Therapeutic VHH antibodies against Staphylococcus aureus alpha-hemolysin

Abstract

The present invention is in the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that prevent membrane binding and / or oligomerization of Staphylococcus aureus (HLA) alpha-hemolysin and HLA-mediated hemolysis.

Description

[Technical Field]

[0001] The present invention is in the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that prevent membrane binding and / or oligomerization of Staphylococcus aureus (HLA) alpha-hemolysin and HLA-mediated hemolysis. [Background technology]

[0002] Staphylococcus aureus is one of the most prevalent bacterial pathogens, causing a myriad of illnesses ranging from superficial skin infections to life-threatening pneumonia, bacteremia, and sepsis. In developed countries, S. aureus is the leading cause of bloodstream infections and results in the highest mortality rates. In fact, in the United States, the annual number of deaths caused by S. aureus was greater than the annual number of deaths caused by acquired immunodeficiency syndrome (AIDS), tuberculosis, and viral hepatitis combined (Cheung et al., 2021). The lack of an approved vaccine and complex treatments make S. aureus a major global healthcare problem.

[0003] Staphylococcus 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 easily cause infection upon skin or mucosal damage. The human immune system is typically able to eliminate invading bacteria within minutes (using complement, macrophages, and, upon re-exposure, antibodies). However, pathogenic bacteria utilize virulence factors that enable them to evade the immune response. Virulence factors can generally be categorized as surface and extracellular secreted proteins; these include bacterial toxins, complement-inactivating factors, phagocytosis inhibitors, or proteases that cleave immunoglobulins or the extracellular matrix. Unlike other bacteria that utilize only one or a few virulence factors, S. aureus possesses more than 20 virulence factors to promote its virulence (Cheung et al., 2021).

[0004] Antibiotics are essential for treating bacterial infections, but the rise of antibiotic-resistant infections complicates treatment. Staphylococcus aureus infections are particularly challenging due to the frequent occurrence of multidrug resistance among S. aureus isolates. Methicillin-resistant S. aureus (MRSA) is the most commonly identified antibiotic-resistant pathogen worldwide. Patients with MRSA infections experience longer hospital stays, higher treatment costs, and higher mortality rates (Ippolito et al., 2010). The first MRSA strain was characterized in 1961, just one year after methicillin was approved for clinical treatment (Ippolito et al., 2010; Turner et al., 2019). Since then, numerous strains resistant to an increasing number of antibiotics have been reported. Some clinical isolates have even demonstrated resistance to more than 10 antibiotic classes (Saleh et al., 2022). New antibiotics for MRSA and, more generally, for the treatment of Staphylococcus aureus have been developed or are in development. Currently, vancomycin is used as a last-resort antibiotic in non-critically ill patients (Cheung et al., 2021). However, vancomycin-resistant S. aureus isolates have already been identified, although not widely. Similarly, vancomycin treatment failure is increasing (Lodise et al., 2008; Ye et al., 2020).

[0005] Another drawback of antibiotic therapy is that, even though the treatment is immediately effective, it does not eliminate bacterial toxins already circulating in the body. Antibiotics may even enhance toxin release. Therefore, there is a need for alternative therapeutic strategies that target virulence factors. One such "antitoxin therapy" approach is to use antibodies to neutralize virulence factors, which would stop the toxic effects and "disarm" the pathogen, allowing the immune system to clear the infection.

[0006] Alpha-hemolysin (HLA) or alpha-toxin is the most important and best-known virulence factor of S. aureus. HLA is a 33-kDa secreted precursor protein present in >95% of S. aureus isolates (Kielian et al., 2001). Upon binding to target membranes, HLA oligomerizes into heptamers, forming 40-Å-wide pores. These pores allow leakage of ions and other small molecules, destabilizing the membrane and leading to cell lysis. HLA primarily targets leukocytes to evade host cell defense systems and erythrocytes to make iron, a limiting factor, available for bacterial growth. In epithelial and endothelial cells, HLA interacts with ADAM10, a metalloprotease and cell surface receptor containing an E-cadherin domain. This activation disrupts adherens junctions, allowing bacteria to escape the bloodstream and invade soft tissues.

[0007] The direct contribution of HLA to sepsis, pneumonia, and skin lesions has been well documented in various mouse models (Kielian et al., 2001; Bubeck Wardenburg et al., 2007). In addition, in a rat model of pneumonia, the virulence of bacterial strains is strongly correlated with elevated HLA expression (Montgomery et al., 2008). Therefore, HLA-targeting strategies, i.e., by interfering with its expression level or by neutralizing already circulating toxins, offer promising solutions for the development of new (passive immune) treatments for S. aureus infections. Summary of the Invention

[0008] The present disclosure provides single domain VHH antibodies directed against Staphylococcus aureus alpha-hemolysin (HLA), which recognize and / or prevent membrane binding and / or oligomerization of S. aureus HLA and HLA-mediated hemolysis, and are suitable for diagnostic, prophylactic, and / or therapeutic applications.

[0009] In some embodiments, VHH antibodies are monovalent, e.g., present as a single VHH domain or as a fusion to a heterologous protein such as serum albumin, which can be used in a wide variety of formats. In some embodiments, VHH antibodies are multivalent, e.g., present as a bivalent Fc fusion. In some embodiments, VHH antibodies are heteromultimeric VHH antibodies comprising two or more different VHH antibody units.

[0010] Their use in monovalent formats is possible due to their very high affinity. In some embodiments, VHH antibodies have target affinities in the picomolar range or even lower in the monovalent format. In some embodiments, VHH antibodies neutralize HLA with high potency.

[0011] A first aspect of the present disclosure is a VHH antibody directed against the Staphylococcus aureus HLA described herein. Preferred VHH antibodies of the present invention, their names, binding properties, i.e., HLA epitopes, HLA affinity, and HLA neutralization capacity, and their thermostability are listed in Table 1 below:

[0012] JPEG2026505798000001.jpg202166

[0013] A complete list of VHH sequences of VHH antibodies is provided at the end of this specification.

[0014] Another aspect of the invention relates to a set of two or more different VHH antibodies, wherein at least one VHH antibody is as described above.

[0015] The above-mentioned VHH antibodies are suitable for use in medicine (e.g. human medicine), in particular for use in therapy, e.g. in the prevention, treatment and / or alleviation of conditions caused by, associated with and / or accompanied by infection with HLA-positive Staphylococcus aureus (in particular by infection with drug-resistant HLA-positive Staphylococcus aureus), or for use in diagnosis, e.g. for detecting Staphylococcus aureus HLA in patient samples (e.g. in body fluids or tissue samples), or for use in research.

[0016] Yet another aspect of the present invention relates to nucleic acid molecules encoding the above-mentioned VHH antibodies, in particular nucleic acid molecules operably linked to heterologous expression control sequences, vectors comprising said nucleic acid molecules, or recombinant cells or non-human organisms transformed or transfected with said nucleic acid molecules or said vectors.

[0017] Yet another aspect of the present invention relates to a method for the recombinant production of the above-mentioned VHH antibodies, said method comprising the steps of culturing the above-mentioned cells or organisms in a suitable medium and obtaining said VHH antibodies from said cells or organisms or from said medium.

[0018] Yet another aspect of the present invention relates to a method for the prevention, treatment and / or alleviation of a condition caused by, associated with and / or accompanied by infection with HLA-positive Staphylococcus aureus (particularly by infection with drug-resistant HLA-positive Staphylococcus aureus), said method comprising the step of administering an effective amount of the above-mentioned VHH antibody or a 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 with and / or accompanied by infection with HLA-positive Staphylococcus aureus.

[0019] A non-limiting list of VHH antibodies of the present invention and their CDR sequences is shown in Table 2 below:

[0020] JPEG2026505798000002.jpg187166 [Brief explanation of the drawings]

[0021] [Figure 1] [Sequence alignment and highlighting of variable regions] Figure 1 shows the alignment of the VHH sequences in Table 2. Residues that deviate from the consensus sequence are marked with a grey background. The three variable CDR regions are indicated. [Figure 2] [Affinity measurement of VHH antibodies to HLA] Biolayer interferometry (BLI) experiments were performed on an Octet instrument with the indicated VHH antibodies using high-precision streptavidin sensors and biotinylated HLA. The black curves represent fitting using a mass transport model. Actual data points are shown in gray. KD was derived from the global fitting. See Example 1 for details. [Figure 3] [Epitope binning of anti-HLA VHH antibodies revealed four distinct HLA-binding sites] (A) Epitope binning experiments were performed pairwise for the indicated VHH antibodies using biotinylated HLA. When HLA was saturated with Ma7A07, Ma8H03 could no longer bind, suggesting that its binding site was blocked by Ma7A07. Binding of other VHH class antibodies was unaffected. Similar pairwise measurements were performed for all VHH classes. (B) VHHs representative of four different epitopes were sequentially bound to immobilized HLA. [Figure 4]Inhibition of HLA-induced hemolysis by VHH antibodies. Erythrocytes were dispensed into 96-well plates and incubated with buffer, water, or HLA at 37°C for 3 hours, followed by 1 hour at 4°C. The plates were centrifuged to pellet any still-intact cells, and the supernatants were analyzed for absorbance at 412 nm in a plate reader. Lysed cells release their cytoplasmic contents, including heme-bound hemoglobin, which has a maximum light absorbance at 412 nm. Where indicated, HLA was preincubated with VHH antibodies. (A) The indicated antibodies were tested using mouse erythrocytes. (B) The indicated antibodies were tested using human erythrocytes. [Figure 5]High-resolution crystal structure of a dimeric VHH·HLA complex. HLA and VHH were produced in the cytoplasm of Escherichia coli (E. coli) NEB Express or NEB Shuffle Express, respectively, and then purified by Ni-chelate chromatography. Dimeric VHH·HLA complexes were formed in solution by mixing HLA and VHH at a 1:1.2 stoichiometry, and excess VHH was removed by gel filtration. The homogeneous complex was crystallized, and an X-ray diffraction dataset was recorded at the Swiss Light Source synchrotron. The structure was solved by molecular replacement. The resolution and R-free resolution were 2.25 Å and 0.23 for Ma7A07·HLA, 3.00 Å and 0.25 for Bm28C04·HLA, 2.20 Å and 0.24 for Ma7F02·HLA, 2.60 Å and 0.26 for Ma8A05·HLA, and 2.30 Å and 0.23 for Ma7D07·HLA. A-E) Crystal structures of the Ma7A07·HLA (A), Bm28C04·HLA (B), Ma7F02·HLA (C), Ma8A05·HLA (D), and Ma8D07·HLA (E) complexes, ribbon representation. Structurally important domains of HLA, namely the N-terminal stalk and membrane-embedded arm, are labeled. F) The crystal structures shown in A, C, D, and E were superimposed by aligning them with HLA and drawn in surface representation. Note that the four VHHs bind to different parts of the HLA molecule, supporting the epitope binning experiments. G-H) In silico docking of Ma8A05 to membrane-bound HLA. Modeling is based on aligning the HLA in D to one HLA unit in the heptameric HLA structure (PDB ID 3M2L; Banerjee et al., 2010). G) Ma8A05 bound to one HLA is shown in ribbon representation. The remaining six HLA protomers have been omitted for clarity. Note that Ma8A05 binds to an epitope distant from the conformational domain of HLA and can therefore recognize HLA in all its forms. H) The model in G is shown with the membrane-embedded domains of all HLA protomers.Major clashes with neighboring HLA protomers indicate that Ma8A05-binding HLA cannot oligomerize into pore-forming heptamers, explaining why this VHH neutralizes toxic HLA activity. The structures of HLA with either Ma7A07, Ma8D07, or Ma7F02 are also all incompatible with hemolysin forming multimeric pores. [Figure 6] [Sequence alignment of individual anti-HLA VHH classes with HLA-interacting residues indicated.] HLA-interacting residues of VHH antibodies were identified from the crystal structure shown in Figure 5 or by homology modeling based on the corresponding structures. HLA-interacting residues are printed in bold. (A) Ma7A07 class VHH antibody (B) Ma7F02 class VHH antibody (C) Ma8D07 class VHH antibody (D) Ma8A05 class VHH antibody (E) Bm28C04 class VHH antibody [Figure 7] [HLA sequences with VHH-interacting residues indicated] The VHH-HLA structures shown in Figure 5 were analyzed to identify the VHH-interacting residues of HLA. The numbers below the residues indicate which VHH they interact with: 1 for Ma7A07, 2 for Ma7F02, 3 for Ma8D07, 4 for Ma8A05, and 5 for Bm28C04. [Figure 8] Thermal Stability of VHH Antibodies The indicated VHH antibodies were subjected to differential scanning fluorescence (DSF), which utilizes thermal unfolding to expose aromatic / hydrophobic residues that subsequently bind to added SYPRO Orange dye, enhancing its fluorescence. Two replicates of each sample were placed in a thermal cycler. Samples were incubated at 25°C for 5 minutes, then the temperature was increased to 95°C by 1°C every 45 seconds. Fluorescence was measured at the end of each step. [Figure 9] Thermostability of VHHs determined by BLI: The indicated VHH antibodies were incubated at room temperature or at 95°C for 10 min and centrifuged at 20,000g for 20 min. The supernatants were diluted 50-fold (20 nM) and analyzed for HLA binding by BLI. DETAILED DESCRIPTION OF THE INVENTION

[0022] [Embodiments of the Invention] In the following, specific embodiments of the present invention are disclosed as follows:

[0023] 1. VHH antibody that recognizes Staphylococcus aureus alpha-hemolysin (HLA).

[0024] 2. A VHH antibody according to embodiment 1, the VHH antibody competes for binding to HLA with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3; VHH antibody.

[0025] 3. The VHH antibody of embodiment 1, the VHH antibody competes for binding to HLA with the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31; VHH antibody.

[0026] 4. The VHH antibody of embodiment 1, the VHH antibody competes for binding to HLA with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55; VHH antibody.

[0027] 5. The VHH antibody of embodiment 1, the VHH antibody competes for binding to HLA with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63; VHH antibody.

[0028] 6. A VHH antibody recognizing Staphylococcus aureus alpha-hemolysin (HLA), particularly as described in embodiment 1, The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (i) amino acid residues at positions 54 to 61, specifically Y54, K56, E57, M60, and / or H61; (ii) amino acid residues at positions 86 to 96, specifically T86, A88, Y91, R92, V93, Y94, and / or E96; (iii) amino acid residues at positions 233 to 239, specifically A233, D234, D238, and P239; and (iv) amino acid residues at positions 279 to 302, specifically R279, W300, and D302; interacting with, VHH antibody.

[0029] 7. A VHH antibody according to embodiment 1 or 6, A VHH sequence comprising the following amino acid residues that interact with HLA, based on the numbering in Figure 6A: - A, V, or D in position 55; - Y at position 59; - I in position 60; - D in position 62; - K at position 65; - T in position 69; - W at position 103; - R at position 104; - W at position 105; - V at position 106; - P at position 107; and - S at position 108; having VHH antibody.

[0030] 8. A VHH antibody recognizing Staphylococcus aureus alpha-hemolysin (HLA), particularly as described in embodiment 1, The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (i) amino acid residues at positions 38 to 44, specifically T38, D39, I40, G41, S42, N43, and / or T44; (ii) amino acid residues at positions 141 to 143, specifically T141 and / or T143; and (iii) amino acid residues at positions 162 to 169, specifically I162, G163, N165, V166, S167, I168, and / or G169; interacting with, VHH antibody.

[0031] 9. The VHH antibody of embodiment 1 or 8, VHH sequences, including the following amino acid residues that interact with HLA, based on the numbering in Figure 6B: - Y or H in position 32; - F in position 47; - N, T, or S in position 52; - W or Y in position 59; - Y at position 60; - P at position 61; - D in position 62; - S or N at position 104; - Y at position 105; - W at position 106; - W at position 109; - H at position 111; and - E at position 112; having VHH antibody.

[0032] 10. A VHH antibody recognizing Staphylococcus aureus alpha-hemolysin (HLA), particularly as described in embodiment 1, The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (i) amino acid residues at positions 210 to 219, specifically R210, D211, S212, W213, P215, and / or N219; and (ii) amino acid residues at positions 285 to 297, specifically H285, T287, S289, K292, T294, N295, and / or K297; interacting with, VHH antibody.

[0033] 11. A VHH antibody according to embodiment 1 or 10, Based on the numbering in Figure 6C, the VHH sequence contains the following amino acid residues that interact with HLA: - Y at position 29; - K in position 30; - L in position 31; - N at position 32; - A in position 33; - L at position 47; - T at position 50; - S at position 52; - S at position 53; - G in position 54; - F in position 58; - N at position 98; - P at position 99; - T at 100th position; - Y at position 101; - N at position 102; and - H in position 103; having VHH antibody.

[0034] 12. A VHH antibody recognizing Staphylococcus aureus alpha-hemolysin (HLA), particularly as described in embodiment 1, The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (i) amino acid residues at positions 204 to 208, specifically N204, W205, G206, and / or Y208; and (ii) amino acid residues at positions 213 to 227, specifically W213, N214, V216, Q220, M223, K224, T225, R226, and / or N227; interacting with, VHH antibody.

[0035] 13. A VHH antibody according to embodiment 1 or 10, VHH sequence, including the following amino acid residues that interact with HLA, based on the numbering in Figure 6D: - A in position 53; - S at position 57; - D in position 58; - I in position 60; - T at position 61; - Y at position 62; - Y at position 63; - S at position 64; - D in position 65; - A in position 103; - F at position 104; - D in position 105; and - F at position 106; having VHH antibody.

[0036] 14. A VHH antibody recognizing Staphylococcus aureus alpha-hemolysin (HLA), particularly as described in embodiment 1, The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (i) amino acid residues at positions 55 to 59, specifically D55, K56, E57, N58, and / or G59; (ii) amino acid residues at positions 88 to 97, specifically A88, G89, Q90, R92, V93, Y94, S95, E96, and / or E97; and (iii) amino acid residues at positions 240 to 302, specifically K240, R277, R279, W300, and / or D302; interacting with, VHH antibody.

[0037] 15. A VHH antibody according to embodiment 1 or 14, Based on the numbering in Figure 6E, the VHH sequence contains the following amino acid residues that interact with HLA: - N at position 30; - S in position 31; - Y in position 32; - Q in position 44; - R at position 45; - S at position 54; - D in position 100; - H in position 102; - Y at position 104; - A at position 105; - F at position 106; - G in position 107; and - D in position 109; having VHH antibody.

[0038] 16. A VHH antibody recognizing a Staphylococcus aureus HLA polypeptide, comprising: (a) a CDR3 sequence set forth in SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, or 106; (b) a CDR3 sequence having at least 80%, at least 90%, or at least 95% identity to the CDR3 sequence of (a); or (c) a VHH antibody, competes for binding to HLA with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or competes for binding to HLA with the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31, or competes for binding to HLA with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55, or competes for binding to HLA with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63; Including, VHH antibody.

[0039] 17. A VHH antibody according to any of the preceding embodiments, comprising: (a) a combination of CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, 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, 88-90, 92-94, 96-98, 100-102, or 104-106; (b) a combination of CDR1, CDR2, and CDR3 sequences that have at least 80%, at least 90%, or at least 95% identity to the combination of CDR1, CDR2, and CDR3 sequences of (a); or (c) a VHH antibody, competes for binding to HLA with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or competes for binding to HLA with the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31, or competes for binding to HLA with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55, or competes for binding to HLA with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63; Including, VHH antibody.

[0040] 18. The VHH antibody of any one of the preceding embodiments, comprising: (a) a VHH sequence as set forth in SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, or 103; (b) a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the VHH sequence of (a); or (c) a VHH antibody, competes for binding to HLA with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or competes for binding to HLA with the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31, or competes for binding to HLA with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55, or competes for binding to HLA with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63; Including, VHH antibody.

[0041] 19. The VHH antibody of any one of the preceding embodiments, Here, the dissociation constant K D the binding affinity, expressed as , is about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less; VHH antibody.

[0042] 20. The VHH antibody of any one of the preceding embodiments, The VHH antibody neutralizes HLA, specifically by inhibiting HLA-induced hemolysis of red blood cells. VHH antibody.

[0043] 21. A VHH antibody according to embodiment 20, the VHH antibody neutralizes HLA; VHH antibody.

[0044] 22. The VHH antibody of any one of the preceding embodiments, the VHH antibody is stable, in particular thermostable or hyperthermostable, VHH antibody.

[0045] 23. A VHH antibody according to embodiment 22, comprising: the VHH antibody has a melting temperature of at least about 40°C, at least about 50°C, at least about 60°C, at least 80°C, or at least about 95°C when measured under non-reducing conditions; VHH antibody.

[0046] 24. A VHH antibody according to embodiment 22 or 23, the VHH antibody has an aggregation temperature of at least about 40°C, at least about 50°C, at least about 60°C, at least 70°C, or at least about 80°C when measured under non-reducing conditions; VHH antibody.

[0047] 25. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody which is a variant thereof; VHH antibody.

[0048] 26. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7A08 having the VHH sequence shown in SEQ ID NO: 7, or a VHH antibody which is a variant thereof; VHH antibody.

[0049] 27. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody MaB03 having the VHH sequence shown in SEQ ID NO: 11, or a VHH antibody which is a variant thereof; VHH antibody.

[0050] 28. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7B12 having the VHH sequence shown in SEQ ID NO: 15, or a VHH antibody which is a variant thereof; VHH antibody.

[0051] 29. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19, or a VHH antibody which is a variant thereof; VHH antibody.

[0052] 30. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7C02 having the VHH sequence shown in SEQ ID NO: 23, or a VHH antibody which is a variant thereof; VHH antibody.

[0053] 31. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7E01 having the VHH sequence shown in SEQ ID NO: 27, or a VHH antibody which is a variant thereof; VHH antibody.

[0054] 32. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31, or a VHH antibody which is a variant thereof; VHH antibody.

[0055] 33. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7D06 having the VHH sequence shown in SEQ ID NO: 35, or a VHH antibody which is a variant thereof; VHH antibody.

[0056] 34. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma7C11 having the VHH sequence shown in SEQ ID NO: 39, or a VHH antibody which is a variant thereof; VHH antibody.

[0057] 35. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8E03 having the VHH sequence shown in SEQ ID NO: 43, or a VHH antibody which is a variant thereof; VHH antibody.

[0058] 36. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8G02 having the VHH sequence shown in SEQ ID NO: 47, or a VHH antibody which is a variant thereof; VHH antibody.

[0059] 37. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8H03 having the VHH sequence shown in SEQ ID NO: 51, or a VHH antibody which is a variant thereof; VHH antibody.

[0060] 38. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55, or a VHH antibody which is a variant thereof; VHH antibody.

[0061] 39. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8D05 having the VHH sequence shown in SEQ ID NO: 59, or a VHH antibody which is a variant thereof; VHH antibody.

[0062] 40. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, or a VHH antibody which is a variant thereof; VHH antibody.

[0063] 41. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma8D08 having the VHH sequence shown in SEQ ID NO: 67, or a VHH antibody which is a variant thereof; VHH antibody.

[0064] 42. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28F09 having the VHH sequence shown in SEQ ID NO: 71, or a VHH antibody which is a variant thereof; VHH antibody.

[0065] 43. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28H01 having the VHH sequence shown in SEQ ID NO: 75, or a VHH antibody which is a variant thereof; VHH antibody.

[0066] 44. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma31A12 having the VHH sequence shown in SEQ ID NO: 79, or a VHH antibody which is a variant thereof; VHH antibody.

[0067] 45. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Ma31B06 having the VHH sequence shown in SEQ ID NO: 83, or a VHH antibody which is a variant thereof; VHH antibody.

[0068] 46. ​​A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28C04 having the VHH sequence shown in SEQ ID NO: 87, or a VHH antibody which is a variant thereof; VHH antibody.

[0069] 47. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28H07 having the VHH sequence shown in SEQ ID NO: 91, or a VHH antibody which is a variant thereof; VHH antibody.

[0070] 48. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28C09 having the VHH sequence shown in SEQ ID NO: 95, or a VHH antibody which is a variant thereof; VHH antibody.

[0071] 49. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28H12 having the VHH sequence shown in SEQ ID NO: 99, or a VHH antibody which is a variant thereof; VHH antibody.

[0072] 50. A VHH antibody according to any one of embodiments 1 to 24, The VHH antibody is selected from the VHH antibody Bm28A12 having the VHH sequence shown in SEQ ID NO: 103, or a VHH antibody which is a variant thereof; VHH antibody.

[0073] 51. The VHH antibody of any one of the preceding embodiments, the VHH antibody is aglycosylated, or the VHH antibody is glycosylated, VHH antibody.

[0074] 52. The VHH antibody of any one of the preceding embodiments, The VHH antibody is produced in bacteria (e.g., E. coli), or in yeast (e.g., Pichia pastoris), or in human or animal cells (e.g., insect cells or mammalian cells, e.g., CHO cells), VHH antibody.

[0075] 53. The VHH antibody of any one of the preceding embodiments, the VHH antibody is in a monovalent format; VHH antibody.

[0076] 54. A VHH antibody according to any one of embodiments 1 to 53, the VHH antibody is in a multimeric format; VHH antibody.

[0077] 55. A VHH antibody according to embodiment 54, The VHH antibody is in a dimeric format. VHH antibody.

[0078] 56. A VHH antibody according to embodiment 55, the VHH antibody is in a homodimeric or heterodimeric format; VHH antibody.

[0079] 57. The VHH antibody of any one of the preceding embodiments, said VHH antibody is covalently or non-covalently conjugated to a heterologous moiety, such as a label group, a capture group, or an effector group; The heterologous moiety is in particular chosen from a fluorescent group, an enzyme such as biotin, peroxidase, phosphatase or luciferase, a hapten, an affinity tag or a nucleic acid such as an oligonucleotide; VHH antibody.

[0080] 58. The VHH antibody of any one of the preceding embodiments, the VHH antibody is fused to a heterologous polypeptide moiety, or the VHH antibody is fused to an Fc fragment, or the VHH antibody is fused to serum albumin or an albumin binding moiety; VHH antibody.

[0081] 59. The VHH antibody of any one of the preceding embodiments, the VHH antibody is conjugated to one or more non-peptidic polymer moieties, preferably hydrophilic polymer moieties such as polyethylene glycol (PEG); VHH antibody.

[0082] 60. A heteromultimeric VHH antibody, comprising two or more different VHH antibody units, for example two, three or four different VHH antibody units, wherein at least one VHH antibody unit comprises a VHH antibody according to any one of embodiments 1 to 53. Heteromultimeric VHH antibody.

[0083] 61. A heteromultimeric VHH antibody according to embodiment 60, The heteromultimeric VHH antibody comprises at least two VHH antibody units that recognize HLA, specifically different epitopes on HLA. Heteromultimeric VHH antibody.

[0084] 62. A heteromultimeric VHH antibody according to embodiment 60 or 61, The heteromultimeric VHH antibody comprises at least two VHH antibody units selected from the following different VHH antibody classes (i), (ii), (iii) and (iv): (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody that competes with the VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) a VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA; Including, Heteromultimeric VHH antibody.

[0085] 63. A heteromultimeric VHH antibody according to any one of embodiments 60 to 62, The heteromultimeric VHH antibody comprises at least two VHH antibody units, which VHH antibodies are selected from at least two different VHH antibody classes (i), (ii), (iii), (iv) and (v) below: (i) a VHH antibody according to embodiment 6 or 7; (ii) a VHH antibody according to embodiment 8 or 9; (iii) a VHH antibody according to embodiment 10 or 11; (iv) a VHH antibody according to embodiment 12 or 13; and (v) a VHH antibody according to embodiment 14 or 15; Including, Heteromultimeric VHH antibody.

[0086] 64. A set of two or more different VHH antibodies that recognize HLA, 64. At least one VHH antibody according to any one of embodiments 1 to 63, in particular at least one VHH antibody in a monovalent format. set.

[0087] 65. The set described in embodiment 64, The set comprises at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii) and (iv) as follows: (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody that competes with the VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) a VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA; Including, set.

[0088] 66. The set described in embodiment 64 or 65, The set comprises at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii), (iv) and (v) as follows: (vi) a VHH antibody according to embodiment 6 or 7; (vii) a VHH antibody according to embodiment 8 or 9; (viii) a VHH antibody according to embodiment 10 or 11; (ix) a VHH antibody according to embodiment 12 or 13; and (x) a VHH antibody according to embodiment 14 or 15; Including, set.

[0089] 67. A VHH antibody according to any one of embodiments 1 to 63, or a set according to any one of embodiments 64 to 66, for use in medicine, in particular for use in therapy or diagnosis, VHH antibody or set.

[0090] 68. A VHH antibody according to any one of embodiments 1 to 63, or a set according to any one of embodiments 64 to 66, For use in the prevention, treatment, and / or alleviation of conditions caused by, associated with, and / or accompanied by infection with HLA-positive Staphylococcus aureus. VHH antibody or set.

[0091] 69. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to embodiment 67 or 68, The Staphylococcus aureus is drug-resistant HLA-positive Staphylococcus aureus. VHH antibody or set.

[0092] 70. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to embodiment 67 or 68, The condition is selected from a skin infection, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis. VHH antibody or set.

[0093] 71. A VHH antibody according to any one of embodiments 1 to 63, or a set according to any one of embodiments 64 to 66, For the use according to any one of embodiments 67 to 69 in a human subject, VHH antibody or set.

[0094] 72. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to any one of embodiments 67 to 70, administered topically, VHH antibody or set.

[0095] 73. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to any one of embodiments 67 to 71, administered systemically, VHH antibody or set.

[0096] 74. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to any one of embodiments 67 to 72, Administered topically (externally), parenterally (e.g., intravenously), pulmonary (e.g., by inhalation), or nasally (e.g., by spray); VHH antibody or set.

[0097] 75. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for the use according to any one of embodiments 67 to 73, administered as monotherapy, VHH antibody or set.

[0098] 76. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for the use according to any one of embodiments 67 to 74, administered sequentially and / or simultaneously as combination therapy with at least one additional active ingredient; VHH antibody or set.

[0099] 77. A VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, for use according to embodiment 76, administered in combination with an antibiotic against Staphylococcus aureus, such as vancomycin, optionally in combination with an additional antibiotic against a different bacterium; VHH antibody or set.

[0100] 78. A composition comprising, as an active ingredient, a VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66, and a pharmaceutically acceptable carrier; Pharmaceutical compositions.

[0101] 79. A nucleic acid molecule encoding a VHH antibody or a VHH antibody-containing unit according to any one of embodiments 1 to 63, Preferably operably linked to a heterologous expression control sequence, Nucleic acid molecule.

[0102] 80. A vector comprising the nucleic acid molecule described in embodiment 79.

[0103] 81. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule described in embodiment 79 or a vector described in embodiment 80.

[0104] 82. A cell or organism according to embodiment 81, The cell or organism is selected from a bacterium such as E. coli, a Bacillus sp., a unicellular eukaryote (e.g., a yeast such as Pichia pastoris, or Leishmania), an insect cell, a mammalian cell, or a plant cell. Cells or organisms.

[0105] 83. A method for recombinantly producing a VHH antibody according to any one of embodiments 1 to 63, comprising: The method comprises culturing a cell or organism according to embodiment 81 or 82 in a suitable medium, and obtaining the VHH antibody from the cell or organism or from the medium. method.

[0106] 84. The method of embodiment 83, further comprising: The method comprises culturing yeast, such as Pichia pastoris, and obtaining the VHH antibody from the culture medium. method.

[0107] 85. Use of a VHH antibody according to any one of embodiments 1 to 63 or a set according to any one of embodiments 64 to 66 for detecting HLA in a sample.

[0108] 86. The use according to embodiment 85, The sample is a biological sample, for example a body fluid such as saliva, sputum, lung lavage, swab, blood, serum, or plasma, a stool sample, a tissue sample, or a biopsy sample; use.

[0109] 87. The use according to embodiment 85 or 86, the detection comprises binding of at least two different VHH antibodies to the HLA molecule; use.

[0110] 88. The use described in embodiment 87, said detection comprising the binding of at least two different VHH antibodies, for example two, three or four VHH antibodies, each directed against a different epitope, to the HLA molecule; use.

[0111] 89. The use according to any one of embodiments 85 to 88, The detection comprises a sandwich assay, such as a sandwich ELISA. use.

[0112] 90. The use according to any one of embodiments 85 to 89, The detection includes a prior HLA enrichment step. use.

[0113] 91. A method for the prevention, treatment, and / or alleviation of conditions caused by, associated with, and / or accompanied by infection with HLA-positive Staphylococcus aureus, comprising: The method comprises administering an effective amount of a VHH antibody according to any one of embodiments 1 to 63, or a set according to any one of embodiments 64 to 66, or a pharmaceutical composition according to embodiment 78 to a subject in need thereof, in particular a human subject, method.

[0114] [Description of the Invention] [VHH antibody] The present invention relates to VHH antibodies that recognize Staphylococcus aureus alpha-hemolysin (HLA).

[0115] VHH antibodies may be monovalent heavy chain-only antibodies comprising CDR1, CDR2 and CDR3 domains connected by framework regions, and include, but are not limited to: full-length VHH antibodies, e.g., naturally occurring 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., fusion proteins with immunoglobulin or non-immunoglobulin peptides or polypeptides, provided they exhibit the properties according to the invention.

[0116] The VHH antibodies of the present invention may be glycosylated or non-glycosylated.

[0117] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but no standard, unambiguous method exists. Determination of CDR sequences from antibody heavy chain variable regions may be performed according to any method known in the art, including, but not limited to, the Kabat, Chothia, and IMGT methods. The selected CDR set may include sequences identified by multiple methods; for example, some CDR sequences may be determined using Kabat and some using IMGT. According to some embodiments of the present invention, the CDR sequences of VHH variable regions are determined using the Kabat method. CDRs may also be defined through multiple alignments (with a number of other VHH antibodies) to identify hotspots of variation and relate them to standard VHH antibody structures. Furthermore, CDRs can be defined by analyzing the structure of a VHH antibody and determining which portions are loops and which portions are antibody scaffolds. In some cases, residues adjacent to the CDRs may also vary and are therefore included in the definition of the CDR.

[0118] The present invention is also directed to covalent or non-covalent conjugates of VHH antibody molecules with non-proteinaceous structures, such as, for example, a label group, a capture group such as a solid phase binding group, or an effector group such as a toxin. For example, the heterologous moiety may be derived from a fluorescent group, an enzyme such as biotin, peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.

[0119] The VHH antibody of the present invention is 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, such as a CHO cell. In some embodiments, the VHH antibody is non-glycosylated. In some embodiments, the VHH antibody is glycosylated, where the sugar chain structure may be derived from a glycosylation site introduced into the VHH sequence and / or from the fusion partner.

[0120] VHH antibodies according to the invention are characterized by: (i) the CDR3 sequence, (ii) a combination of the CDR1, CDR2 and CDR3 sequences, (iii) the complete VHH sequence, or (iv) by competition with a specific reference antibody. Specific CDR and VHH sequences are shown in the tables, figures and sequence listing.

[0121] The present invention encompasses sequences related to the above sequences. These related sequences are defined by having a minimum identity to a specifically designated amino acid sequence, such as a CDR or VHH sequence. This identity is shown over the entire length of the respective reference sequence and may be determined using well-known algorithms such as BLAST.

[0122] In specific embodiments, the related CDR3 sequence has at least 80%, or at least 90%, or at least 95% identity to the specifically indicated CDR3 sequence, and has, for example, 1, 2, or 3 amino acid substitutions.

[0123] In specific embodiments, the relevant combination of CDR1, CDR2, and CDR3 sequences has at least 80%, or at least 90%, or at least 95% identity to the specifically specified combination of CDR1, CDR2, and CDR3 sequences, and has substitutions of, for example, 1, 2, 3, 4, 5, or 6 amino acids with different amino acids.

[0124] In specific embodiments, the related VHH sequence has at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to the VHH sequence and has, for example, 1, 2, 3, 4, 5, or up to 20 amino acid substitutions.

[0125] The present invention further relates to VHH antibodies that compete with specific VHH antibodies disclosed herein for binding to S. aureus HLA polypeptides. In certain embodiments, the competing VHH antibodies bind to the same or overlapping epitopes on S. aureus HLA polypeptides. For example, the present invention relates to VHH antibodies that compete with, for example, the following reference antibodies: VHH antibodies Ma7A07 or Bm28C04 (binding to epitope 1 on HLA), VHH antibodies Ma7B01 or Ma7F02 (binding to epitope 2 on HLA), VHH antibody Ma8A05 (binding to epitope 4 on HLA), or VHH antibody Ma8D07 (binding to epitope 3 on HLA). Competition can be determined by label-free biolayer interferometry performed as a cross-competition assay or epitope binning assay using a label-free detection system, such as the Octet® system from Sartorius, according to the manufacturer's instructions.

[0126] The present invention further relates to antibodies belonging to a specific class of VHH antibodies characterized by common amino acids or amino acid sequences in the VHH sequences and / or by common amino acids or amino acid sequences that interact with HLA. For each corresponding structure, the interacting amino acid residues were identified by determining the intermolecular contacts of the VHH antibody-HLA structure using the "Find Clashes / Contacts" tool in UCSF Chimera (Pettersen et al., 2004). Residues with interatomic distances ≦4 Å are shown in Figure 7.

[0127] In one embodiment, the VHH antibody belongs to class 1 (Ma7A07 class) VHH antibodies.

[0128] Class 1 VHH antibodies are characterized by binding to HLA epitope 1. As shown in Figure 7, class 1 VHH antibodies interact with amino acid residues on HLA selected from the following, as set forth in SEQ ID NO: 107: (i) amino acid residues at positions 54 to 61, specifically Y54, K56, E57, M60, and / or H61; (ii) amino acid residues at positions 86 to 96, specifically T86, A88, Y91, R92, V93, Y94, and / or E96; (iii) amino acid residues at positions 233 to 239, specifically A233, D234, D238, and P239; and (iv) amino acid residues at positions 279 to 302, specifically R279, W300, and D302.

[0129] In one embodiment, a Class 1 VHH antibody is characterized by having a VHH sequence that includes the following HLA-interacting amino acid residues, based on the numbering in Figure 6A: - A, V, or D in position 55; - Y at position 59; - I in position 60; - D in position 62; - K at position 65; - T in position 69; - W at position 103; - R at position 104; - W at position 105; - V at position 106; - P at position 107, and - S at position 108.

[0130] In one embodiment, the VHH antibody belongs to class 2 (Ma7F02 class) VHH antibody.

[0131] Class 2 VHH antibodies are characterized by binding to HLA epitope 2. As shown in Figure 7, class 2 VHH antibodies interact with amino acid residues on HLA selected from the following, as set forth in SEQ ID NO: 107: (i) amino acid residues at positions 38 to 44, specifically T38, D39, I40, G41, S42, N43, and / or T44; (ii) amino acid residues at positions 141 to 143, specifically T141 and / or T143, and (iii) amino acid residues at positions 162 to 169, specifically I162, G163, N165, V166, S167, I168, and / or G169.

[0132] In one embodiment, class 2 VHH antibodies are characterized by having a VHH sequence that includes the following HLA-interacting amino acid residues, based on the numbering in Figure 6B: - Y or H in position 32; - F in position 47; - N, T, or S in position 52; - W or Y in position 59; - Y at position 60; - P at position 61; - D in position 62; - S or N at position 104; - Y at position 105; - W at position 106; - W at position 109; - H in position 111, and - E at 112th position.

[0133] In one embodiment, the VHH antibody belongs to class 3 (Ma8D07 class) VHH antibodies.

[0134] Class 3 VHH antibodies are characterized by binding to HLA epitope 3. As shown in Figure 7, class 3 VHH antibodies interact with amino acid residues on HLA selected from the following, as set forth in SEQ ID NO: 107: (i) amino acid residues at positions 210 to 219, specifically R210, D211, S212, W213, P215, and / or N219, and (ii) amino acid residues at positions 285 to 297, specifically H285, T287, S289, K292, T294, N295, and / or K297.

[0135] In one embodiment, a Class 3 VHH antibody is characterized by having a VHH sequence that includes the following HLA-interacting amino acid residues, based on the numbering in Figure 6C: - Y at position 29; - K in position 30; - L in position 31; - N at position 32; - A in position 33; - L at position 47; - T at position 50; - S at position 52; - S at position 53; - G in position 54; - F in position 58; - N at position 98; - P at position 99; - T at 100th position; - Y at position 101; - N at position 102, and - H at position 103.

[0136] In one embodiment, the VHH antibody belongs to class 4 (Ma8A05 class) VHH antibody.

[0137] Class 4 VHH antibodies are characterized by binding to HLA epitope 4. As shown in Figure 7, class 4 VHH antibodies interact with amino acid residues on HLA selected from the following, as set forth in SEQ ID NO: 107: (i) amino acid residues at positions 204 to 208, specifically N204, W205, G206, and / or Y208, and (ii) amino acid residues at positions 213 to 227, specifically W213, N214, V216, Q220, M223, K224, T225, R226, and / or N227;

[0138] In one embodiment, a Class 4 VHH antibody is characterized by having a VHH sequence that includes the following HLA-interacting amino acid residues, based on the numbering in Figure 6D: - A in position 53; - S at position 57; - D in position 58; - I in position 60; - T at position 61; - Y at position 62; - Y at position 63; - S at position 64; - D in position 65; - A in position 103; - F at position 104; - D in position 105; and - F at position 106.

[0139] In one embodiment, the VHH antibody belongs to class 5 (Bm28C04 class) VHH antibodies.

[0140] Class 5 VHH antibodies are characterized by binding to HLA epitope 1. As shown in Figure 7, Class 5 VHH antibodies interact with amino acid residues on HLA selected from the following, as set forth in SEQ ID NO: 107: (i) amino acid residues at positions 55 to 59, specifically D55, K56, E57, N58, and / or G59; (ii) amino acid residues at positions 88 to 97, specifically A88, G89, Q90, R92, V93, Y94, S95, E96, and / or E97, and (iii) amino acid residues at positions 240 to 302, specifically K240, R277, R279, W300, and / or D302.

[0141] In one embodiment, Class 5 VHH antibodies are characterized by having a VHH sequence that includes the following HLA-interacting amino acid residues, based on the numbering in Figure 6E: - N at position 30; - S in position 31; - Y in position 32; - Q in position 44; - R at position 45; - S at position 54; - D in position 100; - H in position 102; - Y at position 104; - A at position 105; - F at position 106; - G in position 107; and - D at position 109.

[0142] In a specific embodiment, at least one amino acid of a reference sequence, including an amino acid in the CDR1, CDR2, or CDR3 sequence and / or an amino acid in a framework region, is substituted with another amino acid while retaining the structural integrity and epitope binding ability of the VHH antibody. These exchanges may be conservative (i.e., with a similar amino acid) or non-conservative.

[0143] In more specific embodiments, at least one amino acid of the reference sequence, including amino acids in the CDR1, CDR2, or CDR3 sequence and / or in the framework region, is substituted by a conservative amino acid substitution, i.e., by substituting an amino acid with another amino acid having similar biochemical properties. This includes, for example, substituting an aliphatic amino acid, such as Gly, Ala, Val, Leu, or Ile, for another aliphatic amino acid; substituting a basic amino acid, such as His, Lys, or Arg, for another basic amino acid or for Met; substituting an acidic amino acid or its amide, such as Asp, Glu, Asn, or Gln, for another acidic amino acid or its amide; substituting an aromatic amino acid, such as Phe, Tyr, or Trp, for another aromatic amino acid.

[0144] In a more specific embodiment, the VHH antibody is selected from antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 3 are substituted with another amino acid.

[0145] In a more specific embodiment, the VHH antibody is selected from antibody Ma7A08 having the VHH sequence shown in SEQ ID NO: 7, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 7 are substituted with another amino acid.

[0146] In a more specific embodiment, the VHH antibody is selected from antibody Ma7B03 having the VHH sequence shown in SEQ ID NO: 11, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 11 are substituted with another amino acid.

[0147] In a more specific embodiment, the VHH antibody is selected from antibody Ma7B12 having the VHH sequence shown in SEQ ID NO: 15, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 15 are substituted by another amino acid.

[0148] In a more specific embodiment, the VHH antibody is selected from antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 19 are substituted with another amino acid.

[0149] In a more specific embodiment, the VHH antibody is selected from antibody Ma7C02 having the VHH sequence set forth in SEQ ID NO: 23, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 23 are substituted with another amino acid.

[0150] In a more specific embodiment, the VHH antibody is selected from antibody Ma7E01 having the VHH sequence set forth in SEQ ID NO: 27, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 27 are substituted with another amino acid.

[0151] In a more specific embodiment, the VHH antibody is selected from antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 31 are substituted by another amino acid.

[0152] In a more specific embodiment, the VHH antibody is selected from antibody Ma7D06 having the VHH sequence set forth in SEQ ID NO: 35, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 35 are substituted by another amino acid.

[0153] In a more specific embodiment, the VHH antibody is selected from antibody Ma7C11 having the VHH sequence set forth in SEQ ID NO: 39, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 39 are substituted with another amino acid.

[0154] In a more specific embodiment, the VHH antibody is selected from antibody Ma8E03 having the VHH sequence set forth in SEQ ID NO: 43, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 43 are substituted with another amino acid.

[0155] In a more specific embodiment, the VHH antibody is selected from antibody Ma8G02 having the VHH sequence set forth in SEQ ID NO: 47, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 47 are substituted with another amino acid.

[0156] In a more specific embodiment, the VHH antibody is selected from antibody Ma8H03 having the VHH sequence set forth in SEQ ID NO: 51, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 51 are substituted by another amino acid.

[0157] In a more specific embodiment, the VHH antibody is selected from antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 55 are substituted by another amino acid.

[0158] In a more specific embodiment, the VHH antibody is selected from antibody Ma8D05 having the VHH sequence set forth in SEQ ID NO: 59, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 59 are substituted by another amino acid.

[0159] In a more specific embodiment, the VHH antibody is selected from antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 63 are substituted with another amino acid.

[0160] In a more specific embodiment, the VHH antibody is selected from antibody Ma8D08 having the VHH sequence set forth in SEQ ID NO: 67, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 67 are substituted by another amino acid.

[0161] In a more specific embodiment, the VHH antibody is selected from antibody Bm28F09 having the VHH sequence set forth in SEQ ID NO: 71, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 71 are substituted with another amino acid.

[0162] In a more specific embodiment, the VHH antibody is selected from antibody Bm28H01 having the VHH sequence set forth in SEQ ID NO: 75, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 75 are substituted with another amino acid.

[0163] In a more specific embodiment, the VHH antibody is selected from antibody Ma31A12 having the VHH sequence set forth in SEQ ID NO: 79, or a VHH antibody which is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 79 are substituted with another amino acid.

[0164] In a more specific embodiment, the VHH antibody is selected from antibody Ma31B06 having the VHH sequence set forth in SEQ ID NO: 83, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 83 are substituted with another amino acid.

[0165] In a more specific embodiment, the VHH antibody is selected from antibody Bm28C04 having the VHH sequence set forth in SEQ ID NO: 87, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 87 are substituted with another amino acid.

[0166] In a more specific embodiment, the VHH antibody is selected from antibody Bm28H07 having the VHH sequence set forth in SEQ ID NO: 91, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 91 are substituted with another amino acid.

[0167] In a more specific embodiment, the VHH antibody is selected from antibody Bm28C09 having the VHH sequence set forth in SEQ ID NO: 95, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 95 are substituted with another amino acid.

[0168] In a more specific embodiment, the VHH antibody is selected from antibody Bm28H12 having the VHH sequence set forth in SEQ ID NO: 99, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 99 are substituted with another amino acid.

[0169] In a more specific embodiment, the VHH antibody is selected from antibody Bm28A12 having the VHH sequence set forth in SEQ ID NO: 103, or a VHH antibody that is a variant thereof. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 103 are substituted with another amino acid.

[0170] The present invention further relates to a nucleic acid molecule (e.g., a DNA molecule) encoding the VHH described above, or a vector comprising the nucleic acid molecule described above operably linked to an expression control sequence (particularly a heterologous expression control sequence). The present invention also relates to a cell comprising the above-described nucleic acid molecule or vector. Vectors for recombinant production of VHH antibodies are well known in the art. In one embodiment, the vector is an extrachromosomal vector. In another embodiment, the vector is a vector for genomic integration. The cell may be a known host cell for producing antibodies or antibody fragments, such as a prokaryotic cell (e.g., an E. coli or Bacillus cell), a yeast cell (particularly a Pichia yeast cell), an insect cell (e.g., an Sf-9 cell), a mammalian cell (e.g., a CHO cell), or a plant cell. In one embodiment, the cell comprises the nucleic acid or vector extrachromosomally. In another embodiment, the cell comprises the nucleic acid or vector integrated into the genome, for example, as an expression cassette integrated into the genome.

[0171] Yet another aspect of the present invention is a method for recombinantly producing a VHH antibody by growing the above-mentioned cells in a culture medium and obtaining said VHH antibody from said cells or said culture medium. Suitable culture media and conditions are well known in the art.

[0172] [Staphylococcus aureus HLA binding] The VHH antibodies of the present invention bind to S. aureus HLA polypeptides. The inventors have identified VHH antibodies that bind with high affinity to different epitopes on S. aureus HLA, as shown in Table 1 (supra).

[0173] In the context of this disclosure, the term "S. aureus HLA" encompasses S. aureus HLA from various strains, such as MRSA-resistant strains such as USA300, USA400 (CC1), USA500 (CC8), or ST80. These and other suitable strains are described, for example, in 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 incorporated herein by reference.

[0174] However, it should be noted that the term "S. aureus HLA" also encompasses naturally occurring variants of S. aureus HLA polypeptides, as well as the genetically modified constructs described herein.

[0175] The inventors performed selection and crystallization experiments using the HLA shown in SEQ ID NO: 1, and Octet assays using Cys-HLA shown in SEQ ID NO: 2 following biotinylation at an ectopic cysteine ​​at the N-terminus.

[0176] In one embodiment, the VHH antibodies of the invention bind to Staphylococcus aureus HLA, where the dissociation constant K D The binding affinity, expressed as a function of the binding affinity, is about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less. The binding affinity can be determined using the polypeptides of SEQ ID NOs: 1 and 2 above, as described in detail in the Examples herein.

[0177] [HLA neutralization] The VHH antibodies of the present invention are capable of neutralizing Staphylococcus aureus HLA, specifically by inhibiting hemolysis of blood cells (eg, red blood cells) as shown in Table 1 (supra).

[0178] In one embodiment, the VHH antibodies of the present invention alter the biological properties of HLA, specifically by inhibiting HLA binding to receptors and / or membranes and / or heptameric pore formation.

[0179] In one embodiment, a VHH antibody, e.g., Ma8A05 or a competing antibody, binds to the HLA hemolysin protomer and disrupts the assembly of the heptameric pore. In a further embodiment, a VHH antibody, e.g., Ma7A07 and Ma8D07, or a competing VHH antibody, disrupts subunit assembly. In a further embodiment, a VHH antibody, e.g., Ma7F02 or a competing VHH antibody, inhibits the conformational change that results in the membrane-inserted β-hairpin.

[0180] In one embodiment, the VHH antibodies of the present invention neutralize HLA at a concentration stoichiometrically corresponding to the concentration of HLA. Neutralization potency can be determined as described in detail in the Examples herein. In Example 3, in vitro lysis of red blood cells was observed at an HLA concentration of about 0.5 to 1 nM. This lysis could be neutralized with a VHH antibody concentration of about 1 nM.

[0181] [Stability] For therapeutic use, anti-HLA antibodies must not only be highly potent in HLA neutralization, but also be developable as biopharmaceuticals, including being sufficiently stable to withstand lengthy, large-scale manufacturing processes as well as transportation and storage (ideally for several years in liquid formulations) without aggregation or loss of activity.

[0182] A good predictor of stability is thermostability, which can be measured, for example, by thermal shift assays or, more particularly, by differential scanning fluorescence. The inventors have identified several VHH antibodies that are thermostable or hyperthermostable, as shown in Table 1 (supra).

[0183] In a specific embodiment, the present invention relates to a VHH antibody that is stable, in particular thermostable or hyperthermostable. Preferably, said VHH antibody has a melting point (melting temperature) of at least about 40°C, at least about 50°C, at least about 60°C, at least about 80°C, at least 90°C, or at least about 95°C, when measured under non-reducing conditions, and / or an aggregation temperature of at least about 40°C, at least about 50°C, at least about 60°C, at least 70°C, or at least about 80°C. Melting and aggregation temperatures are determined as described herein.

[0184] [VHH antibody set] In a further aspect, the present invention relates to a set comprising at least two, three, four, or more of the above-described VHH antibodies. In such a set, the individual VHH antibodies are present at an appropriate molar ratio. Typically, the molar ratio ranges from about 2:1 to about 1:2, specifically about 1.5:1 to about 1:1.5, and more specifically about 1:1. In one embodiment, the VHH antibody set may comprise a single composition, and the VHH antibodies in the set consist of a predetermined number of different types of the above-described VHH antibodies. The VHH antibody set may also comprise multiple compositions, each containing a different type of the above-described VHH antibody. The set of the present invention may not contain other VHH antibodies.

[0185] In a specific embodiment, 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 epitopes of HLA, e.g., a VHH antibody directed against epitope 1, a VHH antibody directed against epitope 2, a VHH antibody directed against epitope 3, and / or a VHH antibody directed against epitope 4, as defined herein.

[0186] For example, the set of VHH antibodies comprises at least two, e.g., two, three, or four complementary VHH antibodies selected from at least two, three, or four different VHH antibody classes (i), (ii), (iii), and (iv) as follows: (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody that competes with the VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 and / or Ma7F02 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) A VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA.

[0187] A specific preferred set of VHH antibodies includes: - the VHH antibody Ma7A07, having the VHH sequence shown in SEQ ID NO: 3; - the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19, - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0188] A further specific set of preferred VHH antibodies includes: - the VHH antibody Ma7A07, having the VHH sequence shown in SEQ ID NO: 3; - the VHH antibody Ma7F02, having the VHH sequence shown in SEQ ID NO: 31; - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0189] A further specific set of preferred VHH antibodies includes: - the VHH antibody Ma8E03, having the VHH sequence shown in SEQ ID NO: 43; - the VHH antibody Ma7F02, having the VHH sequence shown in SEQ ID NO: 31; - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0190] A further specific set of preferred VHH antibodies includes: - the VHH antibody Ma8E03, having the VHH sequence shown in SEQ ID NO: 43; - the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19, - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0191] A further specific set of preferred VHH antibodies includes: - the VHH antibody Bm28C04, having the VHH sequence shown in SEQ ID NO: 87; - the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19, - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0192] A further specific set of preferred VHH antibodies includes: - the VHH antibody Bm28C04, having the VHH sequence shown in SEQ ID NO: 87; - the VHH antibody Ma7F02, having the VHH sequence shown in SEQ ID NO: 31; - the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, and optionally the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55.

[0193] Sets of different VHH antibodies are useful for therapeutic and diagnostic applications, as described in detail herein below.

[0194] [Monovalent and polyvalent VHH antibodies] In certain embodiments, the VHH antibodies of the invention are in a monovalent format, i.e., have a single binding site for a S. aureus polypeptide. In these embodiments, the VHH antibodies may be present naked or may be covalently or non-covalently attached to a heterologous moiety (e.g., a peptidic or non-peptidic moiety).

[0195] In further embodiments, the VHH antibodies of the present invention are in a multimeric (e.g., dimeric, trimeric, or tetrameric) format. In these embodiments, multiple VHH antibody units may be covalently or non-covalently attached to each other via linkers and / or multimerizing (e.g., dimerizing, trimeric, or tetramerizing) moieties. In these embodiments, the VHH antibodies of the present invention may be homomultimeric or heteromultimeric antibodies.

[0196] A heteromultimeric VHH antibody comprises two or more, for example, two, three, or four different VHH antibody units. The different VHH antibody units may all be directed against the same S. aureus polypeptide, specifically HLA, more specifically against different epitopes on HLA (e.g., as defined herein). Alternatively, the different VHH antibody units may be directed against different S. aureus polypeptides, for example, at least one VHH antibody unit may be directed against HLA and at least one VHH antibody unit may be directed against one or more other S. aureus polypeptides (e.g., S. aureus virulence factors).

[0197] In one embodiment, the heteromultimeric VHH antibody comprises two or more different VHH antibody units, for example, two, three, or four different VHH antibody units directed against different Staphylococcus aureus polypeptides or directed against Staphylococcus aureus HLA, wherein at least one VHH antibody unit comprises a VHH antibody described herein.

[0198] In one embodiment, the heteromultimeric VHH antibody comprises at least two VHH antibody units directed against S. aureus HLA selected from the following different VHH antibody classes (i), (ii), (iii) and (iv): (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody that competes with the VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) A VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA.

[0199] In one embodiment, the heteromultimeric VHH antibody comprises at least two VHH antibody units selected from different VHH antibodies belonging to at least two classes of VHH antibody classes 1, 2, 3, 4, and 5 as defined herein.

[0200] In certain embodiments, the VHH antibody may be covalently or non-covalently conjugated to a heterologous moiety selected from a label group, a capture group, or an effector group, specifically a fluorescent group, an enzyme such as biotin, peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide. In further embodiments, the heterologous moiety is selected from human serum albumin, an albumin-binding portion, or an Fc fragment of an immunoglobulin molecule, such as IgA, IgD, IgE, IgG, IgM, or a subtype thereof. In yet further embodiments, the heterologous moiety is selected from one or more nonpeptidic polymer moieties, preferably hydrophilic polymer moieties such as polyethylene glycol (PEG).

[0201] In one embodiment, the VHH antibody is a homodimeric VHH antibody, in which a VHH antibody unit is covalently attached to a dimerization moiety (eg, the Fc fragment of immunoglobulin IgG).

[0202] In one embodiment, the VHH antibody is a heterodimeric VHH antibody, specifically a covalently linked VHH heterodimer comprising a first VHH antibody and a second VHH antibody, wherein the first VHH antibody and the second VHH antibody bind to different epitopes on HLA.

[0203] [Production of VHH antibodies] VHH antibodies, including monomeric and multimeric VHH antibodies, may be produced as described in WO2022 / 023483 and WO2022 / 023484, the contents of which are incorporated herein by reference, or by other methods known in the art.

[0204] VHH antibodies can be recombinantly produced in suitable host cells, such as prokaryotic or eukaryotic host cells or host organisms. To this end, 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.

[0205] In a specific embodiment, the VHH antibody is recombinantly produced in bacteria, such as E. coli or Bacillus. For example, expression in bacteria can involve cytoplasmic and / or periplasmic expression with purification of the VHH antibody from the host cell, or secretory expression with purification of the VHH antibody from the culture medium. In one embodiment, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence that directs expression into the periplasm and / or into the culture medium.

[0206] In more specific embodiments, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism, preferably in yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Hansenula polymorpha), or in an animal cell, in particular an insect cell (e.g., Sf-9 cell), or a mammalian cell (e.g., a human or hamster cell, such as HEK293F, CHO, or COS-7). For example, expression in a eukaryotic host cell or host organism (e.g., yeast) can 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 one embodiment, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence that directs expression into the culture medium.

[0207] VHHs Ma7A07, Ma7B01, Ma7F02, Ma8A05, Ma8D07, and Ma8H03 were converted to a VHH-IgG1-Fc format, transfected into CHO-DG44 cells using transposase to integrate into the active locus, and selected to generate clonal pools. When these pools were compared with conventional IgG1 antibodies and other VHH-IgG1-Fc fusions in fed-batch fermentation, unexpectedly, 2- to 3-fold higher secreted product yields were observed, reaching 14 g / L. These unusually high titers are likely due to the extremely low aggregation tendency and high stability of the VHHs disclosed herein.

[0208] [Treatment use] A further aspect of the present invention is the use of the above-mentioned VHH antibodies in medicine, in particular for therapeutic and / or in vitro or in vivo diagnostic applications. In one embodiment, the VHH antibodies are used in human medicine.

[0209] The VHH antibodies of the present invention are useful for preventing, treating, and / or alleviating conditions caused by, associated with, and / or accompanied by infection with HLA-positive Staphylococcus aureus. In one embodiment, the Staphylococcus aureus is drug-resistant Staphylococcus aureus.

[0210] Exemplary conditions include, but are not limited to, skin infections, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis.

[0211] In therapeutic applications, VHH antibodies are administered to a subject in need thereof, particularly a human subject, in an effective amount, which depends on the particular type of component, e.g., monovalent or multimeric VHH antibody, the type of disease, and the route of administration.

[0212] Typically, VHH antibodies are administered as pharmaceutical compositions comprising an active ingredient and a pharmaceutically acceptable carrier or excipient. Examples of carriers and excipients suitable for formulating antibodies or antibody fragments are well known in the art.

[0213] Depending on the stage and severity of the disease, the pharmaceutical composition may be administered one or more times during the course of the disease, for example, once or more times daily, every other day, twice weekly, or once weekly for an appropriate period of time.

[0214] In certain embodiments, the pharmaceutical composition is administered parenterally, for example, by subcutaneous, intramuscular, or intravenous injection, or by infusion. In further embodiments, the pharmaceutical composition is administered topically (e.g., by topical application), orally, nasally (e.g., by spraying), or pulmonary (e.g., by inhalation as an aerosol).

[0215] In one embodiment, treatment of patients infected with HLA-positive Staphylococcus aureus involves the systemic administration (e.g., by injection) of one or more complementary neutralizing anti-HLA VHH antibodies. In the case of pneumonia, inhalation may be the preferred route of administration. In either case, treatment should reduce HLA-mediated tissue damage and pathogen virulence, allowing the immune system to eliminate the infection.

[0216] Prophylactic use of anti-HLA VHH antibodies is contemplated for subjects who are colonized with Staphylococcus aureus or who have been or may be exposed to the pathogen, and / or who are immunocompromised, preparing for surgery, and / or have suffered an injury that predisposes them to infection.

[0217] Anti-HLA VHH antibodies are also effective against highly antibiotic-resistant strains and in advanced stages of infection (e.g., in advanced sepsis) when the effects of the toxins can no longer be eliminated by antibiotic treatment alone.

[0218] VHH antibodies may be administered alone as monotherapy, or as combination therapy, e.g., sequentially or simultaneously, with additional active ingredients, in particular with additional ingredients useful for the prevention, treatment, and / or alleviation of diseases caused by and / or associated with infection by Staphylococcus aureus and / or optionally a different bacterium. In one embodiment, the VHH antibody is administered in combination with an antibiotic against Staphylococcus aureus, e.g., vancomycin, and optionally in combination with an additional antibiotic against a different bacterium.

[0219] [Diagnostic use] Diagnostic applications include in vitro methods in which a VHH antibody or a set of different VHH antibodies is used to detect HLA in a sample, for example a body fluid such as saliva, sputum, lung lavage fluid, swab, blood, serum or plasma, a stool sample, or a tissue or biopsy sample.

[0220] Diagnostic applications further include in vivo methods in which VHH antibodies are used to detect HLA in a subject, particularly a human patient. In diagnostic applications, VHH antibodies may be labeled for direct detection or may be used in combination with a secondary detection reagent (e.g., biotin / streptavidin), a detection antibody (including a conventional antibody or a VHH antibody) for indirect detection according to techniques established in the art.

[0221] The present invention encompasses the use of anti-HLA VHH antibodies for highly sensitive detection of toxins. The availability of four complementary epitope classes can be used for highly sensitive detection. In one embodiment, the detection format involves the binding of at least two different VHH antibodies, e.g., two, three, or four different VHH antibodies, to an HLA molecule, specifically at least two different VHH antibodies, e.g., two, three, or four VHH antibodies, each directed against a different epitope. In a specific embodiment, the detection involves the use of a set of two, three, or four different VHH antibodies as described above.

[0222] In a specific embodiment, the detection involves a sandwich assay, such as a sandwich ELISA. In such a test format, a first VHH antibody may be immobilized on a solid phase to capture HLA from the sample, and a second VHH antibody is used in labeled form for the actual detection. A third and / or fourth VHH antibody may also be used for a preliminary enrichment step, for example, by a capture and proteolytic release approach (Frey & Gorlich, 2014b; Frey & Gorlich, 2014a; Vera Rodriguez et al., 2019). This allows for rapid and sensitive detection of HLA, which is crucial in life-threatening conditions.

[0223] The present invention will now be explained in more detail by the following figures and examples.

[0224] [HLA sequence] >HLA (SEQ ID NO: 1) used in hemolysis assays and crystallization ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTL KYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0225] >Cys-HLA (SEQ ID NO: 2) used in the Octet assay following biotinylation at the N-terminal ectopic cysteine SECGSSGADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIG HTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0226] >HLA (SEQ ID NO: 104) including the signal sequence used to determine amino acid interactions MKTRIVSSVTTTLLLGSILMNPVAGAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDDT GKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKI DWEKEEMTN

[0227] [VHH antibody sequence] >Ma7A07 (SEQ ID NO: 3) QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSALSIYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAMDLWGKGTLVTVSS

[0228] >Ma7A08 (SEQ ID NO: 7) QVQLVESGGGLVQPGGSLTLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSVLSVYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAMDLWGKGTLVTVSS

[0229] >Ma7B03 (SEQ ID NO: 11) QVQLVESGGGLVQPGGSLRLSCAASGFTLSNYYVGWFRQAPGKQREGVSSIGSSDLSIYIADSVKGRFTISRDNAKNTIYLHMNNLKPDDTGVYYCARGTDSWRWVPSAMDLWGKGTQVTVSS

[0230] >Ma7B12 (SEQ ID NO: 15) QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSALSIYIADSVKGRFTISRDNAKNTVYLHMNNLKPEDTGVYYCARGIDNWRWVPSAMDLWGKGTQVTVSS

[0231] >Ma7B01 (SEQ ID NO: 19) QVQLVESGGGLVQAGDSLSLSCAASGRTFSNYAMGWFRQAPGKERHFVAVINQIGDSTWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWPHEYGYWGQGTQVTVSS

[0232] >Ma7C02 (SEQ ID NO: 23) QVQLVESGGGLVQAGGSLLSCAASGRTFSDYAMGWFRQAAGKDRDFVAVITRSGDSTYYPDSTKGRFTISRDNAKNTMYLQMNSLKPEDTARYYCAATDPTNYWRIWEHEFDYWGQGTQVTVSS

[0233] >Ma7E01 (SEQ ID NO: 27) QVQLVESGGGSVQAGDSLSLSCVASERTFSNYAMGWFRQAPGKERHFVAVINPIGEETWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWEHEFDYWGQGTQVTVSS

[0234] >Ma7F02 (SEQ ID NO: 31) QVQLVESGGGLVQAGGSLRLSCTVSGSIDSLHTMGWFRQAPGKKRDFVAVVSWSGGNTYYPDYAKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAAESGSGNYWKIWEHEFDSWGQGTQVTVSS

[0235] >Ma7D06 (SEQ ID NO: 35) QVQLVESGGGLVQAGGSLRLSCAASGSISGIDTMAWFRQIPGKDREFVAVIKWAAGSTWYPDFVKGRFTVSRDNAKNMVYLQMDSVKPEDTAIYYCAAAAGEKYYYRLFPYEYDYWGQGTQVTVSS

[0236] >Ma7C11 (SEQ ID NO: 39) QVQLVESGGGLVRAGGSLRLSCAASGSISSVNGMGWFRQAPGKERLFVAQVSLLDSSTYYADSVKGRFTISRDDAKNTMYLQMNNVRPEETAVYYCAATKLRIGTAFSSEYDYWGQGTQVTVSS

[0237] >Ma8E03 (SEQ ID NO: 43) QVQLVESGGGLVHPGGSLRLLSCAASGSSLDHYVIGWFRQVPGKEREGVSCISRSGGSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLGWLEYDDWGQGTQVTVSS

[0238] >Ma8G02 (SEQ ID NO: 47) QVQLVESGGGLVLPGGSLLSCAASGSSLDHYVIGWFRQYPGKEREGVSCISRSGGSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLGWVEYDDWGQGTQVTVSS

[0239] >Ma8H03 (SEQ ID NO: 51) QVQLVESGGGLVHPGGSLRLLSCAASGSSLDHYVIGWFRQVPGKQREGISCISSGGSTNYADSVKGRFTVSRDNVKSTVYLQMNSLAPDDSAEYYCAAQRNLGNFCVLGWLEYDDWGQGTQVTVSS

[0240] >Ma8A05 (SEQ ID NO: 55) QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0241] >Ma8D05 (SEQ ID NO: 59) QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDTVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0242] >Ma8D07 (SEQ ID NO: 63) QVQLVESGGGLVQPGGSLRLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGGSTFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQGTQVTVSS

[0243] >Ma8D08 (SEQ ID NO: 67) QVQLVESGGGFVQAGGSLRLLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGGSTFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQGTQVTVSS

[0244] >Bm28F09 (SEQ ID NO: 71) QVQLVESGGGLVEPGGSLTLSCAASGFTSKNYYIGWFRQAPGKYREGVASIGASDGSLYIADSVKGRFTISSDNAKNTVYLHPRNLKPEDSGVYYCATGRDSWRWVPSAMDYWGKGIQVTVSS

[0245] >Bm28H01 (SEQ ID NO: 75) QVQLVESGGGLVQPGGSLTLSCAASGFTLGDRTLGWFRQVAGKQREGVASIASGVLSAYIADSVKGRFTISRDDAKNTIYLHMNNLKPDDTGVYYCARGVDSWRWVPSTMDLWGKGILVTVSS

[0246] >Ma31A12 (SEQ ID NO: 79) QVQLVESGGGLVQPGGSLRLSCAASAASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEVDADYWGQGTQVTVSS

[0247] >Ma31B06 (SEQ ID NO: 83) QVQLVQSGGGLVQPGGTLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0248] >Bm28C04 (SEQ ID NO: 87) QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRSTGGTSYADSVKGRFTISYDNVQNTVDLQMNSLEPEDTAIYYCAGGDGHIYAFGYDYWGQGTQVTVSS

[0249] >Bm28H07 (SEQ ID NO: 91) QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRSTGGTSYADSVKGRFTISYDNVQNTVDLQMSSLEPEDTAIYYCAGGDGHIYAFGYDYWGQGTQVTVSS

[0250] >Bm28C09 (SEQ ID NO: 95) QVQLVESGGSVVQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRSTGKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGYDYWGQGTQVTVSS

[0251] >Bm28H12 (SEQ ID NO: 99) QVQLVESGGSVVQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRSTGKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGYDYWGQGTQVTVSS

[0252] >Bm28A12 (SEQ ID NO: 103) QVQLVESGGGLVQAGGSLRLLSCVVSGRALSSYNLAWFRQSPGKEREWIATIMPSADKAHYPDFLEGRFTISGDNTENTLYLQMNSLKPEDTAIYYCAAREPSYYHALFEYEYNLWGQGTQVTVSS [Example]

[0253] VHH antibodies that inhibit HLA activity were generated by immunizing two alpacas three times at three-week intervals with a non-toxic HLA mutant (His61Thr), preparing an immune library, and performing phage display using HLA as bait.

[0254] The 192 selected clones were sequenced and classified by sequence similarity. Seventeen representatives of all VHH classes were then expressed in E. coli and purified (Ma7 and Ma8 series, see Figure 1 for specific sequences).

[0255] To generate "second-generation" VHH antibodies, alpacas were immunized with HLA variant proteins 16 months after the last immunization, followed by immune library preparation and phage display selection. This yielded additional VHH antibodies, including VHH variants from previously isolated classes (e.g., those belonging to the Ma7A07 and Ma8A05 classes) as well as new VHH classes, such as Bm28C04, Bm28C09, and Bm28A12.

[0256] VHH-HLA interactions were analyzed by biolayer interferometry (BLI; Abdiche et al., 2008 ).

[0257] VHH antibodies belonging to the Ma8A05 and Ma8D07 classes bind to HLA with intermediate affinity, e.g., K D The K values ​​were 600 pM (for Ma8A05) and 800 pM (for Ma8D07) (see Figure 2). Other VHH antibodies showed significantly stronger HLA binding (Table 1 and Figure 2). Ma7B01, Ma7F02, and Bm28C04 had K values ​​of ~50 pM. D No dissociation was detected in Ma7A07 and Ma8H03. D The value is estimated to be ≦10 pM.

[0258] To assess whether and how many of these VHHs have complementary binding sites on HLA molecules, binning experiments were performed using BLI. In these experiments, one VHH antibody was bound to immobilized HLA, and then the binding of another VHH was monitored. An example is shown in Figure 3. When HLA was fully saturated with Ma7A07, Ma8H03 (the VHH with the highest affinity for HLA) was no longer able to bind, while the binding of the other tested VHHs remained unaffected. By repeating this setup with members of all representative VHH classes, we found that the selected VHH antibodies targeted four complementary epitopes (Table 1). Figure 3 shows that up to four VHH antibodies can simultaneously bind to HLA if each is selected to recognize a different complementary epitope.

[0259] In the following experiments, we tested whether these VHH antibodies could neutralize HLA. Red blood cells (RBCs) are highly sensitive to the lytic activity of HLA. Furthermore, the high concentration of heme (a part of hemoglobin that carries oxygen) in RBCs makes cell lysis very easy to detect. Therefore, we performed HLA-induced hemolysis experiments using RBCs. We observed that 30 nM HLA caused complete cell lysis. When tested at a concentration of 100 nM, all anti-HLA VHHs completely protected cells from hemolysis (Figure 4).

[0260] To understand the structural basis of VHH-dependent HLA inhibition, we sought to crystallize HLA·VHH complexes. We solved the X-ray crystal structures of dimeric HLA complexes of four VHH antibodies representing different epitope binders: Ma7A07, Bm28C04, Ma7F02, Ma8A05, and Ma8D07 (Figure 5). Crystallographic analysis confirmed the fact that these VHHs possess four distinct binding sites on HLA. Figure 5 also illustrates an example of the mode of action of Ma8A05: it binds to the hemolysin protomer and disrupts the assembly of the heptameric pore through collisions with adjacent subunits. Further analysis revealed that other VHH antibodies either disrupt subunit assembly (i.e., Ma7A07, Bm28C04, and Ma8D07) or disrupt the conformational changes preceding the generation of the membrane-inserted β-hairpin (i.e., Ma7F02).

[0261] Example 1: Binding of VHH antibodies to HLA Biolayer interferometry (BLI) experiments were performed using a High Precision Streptavidin biosensor and an Octet RED96e instrument (ForteBio / Sartorius) at 25°C 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. For this, HLA was expressed with an ectopic cysteine ​​at its N-terminus and biotinylated via the addition of sulfhydryl-reactive maleimide-PEG11-biotin (Thermo Scientific, 21911).

[0262] Biotinylated HLA was immobilized on the biosensor until a 1 nm wavelength shift / binding signal was achieved. The biosensor was immersed in wells containing 40, 20, 10, 5, 2.5, or 1.25 nM VHH for 10 minutes of association, followed by 30 minutes of incubation in assay buffer for dissociation. Data were subtracted from the reference value, and curves (gray) were fitted using a mass transport model (Octet Data Analysis HT 12.0 software).

[0263] The results are shown in Table 1 and FIG.

[0264] Example 2: Identification of complementary HLA epitopes Epitope binning experiments were performed using a similar setup to that described in Example 1. Biotinylated HLA was immobilized on a biosensor until a wavelength shift / binding signal of 1 nm was achieved. The biosensor was immersed in a well containing 100 nM VHH (VHH1) for 240 seconds, and then incubated with another VHH (VHH2) at 100 nM for 240 seconds. If the binding of VHH2 was blocked in the presence of VHH1, they were considered to be identical epitope binders. By repeating this process for all VHH class representatives, we grouped the VHH antibodies into four distinct epitope binders.

[0265] The results are shown in Table 1 and Figure 3. Ma7A07, Ma7F02, Ma8D07, and Ma8A05 were able to sequentially bind to immobilized HLA.

[0266] Example 3: Erythrocyte Hemolysis Assay Erythrocyte stocks were freshly prepared from blood samples collected from CD1 male mice. 30 nM HLA was diluted in 150 μl of PBS (pH 7.4), 0.4% (w / v) glucose, and 25 mM EDTA (assay buffer) in the presence or absence of 100 nM VHH antibodies. After 30 min of incubation at room temperature, this was distributed across 4 × 10 cells in one well of a standard 96-well plate.8 was added to 50 μL containing red blood cells.

[0267] The plate was incubated at 37°C with 180 rpm shaking for 3 hours, followed by 1 hour at 4°C to complete cell lysis. The plate was then centrifuged in a swing-out rotor for 10 minutes at 400 x g and 4°C. 150 μL of the supernatant was transferred to another 96-well plate. Analysis was performed using a plate reader at 412 nm absorbance.

[0268] The results are shown in Figure 4A. Note that dilution of red blood cells in water led to complete lysis, and that 30 nM HLA produced the same amount of cell lysis. All VHH antibodies tested potently inhibited cell lysis, with levels comparable to those seen in assay buffer.

[0269] Second-generation VHH antibodies were also tested for inhibitory activity as described above and in Figure 4B, except that human red blood cells were used. Because human red blood cells are less sensitive to HLA, 250 nM HLA was used for cell lysis, and 1 μM VHH antibody was used where indicated. Similarly, all VHH antibodies tested inhibited cell lysis.

[0270] Example 4 - Measurement of thermal stability of VHH antibodies VHH antibodies were subjected to differential scanning fluorescence (DSF), which utilizes thermal unfolding to expose aromatic / hydrophobic residues that subsequently bind to added SYPRO Orange dye, enhancing its fluorescence. The assay was performed in a 20 μl volume at a VHH concentration of 1 mg / ml in 50 mM Tris / HCl, 150 mM NaCl (pH 8.0 at 20°C), and 1× dye (diluted from a 5000× stock; Life Technologies). Two replicates of each sample were pipetted into a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with a clear MicroSeal® 'B' Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed in a CFX96 real-time system (C1000 thermal cycler, Bio-Rad). Samples were incubated at 20°C for 5 minutes, followed by a 1°C increase every 45 seconds to 95°C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long-pass filter. The melting temperature is defined as the inflection point of the first melting peak.

[0271] The results are shown in Table 1 and Figure 8. The VHH antibody Re9B09, which is produced in a reducing cytoplasm and therefore lacks disulfide bonds, melts already at 35°C. The neutralizing VHH antibodies Ma8A05 and Ma7F02 melt similarly at 44°C and 62°C, respectively, while Ma7A07 and Ma8E03 showed negligible melting amplitudes and therefore remained stable up to 95°C.

[0272] Example 5 - Testing VHH stability by BLI BLI is highly sensitive to sample concentration and therefore suitable for detecting any loss of active substance in solution, including loss of active nanobodies due to instability. To determine whether VHHs that already melt at 60–70 °C in DSF experiments retain their binding activity after heat treatment, the antibodies were incubated at 95 °C for 10 min (at 1 μM concentration), cooled, and then centrifuged to remove any aggregates that may have formed. When they were tested for HLA binding by BLI, heated and untreated samples of Ma31A12 and Bm28A12 showed no difference and behaved similarly to the heat-stable VHH Ma8E03 (Figure 9). Thus, these nanobodies either only partially melt or robustly refold to their native state after heat treatment. On the other hand, Ma8D07 showed a loss of activity, suggesting that they aggregated to some extent after heat treatment.

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Claims

1. A VHH antibody that recognizes Staphylococcus aureus (S. aureus) hemolysin (HLA), The VHH antibody is (i) VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, for binding to HLA; or (ii) VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO: 19 and / or VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO: 31 for binding to HLA; or (iii) VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO: 55, for binding to HLA; or (iv) VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, for binding to HLA; competing, VHH antibody.

2. A VHH antibody that recognizes Staphylococcus aureus hemolysin (HLA), The VHH antibody comprises an amino acid residue on HLA selected from the following, as set forth in Figure 7 (SEQ ID NO: 107): (a): (i) amino acid residues at positions 54 to 61, specifically Y54, K56, E57, M60, and / or H61; (ii) amino acid residues at positions 86 to 96, specifically T86, A88, Y91, R92, V93, Y94, and / or E96; (iii) amino acid residues at positions 233 to 239, specifically A233, D234, D238, and P239; and (iv) amino acid residues at positions 279 to 302, specifically R279, W300, and D302; (b): (i) amino acid residues at positions 38 to 44, specifically T38, D39, I40, G41, S42, N43, and / or T44; (ii) amino acid residues at positions 141 to 143, specifically T141 and / or T143; and (iii) amino acid residues at positions 162 to 169, specifically I162, G163, N165, V166, S167, I168, and / or G169; (c): (i) amino acid residues at positions 210 to 219, specifically R210, D211, S212, W213, P215, and / or N219; and (ii) amino acid residues at positions 285 to 297, specifically H285, T287, S289, K292, T294, N295, and / or K297; (d): (i) amino acid residues at positions 204 to 208, specifically N204, W205, G206, and / or Y208; and (ii) amino acid residues at positions 213 to 227, specifically W213, N214, V216, Q220, M223, K224, T225, R226, and / or N227; or (e): (i) amino acid residues at positions 55 to 59, specifically D55, K56, E57, N58, and / or G59; (ii) amino acid residues at positions 88 to 97, specifically A88, G89, Q90, R92, V93, Y94, S95, E96, and / or E97; and (iii) amino acid residues at positions 240 to 302, specifically K240, R277, R279, W300, and / or D302; interacting with, VHH antibody.

3. A VHH antibody that recognizes Staphylococcus aureus hemolysin (HLA), The following VHH sequences: (a) Based on the numbering in Figure 6A, it contains the following amino acid residues that interact with HLA: - A, V, or D in position 55; - Y at position 59; - I in position 60; - D in position 62; - King in position 65; - T in position 69; - W in position 103; - R in position 104; - W in position 105; - V in position 106; - P in position 107; and - S in position 108; (b) Based on the numbering in Figure 6B, it contains the following amino acid residues that interact with HLA: - Y or H at position 32; - F in position 47; - N, T, or S at position 52; - W or Y at position 59; - Y at position 60; - P in position 61; - D in position 62; - S or N at position 104; - Y at position 105; - W in position 106; - W in position 109; - H in position 111; and - E in position 112; (c) Based on the numbering in Figure 6C, it contains the following amino acid residues that interact with HLA: - Y in position 29; - King in position 30; - L in position 31; - N in position 32; - A in position 33; - L at position 47; - T in position 50; - S in position 52; - S in position 53; - G in position 54; - F in position 58; - N at position 98; - P in position 99; - T in position 100; - Y at position 101; - N at position 102; and - H in position 103; (d) Based on the numbering in Figure 6D, it contains the following amino acid residues that interact with HLA: - A in position 53; - S in position 57; - D in position 58; - I in position 60; - T in position 61; - Y at position 62; - Y at position 63; - S in position 64; - D in position 65; - A in position 103; - F in position 104; - D in position 105; and - F in position 106; or (e) Based on the numbering in Figure 6E, it contains the following amino acid residues that interact with HLA: - N in position 30; - S in position 31; - Y in position 32; - Q in position 44; - R in position 45; - S in position 54; - D in position 100; - H in position 102; - Y at position 104; - A in position 105; - F in position 106; - G in position 107; and - D in position 109; having VHH antibody.

4. A VHH antibody recognizing a Staphylococcus aureus HLA polypeptide: （１）： (a) a CDR3 sequence set forth in SEQ ID NO: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, or 106; (b) a CDR3 sequence having at least 80%, at least 90%, or at least 95% identity to the CDR3 sequence of (a); （２）： (a) a combination of CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4-6, 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, 88-90, 92-94, 96-98, 100-102, or 104-106; (b) a combination of CDR1, CDR2, and CDR3 sequences that have at least 80%, at least 90%, or at least 95% identity to the combination of CDR1, CDR2, and CDR3 sequences of (a); or （３）： (a) a VHH sequence as set forth in SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, or 103; (b) a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity to the VHH sequence of (a); Including, VHH antibody.

5. A VHH antibody according to any one of claims 1 to 4, Here, the dissociation constant K for HLA is Ｄ the binding affinity, expressed as: VHH antibody.

6. A VHH antibody according to any one of claims 1 to 5, the VHH antibody neutralizes HLA; VHH antibody.

7. A VHH antibody according to any one of claims 1 to 6, the VHH antibody is stable, in particular thermostable or hyperthermostable; and the VHH antibody has a melting temperature of at least about 50°C, at least about 60°C, at least about 80°C, or at least about 95°C when measured under non-reducing conditions; VHH antibody.

8. A VHH antibody according to any one of claims 1 to 7, The VHH antibody comprises: (i) VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO: 3, or a VHH antibody which is a variant thereof; (ii) a VHH antibody selected from the VHH antibody Ma7A08 having the VHH sequence shown in SEQ ID NO: 7, or a VHH antibody that is a variant thereof; (iii) a VHH antibody selected from the VHH antibody MaB03 having the VHH sequence shown in SEQ ID NO: 11, or a VHH antibody that is a variant thereof; (iv) a VHH antibody selected from the VHH antibody Ma7B12 having the VHH sequence shown in SEQ ID NO: 15, or a VHH antibody that is a variant thereof; (v) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 19, or a VHH antibody that is a variant thereof; (vi) a VHH antibody selected from the VHH antibody Ma7C02 having the VHH sequence set forth in SEQ ID NO: 23, or a VHH antibody that is a variant thereof; (vii) a VHH antibody selected from the VHH antibody Ma7E01 having the VHH sequence set forth in SEQ ID NO: 27, or a VHH antibody that is a variant thereof; (viii) a VHH antibody selected from the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody which is a variant thereof; (ix) a VHH antibody selected from the VHH antibody Ma7D06 having the VHH sequence set forth in SEQ ID NO: 35, or a VHH antibody which is a variant thereof; (x) a VHH antibody selected from the VHH antibody Ma7C11 having the VHH sequence set forth in SEQ ID NO: 39, or a VHH antibody that is a variant thereof; (xi) a VHH antibody selected from the VHH antibody Ma8E03 having the VHH sequence set forth in SEQ ID NO: 43, or a VHH antibody that is a variant thereof; (xii) a VHH antibody selected from the VHH antibody Ma8G02 having the VHH sequence set forth in SEQ ID NO: 47, or a VHH antibody that is a variant thereof; (xiii) a VHH antibody selected from the VHH antibody Ma8H03 having the VHH sequence set forth in SEQ ID NO: 51, or a VHH antibody that is a variant thereof; (xiv) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody which is a variant thereof; (xv) a VHH antibody selected from the VHH antibody Ma8D05 having the VHH sequence set forth in SEQ ID NO: 59, or a VHH antibody that is a variant thereof; (xvi) a VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody which is a variant thereof; or (xvii) a VHH antibody selected from the VHH antibody Ma8D08 having the VHH sequence set forth in SEQ ID NO: 67, or a VHH antibody which is a variant thereof; or (xviii) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28F09 having the VHH sequence shown in SEQ ID NO: 71, or a variant thereof; or (xix) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28H01 having the VHH sequence shown in SEQ ID NO: 75, or a variant thereof; or (xx) A VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Ma31A12 having the VHH sequence set forth in SEQ ID NO: 79, or a VHH antibody which is a variant thereof; or (xxi) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Ma31B06 having the VHH sequence shown in SEQ ID NO: 83, or a variant thereof; or (xxii) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28C04 having the VHH sequence shown in SEQ ID NO: 87, or a variant thereof; or (xxiii) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28H07 having the VHH sequence shown in SEQ ID NO: 91, or a variant thereof; or (xxiv) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28C09 having the VHH sequence shown in SEQ ID NO: 95, or a variant thereof; or (xxv) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28H12 having the VHH sequence shown in SEQ ID NO: 99, or a variant thereof; or (xxvi) The VHH antibody according to any one of embodiments 1 to 24, wherein the VHH antibody is selected from the VHH antibody Bm28A12 having the VHH sequence shown in SEQ ID NO: 103, or a variant thereof; Selected from: VHH antibody.

9. A heteromultimeric VHH antibody, comprising two or more different VHH antibody units, for example two, three or four different VHH antibody units, which recognize Staphylococcus aureus proteins, in particular HLA; wherein at least one VHH antibody unit comprises a VHH antibody according to any one of claims 1 to 8. Heteromultimeric VHH antibodies.

10. The heteromultimeric VHH antibody according to claim 9, The heteromultimeric VHH antibody comprises at least two VHH antibody units selected from the following different VHH antibody classes (i), (ii), (iii) and (iv): (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence set forth in SEQ ID NO: 3, or a VHH antibody that competes with said VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 1919 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) a VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA; Including, Heteromultimeric VHH antibodies.

11. A set of two or more different VHH antibodies that recognize HLA, comprising at least one VHH antibody according to any one of claims 1 to 10, and The set specifically comprises at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii) and (iv) below: (i) a VHH antibody selected from the VHH antibody Ma7A07 having the VHH sequence set forth in SEQ ID NO: 3, or a VHH antibody that competes with said VHH antibody Ma7A07 for binding to HLA; (ii) a VHH antibody selected from the VHH antibody Ma7B01 having the VHH sequence set forth in SEQ ID NO: 1919 and / or the VHH antibody Ma7F02 having the VHH sequence set forth in SEQ ID NO: 31, or a VHH antibody that competes with said VHH antibody Ma7B01 for binding to HLA; (iii) a VHH antibody selected from the VHH antibody Ma8A05 having the VHH sequence set forth in SEQ ID NO: 55, or a VHH antibody that competes with the VHH antibody Ma8A05 for binding to HLA; and (iv) a VHH antibody selected from the VHH antibody Ma8D07 having the VHH sequence set forth in SEQ ID NO: 63, or a VHH antibody that competes with the VHH antibody Ma8D07 for binding to HLA; Including, set.

12. A VHH antibody according to any one of claims 1 to 10 or a set according to claim 11, for use in medicine, particularly for use in the prevention, treatment and / or alleviation of conditions caused by, associated with and / or accompanied by infection with HLA-positive Staphylococcus aureus; The condition is particularly selected from skin infections, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis. VHH antibody or set.

13. A VHH antibody according to any one of claims 1 to 10 or a set according to claim 11, For use according to claim 10 in a human subject, VHH antibody or set.

14. A VHH antibody according to any one of claims 1 to 10 or a set according to claim 11 for the use according to any one of claims 12 to 13, administered as monotherapy, or administered sequentially and / or simultaneously as a combination therapy with at least one additional active ingredient, for example in combination with an antibiotic against Staphylococcus aureus, such as vancomycin, optionally in combination with an additional antibiotic against a different bacterium; VHH antibody or set.

15. A composition comprising, as an active ingredient, a VHH antibody according to any one of claims 1 to 10 or a set according to claim 11, and a pharmaceutically acceptable carrier. Combination medicines.

16. Use of a VHH antibody according to any one of claims 1 to 10 or a set according to claim 11 for detecting HLA in a sample, in particular in a biological sample.

17. 17. The use according to claim 16, The detection comprises the binding of at least two different VHH antibodies to the HLA molecule, in particular the binding of at least two different VHH antibodies, for example two, three or four VHH antibodies, each directed against a different epitope, to the HLA molecule, use.

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