subject is a human.

[0196] 36. A method for preparing a polypeptide or antibody construct according to any one of embodiments 1 to 21, the method comprising expressing one or more nucleic acids capable of expressing the polypeptide.

[0197] 37. A method for preparing a polynucleotide encoding a polypeptide as described in any one of embodiments 1 to 20, the method comprising recombinantly linking a first nucleotide sequence encoding an Fc region having reduced affinity for SpA with a second nucleotide sequence encoding a binding portion specifically binding to Staphylococcus aureus.

[0198] 38. A method for preparing a polynucleotide encoding a polypeptide as described in any one of embodiments 1 to 20, the method comprising recombinantly linking a first nucleotide sequence encoding an Fc region with a second nucleotide sequence encoding a binding portion specifically binding to Staphylococcus aureus, wherein the polypeptide sequence of the Fc region comprises T307I, Q311R, M428L, N434L, Y436K or any combination thereof.

[0199] Example Example 1 – Screening Fc variants To effectively use therapeutic antibodies in Staphylococcus aureus infections, it is necessary to inhibit Fc-mediated capture of the antibody. To achieve this, mutations in the Fc domain that inhibit SpA-mediated binding were identified.

[0200] Computer screening for FcRn affinity, SpA affinity, and stability A significant challenge in the screening process is 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 therefore useful for the long serum half-life of antibodies. Both SpA / Sbi and FcRn bind to the IgG-Fc domain at the CH2 and CH3 interface and involve some of the same amino acids. Using advanced computer AI-driven screening techniques, computer modeling of the protein complexes Fc:FcRn and Fc:SpA against a large number of Fc variants was performed, and mutagenesis and stability predictions led to the identification of 180 candidate Fc mutants.

[0201] Screening candidates for expression of VHH-Fc fusions Subsequently, 180 identified candidate Fc mutants and controls from the literature (Chen et al., PNAS 2022) were expressed and characterized as VHH-Fc fusions, where the VHH targets the placeholder target antigen (e.g., for subsequent replacement with Staphylococcus aureus-specific VHH). The characterization included testing for both SpA binding and FcRn binding. Of the 180 candidate proteins from the literature and controls, 32 proteins were further characterized for their functional capabilities. Other candidates failed the initial tests for SpA binding and / or FcRn binding. Through several rounds of testing, three candidate mutants of further interest were identified: Fc049, Fc067, and Fc136, and two control mutants from the literature: Fc183 and Fc185. Details of the Fc domains of these proteins are provided in Table 1.

[0202] Table 1: Candidate mutants after initial screening and literature comparison It is noteworthy that the candidate mutants identified in Table 1 do not contain any substitution mutations identical to the control mutants from the literature (although some amino acid positions are the same, the specific substitution mutations are different). It is also noteworthy that none of the candidate mutants identified in Table 1 possess the H435R mutation of the control mutants. Furthermore, it is noteworthy that Fc136 is the only mutant identified (from any of the 180 screened mutants) with the M428L mutation.

[0203] Expression and stability tests The expression and stability of candidate mutants and control mutants were tested, and the results were analyzed from Western blotting.

[0204] All candidate mutants tested were well expressed. The selected lead candidate mutants listed in Table 1 did not show aberrant fragmentation in Western blots. However, Fc183 showed sheet-like bands in Western blots, suggesting that this protein is potentially responsible for expression and stability.

[0205] FcRn combined with testing FcRn binding of the Fc mutants listed in Table 1 was tested by ELISA at pH 6 and pH 7. Several other variants from the literature were also tested. For efficient intracellular antibody recycling, FcRn binding occurring at pH 6 but weaker binding at pH 7 is favorable.

[0206] At pH 6, the Fc mutants listed in Table 1 showed binding to FcRn, similar to the WT control (unmodified VHH-Fc fusion). Binding to FcRn at pH 6 was considered acceptable for all the mutants tested in Table 1.

[0207] However, two additional Fc mutants from the literature were tested and failed at this screening stage because they exhibited similarity to FcRn at pH 6. weak Both Fc182 (from Chen et al. (2022) "AESP" containing mutations S254A, Q311E, L432S, and N434P) and Fc184 (from Chen et al. (2022) "AESP-RV" containing mutations S254A, Q311E, L432S, N434P, T307R, and A378V) showed unacceptably weak binding to FcRn at pH 6.

[0208] At pH 7, binding to FcRn was considered acceptable for all Fc mutants listed in Table 1, with notable exception Fc185. Fc185 exhibited strong binding to FcRn at pH 7, the extent of which may negatively impact the antibody half-life of Fc185. These results are presented in... Figure 1 middle.

[0209] In addition, two other Fc mutants from the literature were tested and failed at this screening stage because they exhibited strong binding to FcRn at pH 7. Fc186 (“R-QVV” from Chen et al. (2022), containing mutants H435R, T307Q, Q311V, and A378V) and Fc187 (“R-DDRVV” from Chen et al. (2022), containing mutants H435R, T256D, N286D, T307R, Q311V, and A378V) both showed unacceptably strong binding to FcRn at pH 7, indicating a short serum half-life.

[0210] Example 2 – Testing Antibody Effector Function FcγR receptor binding assay Antibody effector function is primarily mediated by the interaction between the Fc domain and the associated Fc receptor. In the case of IgG antibodies, where the Fc domain is used in these experiments, the Fcγ receptor (FcγR) is particularly relevant. As an approximation of Fc effector function, binding to the Fcγ receptor is detected by ELISA (see Figure 2).

[0211] The binding of the candidate mutant Fc136 to the Fcγ receptor was comparable to that of the wild-type control, indicating that the inserted mutation did not negatively affect Fcγ receptor binding. Binding was conserved for activating receptors (such as FcγRI (Fig. 2A), FcγRIIa (Fig. 2B), and FcγRIIIa (Fig. 2D)) and inhibitory receptors (such as FcγRIIb / c (Fig. 2C)). High-affinity polymorphisms of FcγRIIa and FcγRIIIa were used in these tests.

[0212] The literature shows that mutant Fc185 exhibits the lowest maximum effect on both FcγRI and FcγRIIa. Mutant Fc183 exhibits the lowest maximum effect on FcγRIIb / c.

[0213] These tests confirmed that mutations in Fc136 did not significantly reduce FcγR binding.

[0214] Functional testing of Fc mutants Functional tests were performed on the Fc mutants listed in Table 1, including cell binding assays, ADCC assays, ADCP assays, and C1q recruitment assays.

[0215] Cell binding was assessed using a FlpIn CHO cell line overexpressing the target antigen, an experimentally validated cell line based on a commercially available FlpIn CHO system (https: / / www.thermofisher.com / order / catalog / product / de / en / R75807). ADCC was measured using a kit from Promega GmbH (https: / / www.promega.de / en / products / reporter-bioassays / fc-effector-activity-bioassays / adcc-bioassays / ?catNum=G7010). ADCP was also measured using a kit from Promega GmbH (https: / / www.promega.de / en / products / reporter-bioassays / fc-effector-activity-bioassays / adcp-bioassays / ?catNum=G9991). A C1q recruitment assay was designed based on available literature (Pawluczkowycz et al., J Immunol 1;183(1):749-58 (2009) [Pubmed ID: 19535640]).

[0216] The results of the functional test are shown in Figure 3.

[0217] It was determined that the binding to the cell surface of cell lines overexpressing the target antigen was not affected by the insertional mutation (Figure 3C).

[0218] However, some differences in Fc effector function were observed. Notably, Fc136 performed at least as well as the wild-type protein in ADCC and ADCP reporter assays. Furthermore, Fc136 showed a favorable strong signal in C1q recruitment assays. Fc049 was also noted to show ADCC signaling, but weaker than that of Fc136.

[0219] These tests confirmed that the mutation did not significantly reduce cell binding. Further testing confirmed that Fc136 performed well in all assays compared to mutants tested, and that Fc136 showed a favorable strong signal in the C1q recruitment assay. C1q recruitment to target cells bound by IgG antibodies / VHH-Fc fusion proteins initiates the classical complement pathway, thereby promoting complement-dependent cytotoxicity (CDC). Therefore, these tests indicate that Fc136 can induce enhanced CDC.

[0220] Example 3 – Testing Inhibited Sbi, SpA, and SpG Binding Compared with the wild-type control (Fc001 / WT), the inhibition of binding of Fc mutants, including Fc136, to Staphylococcus aureus secondary immunoglobulin-binding protein (Sbi), as well as SpA and SpG, was further tested by ELISA.

[0221] The ELISA results are shown in Figure 4. Fc136 showed inhibited binding to Sbi, SpA, and SpG, indicating that Fc136 has reduced affinity for Sbi, SpA, and SpG. In particular, Fc136 exhibited significantly reduced affinity for Sbi, SpA, and SpG compared to the wild-type equivalent protein.

[0222] Example 4 – Further confirmation using purified peptides The VHH-Fc fusion mutants Fc136, Fc183, and Fc185, as well as the wild-type control (WT), were further tested in purified form. This was done to further confirm that the Fc136 mutant no longer binds to staphylococcal protein A and streptococcal protein G, while retaining other characteristics similar to the wild-type control (and is therefore desirable).

[0223] Protein production VHH-Fc fusions were generated via transient transfection of ExpiCHO cells. Cell culture supernatants were harvested 8 days post-transfection and subsequently purified by affinity chromatography (FLAG-tagged) and preparative size exclusion chromatography. This yielded a high-purity protein solution (>95%, confirmed by SDS-PAGE) in which the target protein ran at approximately 95 kDa under non-reducing conditions and approximately 45 kDa under reducing conditions. The reducing conditions resulted in the reduction of disulfide bonds in the hinge region of the antibody construct, leading to a single protein chain. The protein was normalized to 2 mg / mL in sterile PBS.

[0224] ELISA testing interactions with target antigens, Fc receptors, SpA, SpG, and Sbi. Uniform binding of the VHH-Fc fusion compound was detected on the target antigen, with EC50 values ​​ranging from 0.39 to 0.51 nM (Figure 5A).

[0225] The binding of FcγRI (CD64), FcγRIIa (CD32a) and FcγRIIIa (CD16) is equivalent between Fc136 and WT (Fc001) (Figs. 5B-5D).

[0226] All candidate Fc mutants (Fc136, Fc183, Fc185) showed reduced binding to protein A, with binding detected only at very high concentrations – EC50 values ​​increased by >1000-fold (Figure 6A).

[0227] In contrast, only Fc136 showed reduced binding to Streptococcus protein G, while the candidates Fc183 and Fc185, which contain H435R, showed binding to protein G comparable to WT (Figure 6B).

[0228] Furthermore, all candidate Fc mutants (Fc136, Fc183, Fc185) showed reduced binding to Sbi, with binding also detected only at very high concentrations (Figure 6A).

[0229] In summary, further studies using the purified peptide confirmed that the Fc136 mutant VHH-Fc fusion exhibits reduced affinity for SpA, SpG, and Sbi, while maintaining the same target antigen and Fc receptor binding properties as the wild-type VHH-Fc fusion. Specifically, Fc136 exhibits significantly reduced affinity for SpA, SpG, and Sbi compared to the equivalent wild-type peptide.

[0230] Example 5 – Improved half-life relative to wild-type and literature peptides Pharmacokinetic studies were performed to evaluate the in vivo binding of the identified Fc mutant to human FcRn. For this purpose, NOD.Cg- expressing human FcRn was selected instead of mouse FcRn. Fcgrt tm1Dcr Prkdc scid Il2rg tm1Wjl Tg(FCGRT)32Dcr / J (NSG FcRn- / - hFcRn (32) Tg) mice were used as model organisms because the binding of the Fc mutant to human FcRn and to mouse FcRn showed different patterns.

[0231] Five groups (each group consisting of four mice) were injected with one of the following samples: • Fc136: 250 µg in 150 µL PBS • Fc183: 250 µg in 150 µL PBS • Fc185: 250 µg in 150 µL PBS • Fc001: 250 µg in 150 µL PBS • PBS: 150 µL Blood was drawn at different time points within an 18-day timeframe. The concentration of VHH-Fc in the serum was then quantified using ELISA. Figure 7 And the results shown in Table 1 below.

[0232] Fc136 consistently showed the highest serum concentrations compared to all other tested samples. The serum concentrations of Fc136 were significantly higher than those observed for the corresponding WT protein (Fc001). Fc136 also showed significantly higher concentrations than Fc185. Fc183 showed no significant difference from the wild type in terms of half-life and serum concentration.

[0233] Table 2: Serum half-life of Fc mutants as determined by the two-phase decay model

Claims

1. A polypeptide comprising a protein against Staphylococcus aureus. A (SpA) has a crystallizable fragment (Fc) region with reduced affinity, wherein the polypeptide sequence of the Fc region comprises T307I, Q311R, M428L, N434L, Y436K or any combination thereof.

2. The polypeptide of claim 1, wherein the polypeptide sequence in the Fc region comprises the substitution mutation M428L, and optionally comprises T307I, Q311R, N434L, Y436K or any combination thereof.

3. The polypeptide of claim 1 or 2, wherein the polypeptide sequence in the Fc region comprises all of the substitution mutations T307I, Q311R, M428L, N434L, and Y436K.

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

5. The polypeptide of any one of claims 1 to 4, wherein the Fc region of the polypeptide has a reduced affinity for Staphylococcus aureus second immunoglobulin-binding protein (Sbi), and optionally wherein the Fc region of the polypeptide has a 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 a reduced affinity for SpA, Sbi, and / or SpG relative to the Fc region of a polypeptide not containing the substitution mutation, optionally wherein the Fc-mediated effector function of the polypeptide is 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 a neonatal Fc receptor (FcRn), and optionally wherein the in vivo half-life of the polypeptide is equivalent to the in vivo half-life of a polypeptide containing an Fc region without the substitution mutation.

8. The polypeptide of any one of claims 1 to 7, wherein the Fc region of the polypeptide binds to the Fcγ receptor (FcγR), and 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 a polypeptide as described in any one of claims 1 to 10, optionally wherein the antibody construct is a full-length immunoglobulin, a construct comprising an Fc region dimer, scFv-Fc, Fab-Fc, Fab'-Fc, F(ab)'2-Fc, Fv-Fc, sdAb-Fc, or VHH-Fc.

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

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

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

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

16. The polypeptide, antibody, one or more nucleic acid sequences, cells, or compositions according to any one of claims 1 to 15, for use in a treatment or diagnostic method, optionally wherein the treatment is the treatment or prevention of Staphylococcus aureus infection.

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