Therapeutic variable domain of heavy chain (VHH) antibody fusions co-neutralizing alpha- and gamma-hemolysins from staphylococcus aureus

Abstract

The present invention pertains to the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that neutralize cytotoxic gamma-hemolysins and optionally the leukocidin LukF of S. aureus. These VHH antibodies are useful in an approach for co-neutralizing alpha- and gamma-hemolysins and the LukSF toxin pair by multimeric VHH-fusions. The invention provides an implementation of this approach.

Description

[0001] Therapeutic variable domain of heavy chain (VHH) antibody fusions co-neutralizing alpha- and gamma-hemolysins from Staphylococcus aureus

[0002] Field of the invention

[0003] The present invention pertains to the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that neutralize cytotoxic gammahemolysins of S. aureus and optionally leukocidin F (LukF). These VHH antibodies are useful in an approach for co-neutralizing alpha- and gamma-hemolysins and the LukSF toxin pair by multimeric VHH-fusions. The invention provides an implementation of this approach.

[0004] Background

[0005] Staphylococcus aureus is one of the most prevalent bacterial pathogens, causing a wide range of diseases, including superficial skin infections, life-threatening pneumonia, bacteremia, and sepsis. In developed countries, S. aureus is the most common cause of blood infections. In the US, the number of deaths from S. aureus was greater than those caused by AIDS, tuberculosis, and viral hepatitis together (Cheung et al., 2021 ). The lack of an approved vaccine and complicated treatments make S. aureus a major global health problem.

[0006] S. aureus is a commensal bacterium, with an estimated 30% of the population being carriers (Tong et al., 2015; Saleh et al., 2022). However, it can readily cause infections upon injuries to the skin or mucosal membranes. The human immune system can normally eliminate invading bacteria within minutes, using complement, macrophages, and - after repeated contact - also antibodies. Virulent bacteria, however, use virulence factors to evade the immune response. Virulence factors comprise mostly surface and extracellular secreted proteins, including bacterial toxins, complement-inactivating factors, inhibitors of phagocytosis, or proteases that cleave immunoglobulins or extracellular matrix. Unlike other bacteria, which deploy only one or a few, S. aureus employs >20 virulence factors to promote its pathogenicity (Cheung et al., 2021 ).

[0007] Antibiotics are key to treating bacterial infections, but the rise of antibiotic-resistant infections makes therapies increasingly unreliable. The frequent occurrence of multi- resistant S. aureus isolates poses a particular challenge. MRSA (methicillin-resistant S. aureus) is the most commonly identified antibiotic-resistant pathogen worldwide. Infected patients have longer hospital stays, higher treatment costs, and higher mortality (Ippolito et al., 2010). The first MRSA strain was characterized in 1961 , just one year after the introduction of methicillin for clinical treatments (Ippolito et al., 2010; Turner et al., 2019). Since then, a plethora of strains have been characterized that are resistant to an increasing number of antibiotics. Some clinical isolates are resistant to >10 antibiotic classes (Saleh et al., 2022). New antibiotics for MRSA and, more generally, for S. aureus therapy have been developed or are in the pipeline. Currently, vancomycin is an antibiotic of last resort in non-critically ill patients (Cheung et al., 2021 ). Yet, although not widespread, vancomycin-resistant S. aureus isolates have emerged and vancomycin treatments are failing more frequently (Lodise et al., 2008; Ye et al., 2020).

[0008] Another limitation of antibiotic therapy is that even if the bacteria are killed, it cannot remove already circulating bacterial toxins from the body. Antibiotics may even enhance toxin release. Therefore, there is a need for alternative treatment strategies that target virulence factors. One such “anti-toxin treatment” approach is to use antibodies to neutralize virulence factors, which should stop the toxin effects, "disarm" the pathogen and allow the immune system to clear the infection.

[0009] Pore-forming toxins (PFTs) are key virulence factors of S. aureus. They are secreted as (water-) soluble monomers, bind to specific membrane receptors on target cells, and assemble into 14-40 A wide, [3-barrel transmembrane pores. Such pores cause leakage of ions and other small molecules, destabilize the membranes, and lead to cell lysis. PFTs mainly target leukocytes to evade the host cell defense system, as well as erythrocytes to make the otherwise limiting iron available for bacterial growth (Spaan et al., 2013; Cassat & Skaar, 2013). S. aureus deploys six different toxin pores, all of which adopt [3-barrel transmembrane structures: an alpha-hemolysin pore, two kinds of gamma-hemolysin pores, and three kinds of leukocidin pores. Alpha-hemolysin (alpha-toxin, Hla) is the best-characterized cytotoxin of S. aureus, targeting a wide range of host cells including erythrocytes, monocytes, epithelial and endothelial cells (Divyakolu et al., 2019). Hla assembles into homo-heptameric pores.

[0010] Gamma-hemolysins (Hlg or gamma-toxins) on the other hand, are bicomponent PFTs with HlgB as the class F (fast-eluting in chromatography columns) component and either HlgA or HlgC as the class S (slowly eluting) component. The S component first binds to a unique receptor and then recruits the F component to form a heterooctameric complex that rearranges to a [3-barrel transmembrane pore (Staali & Colin, 2021 ). Leukocidins are related to gamma-toxins. LukF and LukS make one leukocidin pair (LukSF), LukD and LukE a second pair (LukDE), and LukG and LukH a third one (LukGH). The structures of these pores are highly conserved, and similar to the heptameric pores of Hla. Indeed, all precursor proteins have a molecular weight of 33 kDa and are structurally almost identical. However, the sequence identity between the individual components is only 25-35%, with class F components being more similar to Hla than the class S components.

[0011] Hlgs are present in 99% of S. aureus isolates (Prevost et al., 1995). HlgA uses human chemokine receptors including the Duffy antigen / receptor for chemokines (DARC) expressed in erythrocytes and endothelial cells (Spaan et al., 2015a), as well as CXCR1 and CXCR2 expressed in granulocytes and macrophages (Spaan et al., 2014). HlgC instead uses C5aR1 (complement C5a receptor), expressed in leukocytes, as the major receptor (Spaan et al., 2015b; Zimmermann-Meisse et al., 2017). Consequently, HlgA / HlgB (HlgAB) and HlgC / HlgB (HlgCB) toxins extend the virulence of S. aureus by targeting different receptors.

[0012] Nanobodies (VHH antibodies) comprise a novel class of therapeutic proteins based on monovalent, camelid-derived heavy chain-only antibodies. In contrast to monoclonal IgGs, which are manufactured in mammalian cells, nanobodies can also be produced in bacteria or yeast. Apart from their low molecular weight, certain nanobodies proved to be hyper-thermostable and display a particularly high affinity for their targets (Guttler et al., 2021 ). To effectively treat sepsis caused by S. aureus, several pore-forming toxins should be neutralized. The most economical approach is to do this with a single molecule, for example by a tandem fusion of different VHH antibodies, each neutralizing a different toxin.

[0013] PCT / EP2024 / 052663, the content of which is herein incorporated by reference, discloses VHH antibodies that specifically bind and neutralize S. aureus Hla.

[0014] It was the objective of this invention to provide VHH antibodies against S. aureus Hlg components and VHH fusions that effectively co-neutralize alpha- and gammahemolysin toxins.

[0015] Summary of the invention

[0016] The present disclosure provides single-domain VHH antibodies specifically binding (recognizing) an S. aureus gamma-hemolysin (Hlg). They recognize and / or prevent membrane-binding and / or oligomerization of and hemolysis by S. aureus Hlg and are suitable for diagnostic, prophylactic and / or therapeutic applications.

[0017] In certain embodiments, the VHH antibody specifically binds both the B-component of Hlg and the leukocidin LukF. This VHH antibody blocks the toxicity of the HlgAB, HlgCB, and LukSF toxin pairs.

[0018] In certain embodiments, the VHH antibody is in monovalent form, e.g., as a single VHH domain or as a fusion to a heterologous protein such as serum albumin useable in a wide range of formats. In certain embodiments, the VHH antibody is in multivalent form, e.g., as bivalent Fc-fusion. In certain embodiments, the VHH antibody is a hetero-multimeric VHH antibody comprising of two or more, e.g. 2, 3 or 4 different VHH antibody units.

[0019] The use in a monovalent format is possible due to their very high affinity. In certain embodiments, the VHH antibodies have - in the monovalent format - a target affinity in picomolar or even low picomolar range. In certain embodiments, the VHH antibodies neutralize Hlg with high potency. In certain embodiments, the VHH antibodies neutralize both Hlg, including HlgAB and HlgBC, and LukSF with high potency. A first aspect of the present disclosure is a VHH antibody directed against S. aureus Hlg as described herein. In certain embodiments, the VHH antibody is directed against HlgB. In certain embodiments, the VHH antibody is directed against HlgB and against LukF.

[0020] Preferred VHH antibodies of the present invention, their designations, binding characteristics, i.e., Hlg epitope, Hlg affinity, and Hlg neutralization as well as their thermostabilities are listed in the following Table 1 :

[0021] Table 1

[0022] Table 1: Anti-HIg VHHs, their thermostability, affinities for respective Hlg component (expressed as KD values) and inhibitory properties. A list of complete VHH sequences of anti-HIg VHH antibodies is shown at the end of the specification.

[0023] A further aspect of the present invention relates to a set of two or more different VHH antibodies, wherein at least one VHH antibody is as described above.

[0024] A VHH antibody described above is suitable for use in medicine, e.g., human medicine, particularly for use in therapy, e.g., in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with Hlg-positive S. aureus, particularly by an infection with a drug-resistant Hlg-positive S. aureus, or in diagnostics, e.g., for detecting S. aureus Hlg in a patient sample, e.g., in a body fluid or tissue sample, or in research.

[0025] Still a further aspect of the invention relates to a nucleic acid molecule encoding a VHH antibody as described above, particularly in operative linkage with a heterologous expression control sequence, a vector comprising said nucleic acid molecule or a recombinant cell or non-human organism transformed or transfected with said nucleic acid molecule or said vector.

[0026] Still a further aspect of the invention relates to a method for the recombinant production of a VHH antibody as described above, comprising cultivating a cell or an organism as described above in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium.

[0027] Still a further aspect of the invention relates to a method for the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with Hlg-positive S. aureus, particularly by an infection with a drug-resistant Hlg-positive S. aureus, comprising administering an effective dose of the VHH antibody as described above or the set of at least two different VHH antibodies as described above to a subject in need thereof, particularly to a human subject suffering from a disorder caused by, associated and / or accompanied by an infection with Hlg- positive S. aureus. A non-limitative list of Anti-HIg VHH antibodies of the present invention and their CDR sequences is shown in the following Table 2:

[0028] Table 2

[0029] Table 2: Anti-HIg VHHs and their CDR regions. Note that CDRs include here also variable residues that flank the actual CDR loops

[0030] Still a further aspect of the present invention relates to a hetero-multimeric VHH antibody comprising a first VHH antibody unit which recognizes Hlg, particularly an anti-HIg VHH antibody as herein described above, and a second VHH antibody unit which recognizes Hla, particularly an anti-Hla antibody as described in PCT / EP2024 / 052663.

[0031] In certain embodiments, the hetero-multimeric VHH antibody is a tandem fusion of VHH antibody units comprising a first unit which is an anti-HIg VHH antibody, and second unit which is an anti-Hla VHH antibody wherein the first unit is covalently fused to the second unit, particularly via a linker. In certain embodiments, the tandem fusion is further fused to a heterologous polypeptide, e.g., an immunoglobulin Fc fragment.

[0032] In certain embodiments, the first unit of the hetero-multimeric antibody is an anti-HlgB VHH antibody. In certain embodiments, the first unit of the hetero-multimeric antibody is an anti-HlgB VHH antibody that also specifically binds to LukF.

[0033] A non-limitative list of anti-Hla VHH antibodies and their CDR sequences is shown in the following Table 3:

[0034] Table 3 Table 3: Anti-Hla VHH antibodies and their CDR regions. Note that CDRs include here also variable residues that flank the actual CDR loops.

[0035] Embodiments of the invention

[0036] In the following, specific embodiments of the invention are disclosed as follows:

[0037] 1 . A VHH antibody recognizing an S. aureus gamma-hemolysin (Hlg).

[0038] 2. The VHH antibody of embodiment 1 which recognizes S. aureus gammahemolysin component A (HlgA), S. aureus gamma-hemolysin component B (HlgB), or S. aureus gamma-hemolysin component C (HlgC).

[0039] 3. The VHH antibody of embodiment 1 or 2 which recognizes S. aureus gammahemolysin component B (HlgB).

[0040] 4. The VHH antibody of embodiment 3 which further recognizes S. aureus leukocidin component F (LukF).

[0041] 5. The VHH antibody of any one of embodiments 1 -4, which binds to the same epitope on HlgB as the VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 and / or the VHH antibody Bm29E06 having a VHH sequence as shown in SEQ. ID NO: 13.

[0042] 6. The VHH antibody of any one of embodiments 1 -4, which binds to the same epitope on HlgB as the VHH antibody Bm29B04, as shown in Figure 11 , and having a VHH sequence as shown in SEQ. ID NO: 21.

[0043] 7. The VHH antibody of embodiment 1 or 2, which binds to the same epitope on HlgA as the VHH antibody Bm31G03 having a VHH sequence as shown in SEQ. ID NO: 37 and / or the VHH antibody Bm31 G12 having a VHH sequence as shown in SEQ. ID NO: 49. The VHH antibody of embodiment 1 or 2, which binds to the same epitope on HlgC as the VHH antibody Bm32A03 having a VHH sequence as shown in SEQ. ID NO: 73. The VHH antibody of embodiment 1 or 2, which binds to the same epitope on HlgC as the VHH antibody Bm32A08 having a VHH sequence as shown in SEQ. ID NO: 85. A VHH antibody recognizing an S. aureus HlgB polypeptide, comprising

[0044] (a) a CDR3 sequence as shown in SEQ. ID NO: 8, 12, 16, 20, 24, 28, 32, or 36,

[0045] (b) a CDR3 sequence, which has an identity of at least 80%, at least 90% or at least 95% to a CDR3 sequence of (a). A VHH antibody recognizing an S. aureus HlgA polypeptide, comprising

[0046] (a) a CDR3 sequence as shown in SEQ. ID NO: 40, 44, 48, 52, 56, 60, 64, 68, or 72,

[0047] (b) a CDR3 sequence, which has an identity of at least 80%, at least 90% or at least 95% to a CDR3 sequence of (a). A VHH antibody recognizing an S. aureus HlgC polypeptide, comprising

[0048] (a) a CDR3 sequence as shown in SEQ. ID NO: 76, 80, 84, 88, or 92,

[0049] (b) a CDR3 sequence, which has an identity of at least 80%, at least 90% or at least 95% to a CDR3 sequence of (a). The VHH antibody of any of the preceding embodiments, comprising

[0050] (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88 or 90-92; or

[0051] (b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a). The VHH antibody of any one of the preceding embodiments, comprising (a) a VHH sequence as shown in SEQ. ID NO: 5, 9, 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, or 89;

[0052] (b) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a). The VHH antibody of any one of the preceding embodiments, wherein the binding affinity expressed as dissociation constant KD to HlgC, HlgA or HlgB is about 1 nM or less, about 0.1 nM or less, or about 0.05 nM or less. The VHH antibody of any one of the preceding embodiments, wherein the binding affinity expressed as dissociation constant KD to HlgB and LukF is about 1 nM or less, about 0.1 nM or less, or about 0.05 nM or less. The VHH antibody of any one of the preceding embodiments, which neutralizes Hlg, particularly by inhibiting Hlg-induced hemolysis of erythrocytes and / or by inhibiting Hlg-induced cytotoxicity. The VHH antibody of any one of the preceding embodiments, which neutralizes Hlg and LukSF, particularly by (i) inhibiting Hlg-induced hemolysis of erythrocytes and / or by inhibiting Hlg-induced cytotoxicity and (ii) by inhibiting LukSF-induced membrane disruption of macrophages and / or by inhibiting LukSF-induced cytotoxicity. The VHH antibody of any one of the preceding embodiments, which is stable, particularly thermostable, or hyperthermostable. The VHH antibody of embodiment 19, which has a melting temperature of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least 80°C or of at least about 95°C when measured under non-reducing conditions. The VHH antibody of embodiment 19 or 20, which has a melting temperature of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least 80°C or of at least about 95°C when measured under reducing conditions. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29H04 having a VHH sequence as shown in SEQ. ID NO: 9 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29E06 having a VHH sequence as shown in SEQ. ID NO: 13 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29F05 having a VHH sequence as shown in SEQ. ID NO: 17 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof, including a humanized variant The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm29D07 having a VHH sequence as shown in SEQ. ID NO: 29 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 D04 having a VHH sequence as shown in SEQ. ID NO: 33 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 G03 having a VHH sequence as shown in SEQ. ID NO: 37 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 G04 having a VHH sequence as shown in SEQ. ID NO: 41 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 F09 having a VHH sequence as shown in SEQ. ID NO: 45 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 G12 having a VHH sequence as shown in SEQ. ID NO: 49 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31A09 having a VHH sequence as shown in SEQ. ID NO: 53 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 D02 having a VHH sequence as shown in SEQ. ID NO: 57 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 D04 having a VHH sequence as shown in SEQ. ID NO: 61 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31A08 having a VHH sequence as shown in SEQ. ID NO: 65 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm31 B04 having a VHH sequence as shown in SEQ. ID NO: 69 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm32A03 having a VHH sequence as shown in SEQ. ID NO: 73 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm32B07 having a VHH sequence as shown in SEQ. ID NO: 77 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm32D01 having a VHH sequence as shown in SEQ. ID NO: 81 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm32A08 having a VHH sequence as shown in SEQ. ID NO: 85 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -21 , which is selected from VHH antibody Bm32F03 having a VHH sequence as shown in SEQ. ID NO: 89 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of the preceding embodiments, which is nonglycosylated, or which is glycosylated. The VHH antibody of any one of the preceding embodiments, which is produced in a bacterium, e.g., E. coli, or in a yeast, e.g., Pichia pastoris, or in a human or animal cell, e.g., an insect cell or a mammalian cell, e.g., a CHO cell. The VHH antibody of any one of the preceding embodiments, which is in a monovalent format. The VHH antibody of any one of embodiments 1 -45, which is in a multimeric format. The VHH antibody of embodiment 47, which is in a dimeric format. The VHH antibody of embodiment 48, which is in a homodimeric or in a heterodimeric format. The VHH antibody of any one of the preceding embodiments, which is covalently or non-covalently conjugated to a heterologous moiety, e.g., a labeling group, a capture group, or an effector group, wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide. The VHH antibody of any one of the preceding embodiments, which is fused to a heterologous polypeptide moiety, or which is fused to an Fc Fragment, or which is fused to serum albumin or an albumin-binding moiety. The VHH antibody of any one of the preceding embodiments, which is conjugated to one or several non-peptidic polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG). A hetero-multimeric VHH antibody comprising of two or more different VHH antibody units, e.g., 2, 3 or 4 different VHH antibody units wherein at least one VHH antibody unit comprises a VHH antibody of any of embodiments 1 -52. The hetero-multimeric VHH antibody of embodiment 53, which is a tandem fusion of VHH antibody units comprising a first anti-HIg VHH antibody unit, and a second VHH antibody wherein the first unit is covalently fused to the second unit. The hetero-multimeric VHH antibody of embodiment 53 or 54, which comprises at least two VHH antibody units recognizing different Hlg polypeptides, particularly selected from HlgA, HlgB, and HlgC. The hetero-multimeric VHH antibody of embodiment 53 or 54, which comprises at least two VHH antibody units recognizing different epitopes on the same Hlg polypeptide, particularly different epitopes on HlgA, HlgB, or HlgC. The hetero-multimeric VHH antibody of any one of embodiments 53-56, which comprises a first VHH antibody unit which recognizes HlgB. The hetero-multimeric VHH antibody of embodiment 57 wherein the first VHH antibody unit further recognizes S. aureus leukocidin component F (LukF). The hetero-multimeric VHH antibody of any one of embodiments 53, 54, 57 or 58, which comprises a first VHH antibody unit which recognizes Hlg, particularly HlgB, and a second VHH antibody unit which recognizes Hla. The hetero-multimeric VHH antibody of any one of embodiments 53-59 wherein the VHH antibody units are covalently fused to each other via a linker. The hetero-multimeric VHH antibody of embodiment 60 wherein the linker has a length of 5-50 amino acids, particularly a length of 10-30 amino acids. The hetero-multimeric VHH antibody of embodiment 60 or 61 wherein the linker substantially consists of amino acids G, E, S and T. The hetero-multimeric VHH antibody of any one of embodiments 53-62 which further comprises a heterologous polypeptide, e.g., an immunoglobulin Fc fragment. The hetero-multimeric VHH antibody of embodiment 63 wherein the heterologous polypeptide is at the C-terminus of the hetero-multimeric VHH antibody. The hetero-multimeric VHH antibody of any one of embodiments 53-64 wherein the anti-HIg VHH antibody comprises

[0053] (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88 or 90-92; or

[0054] (b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a). The hetero-multimeric VHH antibody of any one of embodiments 53-65 wherein the anti-HIg VHH antibody comprises

[0055] (a) a VHH sequence as shown in SEQ. ID NO: 5, 9, 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, or 89;

[0056] (b) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a). The hetero-multimeric VHH antibody of any one of embodiments 53-66 comprising

[0057] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant; or

[0058] (ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0059] (iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant. The hetero-multimeric VHH antibody of any one of embodiments 59-67 wherein the anti-Hla VHH antibody comprises

[0060] (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 94-96, 98-100, 102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-136, 138-140, 142-144, 146-148, 150-152, 154-156, 158-160, 162-164, 166-168, 170-172, 174-176, 178-180, 182-184, 186-188, 190-192 or 194-196; or

[0061] (b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a). The hetero-multimeric VHH antibody of any one of embodiments 59-68 wherein the anti-Hla VHH antibody comprises

[0062] (c) a VHH sequence as shown in SEQ. ID NO: 93, 97, 101 , 105, 109, 113, 117, 121 , 125, 129, 133, 137, 141 , 145, 149, 153, 157, 161 , 165, 169, 173, 177, 181 , 185, 189 or 193;

[0063] (d) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a). The hetero-multimeric VHH antibody of any one of embodiments 59-69 comprising the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof. The hetero-multimeric VHH antibody of any one of embodiments 59-70 comprising

[0064] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3, or

[0065] (ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3, or

[0066] (iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3. The hetero-multimeric VHH antibody of any one of embodiments 59-71 comprising

[0067] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0068] (ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0069] (iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant. A set of two or more different VHH antibodies comprising at least one VHH antibody of any one of embodiments 1 -72. The set of embodiment 73 comprising

[0070] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0071] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3, or

[0072] (i) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0073] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3, or (i) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0074] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3. The set of embodiment 73 or 74 comprising

[0075] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0076] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0077] (i) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0078] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0079] (i) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0080] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for use in medicine, particularly for use in therapy or diagnostics. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for use in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with an Hlg-positive, optionally LukF-positive and optionally Hla-positive S. aureus. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-77 wherein the S. aureus is a drug-resistant Hlg-positive, optionally LukF-positive and optionally Hla-positive S. aureus. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-78 wherein the S. aureus is a drug-resistant Hlg-positive, optionally LukF-positive and optionally Hla-positive S. aureus. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-79, wherein the condition is selected from a skin infection, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-80 in a human subject. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-81 , which is administered locally. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-82, which is administered systemically. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-82, which is administered topically, parenterally, e.g., intravenously, pulmonally, e.g., by inhalation, or nasally, e.g., by spraying. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-84, which is administered as a monotherapy. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-84, which is administered sequentially and / or simultaneously as a combination therapy with at least one further active agent. The VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for the use of any one of embodiments 76-84 or 86, which is administered in combination with an antibiotic against S. aureus, e.g., vancomycin, and optionally with a further antibiotic against a different bacterium. A pharmaceutical composition comprising as an active agent a VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75, and a pharmaceutically acceptable carrier. A nucleic acid molecule encoding a VHH antibody or a VHH antibody containing unit according to any one of embodiments 1 -72, preferably in operative linkage with a heterologous expression control sequence. A vector comprising a nucleic acid molecule according to embodiment 89. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule according to embodiment 89 or a vector according to embodiment 90. The cell or organism of embodiment 91 , which is selected from a bacterium such as E. coli Bacillus sp., a unicellular eukaryotic organism, e.g., yeast such as Pichia pastoris, or Leishmania, an insect cell, a mammalian cell, or a plant cell. A method for recombinant production of a VHH antibody of any one of embodiments 1 -72, comprising cultivating a cell or an organism of embodiment 91 or 92 in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium. The method of embodiment 93, comprising cultivating a yeast such as Pichia pastoris and obtaining the VHH antibody from the medium. Use of the VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 for detecting Hlg and / or LukF and optionally Hla in a sample. The use of embodiment 95, wherein the sample is a biological sample, e.g., a body fluid such as saliva, sputum, a lung lavage, a swab, blood, serum or plasma, a stool sample, a tissue sample, or a biopsy sample. The use of embodiment 95 or 96, wherein the detection comprises the binding of at least 2 different VHH antibodies to an Hlg and / or LukF molecule. The use of embodiment 97, wherein the detection comprises the binding of at least 2 different VHH antibodies, e.g., 2, 3, or 4 VHH antibodies each directed against a different epitope, to an Hlg and / or LukF molecule. The use of any one of embodiments 95-98, wherein the detection comprises a sandwich assay, e.g., a sandwich ELISA. . The use of any one of embodiments 95-99, wherein the detection comprises a prior Hlg enrichment and / or LukF enrichment and optionally Hla enrichment step. . A method for the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with an Hlg- positive and optionally Hla-positive S. aureus comprising administering an effective dose of the VHH antibody of any one of embodiments 1 -72 or the set of any one of embodiments 73-75 or the pharmaceutical composition of embodiment 88 to a subject in need thereof, particularly to a human subject. Description of the Invention

[0081] VHH antibodies

[0082] The present invention relates to a VHH antibody recognizing an S. aureus gammahemolysin (Hlg).

[0083] The VHH antibody may be a monovalent heavy chain-only antibody comprising a CDR1 domain, a CDR2 domain and a CDR3 domain linked by framework regions including, but not being limited to, whole VHH antibodies, e.g., native VHH antibodies comprising framework regions derived from camelids, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, e.g. a fusion protein with an immunoglobulin or non-immunoglobulin peptide or polypeptide, as long as it shows the properties according to the invention.

[0084] The VHH antibody of the present invention may be glycosylated or non-glycosylated.

[0085] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including, but not limited to, the methods known as Kabat, Chothia, and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using Kabat and some using IMGT, for example. According to some embodiments of the present invention, the CDR sequences of a VHH variable region are determined using the Kabat method. CDRs may also be defined through a multiple alignment (with many other VHH antibodies), to identify the hot-spots of variability and relate them to a standard VHH antibody structure. It is also possible to define CDRs by analyzing the structure of the VHH antibody and deciding what is a loop and what is the antibody’s scaffold. In some cases, CDR-adjacent residues are also variable, and are therefore included in the CDR definition. The present invention is also directed to a covalent or non-covalent conjugate of a VHH antibody molecule to a non-proteinaceous structure, for example, a labeling group, a capture group such a solid phase-binding group, or an effector group such as a toxin. For example, the heterologous moiety may be from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.

[0086] The VHH antibody of the present invention is particularly a monoclonal VHH antibody characterized by a specific amino acid sequence. The VHH antibody may be produced in a prokaryotic host cell, a yeast cell or a mammalian cell, e.g., a CHO cell. In certain embodiments, the VHH antibody is non-glycosylated. In certain embodiments, the VHH antibody is glycosylated, wherein a carbohydrate structure may be derived from a glycosylation site introduced into the VHH sequence and / or from a fusion partner.

[0087] A VHH antibody according to the present invention is characterized by (i) a CDR3 sequence, (ii) a combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (iii) by a complete VHH sequence, or (iv) by competition with a specific reference antibody. Specific CDR and VHH sequences are provided in the Tables, Figures and the Sequence Listing.

[0088] According to the present invention, sequences related to the above sequences are encompassed. These related sequences are defined by having a minimum identity to a specifically indicated amino acid sequence, e.g., a CDR or VHH sequence. This identity is indicated over the whole length of the respective reference sequence and may be determined by using well-known algorithms such as BLAST.

[0089] In particular embodiments, a related CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated CDR3 sequence, e.g., a substitution of 1 , 2, or 3 amino acids.

[0090] In particular embodiments, a related combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, e.g., a substitution of 1 , 2, 3, 4, 5 or 6 amino acids by different amino acids.

[0091] In particular embodiments, a related VHH sequence has an identity of least 70%, at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence, e.g., a substitution of 1 , 2, 3, 4, 5 or up to 20 amino acids.

[0092] Further, the invention refers to a VHH antibody, which binds to the same epitope of an S. aureus Hlg polypeptide as a specific VHH antibody disclosed herein. In certain embodiments, a competing VHH antibody binds the same or an overlapping epitope on the S. aureus Hlg polypeptide. For example, the invention refers to a VHH antibody, which binds to the same epitope as a reference antibody, e.g., VHH antibody Bm29C02 or Bm29E06 (binding to epitope 2 on HlgB), VHH antibody Bm29B04 (binding to epitope 1 on HlgB as shown in Fig. 11 ), VHH antibody Bm31 G03 or Bm31 G12 (binding to epitope 1 on HlgA), VHH antibody Bm32A03 (binding to epitope 1 on HlgC) or VHH antibody Bm32A08 (binding to epitope 2 on HlgC). Binding to the same epitope may be determined by competition measurements, e.g., by label-free biolayer interferometry (BLI) performed as a cross-competition or epitope binning assay using a label-free detection system, e.g., an Octet® system from Sartorius, according to the manufacturer's instructions.

[0093] In embodiments, the VHH antibody Bm29B04 binds to an epitope on HlgB which is located within a stretch of amino acids as shown in Fig. 11 :

[0094] NGWGPYGRDSFHPTYGNELFLAGRQSSA (SEQ ID NO: 206) wherein the epitope is particularly defined by 13 bold marked amino acid residues which interact with the VHH antibody or by at least 10, 11 or 12 of these amino acids.

[0095] A corresponding epitope is also found on LukF (against which the VHH antibody Bm29B04 cross-reacts). This epitope is located within a stretch of amino acids (as shown in Fig. 11 ):

[0096] NGWGPYGRDSYHSTYGNEMFLGSRQSNL (SEQ ID NO:207) wherein the epitope is particularly defined by 13 bold marked amino acid residues which interact with the VHH antibody or by at least 10, 11 or 12 of these amino acids.

[0097] The epitope identified in the LukF sequence differs from the epitope identified in the HlgB sequence by only one of the bold marked amino acids (S23 instead of G23).

[0098] Further, the invention refers to a VHH antibody which is a variant of one of the specifically disclosed VHH antibodies. In particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1 , CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by another amino acid, while preserving structural integrity and epitope-binding of the VHH antibody. These exchanges can be conservative (i.e., by a similar amino acid) or nonconservative.

[0099] In further particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1 , CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by a conservative amino acid substitution, i.e. a substitution of an amino acid by another amino acid with similar biochemical properties, for example a substitution of an aliphatic amino acid, e.g. Gly, Ala, Vai, Leu, or lie, for another aliphatic amino acid; a substitution of a basic amino acid, e.g. His, Lys or Arg, against another basic amino acid or against Met; a substitution of an acidic amino acid or an amide thereof, e.g., Asp, Glu, Asn or Gin, against another acidic amino acid or an amide thereof; a substitution of an aromatic amino acid, e.g., Phe, Tyr or Trp, against another aromatic amino acid.

[0100] In certain embodiments, the VHH antibody is a humanized VHH variant which differs from the original VHH sequence by at least one framework mutation, e.g., by 1 to 15 or 1 to 10 framework mutations, wherein the sequence identity to a human immunoglobulin variable region, e.g., a human germline immunoglobulin variable region is increased. In particular embodiments, a humanized VHH variant comprises humanized framework regions wherein at least one amino acid, e.g.,1 to 10 amino acids in the framework regions, i.e., the regions outside the hypervariable CDR1 , CDR2 and CDR3 regions are replaced by other amino acids found in a human framework region.

[0101] Methods of humanizing antibody framework sequences are well known in the art as described in a review article by Rossotti et al. (FEBS J. 289 (2022), 4304-4327) and the citations listed therein, the contents of which are herein incorporated by reference. The humanization changes the framework but keeps the paratope intact. In particular embodiments, the combination of CDR1 , CDR2 and CDR3 sequences remains unaltered. Thus, a humanized VHH antibody variant of the present invention may comprise a combination of CDR1 , CDR2 and CDR3 sequences of the respective camelid VHH antibody and humanized framework regions.

[0102] In particular embodiments, a humanized VHH antibody variant of the present invention may comprise a VHH sequence as described herein wherein at least one and up to 15 or up to 10 amino acids of the original camelid framework sequence are replaced by amino acids present at a corresponding position in a framework sequence of a variable domain of a human antibody heavy chain, particularly of a human heavy IgG chain, e.g., a germline version of a variable domain of a heavy IgG chain. Tolerability of individual amino acid replacements at individual positions may be determined in a straightforward manner by testing the characteristics of the VHH antibody variants as described herein below in Example 1 (Antigen binding), Example 2 (Identification of Hlg epitopes), Examples 3, 4 and 7 (Inhibition of erythrocyte hemolysis), Example 5 (Inhibition of cytotoxicity), Example 6 (Thermostability), and Examples 8 and 9 (Inhibition of cell membrane disruption). A first test can also be done computationally on the basis of the here disclosed x-ray structure of the VHH-HlgB complex.

[0103] In further particular embodiments, a humanized VHH antibody variant of the present invention comprises a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88 or 90-92; and has an overall amino sequence identity of at least 80%, at least 90% or at least 95% and up to 99% to the VHH sequence as shown in SEQ. ID NO: 5, 9, 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, or 89. In further particular embodiments, the VHH antibody is selected from antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 5 are replaced by another amino acid.

[0104] In further particular embodiments, the VHH antibody is selected from antibody Bm29H04 having a VHH sequence as shown in SEQ. ID NO: 9 or a VHH antibody, which is a variant thereof including a humanized VHH variant . In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 9 are replaced by another amino acid.

[0105] In further particular embodiments, the VHH antibody is selected from antibody Bm29E06 having a VHH sequence as shown in SEQ. ID NO: 13 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 13 are replaced by another amino acid.

[0106] In further particular embodiments, the VHH antibody is selected from antibody Bm29F05 having a VHH sequence as shown in SEQ. ID NO: 17 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 17 are replaced by another amino acid.

[0107] In further particular embodiments, the VHH antibody is selected from antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 21 are replaced by another amino acid.

[0108] In further particular embodiments, the VHH antibody is selected from antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 25 are replaced by another amino acid.

[0109] In further particular embodiments, the VHH antibody is selected from antibody Bm29D07 having a VHH sequence as shown in SEQ. ID NO: 29 or a VHH antibody, which is a variant thereof including a humanized VHH variant . In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 29 are replaced by another amino acid.

[0110] In further particular embodiments, the VHH antibody is selected from antibody Bm31 D04 having a VHH sequence as shown in SEQ. ID NO: 33 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 33 are replaced by another amino acid.

[0111] In further particular embodiments, the VHH antibody is selected from antibody Bm31 G03 having a VHH sequence as shown in SEQ. ID NO: 37 or a VHH antibody, which is a variant thereof including a humanized VHH variant . In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 37 are replaced by another amino acid.

[0112] In further particular embodiments, the VHH antibody is selected from antibody Bm31 G04 having a VHH sequence as shown in SEQ. ID NO: 41 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 41 are replaced by another amino acid.

[0113] In further particular embodiments, the VHH antibody is selected from antibody Bm31 F09 having a VHH sequence as shown in SEQ. ID NO: 45 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 45 are replaced by another amino acid. In further particular embodiments, the VHH antibody is selected from antibody Bm31 G12 having a VHH sequence as shown in SEQ. ID NO: 49 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 49 are replaced by another amino acid.

[0114] In further particular embodiments, the VHH antibody is selected from antibody Bm31A09 having a VHH sequence as shown in SEQ. ID NO: 53 or a VHH antibody, which is a variant thereof including a humanized VHH variant . In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 53 are replaced by another amino acid.

[0115] In further particular embodiments, the VHH antibody is selected from antibody Bm31 D02 having a VHH sequence as shown in SEQ. ID NO: 57 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 57 are replaced by another amino acid.

[0116] In further particular embodiments, the VHH antibody is selected from antibody Bm31A04 having a VHH sequence as shown in SEQ. ID NO: 61 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 61 are replaced by another amino acid.

[0117] In further particular embodiments, the VHH antibody is selected from antibody Bm31A08 having a VHH sequence as shown in SEQ. ID NO: 65 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 65 are replaced by another amino acid.

[0118] In further particular embodiments, the VHH antibody is selected from antibody Bm31 B04 having a VHH sequence as shown in SEQ. ID NO: 69 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 69 are replaced by another amino acid.

[0119] In further particular embodiments, the VHH antibody is selected from antibody Bm32A03 having a VHH sequence as shown in SEQ. ID NO: 73 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 73 are replaced by another amino acid.

[0120] In further particular embodiments, the VHH antibody is selected from antibody Bm32B07 having a VHH sequence as shown in SEQ. ID NO: 77 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 77 are replaced by another amino acid.

[0121] In further particular embodiments, the VHH antibody is selected from antibody Bm32D01 having a VHH sequence as shown in SEQ. ID NO: 81 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 81 are replaced by another amino acid.

[0122] In further particular embodiments, the VHH antibody is selected from antibody Bm32A08 having a VHH sequence as shown in SEQ. ID NO: 85 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 85 are replaced by another amino acid.

[0123] In further particular embodiments, the VHH antibody is selected from antibody Bm32F03 having a VHH sequence as shown in SEQ. ID NO: 89 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 89 are replaced by another amino acid. In further particular embodiments, the VHH antibody is selected from an antibody having a VHH sequence as shown in SEQ. ID NO: 5, 9, 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, or 89 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 13 are replaced by another amino acid.

[0124] Further, the present invention relates to a nucleic acid molecule, e.g., a DNA molecule, encoding a VHH as indicated above, or a vector, comprising said nucleic acid molecule as indicated above in operative linkage with an expression control sequence, particularly with a heterologous expression control sequence. Furthermore, the invention relates to a cell comprising a nucleic acid molecule or a vector as described above. Vectors for the recombinant production of VHH antibodies are well-known in the art. In certain embodiments, the vector is an extrachromosomal vector. In other embodiments, the vector is a vector for genomic integration. The cell may be a known host cell for producing antibodies or antibody fragments, e.g., a prokaryotic cell such as an E. coli or a Bacillus sp. cell, a yeast cell, particularly a Pichia yeast cell, an insect cell, e.g., an Sf-9 cell, or a mammalian cell, e.g., a CHO cell, or a plant cell. In certain embodiments, the cell comprises the nucleic acid or the vector extrachromosomally. In other embodiments, the cell comprises the nucleic acid or the vector integrated into the genome, e.g., as a genomically integrated expression cassette.

[0125] Still a further aspect of the present invention is a method of recombinantly producing a VHH antibody by growing a cell as described above in a culture medium and obtaining the VHH antibody from the cell or the culture medium. Suitable culture media and culture conditions are well known in the art.

[0126] Binding to S. aureus toxins Hlg and LukF

[0127] The VHH antibodies of the present invention bind to an S. aureus Hlg polypeptide, i.e. , HlgA, HlgB or HlgC. The inventors have identified VHH antibodies, which bind with high affinity to different epitopes on S. aureus HlgA, HlgB or HlgC, as shown in Table 1 , supra. The inventors further found that VHH antibodies Bm29B04 having a VHH sequence as shown in SEQ ID NO: 21 and Bm29B05 having a VHH sequence as shown in SEQ ID NO: 25 also bind the S. aureus toxin LukF with high affinity.

[0128] In the context of the present disclosure the term “S. aureus Hlg” encompasses S. aureus Hlg from different strains, e.g., MRSA-resistant strains, such as USA300, USA400 (CC1 ), USA500 (CC8) or ST80. These and other suitable strains are e.g., described by Diep et al. (2006); Cortes et al. (2017); Earls et al. (2019); Frisch et al. (2018) and Mairi et al. (2020), the contents of which are herein incorporated by reference.

[0129] In the context of the present disclosure the term “S. aureus LukF” encompasses S. aureus LukF from different strains, e.g., MRSA-resistant strains, such as ST80. These and other suitable strains are e.g., described in Witte et al. (2005) and Mairi et al. (2020), the contents of which are herein incorporated by reference.

[0130] It should be noted, however, that the terms “S. aureus Hlg” and “S. aureus LukF” also encompass naturally occurring variants of S. aureus Hlg and LukF polypeptides and genetically modified constructs as described herein.

[0131] The inventors have performed their selection and crystallization experiments with HlgA shown in SEQ ID NO: 1 , HlgB shown in SEQ ID NO: 2 and HlgC shown in SEQ ID NO: 3.

[0132] Further, the inventors have performed experiments with Hla shown in SEQ ID NO: 4 when determining the binding characteristics of tandem fusions directed to both Hlg and Hla.

[0133] In certain embodiments, the VHH antibody of the present invention binds to a S. aureus Hlg polypeptide, i.e., HlgA, HlgB or HlgC, wherein the binding affinity expressed as dissociation constant KD is about 1 nM or less, of about 0.1 nM or less, or of about 0.05 nM or less. The binding affinity may be determined as described herein in detail in the Examples using the polypeptides of SEQ. ID NO: 1 , 2 and 3 as described above. In certain embodiments, the VHH antibody of the present invention binds to a S. aureus LukF polypeptide, wherein the binding affinity expressed as dissociation constant KD is about 1 nM or less, of about 0.1 nM or less, or of about 0.05 nM or less. The binding affinity may be determined as described herein in detail in the Examples using the polypeptide of SEQ. ID NO: 200 as described above.

[0134] Neutralization of S. aureus toxins Hlg and LukF

[0135] The VHH antibodies of the present invention are capable of neutralizing S. aureus Hlg, particularly by inhibiting the hemolysis of blood cells, e.g., erythrocytes, and / or by inhibiting cytotoxic activity against HeLa cells as shown in Table 1 , supra.

[0136] Further, the VHH antibodies Bm29B04 as shown in Figure 19 and Bm29B05 neutralize not only Hlg but also the LukSF toxin pair.

[0137] The VHH antibodies of the present invention may block the biological activity of Hlg components or Hlg toxins and optionally Hla toxins, and optionally the biological activity of LukSF components or of the LukF toxin and / or inhibit receptor or membrane binding of Hlg components or Hlg toxins and optionally Hla toxins, and optionally the biological activity of LukSF components or of the LukF toxin and / or formation of oligomeric pores.

[0138] In certain embodiments, the VHH antibody of the present invention neutralizes HlgAB and inhibits HlgAB-mediated hemolysis of red blood cells. In particular embodiments, the VHH antibody recognizes HlgA, more particularly epitope 1 of HlgA, such as Bm31A04, Bm31A09, Bm31 D04, Bm31 G03 and Bm31 G04 including variants thereof. In further particular embodiments, the VHH antibody recognizes HlgB, more particularly epitope 1 or 2 of HlgB such as Bm29C02, Bm29H04, Bm29E06, Bm29F05, Bm29B04, or Bm29B05 including variants thereof. In certain embodiments, the VHH antibody of the present invention neutralizes HlgBC and inhibits HlgBC-mediated hemolysis of red blood cells. In particular embodiments, the VHH antibody recognizes HlgB, more particularly epitope 1 or 2 of HlgB such as Bm29C02, Bm29H04, Bm29E06, Bm29F05, Bm29B04, or Bm29B05 including variants thereof.

[0139] In certain embodiments, the VHH antibody of the present invention neutralizes HlgBC and inhibits HlgBC-mediated cytotoxicity against HeLa cells. In particular embodiments, the VHH antibody recognizes HlgC, more particularly epitope 1 or 2 of HlgC, such as Bm32A03, Bm32B07, and Bm32A08 including variants thereof. In further particular embodiments, the VHH antibody recognizes HlgB, more particularly epitope 1 or 2 of HlgB such as Bm29C02, Bm29H04, Bm29E06, Bm29F05, Bm29B04, or Bm29B05 including variants thereof.

[0140] In certain embodiments, the VHH antibody of the present invention neutralizes LukSF and inhibits LukSF-mediated cytotoxicity against macrophages. In particular embodiments, the VHH antibody recognizes LukF, more particularly an epitope as shown in Fig. 11 such as Bm29B04, or Bm29B05 including variants thereof.

[0141] In certain embodiments, the VHH antibody of the present invention is a tandem fusion and neutralizes HlgAB, HlgBC and Hla. In particular embodiments, the tandem fusion comprises two VHH units one of them recognizing HlgB and another recognizing Hla, e.g., a tandem fusion comprising the anti-HlgB VHH antibody Bm29C02 including variants thereof and an anti-Hla VHH antibody as shown in Table 3, such as Ma7A07 including variants thereof.

[0142] In certain embodiments, the VHH antibody of the present invention is a tandem fusion and neutralizes HlgAB, HlgBC, Hla and LukSF. In particular embodiments, the tandem fusion comprises two VHH units, one of them recognizing HlgB and LukF and another recognizing Hla, e.g., a tandem fusion comprising the anti-HlgB VHH antibody Bm29B04 including variants thereof or the anti-HlgB VHH antibody Bm29B05 including variants thereof and an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3, such as Ma7A07 including variants thereof. Stability

[0143] For the intended therapeutic application, the anti-HIg antibodies should not only be highly potent in Hlg neutralization, but also, they should be developable as biological drugs. This includes that they are stable enough to survive a lengthy, large-scale production process as well as transportation and storage (ideally for years in liquid formulation) without aggregation or loss of activity.

[0144] A good predictor for stability is thermostability, which can be measured, e.g., by thermal shift assays or specifically by differential scanning fluorimetry. The present inventors have identified several thermostable or hyperthermostable VHH antibodies as shown in Table 1 , supra.

[0145] In a particular embodiment, the invention relates to a VHH antibody, which is stable, particularly thermostable, or hyperthermostable. Preferably, the VHH antibody has a melting point (melting temperature) of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least about 80°C, of at least 90°C or of at least about 95°C, when measured under non-reducing and / or reducing conditions, e.g., in the absence of a reducing agent or the presence of a reducing agent such as dithiothreitol (DTT). Melting temperatures are determined as described herein.

[0146] Sets of VHH antibodies

[0147] In a further aspect, the present invention relates to a set comprising at least 2, 3, 4 or more different VHH antibodies wherein the set comprises at least one VHH antibody as described above. In such a set, the individual VHH antibodies are present in suitable molar ratios. Typically, the molar ratios are in the range of about 2:1 to about 1 :2, particularly about 1.5:1 to about 1 :1.5, even more particularly about 1 :1. In certain embodiments, the VHH antibody set may comprise a single composition wherein the VHH antibodies in said set consist of a predetermined number of different species of VHH antibodies as described above. The VHH antibody set may comprise a plurality of compositions each comprising a different species of VHH antibody, e.g., as described above. The set of the present invention may be free from other VHH antibodies. In particular embodiments, the set of VHH antibodies comprises at least two, e.g., two, three or four complementary VHH antibodies, i.e. , VHH antibodies directed against different Hlg polypeptides, directed against different epitopes of a Hlg polypeptide and / or directed against different S. aureus toxins or virulence factors .

[0148] In certain embodiments, the set of VHH antibodies comprises complementary VHH antibodies directed against different Hlg polypeptides, e.g., at least one VHH antibody against HlgA and at least one VHH antibody against HlgB, at least one VHH antibody against HlgB and at least one VHH antibody against HlgC, or at least one VHH antibody against HlgA, at least one VHH antibody against HlgB and at least one VHH antibody against HlgC.

[0149] In certain embodiments, the set of VHH antibodies comprises complementary VHH antibodies directed against different epitopes on a Hlg polypeptide, e.g., at least one VHH antibody against epitope 1 of HlgB and at least one VHH antibody against epitope 2 of HlgB, or at least one VHH antibody against epitope 1 of HlgC and at least one VHH antibody against epitope 2 of HlgC.

[0150] In further embodiments, the set of VHH antibodies comprises complementary VHH antibodies directed against different toxins or virulence factors, of S. aureus, e.g., pore-forming toxins of S. aureus. For example, the set of VHH antibodies may comprise at least one VHH antibody against a Hlg polypeptide as described herein, e.g., a HlgB polypeptide, and at least one VHH antibody against another pore-forming toxin. In certain embodiments, the set comprises at least one VHH antibody against S. aureus Hla, particularly a VHH antibody described in PCT / EP2024 / 052663 as shown in Table 3, or at least one VHH antibody against Leukocidin S (LukS), Leukocidin F (LukF), Leukocidin E (LukE), Leukocidin D (LukD), Leukocidin G (LukG=LukL1 ), or Leukocidin H (LukH=LukL2).

[0151] In particular embodiments, the anti-Hla VHH antibody may comprise

[0152] (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 94-96, 98-100, 102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-136, 138-140, 142-144, 146-148, 150-152, 154-156, 158-160, 162-164, 166-168, 170-172, 174-176, 178-180, 182-184, 186-188, 190-192 or 194-196; or

[0153] (b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a).

[0154] In further particular embodiments, the anti-Hla VHH antibody may comprise

[0155] (a) a VHH sequence as shown in SEQ. ID NO: 93, 97, 101 , 105, 109, 113, 117, 121 , 125, 129, 133, 137, 141 , 145, 149, 153, 157, 161 , 165, 169, 173, 177, 181 , 185, 189 or 193; or

[0156] (b) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).

[0157] Sets of different VHH antibodies are useful for therapeutic and diagnostic applications as described herein in detail below.

[0158] In certain embodiments, a set of VHH antibodies comprises:

[0159] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0160] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3.

[0161] In further embodiments, a set of VHH antibodies comprises:

[0162] (i) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0163] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3.

[0164] In further embodiments, a set of VHH antibodies comprises: (i) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0165] (ii) an anti-Hla VHH antibody, particularly an anti-Hla VHH antibody as shown in Table 3.

[0166] In certain more specific embodiments, a set of VHH antibodies comprises:

[0167] (i) the anti-HlgB VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0168] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

[0169] In further more specific embodiments, a set of VHH antibodies comprises:

[0170] (i) the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0171] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

[0172] In further more specific embodiments, a set of VHH antibodies comprises:

[0173] (i) the anti-HlgB VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 215 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0174] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

[0175] In further embodiments, a set of VHH antibodies comprises: (i) the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0176] (ii) the anti-Hla VHH antibody Ma31A12 having a VHH sequence as shown in SEQ. ID NO: 169 or a VHH antibody, which is a variant thereof including a humanized variant , and / or

[0177] (iii) the anti-Hla VHH antibody Ma8E03 having a VHH sequence as shown in SEQ. ID NO: 133 or a VHH antibody, which is a variant thereof including a humanized variant .

[0178] In certain embodiments, a set of VHH antibodies comprises:

[0179] (i) the anti-HlgB VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant,

[0180] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant,

[0181] (iii) the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0182] (Iv) the anti-Hla VHH antibody Ma31A12 having a VHH sequence as shown in SEQ. ID NO: 169 or a VHH antibody, which is a variant thereof including a humanized variant.

[0183] In certain embodiments, a set of VHH antibodies comprises:

[0184] (i) the anti-HlgB VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant,

[0185] (ii) the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant,

[0186] (iii) the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof, including a humanized variant and (iv) the anti-Hla VHH antibody Ma8E03 having a VHH sequence as shown in SEQ. ID NO: 133 or a VHH antibody, which is a variant thereof including a humanized variant.

[0187] In certain embodiments, a set of VHH antibodies comprises at least one heteromultimeric VHH antibody comprising two or more different VHH antibody units, e.g.:

[0188] (i) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant;

[0189] (ii) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant;

[0190] (iii) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant;

[0191] (iv) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma31A12 having a VHH sequence as shown in SEQ. ID NO: 169 or a VHH antibody, which is a variant thereof including a humanized variant; and / or

[0192] (v) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma8E03 having a VHH sequence as shown in SEQ. ID NO: 133 or a VHH antibody, which is a variant thereof including a humanized variant.

[0193] For example, a set may comprise the hetero-multimeric VHH antibody Bm29C02 / Ma7A07, the monomeric VHH antibody Bm29B04 and at least one of the monomeric VHH antibodies Ma31A12 and Ma8E03.

[0194] Further, a set may comprise at least one of the hetero-multimeric VHH antibodies Bm29B04 / Ma31A12 and Bm29B04 / Ma8E03, the monomeric VHH antibody Bm29C02 and the monomeric VHH antibody Ma7A07.

[0195] Further, a set may comprise at least one of the hetero-multimeric VHH antibodies Bm29B04 / Ma31A12 and Bm29B04 / Ma8E03, and the hetero-multimeric VHH antibody Bm29C02 / Ma7A07.

[0196] Further, the set may comprise at least one of the hetero-multimeric VHH antibodies Bm29B04 / Ma7A07 and Bm29B05 / Ma7A07.

[0197] Monovalent and multivalent VHH antibodies

[0198] In certain embodiments, the VHH antibody of the present invention is in a monovalent format, i.e., it has a single binding site for an S. aureus polypeptide. In these embodiments, the VHH antibody may be present as such or covalently or non- covalently attached to a heterologous moiety, e.g., a peptidic or non-peptidic moiety.

[0199] In further embodiments, the VHH antibody of the present invention is in in a multimeric, e.g., dimeric, trimeric or tetrameric format. In these embodiments, several VHH antibody units may be covalently or non-covalently attached to each other together via a linker and / or a multimerization, e.g., dimerization, trimerization or tetramerization moiety. In these embodiments, the VHH antibody of the present invention may be a homo-multimeric or hetero-multimeric antibody. In certain embodiments, the VHH antibody is a homodimeric VHH antibody, wherein a VHH antibody unit is covalently attached to a dimerization moiety, e.g., an immunoglobulin IgG Fc fragment.

[0200] In certain embodiments, the VHH antibody is a heterodimeric VHH antibody, particularly a covalently linked VHH heterodimer comprising a first VHH antibody unit and a second VHH antibody unit wherein the first VHH antibody unit and the second VHH antibody unit bind to different antigens or different epitopes on the same antigen.

[0201] In certain embodiments, the multimeric VHH antibody is a hetero-multimeric VHH antibody comprising two or more, e.g., 2, 3 or 4 different VHH antibody units, i.e. , VHH antibody units directed against different Hlg polypeptides, directed against different epitopes of a Hlg polypeptide and / or directed against different S. aureus toxins or virulence factors, e.g., S. aureus Hla. In certain embodiments, the at least one VHH antibody unit comprises an anti-HIg VHH antibody as described herein.

[0202] In particular embodiments, the hetero-multimeric VHH antibody is a tandem fusion of VHH antibody units comprising a first anti-HIg VHH antibody unit, particularly an anti- HIg VHH antibody unit as described herein and a second VHH antibody wherein the first unit is covalently fused to the second unit.

[0203] The VHH antibody units are covalently fused to each other either directly or via a linker. In certain embodiments, the linker has a length of 5-50 amino acids, particularly a length of 10-30 amino acids. In certain embodiments, the linker is a flexible linker which may substantially consist or consist of amino acids selected from G, E, S and T.

[0204] In certain embodiments, the hetero-multimeric VHH antibody comprises at least two VHH antibody units directed against different Hlg polypeptides selected from HlgA, HlgB and HlgC, e.g., at least one VHH antibody unit against HlgA and at least one VHH antibody unit against HlgB, at least one VHH antibody unit against HlgB and at least one VHH antibody unit against HlgC, or at least one VHH antibody unit against HlgA, at least one VHH antibody unit against HlgB and at least one VHH antibody unit against HlgC. In certain embodiments, the hetero-multimeric VHH antibody comprises at least two VHH antibody units directed against different epitopes on a Hlg polypeptide, e.g., at least one VHH antibody unit against epitope 1 of HlgB and at least one VHH antibody unit against epitope 2 of HlgB, or at least one VHH antibody unit against epitope 1 of HlgC and at least one VHH antibody unit against epitope 2 of HlgC.

[0205] In further embodiments, the hetero-multimeric VHH antibody comprises at least two VHH antibody units directed against different toxins or virulence factors, of S. aureus, e.g., pore-forming toxins of S. aureus. For example, the hetero-multimeric VHH antibody may comprise at least one VHH antibody unite against a Hlg polypeptide as described herein, e.g., a HlgB polypeptide, and at least one VHH antibody unit against another pore-forming toxin. In certain embodiments, the hetero-multimeric VHH antibody comprises at least one VHH antibody unit against S. aureus alpha hemolysin (Hla), particularly a VHH antibody unit described in PCT / EP2024 / 052663, or at least one VHH antibody unit against Leukocidin S (LukS), Leukocidin F (LukF), Leukocidin E (LukE) or Leukocidin D (LukD).

[0206] In certain embodiments, the hetero-multimeric VHH antibody comprises a VHH unit recognizing HlgB and optionally LukF selected from:

[0207] (i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant; or

[0208] (ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0209] (iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant.

[0210] In certain embodiments, the hetero-multimeric VHH antibody comprises a VHH unit recognizing Hla selected from the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant. For example, the hetero-multimeric VHH antibody comprises two VHH antibody units which are:

[0211] (i) the anti-HlgB VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0212] (ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or

[0213] (iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

[0214] In specific embodiments, a hetero-multimeric VHH antibody is provided as shown in SEQ ID NO: 197, 198 or 199.

[0215] In a further embodiment, a hetero-multimeric VHH antibody may comprise two VHH antibody units which are:

[0216] (i) the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 9 or a VHH antibody, which is a variant thereof including a humanized variant, and

[0217] (ii) the anti-Hla VHH antibody Ma31A12 having a VHH sequence as shown in SEQ. ID NO: 169 or a VHH antibody, which is a variant thereof including a humanized variant.

[0218] In a further embodiment, a hetero-multimeric VHH antibody may comprise two VHH antibody units which are:

[0219] (i) a hetero-multimeric VHH antibody comprising the anti-HlgB VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 9 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti- Hla VHH antibody Ma8E03 having a VHH sequence as shown in SEQ. ID NO: 133 or a VHH antibody, which is a variant thereof including a humanized variant.

[0220] In certain embodiments, the VHH antibody including the hetero-multimeric VHH antibody may be covalently or non-covalently conjugated to a heterologous moiety, which is selected from a labeling group, a capture group, or an effector group, and wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.

[0221] In further embodiments, the heterologous moiety is selected from human serum albumin, an albumin-binding moiety, or an Fc fragment of an immunoglobulin molecule, e.g., IgA, IgD, IgE, IgG, IgM, or a subtype thereof. In still further embodiments, the heterologous moiety is selected from one or several non-peptidic polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG).

[0222] In specific embodiments, a hetero-multimeric VHH antibody is provided which further comprises a heterologous polypeptide, e.g., an immunoglobulin Fc fragment and wherein the heterologous polypeptide may be at the C-terminus of the hetero- multimeric VHH antibody.

[0223] Production of VHH antibodies

[0224] VHH antibodies including monomeric and multimeric VHH antibodies may be produced as described in WO 2022 / 023483 and WO 2022 / 023484, the contents of which are herein incorporated by reference, or by other methods well known in the art.

[0225] A VHH antibody may be recombinantly produced in a suitable host cell, e.g., in a prokaryotic or eukaryotic host cell or host organism. For this purpose, a nucleic acid molecule encoding the VHH antibody is introduced into the host cell or host organism and expressed in the host cell or host organism. The nucleic acid molecule may encode a monomeric VHH antibody or a subunit of a multimeric VHH antibody.

[0226] In a particular embodiment, the VHH antibody is recombinantly produced in a bacterium, e.g., E. coli or Bacillus. For example, expression in a bacterium may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or secretory expression and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression to the periplasm and / or into the culture medium.

[0227] In a further particular embodiment, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism, preferably in yeast, e.g., in Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Hansenula polymorpha, or in an animal cell, particularly in an insect cell, e.g., an Sf-9 cell, or a mammalian cell, e.g., human or a hamster cell such as HEK293F, CHO or COS-7. For example, expression in a eukaryotic host cell or host organism, e.g., yeast may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or preferably secretion from the host cell and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression into the culture medium.

[0228] Therapeutic applications

[0229] Still a further aspect of the present invention is the use of a VHH antibody as described above in medicine, particularly for therapeutic and / or in vitro or vivo diagnostic use. In certain embodiments, the VHH antibody is used in human medicine.

[0230] The VHH antibody of the present invention is useful in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with an Hlg-positive and optionally Hla-positive e S. aureus. In certain embodiments, the S. aureus is a drug-resistant S. aureus. Exemplary conditions include, but are not limited to a skin infection, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis.

[0231] In therapeutic applications, the VHH antibody is administered in an effective amount to a subject in need thereof, particularly to a human subject. The dose will depend on the specific type of agent, e.g., monovalent VHH antibody or multimeric VHH antibody, the type of disease, and the route of administration.

[0232] Typically, the VHH antibody is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well-known in the art.

[0233] Depending on the stage and the severity of the disorder, the pharmaceutical composition may be administered once or several times during the course of the disorder. For example, it may be administered once or several times daily, each second day, two times weekly or weekly for a suitable period of time.

[0234] In certain embodiments, the pharmaceutical composition is administered parenterally, e.g., by subcutaneous, intramuscular, or intravenous injection or by infusion. In further embodiments, the pharmaceutical composition is administered locally, e.g., topically, orally, nasally, for example by spraying or intrapulmonary, for example by inhalation as an aerosol.

[0235] In certain embodiments, treatment of a patient infected by Hlg-positive, optionally LukSF positive and optionally Hla-positive S. aureus involves a systemic application of one or several complementary neutralizing anti-HIg VHH antibodies including hetero-multimeric VHH antibodies, for example, by route of injection. In the case of pneumonia, inhalation might be a preferred application route. In the case of colonization of the nose, application of a spray is possible. In all cases, the treatment should reduce tissue damage by Hlg and / or LukSF and the virulence of the pathogen to allow the immune system to clear the infection. Prophylactic use of anti-HIg VHH antibodies including hetero-multimeric anti-Hlg / Hla or anti-Hlg / Hla / LukSF VHH antibodies is considered for a subject that is colonized by S. aureus or has been exposed or is likely exposed to the pathogen and / or a subject that is immune-compromised, prepared for surgery and / or has suffered injuries that make an infection likely.

[0236] Anti-HIg VHH antibodies including hetero-multimeric anti-Hlg / Hla or anti- Hlg / Hla / LukSF VHH antibodies will be effective also against highly antibiotic-resistant strains, and in an advanced state of infection, e.g., in an advanced state of sepsis, where the toxin’s action can no longer be cleared by antibiotic treatment alone.

[0237] In certain embodiments, a single tandem fusion VHH is administered. In further embodiments, a combination (set) of VHH fusions directed against different epitope classes is administered In further embodiments, a combination (set) of VHH fusions directed against different pore-forming toxins is administered. In further embodiments, a combination (set) of VHH fusions directed against different virulence factors of S. aureus is administered.

[0238] The VHH antibody may be administered alone as a monotherapy or together, e.g., sequentially, or simultaneously, with a further active agent as a combination therapy, particularly with a further agent that is useful in the prevention, treatment, and / or mitigation of a disorder caused by and / or associated with an infection with S. aureus and / or optionally a different bacterium. In certain embodiments, the VHH antibody is administered in combination with an antibiotic against S. aureus, e.g., vancomycin, and optionally with a further antibiotic against a different bacterium.

[0239] Diagnostic applications

[0240] Diagnostic applications include in vitro methods wherein a VHH antibody or a set of different VHH antibodies is used for detecting Hlg and / or LukF in a sample, e.g., in a body fluid such as saliva, sputum, a lung lavage, a swab, blood, serum or plasma, stool samples or in a tissue or biopsy sample. Diagnostic applications further include in vivo methods wherein a VHH antibody is used for detecting Hlg and / or LukF in a subject, particularly in a human patient. For diagnostic applications, the VHH antibody may carry a label for direct detection or used in combination with secondary detection reagents, e.g., biotin / streptavidin, detection antibodies, including conventional antibodies or VHH antibodies, for indirect detection according to established techniques in the art.

[0241] The present invention encompasses the use of anti-HIg VHH antibodies including hetero-multimeric VHH antibodies for detecting the toxin with high sensitivity. The availability of different complementary epitope classes can be exploited for highly sensitive detection. In certain embodiments, the detection format comprises the binding of at least 2 different VHH antibodies to an Hlg molecule, particularly the binding of at least 2 different VHH antibodies each directed against a different epitope. In particular embodiments, the detection comprises the use of a set of 2, 3 or 4 different VHH antibodies as described above.

[0242] In particular embodiments, the detection comprises a sandwich assay, e.g., a sandwich ELISA. In such a test format, VHH antibodies directed against several complementary epitope classes can be exploited for highly sensitive detection. More specifically, a first VHH antibody may be immobilized to a solid phase for capturing Hlg from the sample, and a second VHH antibody is employed in labelled form for the actual detection. A third and / or fourth VHH antibody can also be used for a prior enrichment step, e.g., by a capture and proteolytic release-approach (Frey & Gdrlich, 2014b; Frey & Gdrlich, 2014a; Vera Rodriguez et al., 2019). This allows a rapid and sensitive detection of Hlg which is highly important in life-threatening conditions.

[0243] Further, the present invention is explained in more detail by the following Figures and Examples.

[0244] Figure Legends

[0245] Figure 1 : Sequence alignment and highlighting of variable regions The figure shows an alignment of sequences from selected VHH antibodies listed in Table 2. Residues that deviate from the consensus are highlighted by a gray background. The three variable CDR regions are indicated.

[0246] Figure 2: Affinities of anti-HlgB VHHs for HlgB as measured by Bio-layer interferometry (BLI)

[0247] BLI experiments were performed for HlgB with the indicated VHH antibodies. Curves (grey) were fitted with a mass transport model (black). Calculated dissociation constants (KDS) are shown above the curves.

[0248] Figure 3: Affinities of anti-HlgA VHHs for HlgA

[0249] HlgA affinities for the binding of indicated VHHs were measured by BLI.

[0250] Figure 4: Affinities of anti-HlgC VHHs for HlgC

[0251] HlgC affinities of indicated VHHs were measured by BLI.

[0252] Figure 5: Dose dependence of toxin-induced hemolysis

[0253] Human erythrocytes were dispersed in 96 well plates and incubated with buffer, water (for a hypoosmotic shock) or with indicated amount of toxins for 150 min at 37°C followed by 30 min at 4°C. The plate was centrifuged to pellet intact cells. The supernatants were analyzed on a plate reader for absorbance at 412 nm. Lysed cells release their cytoplasmic contents including heme-bound hemoglobin, which has a maximum absorbance at 412 nm. Dashed lines indicate complete lysis (as triggered by osmotic shock) and baseline absorbance (buffer).

[0254] Figure 6: Inhibition of HlgAB-induced hemolysis by anti-HlgA VHH antibodies.

[0255] Hemolysis experiment was performed as described in Figure 5 using buffer, water or HlgAB toxin. Where indicated, HlgA was pre-incubated with VHH antibodies before mixing with HlgB. Figure 7: Inhibition of HlgAB-induced hemolysis by anti-HlgB VHH antibodies.

[0256] Hemolysis experiment was performed as described in Figure 5 using buffer, water or HlgAB toxin. Where indicated, HlgB was pre-incubated with VHH antibodies before mixing with HlgA.

[0257] Figure 8: Inhibition of HlgCB-induced hemolysis by anti-HlgB VHH antibodies.

[0258] Hemolysis experiment was performed as described in Figure 5 using buffer, water or HlgCB toxin. Where indicated, HlgB was pre-incubated with VHH antibodies before mixing with HlgC.

[0259] Figure 9: Inhibition of HlgCB-induced cell lysis by anti-HlgC VHH antibodies.

[0260] HeLa cells were dispersed in culture medium into an 8-well ibidi plate. HlgB and HlgC, or HlgB and HlgC pre-incubated with the indicated VHHs were then added as indicated. After overnight incubation at 37 °C and 5% CO2, the cells were examined by bright field microscopy.

[0261] Figure 10: Crystal structure of heterodimeric HlgB«Bm29B04 complex

[0262] HlgB and Bm29B04 were produced in the cytoplasm or periplasm of E. coli NEB Express respectively, and then purified by Ni-chelate chromatography. Dimeric HlgB*VHH complex was formed in solution by mixing HlgB and VHH in a 1 :1.2 stoichiometry and excess VHH was removed by size exclusion chromatography. The homogenous complex was crystallized, an x-ray diffraction dataset was collected at the European Synchrotron Radiation Facility (ESRF), and the structure was solved by molecular replacement to a resolution of 2.1 A and an Rfree of 0.22.

[0263] A) Crystal structure of the complex in ribbon representation.

[0264] B-C) Docking Bm29B04 onto the octameric (pre-pore) HlgAB toxin. Modelling is based on the alignment of the complex in A with a single HlgB unit in the octameric structure (PDB ID 4P1Y; Yamashita et al., 2014). Docked Bm29B04 is shown in two different orientations. Major clashes with neighboring HlgA subunits as well as with membrane-embedding domains indicate that the Bm29B04-bound HlgB cannot oligomerize into a pore-forming octamer or insert itself into membranes.

[0265] Figure 11 : Sequence alignment of PFTs

[0266] Figure shows an alignment of the amino acid sequences of the 10 pore-forming toxins from S. aureus. Bm29B04-interacting residues of HlgB (i.e., the epitope) were identified from the crystal structure shown in Figure 10; they are indicated by lines above the alignment and by marking them as bold underlined characters in the sequence. Note that the epitope is well conserved between HlgB and LukF - consistent with the cross-reaction of this VHH antibody.

[0267] Figure 12: Cross-reaction of Bm29B04 class members with LukF

[0268] LukF affinities of indicated VHHs as estimated by BLI.

[0269] Figure 13: Thermal stability of anti-HlgB VHH antibodies.

[0270] VHH antibodies were subjected to Differential scanning fluorimetry (DSF) as detailed in Example 6. Thermal unfolding is here measured as an enhanced fluorescence of added SYBR Orange following a stepwise increase in temperature with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak.

[0271] Figure 14: Thermal stability of anti-HlgA VHH antibodies.

[0272] Anti-HlgA VHH antibodies were subjected to DSF as detailed in Figure 13 and Example 6.

[0273] Figure 15: Thermal stability of anti-HlgC VHH antibodies.

[0274] Anti-HlgC VHH antibodies were subjected to DSF as detailed in Figure 13 and Example 6. Figure 16: Inhibition of hemolysis by tandem VHH (I).

[0275] Hemolysis experiment was performed as described in Figure 5 using Hla, HlgAB or HlgCB toxins. Where indicated, toxins were pre-incubated with buffer, anti-Hla VHH Ma7A07, anti-HlgB VHH Bm29C02 or an Ma7A07-Bm29C02 tandem fusion. Dashed lines indicate complete lysis (as triggered by osmotic shock) and baseline absorbance when buffer instead of a lytic agent had been added.

[0276] Figure 17: Inhibition of hemolysis by tandem VHH (II).

[0277] Hemolysis experiment was performed as described in Figure 16. Where indicated, toxins were pre-incubated with buffer, anti-Hla VHH Ma7A07, anti-HlgB VHH Bm29B04, and Bm29B04-Ma7A07 or Ma7A07-Bm29B04 tandem fusions. Dashed lines indicate complete lysis and baseline absorbance.

[0278] Figure 18: Dose dependence of toxin-induced cytoplasmic leakage.

[0279] THP1 cells were seeded in 96-well plates, differentiated with PMA (phorbol myristate acetate), and incubated with buffer, lysis solution or with indicated concentrations of toxins for 180 minutes at 37 °C (in cell culture medium +5% CO2), followed by 20 minutes at 22 °C. Lysed cells release their cytoplasmic contents including LDH (lactate dehydrogenase). LDH is quantified by a coupled enzymatic assay (CytoTox-ONETM), whereby LDH generates NADH from lactate and NAD+, and NADH is used by the helper enzyme diaphorase to convert resazurin into fluorescent resorufin (quantified by fluorescence with 571 nm excitation and 585 nm emission). Dashed lines indicate complete lysis (as induced by a lysis solution) and base fluorescence (buffer).

[0280] Figure 19: Block of LukSF-induced membrane damage by tandem VHH antibodies.

[0281] Membrane integrity loss experiment was performed as described in Figure 19 using LukSF toxin. Where indicated, toxins were pre-incubated with buffer, anti-Hla VHH Ma7A07, anti-HlgB VHH Bm29B04, Bm29B04-Ma7A07 or Ma7A07-Bm29B04 tandem fusions. Sequences of Hla and Hlg components used for assays as described

[0282] >Hlg-A (UniProt P0A074 (30-309)) (SEQ ID NO. 1)

[0283] ENKIEDIGQGAEIIKRTQDITSKRLAITQNIQFDFVKDKKYNKDALWKMQGFISSRTT YSDLKKYPYIKRMIWPFQYNISLKTKDSNVDLINYLPKNKIDSADVSQKLGYNIGGNF QSAPSIGGSGSFNYSKTISYNQKNYVTEVESQNSKGVKWGVKANSFVTPNGQVSA YDQYLFAQDPTGPAARDYFVPDNQLPPLIQSGFNPSFITTLSHERGKGDKSEFEITY GRNMDATYAYVTRHRLAVDRKHDAFKNRNVTVKYEVNWKTHEVKIKSITPK

[0284] >Hlg-B (UniProt P0A077 (36-325)) (SEQ ID NO. 2)

[0285] EGKITPVSVKKVDDKVTLYKTTATADSDKFKISQILTFNFIKDKSYDKDTLVLKATGNI NSGFVKPNPNDYDFSKLYWGAKYNVSISSQSNDSVNWDYAPKNQNEEFQVQNTL GYTFGGDISISNGLSGGLNGNTAFSETINYKQESYRTTLSRNTNYKNVGWGVEAHK IMNNGWGPYGRDSFHPTYGNELFLAGRQSSAYAGQNFIAQHQMPLLSRSNFNPEF LSVLSHRQDGAKKSKITVTYQREMDLYQIRWNGFYWAGANYKNFKTRTFKSTYEID WENHKVKLLDTKETENNK

[0286] >Hlg-C (UniProt Q07227 (30-315)) (SEQ ID NO. 3)

[0287] ANDTEDIGKGSDIEIIKRTEDKTSNKWGVTQNIQFDFVKDKKYNKDALILKMQGFISS RTTYYNYKKTNHVKAMRWPFQYNIGLKTNDKYVSLINYLPKNKIESTNVSQTLGYNI GGNFQSAPSLGGNGSFNYSKSISYTQQNYVSEVEQQNSKSVLWGVKANSFATES GQKSAFDSDLFVGYKPHSKDPRDYFVPDSELPPLVQSGFNPSFIATVSHEKGSSDT SEFEITYGRNMDVTHAIKRSTHYGNSYLDGHRVHNAFVNRNYTVKYEVNWKTHEIK

[0288] VKGQN

[0289] >Hla (UniProt P09616 (27-319)) (SEQ ID NO. 4)

[0290] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTK GTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMS TLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIF NNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGF SPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSS

[0291] ERYKIDWEKEEMTN

[0292] >LukF (UniProt Q5FBD2 (25-325)) (SEQ ID NO: 200)

[0293] AQHITPVSEKKVDDKITLYKTTATSDSDKLKISQILTFNFIKDKSYDKDTLILKAAGNIY SGYTKPNPKDTISSQFYWGSKYNISINSDSNDSVNWDYAPKNQNEEFQVQQTVG YSYGGDINISNGLSGGGNGSKSFSETINYKQESYRTSLDKRTNFKKIGWDVEAHKI MNNGWGPYGRDSYHSTYGNEMFLGSRQSNLNAGQNFLEYHKMPVLSRGNFNPE FIGVLSRKQNAAKKSKITVTYQREMDRYTNFWNQLHWIGNNYKDENRATHTSIYEV

[0294] D WE N HTVKL I DTQ S KE KN P M S

[0295] >LukS (UniProt Q53746 (30-312)) (SEQ ID NO: 201 ) NNIENIGDGAEWKRTEDTSSDKWGVTQNIQFDFVKDKKYNKDALILKMQGFINSKT TYYNYKNTDHIKAMRWPFQYNIGLKTNDPNVDLINYLPKNKIDSVNVSQTLGYNIGG NFNSGPSTGGNGSFNYSKTISYNQQNYISEVEHQNSKSVQWGIKANSFITSLGKMS GHDPNLFVGYKPYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSEFE ITYGRNMDVTHATRRTTHYGNSYLEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGH N

[0296] >LukD (UniProt Q2FXB1 (27-327)) (SEQ ID NO: 202)

[0297] AQHITPVSEKKVDDKITLYKTTATSDNDKLNISQILTFNFIKDKSYDKDTLVLKAAGNI NSGYKKPNPKDYNYSQFYWGGKYNVSVSSESNDAVNVVDYAPKNQNEEFQVQQ TLGYSYGGDINISNGLSGGLNGSKSFSETINYKQESYRTTIDRKTNHKSIGWGVEAH KIMNNGWGPYGRDSYDPTYGNELFLGGRQSSSNAGQNFLPTHQMPLLARGNFNP

[0298] EFISVLSHKQNDTKKSKIKVTYQREMDRYTNQWNRLHWVGNNYKNQNTVTFTSTY EVDWQNHTVKLIGTDSKETNPGV

[0299] >LukL2 (UniProt Q2FFA2 (33-351 )) (SEQ ID NO: 203)

[0300] DSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAPDDIGKNGKITKRTETVYDEKTNIL QNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHKEEKNSNWLKYPSEYHVDFQV KRNRKTEILDQLPKNKISTAKVDSTFSYSSGGKFDSTKGIGRTSSNSYSKTISYNQQ NYDTIASGKNNNWHVHWSVIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFA

[0301] SKYRYPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILE KNKDGQRLIVTYEVDWKNKTVKWDKYSDDNKPYKEG

[0302] >LukE (UniProt Q2FXB0 (24-306)) (SEQ ID NO: 204)

[0303] NTNIENIGDGAEVIKRTEDVSSKKWGVTQNVQFDFVKDKKYNKDALIVKMQGFINSR

[0304] TSFSDVKGSGYELTKRMIWPFQYNIGLTTKDPNVSLINYLPKNKIETTDVGQTLGYNI

[0305] GGNFQSAPSIGGNGSFNYSKTISYTQKSYVSEVDKQNSKSVKWGVKANEFVTPDG

[0306] KKSAHDRYLFVQSPNGPTGSAREYFAPDNQLPPLVQSGFNPSFITTLSHEKGSSDT

[0307] SEFEISYGRNLDITYATLFPRTGIYAERKHNAFVNRNFWRYEVNWKTHEIKVKGHN

[0308] >LukL1 (UniProt Q2FFA3 (30-338)) (SEQ ID NO: 205)

[0309] KINSEIKQVSEKNLDGDTKMYTRTATTSDSQKNITQSLQFNFLTEPNYDKETVFIKAK

[0310] GTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKNQDESREV

[0311] KYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWK

[0312] VEAHLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVS

[0313] EGFNPEFLAVMSHDKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHV

[0314] DKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK

[0315] Anti-HIg VHH antibody sequences

[0316] >Bm29C02 (SEQ ID NO. 5)

[0317] QVQLVESGGGLVQPGGSLRLSCTASGFTLDDWAIGWFRQAPGKEREGVSCTSSR ERSSYYRDSVKGRFTISRDSTSNTVYLQMNELTLEDTAVYYCAADPQPYPCSLGQL GRYTTWGQGTQVTVSS >Bm29H04 (SEQ ID NO. 9)

[0318] QVQLVESGGGLVQPGGSLRLSCTASGVSLDDFAIGWFRQAPGKEREGVSCSSSR DRSEYYRDSVKGRFTISRDSQKNTVDLQMNSLTIEDTAVYYCAADPQPYPCSVGSL DRYNSWGQGTLVTVSS

[0319] >Bm29E06 (SEQ ID NO. 13)

[0320] QVQLVESGGGLVQPGGSLRLSCTASGVSLYDFAVGWFRQAPGKEREGVSCSSSR DRSEYYRDSVKGRFDIVRDNLKNIIYLQMDNLTIEDTAVYFCAADSQPYPCSLGSLE RYDSWGQGTQVTVSS

[0321] >Bm29F05 (SEQ ID NO. 17)

[0322] QVQLVESGGGLVQPGGSLRLSCTASGVSLYDFAVGWWRQAPGKEREGVSCTSS RDRSEYYRDSVKGRFTIVRDNLKNTIYLQMDSLTIEDTAVYFCAADSQPYPCSLGSL ERYDSWGQGIQVTVSS

[0323] >Bm29B04 (SEQ ID NO. 21 )

[0324] QVQLVESGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKEREAVACISNGD GVANVPNSMKGRFTISSDSAKNTVHLRADSLKADDTGVYYCAAVKERLCVHEGME YWGKGTQVTVSS

[0325] >Bm29B05 (SEQ ID NO. 25)

[0326] QVQLVESGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKEREAVACISNGD GVTNYPNSMRGRFTISSDSAKNTVYLQTDSLKPDDTGVYYCATAKERLCVQGGME YWGKGTLVTVSS

[0327] >Bm29D07 (SEQ ID NO. 29)

[0328] QVQLVESGGGLVQPGGSLRLSCTASSIIVSYYAIGWFRQAPGKGREGVSRITVPAG NLYYADSVKGRFSITRDDVKNTVYLQMNSFQVEDTATYYCAVSYGDSWVDPSYVD HWGQGIQVTVSS

[0329] >Bm31 D04 (SEQ ID NO. 33)

[0330] QVQLVESGGGLVQPGGSLRLSCAASGIIFDFYDMGWYRQAPGKQRELVAGISISGS TTYADSVKGRFTISRDNAKNTIYLQMNSLKPEDTAVYYCNAHHVDSDYLRGYDYFG QGTQVTVSS

[0331] >Bm31 G03 (SEQ ID NO. 37)

[0332] QVQLVESGGGLVQAGGSLRLSCAASGIIFSFYDMGWYRQAPGKQREWATISISGR TTYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYICNAQHVDSDYLAGYDYFG QGTQVTVSS

[0333] >Bm31 G04 (SEQ ID NO. 41 )

[0334] QVQLVESGGGLVQPGGSLRLSCAASGITFTFYDMGWYRQAPGKQRELVATISISSS

[0335] TTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAQHVDSDYLAGYDYF GQGTQVTVSS >Bm31 F09 (SEQ ID NO. 45)

[0336] QVQLVESGGALVQPGGSLRLSCAASGFTFDDYVMSWVRQAPGTGLEWVSSIHTA SSDTYYADSVKDRFTISTDNAKNTLYLQMNSLKPEDTAVYYCAADKAPLTIATMTRD EYDYWGQGTQVTVSS

[0337] >Bm31 G12 (SEQ ID NO. 49)

[0338] QVQLVESGGGLVQPGGSLRLSCAAAGFTVDDYVMSWVRQVPGKGREWVSSIHSY SSDTYYADSVKGRFTISTDNAKNTLYLQMNSLKPEDTAVYYCAADKDPLTIATMTRY EYDYWGQGTQVTVSS

[0339] >Bm31A09 (SEQ ID NO. 53)

[0340] QVQLVQSGGGLVQAGGSLRLSCAASGSTGSIYVMGWFRQAPGKQRELVASITRG EGSANYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCHADTGLGDYSDYA PHGYKYDYWGQGTQVTVSS

[0341] >Bm31 D02 (SEQ ID NO. 57)

[0342] QVQLVESGGGLVQAGGSLRLSCAASGSTGSIYVMGWFRQAPGKQRELVASITRGE

[0343] GSANYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCHADTGLGDYSDYAP HGYKYDYWGQGTQVTVSS

[0344] >Bm31A04 (SEQ ID NO. 61 )

[0345] QVQLVQSGGGLVQPGGSLRLSCAASESISNIYVMGWFRQAPGKQRELVAAITSSN NTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADWGLGGYSDYGEY DYWGQGTQVTVSS

[0346] >Bm31A08 (SEQ ID NO. 65)

[0347] QVQLVESGGGLAQPGGSLTLSCAASGWWSKYVMSWVRQVPEKGFEWVSGIKSD

[0348] GSDTYYADSVKGRFTVSGDLAKKTVYLQLNSLKPEDTALYYCVLEYGTSWPPRVE EYDYWGQGTQVTVSS

[0349] >Bm31 B04 (SEQ ID NO. 69)

[0350] QVQLVESGGGLVQPGGSLRLSCAASGSSFSIYGMSWYRQAPGNQRELVALINVGD WTTYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCKADLSVSGSSFIRTYW GQGTQVTVSS

[0351] >Bm32A03 (SEQ ID NO. 73)

[0352] QVQLVESGGALVQTGGSLRLSCVTSGSISSIYAMPWFRQAPGKEREFVAAISWHT NLAYYADSVKGRFTISKDNAKNTVYLQMDSLKPEDTATYYCNVPSKEEYDRTSYYP TGGSWGQGTQVTVSS

[0353] >Bm32B07 (SEQ ID NO. 77) QVQLVESGGGLVQTGGSLRLSCATSGRISSIYAMPWFRQAPGKEREFVAAISWHT DLAYYADSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCNIPSKEEYDRNYYYP TGGSWGQGTQVTVSS

[0354] >Bm32D01 (SEQ ID NO. 81 )

[0355] QVQLVESGGGLVQTGGSLRLSCATSGRISSIYAMPWFRQAPGKEREFVAAISWHT DLTYYADSVKDRFTISKDNAKATVYLQMNNLQPEDTAVYYCNVPSKEQYDRTSYYP TGGSWGQGTQVTVSS

[0356] >Bm32A08 (SEQ ID NO. 85)

[0357] QVQLVESGGGLVQAGGSLRVSCAASGDWFIINSMAWFRQAPGKQREQVATISHG GIPRYADSVKGRFTISRDNSKGAVYLQMNNLKSDDTAMYVCAAGGLDYGFPDPTP EEFDYWGRGTQVTVSS

[0358] >Bm32F03 (SEQ ID NO.89)

[0359] QVQLVESGGGLVQAGGSLRVSCAASGDWFIINSMAWFRQAPGKQREQVATISHG GIPRYADSVKGRFTISRDDSKDAVYLQMNNLKSDDTAMYVCAAGGLDYGFPDPTP EEFDYWGRGTQVTVSS

[0360] Anti-Hla VHH antibody sequences

[0361] >Ma7A07 (SEQ ID NO: 93)

[0362] QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASS ALSIYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAM DLWGKGTLVTVSS

[0363] >Ma7A08 (SEQ ID NO: 97)

[0364] QVQLVESGGGLVQPGGSLTLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSV LSVYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAM DLWGKGTLVTVSS

[0365] >Ma7B03 (SEQ ID NO: 101 )

[0366] QVQLVESGGGLVQPGGSLRLSCAASGFTLSNYYVGWFRQAPGKQREGVSSIGSS DLSIYIADSVKGRFTISRDNAKNTIYLHMNNLKPDDTGVYYCARGTDSWRWVPSAM DLWGKGTQVTVSS

[0367] >Ma7B12 (SEQ ID NO: 105)

[0368] QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASS ALSIYIADSVKGRFTISRDNAKNTVYLHMNNLKPEDTGVYYCARGIDNWRWVPSAM DLWGKGTQVTVSS

[0369] >Ma7B01 (SEQ ID NO: 109)

[0370] QVQLVESGGGLVQAGDSLSLSCAASGRTFSNYAMGWFRQAPGKERHFVAVINQIG DSTWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWPH EYGYWGQGTQVTVSS

[0371] >Ma7C02 (SEQ ID NO: 113) QVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAMGWFRQAAGKDRDFVAVITRS GDSTYYPDSTKGRFTISRDNAKNTMYLQMNSLKPEDTARYYCAATDPTNYWRIWE HEFDYWGQGTQVTVSS

[0372] >Ma7E01 (SEQ ID NO: 117)

[0373] QVQLVESGGGSVQAGDSLSLSCVASERTFSNYAMGWFRQAPGKERHFVAVINPIG EETWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWEH EFDYWGQGTQVTVSS

[0374] >Ma7F02 (SEQ ID NO: 121 )

[0375] QVQLVESGGGLVQAGGSLRLSCTVSGSIDSLHTMGWFRQAPGKKRDFVAWSWS GGNTYYPDYAKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAAESGSGNYWKIW EHEFDSWGQGTQVTVSS

[0376] >Ma7D06 (SEQ ID NO: 125)

[0377] QVQLVESGGGLVQAGGSLRLSCAASGSISGIDTMAWFRQIPGKDREFVAVIKWAA GSTWYPDFVKGRFTVSRDNAKNMVYLQMDSVKPEDTAIYYCAAAAGEKYYYRLFP YEYDYWGQGTQVTVSS

[0378] >Ma7C11 (SEQ ID NO: 129)

[0379] QVQLVESGGGLVRAGGSLRLSCAASGSISSVNGMGWFRQAPGKERLFVAQVSLL DSSTYYADSVKGRFTISRDDAKNTMYLQMNNVRPEETAVYYCAATKLRIGTAFSSE YDYWGQGTQVTVSS

[0380] >Ma8E03 (SEQ ID NO: 133)

[0381] QVQLVESGGGLVHPGGSLRLSCAASGSSLDHYVIGWFRQVPGKEREGVSCISRSG

[0382] GSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLG WLEYDDWGQGTQVTVSS

[0383] >Ma8G02 (SEQ ID NO: 137)

[0384] QVQLVESGGGLVLPGGSLRLSCAASGSSLDHYVIGWFRQYPGKEREGVSCISRSG

[0385] GSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLG WVEYDDWGQGTQVTVSS

[0386] >Ma8H03 (SEQ ID NO: 141 )

[0387] QVQLVESGGGLVHPGGSLRLSCAASGSSLDHYVIGWFRQVPGKQREGISCISSGG STNYADSVKGRFTVSRDNVKSTVYLQMNSLAPDDSAEYYCAAQRNLGNFCVLGW LEYDDWGQGTQVTVSS

[0388] >Ma8A05 (SEQ ID NO: 145)

[0389] QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISL SDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADA DYWGQGTQVTVSS

[0390] >Ma8D05 (SEQ ID NO: 149)

[0391] QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISL SDEITYYSDTVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADA DYWGQGTQVTVSS

[0392] >Ma8D07 (SEQ ID NO: 153) QVQLVESGGGLVQPGGSLRLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGG STFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQG TQVTVSS

[0393] >Ma8D08 (SEQ ID NO: 157)

[0394] QVQLVESGGGFVQAGGSLRLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGG STFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQG TQVTVSS

[0395] >Bm28F09 (SEQ ID NO: 161 )

[0396] QVQLVESGGGLVEPGGSLTLSCAASGFTSKNYYIGWFRQAPGKYREGVASIGASD GSLYIADSVKGRFTISSDNAKNTVYLHPRNLKPEDSGVYYCATGRDSWRWVPSAM DYWGKGIQVTVSS

[0397] >Bm28H01 (SEQ ID NO: 165)

[0398] QVQLVESGGGLVQPGGSLTLSCAASGFTLGDRTLGWFRQVAGKQREGVASIASG VLSAYIADSVKGRFTISRDDAKNTIYLHMNNLKPDDTGVYYCARGVDSWRWVPST MDLWGKGILVTVSS

[0399] >Ma31A12 (SEQ ID NO: 169)

[0400] QVQLVESGGGLVQPGGSLRLSCAASAASGFTLDLYTIAWFLQAPGKEREGVSAISL SDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEVDA DYWGQGTQVTVSS

[0401] >Ma31 B06 (SEQ ID NO: 173)

[0402] QVQLVQSGGGLVQPGGTLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISL SDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADA DYWGQGTQVTVSS

[0403] >Bm28C04 (SEQ ID NO: 177)

[0404] QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRST GGTSYADSVKGRFTISYDNVQNTVDLQMNSLEPEDTAIYYCAGGDGHIYAFGYDY WGQGTQVTVSS

[0405] >Bm28H07 (SEQ ID NO: 181 )

[0406] QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRST GGTSYADSVKGRFTISYDNVQNTVDLQMSSLEPEDTAIYYCAGGDGHIYAFGYDY WGQGTQVTVSS

[0407] >Bm28C09 (SEQ ID NO: 185)

[0408] QVQLVESGGSWQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRST GKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGY DYWGQGTQVTVSS

[0409] >Bm28H12 (SEQ ID NO: 189)

[0410] QVQLVESGGSWQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRST GKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGY DYWGQGTQVTVSS

[0411] >Bm28A12 (SEQ ID NO: 193) QVQLVESGGGLVQAGGSLRLSCWSGRALSSYNLAWFRQSPGKEREWIATIMPSA

[0412] DKAHYPDFLEGRFTISGDNTENTLYLQMNSLKPEDTAIYYCAAREPSYYHALFEYEY

[0413] NLWGQGTQVTVSS

[0414] Tandem fusions

[0415] >Ma7A07-Bm29C02-Fc fusion (SEQ ID NO. 197)

[0416] ETGGSQVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVS SIASSALSIYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWV PSAMDLWGKGTLVTVSSTSTSGEGEGGGEGGSQVQLVESGGGLVQPGGSLRLS CTASGFTLDDWAIGWFRQAPGKEREGVSCTSSRERSSYYRDSVKGRFTISRDSTS

[0417] NTVYLQMNELTLEDTAVYYCAADPQPYPCSLGQLGRYTTWGQGTQVTVSSGTENL

[0418] YFQGKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKATPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

[0419] HYTQKSLSLSPGKSGTSHHHHHH

[0420] >Ma7A07-Bm29B04-tandem pDG05587 (SEQ ID NO. 198)

[0421] AEDGSQVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVS SIASSALSIYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWV PSAMDLWGKGTLVTVSSTSESGETGGSEGSQVQLVESGGGLVQAGGSLRLSCAA SGFTFDDYVIGWFRQAPGKEREAVACISNGDGVANVPNSMKGRFTISSDSAKNTV

[0422] HLRADSLKADDTGVYYCAAVKERLCVHEGMEYWGKGTQVTVSSTSGSGTEGSEG LNDIFEAQKIEWHEGGEGGEGGEGGHHHHSHHSHHHHHH

[0423] >Bm29B04-Ma7A07-tandem pDG05590 (SEQ ID NO. 199)

[0424] AEDGSQVQLVESGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKEREAVA CISNGDGVANVPNSMKGRFTISSDSAKNTVHLRADSLKADDTGVYYCAAVKERLCV HEGMEYWGKGTQVTVSSTSESGETGGSEGSQVQLVESGGGLVQPGGSLRLSCA

[0425] ASGFTLGDYTLGWFRQAPGKQREGVSSIASSALSIYIADSVKGRFTISRDDAKNTIYL HMNNLKPEDTGVYYCARGVDSWRVWPSAMDLWGKGTLVTVSSTSGSGTEGSEG LNDIFEAQKIEWHEGGEGGEGGEGGHHHHSHHSHHHHHH Examples

[0426] The primary aim was to obtain VHH antibodies that inhibit Gamma-Hemolysin (Hlg) activity. To this end, the inventors recombinantly produced Hlg components and immunized one alpaca with the A and C components (HlgA and HlgC) and another alpaca with the B component (HlgB) and LukF four times at three-weeks intervals. Please note that toxicity requires AB or BC components together or LukF combined with LukS, so the immunogens used were not toxic for the animal.

[0427] One week after the final immunization, blood samples were taken, lymphocytes were isolated, and mRNAs were purified and reverse transcribed. VHH-coding regions were amplified by nested PCR and cloned as cDNAs into an M13 phagemid, yielding libraries with >100 million independent clones.

[0428] In the next steps, two rounds of phage display were performed using the individual components as baits. Selected VHH antibodies were classified by sequence similarity, cloned into E. coli expression vectors, produced by periplasmic expression, and purified. The Bm29 series targets HlgB, whereby the Bm29B04 / Bm29B05 class also cross-reacts with LukF. The Bm31 series targets HlgA, and the Bm32 series targets HlgC (see Figure 1 for specific sequences).

[0429] The inventors analyzed interactions between the VHHs and Hlg components by BioLayer Interferometry (BLI; (Abdiche et al., 2008)). Results are shown in Figures 2-4, Figure 12, as well as in Table 1. These measurements revealed extremely high affinities (up to 10 pM, please note that the BLI setup does not allow to discern affinities below 10 pM) with high on-rates (mostly around 105- 106M’1s’1), and non-detectable or nearly non-detectable dissociation rates for most of the VHHs.

[0430] To assess if and how many complementary binding sites these VHHs have on the respective Hlg components, the inventors performed binning experiments using BLI. In these experiments, one Hlg component was allowed to bind to immobilized VHH, and then the binding of another VHH was monitored. ‘Binning’ identified three complementary epitopes (epitopes 1 -3) for HlgB, and two complementary epitopes for HlgA and HlgC, where epitope 1 -binding VHH antibodies could bind simultaneously with either epitope 2-binders or epitope 3-binders. Likewise, epitope 2- and 3-binders could also bind simultaneously.

[0431] In the following experiments, it was tested whether these VHH antibodies could neutralize Hlg. Red blood cells (RBCs or erythrocytes) are susceptible to HlgAB’s lytic activity. Moreover, the high concentration of heme (as part of oxygen-carrying hemoglobin) in RBCs makes the detection of cell lysis very straightforward. Therefore, the inventors performed HlgAB-induced hemolysis experiments using human RBCs. The inventors observed that 50 nM HlgAB led to complete cell lysis (Figure 5).

[0432] HlgCB does not primarily target human erythrocytes as these cells lack an HlgC receptor. Nevertheless, in vitro, high concentrations of HlgCB shows hemolytic activity probably through weak binding to the HlgA receptor DARC (Spaan et al., 2015a). In fact, the inventors found that 500 nM HlgCB was sufficient for complete hemolysis (Figure 5).

[0433] When 150 nM anti-HlgA VHHs were pre-incubated with 50 nM HlgA, all epitope 1 - binders impeded HlgAB-induced toxicity. Bm31A04, Bm31A09, Bm31 D04, Bm31 G03 and Bm31 G04 protected cells from hemolysis completely (Figure 6 and Table 1 ).

[0434] All epitope 1 - and 2-binders of anti-HlgB VHHs also blocked HlgAB-induced hemolysis completely when pre-incubated with HlgB. Bm29D07, the only epitope 3-binder, failed to protect cells (Figure 7 and Table 1 ).The inventors also tested these anti-HlgB VHHs in the HlgCB-induced hemolysis experiments and found that all epitope 1 - and 2- binders potently inhibited cell lysis (Figure 8). However, when anti-HlgC VHHs were tested for hemolysis inhibition, all VHHs failed to protect cells from hemolysis in this assay.

[0435] In human hosts, HlgCB targets leukocytes and other C5aR1 -expressing cells, rather than RBCs. It was thus well possible that HlgC-C5aR1 and HlgC-DARC interactions had different modes of action and / or receptor recognition mechanisms. Therefore, the inventors tested HlgCB also in a C5aR1 context. HeLa cells were reported to be affected by HlgCB toxin (Thelestam et al., 1973). Indeed, when added to cultured Hela cells, HlgCB caused death and detachment of the cells from the plate surface (Figure 9). Preincubation of the HlgCB toxin with anti-HlgC VHHs prevented cell death. Bm32A03, Bm32A08 and Bm32B07 were particularly effective in neutralizing toxicity and protecting cells. When tested in analogous conditions, epitope 1 - and 2-binders amongst the anti-HlgB VHH antibodies also blocked cell death completely. The consistent results in RBCs and HeLa cells might indicate that the neutralizing activity of anti-HlgB VHH antibodies is independent of the target receptor but acts directly on the HlgC interaction.

[0436] To understand the structural basis of the VHH-dependent Hlg inhibition, the inventors aimed at crystallizing Hlg subunibVHH complexes. The X-ray crystal structure of heterodimeric HlgB*Bm29B04 complex was solved (Figure 10). Analysis of the structure showed that Bm29B04 binds and shields HlgB residues (e.g., Trp176 and Arg197) that are essential for phospholipid binding (Guillet et al., 2004). Comparison of this structure with the octameric (pre-pore) HlgAB structure (Yamashita et al., 2014) indicated that Bm29B04 blocks Hlg in three ways. First, it prevents binding of the HlgB subunit to the membrane. Second, it clashes with the neighboring subunits and blocks assembly of the octameric pores. Finally, it sterically excludes the conformational movement of the membrane-inserting arm.

[0437] Further analysis of the HlgB*Bm29B04 structure revealed that the Bm29B04- interacting residues of HlgB are part of the most conserved region among the poreforming toxins (Figure 11 ). With 12 out of 13 residues being identical, LukF is the most similar toxin subunit. In order to test if Bm29B04 (and Bm29B05) could cross-react with LukF, the inventors analyzed VHH-LukF interactions by BLI. Bm29B04 and Bm29B05 bound LukF tightly with KD values of 200 pM and 160 pM, respectively (Figure 12).

[0438] In a following experiment, the investors tested whether Bm29B04 could cross- neutralize leukocidin LukF. The LukSF toxin pair is very toxic for human macrophages (Tromp et al., 2018). When added to the THP-1 monocyte cell line, it disrupted membrane integrity and caused a release of the cytoplasmic contents (Figure 18). When Bm29B04 was pre-incubated with the LukF-LukS toxin pair, it neutralized the toxin and protected the cells (Figure 19). The anti-HIg VHH antibodies were also tested for their thermostability by thermal shift assays or specifically by differential scanning fluorimetry (Goldberg et al., 2011 ). The method exploits that melting (thermal unfolding) of a protein exposes aromatic / hydrophobic residues (from its hydrophobic core), which then bind and enhance the fluorescence of the added SYPRO Orange dye.

[0439] Figures 10-12 show such analyses for the disclosed VHH antibodies, with slow heating from 20°C to 95°C. Re9B09 (described in WQ2022 / 023483), which was expressed in the cytoplasm of NEB Express cells and hence lacks disulfide bonds, melts already at 35 °C.

[0440] The majority of VHHs (Bm29C02, Bm29H04, Bm29F05, Bm29B04, Bm29B05, Bm31 D04, Bm31 G03, Bm31 G04, Bm31 F09, Bm31 G12, Bm31A09, Bm31 D02, Bm31A04, Bm32A03, Bm32B07, Bm32F03; see Table 1 ) showed only a negligible unfolding signal and thus no melting (the small fluorescence peak did not or hardly exceed the measured fluorescence at 20°C). The very low melting amplitudes indicate resistance to melting and thus full thermal stability. We observed that most of the VHHs retained their thermostability even in the presence of the disulfide bond-reducing agent DTT (dithiothreitol) (Figures 13-15, and Table 1 ).

[0441] Having generated VHH antibodies against Hla (disclosed in PCT / EP2024 / 052663) and Hlg toxins, the inventors explored whether a single polypeptide, i.e. , tandem fusion of two VHH antibodies, could neutralize multiple toxins. Figures 7 and 8 show that anti- HlgB VHHs can block both HlgAB- and HlgCB-induced hemolysis. The inventors therefore reasoned that a fusion of a single anti-Hla VHH antibody with a single anti- HlgB VHH antibody could block three toxins simultaneously.

[0442] Ma7A07 (SEQ ID NO: 93) is a hyperthermostable VHH antibody that binds Hla with a <10 pM affinity as described in PCT / EP2024 / 052663. Bm29C02 (SEQ ID NO: 5) is also hyperthermostable and binds HlgB with 10 pM affinity (Table 1 and Figure 2). Hence, Ma7A07 was fused trough a 15 amino acid linker in tandem with Bm29C02 and a human lgG1 Fc region. The tandem was then produced recombinantly in Expi293FTMhuman cells and tested in hemolysis inhibition assays. While individual VHH antibodies blocked only their cognate toxins, the resulting fusion fully protected RBCs from all toxins, namely Hla, HlgAB, and HlgCB (Figure 16).

[0443] Moreover, given that Bm29B04 cross-reacts with LukF, it was likely that a similar tandem could block even four different toxins. To test this idea, Bm29B04 was also fused in tandem with Ma7A07 and tested in toxin neutralization assays. This fusion protected not only RBCs against Hla, HlgAB, and HlgCB toxins (Figure 17), but also macrophages from LukSF toxin (Figure 19).

[0444] Example 1 : Kinetic measurements by Bio-layer interferometry (BLI)

[0445] For affinity measurements, VHHs were produced with a C-terminal Avi-Biotin-tag and enzymatically biotinylated by recombinant BirA (Beckett et al., 1999). They were immobilized at a concentration of 100 nM for 120 seconds on High Precision Streptavidin biosensors of an Octet RED96e instrument (ForteBio / Sartorius), using Phosphate-Buffered Saline (PBS) pH 7.4, 0.02% (w / v) Tween 20 and 0.1 % (w / v) bovine serum albumin (BSA) as the assay buffer. 50, 25, 12.5, 6.25 or 3.125 nM of the indicated Hlg component were then allowed to bind for 450 seconds, followed by a dissociation step of 1800 seconds. Binding and dissociation were recorded as wavelength shifts (in nm). Baselines were recorded by measuring a ‘minus Hlg control’ in parallel. On-rates, off-rates, and dissociation constants (KDS) were calculated using the Octet Data Analysis HT 12.0 software by fitting a mass transport model to the data. This revealed very tight binding of the analyzed VHHs to their cognate Hlg components.

[0446] The results are summarized in Table 1 and shown in Figures 2-4 and 12.

[0447] Example 2: Identification of complementary Hlg epitopes

[0448] Epitope binning experiments were performed in a similar setup as described in Example 1. One biotinylated VHH (VHH1 ) was immobilized on biosensors. The biosensors were dipped into wells containing 100 nM of the corresponding Hlg component for 300 seconds and then incubated for 300 seconds with a second VHHs (VHH2) at 100 nM concentration. When VHH2 showed no binding, VHH1 and VHH2 were considered to belong to the same epitope class. By repeating this for all VHH class representatives, VHH antibodies were classified by their epitope class. Three epitopes were identified for HlgB, and two epitopes for HlgA and HlgC each.

[0449] The results are summarized in Table 1 .

[0450] Example 3: Erythrocyte hemolysis assays

[0451] 150 pl of the indicated Hla, HlgAB, and HlgCB dilutions (10 nM to 1 pM) in assay buffer (PBS pH 7.4, 0.4% w / v glucose, 25 mM EDTA) were pipetted in duplicate into a 96- well plate before 50 pL erythrocytes (4x108cells) were added to each well. The erythrocytes (AB RH+) were obtained from the German Red Cross.

[0452] The plate was incubated for 2.5 hours with shaking at 900 rpm at 37°C, followed by another 30 min incubation at 4°C. The plate was then centrifuged for 10 minutes in a swing-out rotor at 400 g and 4°C. 100 pL of the supernatant was transferred to another 96-well plate. The concentrations of released hemoglobin were measured photometrically at 412 nm (OD412). To determine baseline and maximum absorbance values, cells were incubated with buffer alone or water (for hypo-osmotic lysis).

[0453] The results are shown in Figure 5. Note that diluting erythrocytes in water leads to complete lysis, and that 50 nM HlgAB resulted in the same complete cell lysis. On the other hand, 500 nM HlgCB was required for complete lysis. This is consistent with the fact that HlgC binds DARC 10-fold weaker than HlgA (KDS 229 nM vs 29 nM; (Spaan et al., 2015a)). Hla required the highest concentration (1 pM) for complete hemolysis.

[0454] Example 4: Hemolysis inhibition assays

[0455] To assess if the disclosed anti-HIg VHH antibodies neutralize toxin activity, hemolysis inhibition assays were performed using human erythrocytes.

[0456] To test for HlgAB neutralization, HlgA or HlgB (66.6 nM each) was pre-incubated with 200 nM of anti-HlgA or anti-HlgB VHH antibodies for 30 min, before addition to erythrocytes. Cells incubated with buffer, water, and HlgAB in the absence of any VHH served as controls. Each condition was assayed in quadruplicate. Erythrocytes were processed, and hemoglobin levels in the supernatant were measured, as described in Example 3. Please note that the concentrations of active toxin and VHH s in the final assay solution were 50 nM and 150 nM, respectively.

[0457] For HlgCB neutralization, 666 nM HlgB was pre-incubated with 2 pM anti-HlgB VHH antibodies for 30 min, and then mixed with 666 nM HlgC before the hemolysis assay. The data showed that all epitope 1 -binders of HlgA and all epitope 1 - and 2-binders of HlgB blocked Hlg-AB-induced toxicity. Remarkably, all Bm29B04, Bm29C02, Bm31 D04, and Bm31 G03 class members completely blocked hemolysis (Figures 6 and 7, and Table 1 ). Similarly, Bm29B04 and Bm29C02 class members of anti-HlgB VHH antibodies fully protected cells from HlgCB-induced hemolysis (Figure 8 and Table 1 ).

[0458] Example 5: HeLa-based cytotoxicity inhibition assays

[0459] Erythrocytes are not the primary target of the HlgCB toxin because these cells lack the main receptor, and the affinity of HlgCB for DARC is relatively weak. The toxin rather targets leukocytes and other tissues, including HeLa cells, which express C5aR1. To test whether the anti-HlgB and anti-HlgC VHH antibodies protect cells from the HlgCB toxin, neutralization assays were performed with cultured HeLa cells.

[0460] To assess neutralization, 240 pM HlgC was pre-incubated with 1 .2 pM of the indicated anti-HlgC VHH antibodies for 30 min. The mixes were supplemented with 240 nM HlgB, before adding 50 pL of them to 250 pL medium with cultured HeLa cells (yielding final toxin and VHH concentrations of 40 nM and 200 nM, respectively). As controls, cells treated with media alone or with HlgCB in the absence of any VHH. Each condition was assayed in duplicate. Cells were incubated overnight at 37°C in cell culture medium with 5% CO2, and then imaged using a (20x objective) brightfield microscope.

[0461] Almost all of the toxin-treated cells died and detached from the surface after overnight incubation, whereas untreated cells proliferated normally. The images showed that pre-incubation of the toxin with anti-HlgC VHH antibodies prevented cell death. In particular Bm32A03, Bm32A08, and Bm32B07 were very effective at protecting cells, as corresponding cells were not different from untreated cells (Figure 9).

[0462] Anti-HlgB VHH antibodies were also tested under identical conditions. Similar to the results shown in Example 4 and Figure 8, all epitope 1 - and 2-binders potently prevented cell death.

[0463] Example 6 - Thermostability measurement of VHH antibodies

[0464] VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposes aromatic / hydrophobic residues, which then bind and enhance the fluorescence of the added SYPRO Orange dye. Assays were performed in a volume of 20 pl, at 1 mg / ml VHH concentration in 50 mM Tris / HCI, 150 mM NaCI (pH 8.0 at 20°C), and 1x dye (diluted from a 5000x stock; Life Technologies), in the absence or presence of 10 mM DTT (dithiothreitol). Two sample replicates were pipetted into a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal (Bio-Rad), briefly centrifuged to remove air bubbles, and placed in a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 20°C and then heated in 1 °C increments of 45 seconds up to 95°C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak.

[0465] The results are shown in Table 1 and Figure 13-15. VHH antibody Re9B09, produced in reducing cytosol and hence lacking disulfide bonds, melts already at 35°C. VHH antibodies Bm29D07, Bm29E06, Bm31A08, and Bm31 B04 melt similarly at 44°C, 58°C, 67°C and 72°C, respectively, while majority of VHH antibodies showed only a negligible melting amplitude and thus remained stable throughout 95°C. Remarkably, Bm28B04, Bm29C02, Bm31A09, Bm31 G03, Bm32A03, and Bm32A08 were resistant to melting even in the presence of disulfide-reducing DTT.

[0466] Example 7: Inhibition of toxin-induced hemolysis by VHH tandems Anti-Hla VHH Ma7A07, anti-HlgB VHH Bm29C02, and a Ma7A07-Bm29C02-Fc tandem were used in neutralization experiments of the Hla, HlgAB, and HlgCB toxins. 1 pM HLA, 50 nM HlgAB, or 500 nM HlgCB was pre-incubated with a 3-fold molar excess of VHH antibodies. Otherwise experiments were performed analogously to those described in Examples 3 and 4, and Figures 5-8. Figure 16 documents the results. While Ma7A07 neutralized only Hla and Bm29C02 only Hlgs, the tandem VHH blocked all toxins (Hla and Hlg) and fully protected cells from lysis.

[0467] In a second set of experiments, Bm29B04-Ma7A07 and Ma7A07-Bm29B04 tandems were tested in hemolysis experiments with the Hla, HlgAB, and HlgCB toxins analogously as described above. The results are shown in Figure 17. While individual VHHs neutralized their respective toxins only, both tandem fusions blocked all toxins and protected the erythrocytes from lysis.

[0468] Example 8: Cell membrane integrity assay

[0469] LukSF is a toxin that targets leukocytes expressing C5aR1 C5aR2. In addition, CD45 has been described as a receptor for LukF, and its absence leads to reduced sensitivity by LukSF on humanized murine models (Tromp, et al., 2018). To test any cross-reactive effect of anti-HlgB VHH antibodies, neutralization assays were performed with macrophages differentiated from THP-1 cells, obtained from the Leibniz Institute DSMZ (ACC16)

[0470] THP1 cells (2.5x104cells) were seeded in 96-well plates and differentiated with 200 pL of a Phorbol 12-mystriate 13-acetate (PMA) 20 ng / mL solution in RPMI-1640 medium. After 48 hours, the PMA was washed away and cells were incubated in fresh medium for another 24 hours. 150 pl of the indicated LukSF dilutions (5 pM to 100 nM) in fresh medium were added to the differentiated cells.

[0471] The plate was incubated for 3 hours at 37 °C, and 5% CO2, followed by a 20 minutes incubation at 22 °C. 50 pL of the supernatant was transferred to a black 96-well plate and mixed with 50 pL of CytoTox-ONE™ reagent from the cell viability kit CytoTox- ONETMHomogeneous Membrane Integrity Assay (Promega) for 10 minutes at 22 °C. Membrane integrity loss is measured by the leakage of LDH in a coupled enzymatic assay. The reagent contains a mix of diaphorase and resazurin. Released LDH from disrupted cells converts lactate into pyruvate, generating NADH, which then serves as a coenzyme for diaphorase. This enzyme reduces rezasurin to the fluorescent compound resofurin with an excitation and emission wavelengths of 571 and 585 nm, respectively.

[0472] Stop solution was added after the 10 minutes and fluorescence signal was measured within the hour. For calibration, cells were incubated with medium alone to determine a baseline or a with a Triton X-100 solution to obtain numbers for a complete membrane disruption. Percentage of LDH release was calculated as follows:

[0473] (Experimental — baseline)

[0474] LDH release % = 100 X

[0475] (Maximum LDH Release — baseline)

[0476] The results are shown in Figure 18. Triton X-100 leads to complete disruption of macrophages; LukSF is a potent toxin that requires less than 2 nM for a similar level of cytoplasmic leakage (Figure 18). Additionally, after the 3-hour incubation time treated cells detached from the well bottom, swelling and becoming round.

[0477] Example 9: Inhibition of membrane integrity loss by VHH tandem

[0478] To test whether LukF-cross-reacting anti-HlgB VHH antibodies could protect cells from LukSF toxin, cell viability assays were performed with macrophages as described in example 8.

[0479] To test for LukSF neutralization, 20 nM LukF was pre-incubated with 200 nM of anti- Hla (i.e., Ma7A07), anti-HlgB (i.e., Bm29B04), or tandem VHH antibodies for 120 minutes in fresh RPMI-1640 medium. Then, 20 nM LukS was added before addition to the macrophages. As controls, cells were incubated with lysis buffer, just medium, or medium plus LukSF in the absence of any VHH. LDH release was then measured as described in Example 8. Each condition was assayed in nine replicates.

[0480] The data shows pre-incubation with either tandem Ma7A07-Bm29B04 VHHs, or Bm29B04 stopped cytoplasmic leakage by LukSF. This demonstrates an anti-HlgB VHH antibody can protect against an additional leukocidin pair besides the Hlg toxins. Additionally, the experiment demonstrates that tandem context of the VHHs does not impede a neutralization of the cognate toxin (Figure 19).

[0481] References

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Claims

Claims1. A hetero-multimeric VHH antibody comprising of two or more different VHH antibody units, e.g., 2, 3 or 4 different VHH antibody units covalently fused to each other wherein at least one VHH antibody unit recognizes an S. aureus gamma hemolysin (Hlg).

2. The hetero-multimeric VHH antibody of claim 1 , wherein the at least one VHH antibody unit recognizing an S. aureus gamma hemolysin (Hlg) is directed against Hlg component HlgB, HlgA or HlgC, particularly HlgB.

3. The hetero-multimeric VHH antibody of claim 1 or 2, wherein the at least one VHH antibody unit recognizing an S. aureus gamma hemolysin (Hlg) comprises(a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88 or 90-92; or(b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a).

4. The hetero-multimeric VHH antibody of any one of claims 1 -3, wherein the at least one VHH antibody unit recognizing an S. aureus gamma hemolysin (Hlg) comprises(a) a VHH sequence as shown in SEQ. ID NO: 5, 9, 13, 17, 21 , 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61 , 65, 69, 73, 77, 81 , 85, or 89;(b) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).

5. The hetero-multimeric VHH antibody of any one of claims 1 -4 wherein the at least one VHH antibody unit recognizing an Hlg comprises(i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant;(ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, or(iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant..

6. The hetero-multimeric VHH antibody of any one of claims 1 -5, which comprises(i) at least two VHH antibody units recognizing different Hlg polypeptides selected from HlgA, HlgB, and HlgC;(ii) at least two VHH antibody units recognizing different epitopes on the same Hlg polypeptide selected from HlgA, HlgB, or HlgC;(iii) a first VHH antibody unit which recognizes Hlg and a second VHH antibody unit which recognizes a different toxin or virulence factor from S. aureus, or(iv) a first VHH antibody unit which recognizes both Hlg and leukocidin F (LukF).

7. The hetero-multimeric VHH antibody of claim 6 wherein the second VHH antibody unit recognizes S. aureus alpha hemolysin (Hla).

8. The hetero-multimeric VHH antibody of claim 7 wherein the second VHH antibody unit recognizing Hla comprises(i) (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 94-96, 98-100, 102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-136, 138-140, 142-144, 146-148, 150-152, 154-156, 158-160, 162-164, 166-168, 170-172, 174-176, 178-180, 182-184, 186-188, 190-192 or 194-196; or(b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a) or(ii) (a) a VHH sequence as shown in SEQ. ID NO: 93, 97, 101 , 105, 109, 113, 117, 121 , 125, 129, 133, 137, 141 , 145, 149, 153, 157, 161 , 165, 169, 173, 177, 181 , 185, 189 or 193; or(b) a sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).

9. The hetero-multimeric VHH antibody of claim 7 or 8, wherein the at least one VHH antibody unit recognizing Hla comprises the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

10. The hetero-multimeric VHH antibody of any one of claims 1 -9 wherein the VHH antibody units are covalently fused to each other via a linker, wherein the linker has a length of 5-50 amino acids, more particularly a length of 10-30 amino acids.

11. The hetero-multimeric VHH antibody of any one of claims 1 -10, which is covalently or non-covalently conjugated to a heterologous moiety, and which is particularly fused to an immunoglobulin Fc fragment.

12. The hetero-multimeric VHH antibody of any one of claims 1 -11 , which neutralizes HlgAB, HlgBC and Hla or which neutralizes HlgAB, HlgBC, Hla and LukSF.

13. The hetero-multimeric VHH antibody of any one of claims 1 -12 comprising(i) the anti-HIg VHH antibody Bm29C02 having a VHH sequence as shown in SEQ. ID NO: 5 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or(ii) the anti-HIg VHH antibody Bm29B04 having a VHH sequence as shown in SEQ. ID NO: 21 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHHsequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant, or(iii) the anti-HIg VHH antibody Bm29B05 having a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof including a humanized variant, and the anti-Hla VHH antibody Ma7A07 having a VHH sequence as shown in SEQ. ID NO: 93 or a VHH antibody, which is a variant thereof including a humanized variant.

14. A set of two or more different hetero-multimeric VHH antibodies of any one of claims 1 -13.

15. The hetero-multimeric VHH antibody of any one of claims 1 -13 or the set of claim 14 for use in medicine.

16. The hetero-multimeric VHH antibody of any one of claims 1 -13 or the set of claim 14 for use for use in therapy or diagnostics.

17. The hetero-multimeric VHH antibody of any one of claims 1 -13 or the set of claim 14 for use in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with an Hlg- positive, optionally LukSF-positive and optionally Hla-positive S. aureus.

18. Use of the hetero-multimeric VHH antibody of any one of claims 1 -13 or the set of claim 14 for detecting Hlg, optionally LukF and optionally Hla in a sample, wherein the sample particularly is a biological sample, e.g., a body fluid such as saliva, sputum, a lung lavage, a swab, blood, serum or plasma, a stool sample, a tissue sample, or a biopsy sample.

19. A method for the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with an Hlg-positive, optionally LukSF-positive and optionally Hla-positive S. aureus comprising administering an effective dose of the VHH antibody of any one of claims 1 -13 or the set of claim 14 to a subject in need thereof, particularly to a human subject.

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