Therapeutic VHH antibodies against alpha-hemolysin from staphylococcus aureus

Abstract

The present invention is in the fields of antibody technology, medicine, pharmacology, infection biology and medical diagnosis. More particularly, the present disclosure provides VHH antibodies that prevent membrane binding and / or oligomerization of Staphylococcus aureus (S. aureus) alpha-hemolysin (HLA), as well as the hemolysis effects caused thereby.

Description

Technical Field

[0001] This invention belongs to the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, this disclosure provides a VHH antibody that inhibits the membrane binding and / or oligomerization of Staphylococcus aureus α-hemolysin (HLA) and the resulting hemolysis. Background Technology

[0002] Staphylococcus aureus is one of the most prevalent bacterial pathogens, causing a wide range of illnesses, from superficial skin infections to life-threatening pneumonia, bacteremia, and sepsis. In developed countries, Staphylococcus aureus is the leading cause of bloodstream infections and has the highest mortality rate. In fact, in the United States, deaths from Staphylococcus aureus each year exceed the combined deaths from acquired immunodeficiency syndrome (AIDS), tuberculosis, and viral hepatitis (Cheung et al., 2021). The lack of approved vaccines and complex treatments make Staphylococcus 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 readily cause infection after skin or mucous membrane injury. Under normal circumstances, the human immune system can eliminate invading bacteria within minutes (using complement, macrophages, and, upon repeated exposure, antibodies). However, highly virulent bacteria employ virulence factors that allow them to evade the immune response. Virulence factors can generally be classified as surface and extracellular secretory proteins; these include bacterial toxins, complement-inactivating factors, inhibitors of phagocytosis, or proteases that cleave immunoglobulins or the extracellular matrix. Unlike other bacteria that employ only one or a few virulence factors, Staphylococcus aureus possesses >20 virulence factors to promote its pathogenicity (Cheung et al., 2021).

[0004] Antibiotics are key to treating bacterial infections; however, the increasing prevalence of antibiotic-resistant infections complicates treatment. Staphylococcus aureus infections are particularly challenging due to the frequent occurrence of multidrug resistance in Staphylococcus aureus isolates. Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified antibiotic-resistant pathogen globally. Patients infected with MRSA experience longer hospital stays, incur higher treatment costs, and exhibit higher mortality rates (Ippolito et al., 2010). The first MRSA strain was characterized in 1961, just one year after methicillin was approved for clinical use (Ippolito et al., 2010; Turner et al., 2019). Since then, far too many strains resistant to an ever-growing number of antibiotics have been reported. Some clinical isolates have even shown resistance to ≥10 classes of antibiotics (Saleh et al., 2022). Novel antibiotics for the treatment of MRSA, and more broadly for Staphylococcus aureus, have been developed or are under development. Currently, vancomycin is used as a last-resort antibiotic in non-critical patients (Cheung et al., 2021). However, although not widely distributed, vancomycin-resistant Staphylococcus aureus isolates have been identified. Similarly, an increasing number of cases fail to respond to vancomycin treatment (Lodise et al., 2008; Ye et al., 2020).

[0005] Another drawback of antibiotic therapy is that even if the treatment is immediately effective, it cannot remove circulating bacterial toxins from the body. Antibiotics may even enhance toxin release. Therefore, there is a need for alternative treatment strategies that target virulence factors. One such "antitoxin therapy" approach would use antibodies to neutralize virulence factors, which should stop the toxin effect, "disarm" the pathogen, and allow the immune system to clear the infection.

[0006] Alpha-hemolysin (HLA), or alpha-toxin, is the most important and well-known virulence factor in Staphylococcus aureus. HLA is a 33 kDa secretory precursor protein found in >95% of Staphylococcus aureus isolates (Kielian et al., 2001). Upon binding to the target membrane, HLA oligomerizes into heptamers and forms... Wide pores. Such pores allow leakage of ions and other small molecules, destabilizing the membrane and leading to cell lysis. HLA primarily targets leukocytes to evade the host cell's defense system and targets erythrocytes, thus making previously limited iron available for bacterial growth. In epithelial and endothelial cells, HLA interacts with ADAM10 (a cell surface receptor with metalloproteinase and E-cadherin domains), the activation of which breaks adhesion junctions, allowing bacteria to leave the bloodstream and invade soft tissues.

[0007] The direct contribution of HLA to sepsis, pneumonia, and skin lesions has been well demonstrated in various rodent models (Kielian et al., 2001; Bubeck Wardenburg et al., 2007). Furthermore, in rat models of pneumonia, bacterial pathogenicity is highly correlated with elevated HLA expression (Montgomery et al., 2008). Therefore, strategies targeting HLA (whether by interfering with its expression levels or by neutralizing already circulating toxins) offer promising solutions in progress for developing novel (passive immunotherapy) therapies against Staphylococcus aureus infections. Invention Overview

[0009] This disclosure provides single-domain VHH antibodies against Staphylococcus aureus α-hemolysin (HLA). They recognize and / or inhibit membrane binding and / or oligomerization of Staphylococcus aureus HLA and the resulting hemolysis, and are suitable for diagnostic, preventive, and / or therapeutic applications.

[0010] In some embodiments, the VHH antibody is in a monovalent form, such as as a single VHH domain, or as a fusion with a heterologous protein (e.g., serum albumin), and is available in a wide variety of styles. In some embodiments, the VHH antibody is in a multivalent form, such as as a bivalent Fc-fusion. In some embodiments, the VHH antibody is a heteromultimeric VHH antibody comprising two or more distinct VHH antibody units.

[0011] Due to its very high affinity, use in a monovalent form is possible. In some embodiments, the VHH antibody (in a monovalent form) has target affinity in the pM or even low pM range. In some embodiments, the VHH antibody neutralizes HLA with high potency.

[0012] The first aspect of this disclosure is the VHH antibody against Staphylococcus aureus HLA described herein. Preferred VHH antibodies of the present invention, their names, binding characteristics (i.e., HLA epitopes, HLA affinity, and HLA neutralization), and their thermal stability are listed in Table 1 below.

[0013] Table 1: Thermostability and Affinity (in KJ) of Anti-HLA VHH Antibodies D (Value representation) and inhibition characteristics.

[0014]

[0015] A list of the complete VHH sequences of the VHH antibodies is shown at the end of the instruction manual.

[0016] A further aspect of the invention relates to a set of two or more different VHH antibodies, wherein at least one VHH antibody is described above.

[0017] The VHH antibodies described above are suitable for use in medicine (e.g., human medicine), particularly for the prevention, treatment, and / or mitigation of symptoms caused by, associated with, and / or accompanied by HLA-positive Staphylococcus aureus infection, especially drug-resistant HLA-positive Staphylococcus aureus infection; or for use in diagnostics, such as for the detection of Staphylococcus aureus HLA in patient samples (e.g., in body fluids or tissue samples); or for use in research.

[0018] Another further aspect of the invention relates to nucleic acid molecules encoding the VHH antibodies described above, particularly in an operative link with a heterologous expression regulatory sequence, a vector containing the nucleic acid molecule, or recombinant cells or non-human objects transformed or transfected with the nucleic acid molecule or the vector.

[0019] Another further aspect of the invention relates to a method for recombinantly generating the VHH antibody described above, comprising culturing the cells or organisms described above in a suitable culture medium, and obtaining the VHH antibody from the cells or organisms or from the culture medium.

[0020] Another further aspect of the invention relates to a method for preventing, treating, and / or alleviating symptoms caused by, associated with, and / or accompanied by HLA-positive Staphylococcus aureus infection, particularly said HLA-positive Staphylococcus aureus infection being drug-resistant HLA-positive Staphylococcus aureus infection, said method comprising administering to a subject in need, particularly to a subject suffering from symptoms caused by, associated with, and / or accompanied by HLA-positive Staphylococcus aureus infection, an effective dose of the VHH antibody described above or a kit of at least two different VHH antibodies described above.

[0021] A non-limiting list of the VHH antibodies of the present invention and their CDR sequences is shown in Table 2 below.

[0022] Table 2: Anti-HLA VHH antibodies and their CDR regions. Note: The CDR here also includes variable residues flanking the actual CDR ring.

[0023] Embodiments of the present invention

[0024] The following sections disclose specific embodiments of the present invention:

[0025] 1. VHH antibody that recognizes Staphylococcus aureus α-hemolysin (HLA).

[0026] 2. The VHH antibody according to embodiment 1, which competitively binds to HLA with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3.

[0027] 3. The VHH antibody according to embodiment 1, which competes with 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.

[0028] 4. The VHH antibody according to embodiment 1, which competitively binds to HLA with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0029] 5. The VHH antibody according to embodiment 1, which competitively binds to HLA with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63.

[0030] 6. A VHH antibody (particularly according to embodiment 1) that recognizes Staphylococcus aureus α-hemolysin (HLA), which, in conjunction with a selection from the following, is used in accordance with... Figure 7 HLA amino acid residue interactions of (SEQ ID NO:107):

[0031] (i) Amino acid residues at positions 54-61, especially Y54, K56, E57, M60 and / or H61;

[0032] (ii) Amino acid residues at positions 86-96, especially T86, A88, Y91, R92, V93, Y94 and / or E96;

[0033] (iii) Amino acid residues at positions 233-239, especially A233, D234, D238, and P239; and

[0034] (iv) Amino acid residues at positions 279-302, especially R279, W300 and D302.

[0035] 7. The VHH antibody according to embodiment 1 or 6, having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6A Number:

[0036] - At position 55, A, V, or D;

[0037] -Y at position 59;

[0038] -I at position 60;

[0039] -D at position 62;

[0040] -K at position 65;

[0041] -T at position 69;

[0042] -W at position 103;

[0043] -R at position 104;

[0044] -W at position 105;

[0045] -V at position 106;

[0046] - P at position 107; and

[0047] -S at position 108.

[0048] 8. A VHH antibody (particularly according to embodiment 1) that recognizes Staphylococcus aureus α-hemolysin (HLA), which, in conjunction with a selection from the following, is used in accordance with... Figure 7 HLA amino acid residue interactions of (SEQ ID NO:107):

[0049] (i) Amino acid residues at positions 38-44, especially T38, D39, I40, G41, S42, N43 and / or T44;

[0050] (ii) amino acid residues at positions 141-143, particularly T141 and / or T143; and

[0051] (iii) Amino acid residues at positions 162-169, especially I162, G163, N165, V166, S167, I168 and / or G169.

[0052] 9. The VHH antibody according to embodiment 1 or 8, having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6B Number:

[0053] - Y or H at position 32;

[0054] -F at position 47;

[0055] -N, T, or S at position 52;

[0056] - W or Y at position 59;

[0057] -Y at position 60;

[0058] -P at position 61;

[0059] -D at position 62;

[0060] -S or N at position 104;

[0061] -Y at position 105;

[0062] -W at position 106;

[0063] -W at position 109;

[0064] -H at position 111; and

[0065] -E at position 112.

[0066] 10. A VHH antibody (particularly according to embodiment 1) that recognizes Staphylococcus aureus α-hemolysin (HLA), which, in conjunction with a selection from the following, is used in accordance with... Figure 7 HLA amino acid residue interactions of (SEQ ID NO:107):

[0067] (i) amino acid residues at positions 210-219, particularly R210, D211, S212, W213, P215 and / or N219; and

[0068] (ii) Amino acid residues at positions 285-297, especially H285, T287, S289, K292, T294, N295 and / or K297.

[0069] 11. The VHH antibody according to embodiment 1 or 10, having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6C Number:

[0070] -Y at position 29;

[0071] -K at position 30;

[0072] -L at position 31;

[0073] -N at position 32;

[0074] -A at position 33;

[0075] -L at position 47;

[0076] -T at position 50;

[0077] -S at position 52;

[0078] -S at position 53;

[0079] -G at position 54;

[0080] -F at position 58;

[0081] -N at position 98;

[0082] -P at position 99;

[0083] -T at position 100;

[0084] -Y at position 101;

[0085] -N at position 102; and

[0086] -H at position 103.

[0087] 12. A VHH antibody (particularly according to embodiment 1) that recognizes Staphylococcus aureus α-hemolysin (HLA), which, in conjunction with a selection from the following, is used in accordance with... Figure 7 HLA amino acid residue interactions of (SEQ ID NO:107):

[0088] (i) amino acid residues at positions 204-208, particularly N204, W205, G206, and / or Y208; and

[0089] (ii) Amino acid residues at positions 213-227, especially W213, N214, V216, Q220, M223, K224, T225, R226 and / or N227.

[0090] 13. The VHH antibody according to embodiment 1 or 12, having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6D Number:

[0091] -A at position 53;

[0092] -S at position 57;

[0093] -D at position 58;

[0094] -I at position 60;

[0095] -T at position 61;

[0096] -Y at position 62;

[0097] -Y at position 63;

[0098] -S at position 64;

[0099] - D at position 65;

[0100] -A at position 103;

[0101] -F at position 104;

[0102] -D at position 105; and

[0103] -F at position 106.

[0104] 14. A VHH antibody (particularly according to embodiment 1) that recognizes Staphylococcus aureus α-hemolysin (HLA), which, in conjunction with a selection from the following, is used in accordance with... Figure 7 HLA amino acid residue interactions of (SEQ ID NO:107):

[0105] (i) Amino acid residues at positions 55-59, especially D55, K56, E57, N58 and / or G59;

[0106] (ii) amino acid residues at positions 88-97, particularly A88, G89, Q90, R92, V93, Y94, S95, E96 and / or E97; and

[0107] (iii) Amino acid residues at positions 240-302, especially K240, R277, R279, W300 and / or D302.

[0108] 15. The VHH antibody according to embodiment 1 or 14, having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6E Number:

[0109] -N at position 30;

[0110] -S at position 31;

[0111] -Y at position 32;

[0112] -Q at position 44;

[0113] -R at position 45;

[0114] -S at position 54;

[0115] -D at position 100;

[0116] -H at position 102;

[0117] -Y at position 104;

[0118] -A at position 105;

[0119] -F at position 106;

[0120] -G at position 107; and

[0121] - D at position 109.

[0122] 16. A VHH antibody that recognizes Staphylococcus aureus HLA peptides, comprising:

[0123] (a) The CDR3 sequence shown 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;

[0124] (b) A CDR3 sequence that shares at least 80%, at least 90%, or at least 95% identity with the CDR3 sequence of (a); or

[0125] (c) A VHH antibody that competes with VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3 for binding to HLA, or with 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 with VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or with VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63 for binding to HLA.

[0126] 17. The VHH antibody according to any one of the foregoing embodiments, comprising:

[0127] (a) Combinations of CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: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 and 104-106;

[0128] (b) Combinations of CDR1, CDR2, and CDR3 sequences that share at least 80%, at least 90%, or at least 95% identity with the CDR1, CDR2, and CDR3 sequences of (a); or

[0129] (c) A VHH antibody that competes with VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3 for binding to HLA, or with 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 with VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or with VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63 for binding to HLA.

[0130] 18. The VHH antibody according to any one of the foregoing embodiments, comprising:

[0131] (a) VHH sequences shown 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 and 103;

[0132] (b) A sequence that has at least 80%, at least 90%, at least 95%, or at least 99% identity with the VHH sequence of (a); or

[0133] (c) A VHH antibody that competes with VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3 for binding to HLA, or with 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 with VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or with VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63 for binding to HLA.

[0134] 19. The VHH antibody according to any one of the foregoing embodiments, wherein the dissociation constant K is... D The indicated binding affinity to HLA is approximately 1 nM or less, approximately 0.5 nM or less, or approximately 0.1 nM or less.

[0135] 20. The VHH antibody according to any one of the foregoing embodiments, wherein HLA is neutralized, particularly by inhibiting HLA-induced hemolysis of erythrocytes.

[0136] 21. The VHH antibody according to embodiment 20, wherein HLA is neutralized.

[0137] 22. The VHH antibody according to any one of the foregoing embodiments is stable, particularly thermally stable or ultrathermally stable.

[0138] 23. The VHH antibody according to embodiment 22, having a melting temperature of at least about 40°C, 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.

[0139] 24. The VHH antibody according to embodiment 22 or 23, having an aggregation temperature of at least about 40°C, at least about 50°C, at least about 60°C, at least about 70°C, or at least about 80°C when measured under non-reducing conditions.

[0140] 25. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or a VHH antibody as a variant thereof.

[0141] 26. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7A08 having the VHH sequence shown in SEQ ID NO:7, or a VHH antibody as a variant thereof.

[0142] 27. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody MaB03 having the VHH sequence shown in SEQ ID NO:11, or a VHH antibody as a variant thereof.

[0143] 28. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7B12 having the VHH sequence shown in SEQ ID NO:15, or a VHH antibody as a variant thereof.

[0144] 29. The VHH antibody according to any one of embodiments 1-24, wherein the VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO:19, or a variant thereof, is selected from VHH antibody Ma7B01.

[0145] 30. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7C02 having the VHH sequence shown in SEQ ID NO:23, or a VHH antibody as a variant thereof.

[0146] 31. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7E01 having the VHH sequence shown in SEQ ID NO:27, or a VHH antibody as a variant thereof.

[0147] 32. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO:31, or a VHH antibody as a variant thereof.

[0148] 33. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7D06 having the VHH sequence shown in SEQ ID NO:35, or a VHH antibody as a variant thereof.

[0149] 34. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma7C11 having the VHH sequence shown in SEQ ID NO:39, or a VHH antibody as a variant thereof.

[0150] 35. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma8E03 having the VHH sequence shown in SEQ ID NO:43, or a VHH antibody as a variant thereof.

[0151] 36. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma8G02 having the VHH sequence shown in SEQ ID NO:47, or a VHH antibody as a variant thereof.

[0152] 37. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma8H03 having the VHH sequence shown in SEQ ID NO:51, or a VHH antibody as a variant thereof.

[0153] 38. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody as a variant thereof.

[0154] 39. The VHH antibody according to any one of embodiments 1-24, wherein the VHH antibody Ma8D05 having the VHH sequence shown in SEQ ID NO:59, or a variant thereof, is selected from VHH antibody Ma8D05.

[0155] 40. The VHH antibody according to any one of embodiments 1-24, wherein the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or a variant thereof, is selected from VHH antibodies.

[0156] 41. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma8D08 having the VHH sequence shown in SEQ ID NO:67, or a VHH antibody as a variant thereof.

[0157] 42. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Bm28F09 having the VHH sequence shown in SEQ ID NO:71, or a VHH antibody as a variant thereof.

[0158] 43. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Bm28H01 having the VHH sequence shown in SEQ ID NO:75, or a VHH antibody as a variant thereof.

[0159] 44. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma31A12 having the VHH sequence shown in SEQ ID NO:79, or a VHH antibody as a variant thereof.

[0160] 45. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Ma31B06 having the VHH sequence shown in SEQ ID NO:83, or a VHH antibody as a variant thereof.

[0161] 46. ​​The VHH antibody according to any one of embodiments 1-24, wherein the VHH antibody Bm28C04 having the VHH sequence shown in SEQ ID NO:87, or a variant thereof, is selected from VHH antibody Bm28C04.

[0162] 47. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Bm28H07 having the VHH sequence shown in SEQ ID NO:91, or a VHH antibody as a variant thereof.

[0163] 48. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Bm28C09 having the VHH sequence shown in SEQ ID NO:95, or a VHH antibody as a variant thereof.

[0164] 49. The VHH antibody according to any one of embodiments 1-24, wherein the VHH antibody Bm28H12 having the VHH sequence shown in SEQ ID NO:99, or a variant thereof, is selected from VHH antibody Bm28H12.

[0165] 50. The VHH antibody according to any one of embodiments 1-24, which is selected from VHH antibody Bm28A12 having the VHH sequence shown in SEQ ID NO:103, or a VHH antibody as a variant thereof.

[0166] 51. The VHH antibody according to any one of the foregoing embodiments is either non-glycosylated or glycosylated.

[0167] 52. The VHH antibody according to any one of the foregoing embodiments is produced in bacteria (e.g., Escherichia coli), or in yeast (e.g., Pichia pastoris), or in human or animal cells (e.g., insect cells or mammalian cells, such as CHO cells).

[0168] 53. The VHH antibody according to any one of the foregoing embodiments is in a monovalent form.

[0169] 54. The VHH antibody according to any one of embodiments 1-53, which is in a polymeric form.

[0170] 55. The VHH antibody according to embodiment 54 is in a dimer form.

[0171] 56. The VHH antibody according to embodiment 55 is in the form of a homodimer or a heterodimer.

[0172] 57. The VHH antibody according to any one of the foregoing embodiments, which is covalently or non-covalently conjugated to a heterologous portion, such as a tagging group, a capture group, or an effector group, wherein the heterologous portion is particularly selected from fluorescent groups, biotin, enzymes (e.g., peroxidases, phosphatases, or luciferases), haptens, affinity tags, or nucleic acids (e.g., oligonucleotides).

[0173] 58. The VHH antibody according to any one of the foregoing embodiments, wherein it is fused with a heteropeptide portion, or fused with an Fc fragment, or fused with serum albumin or an albumin-binding portion.

[0174] 59. The VHH antibody according to any one of the foregoing embodiments, which is conjugated to one or more non-peptide polymer portions, preferably hydrophilic polymer portions, such as polyethylene glycol (PEG).

[0175] 60. A heteropolymeric VHH antibody comprising two or more different VHH antibody units, such as 2, 3 or 4 different VHH antibody units, wherein at least one VHH antibody unit comprises a VHH antibody according to any one of embodiments 1-53.

[0176] 61. The heteropolymeric VHH antibody according to embodiment 60, comprising at least two VHH antibody units that recognize HLA (specifically, different epitopes on HLA).

[0177] 62. The heteropolymeric VHH antibody according to embodiment 60 or 61, comprising at least two VHH antibody units selected from the following different VHH antibody classes (i), (ii), (iii) and (iv):

[0178] (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA;

[0179] (ii) VHH antibody, selected from 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, or a VHH antibody that competes with VHH antibody Ma7B01 for binding to HLA.

[0180] (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and

[0181] (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

[0182] 63. A heteropolymeric VHH antibody according to any one of embodiments 60-62, comprising at least two VHH antibody units, said VHH antibody unit comprising a VHH antibody selected from at least two different VHH antibody classes (i), (ii), (iii), (iv), and (v) below:

[0183] (i) The VHH antibody according to embodiment 6 or 7;

[0184] (ii) The VHH antibody according to implementation scheme 8 or 9;

[0185] (iii) The VHH antibody according to embodiment 10 or 11;

[0186] (iv) the VHH antibody according to embodiment 12 or 13; and

[0187] (v) VHH antibody according to implementation scheme 14 or 15.

[0188] 64. A kit of two or more different VHH antibodies that recognize HLA, comprising at least one VHH antibody according to any one of embodiments 1-63, particularly at least one VHH antibody in a monovalent form.

[0189] 65. The kit according to embodiment 64, comprising at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii), and (iv) below:

[0190] (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA;

[0191] (ii) VHH antibody, selected from 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, or a VHH antibody that competes with VHH antibody Ma7B01 for binding to HLA.

[0192] (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and

[0193] (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

[0194] 66. The kit according to embodiment 64 or 65, comprising at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii), (iv), and (v) below:

[0195] (i) The VHH antibody according to embodiment 6 or 7;

[0196] (ii) The VHH antibody according to implementation scheme 8 or 9;

[0197] (iii) The VHH antibody according to embodiment 10 or 11;

[0198] (iv) the VHH antibody according to embodiment 12 or 13; and

[0199] (v) VHH antibody according to implementation scheme 14 or 15.

[0200] 67. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use in medicine, particularly for use in therapy or diagnostics.

[0201] 68. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use in the prevention, treatment and / or mitigation of symptoms caused by, associated with and / or accompanied by HLA-positive Staphylococcus aureus infection.

[0202] 69. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use as described in embodiment 67 or 68, wherein the Staphylococcus aureus is a drug-resistant HLA-positive Staphylococcus aureus.

[0203] 70. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use as described in embodiment 67 or 68, wherein the condition is selected from skin infection, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis and / or endocarditis.

[0204] 71. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use in human subjects according to any one of embodiments 67-69.

[0205] 72. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use according to any one of embodiments 67-70, wherein the kit is applied topically.

[0206] 73. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use according to any one of embodiments 67-71, wherein the kit is administered systemically.

[0207] 74. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use according to any one of embodiments 67-72, is administered by means of a surface, parenteral (e.g., intravenous), pulmonary (e.g., by inhalation) or nasal (e.g., by spray).

[0208] 75. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use according to any one of embodiments 67-73, administered as a monotherapy.

[0209] 76. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use according to any one of embodiments 67-74, wherein the kit is administered as a combination therapy sequentially and / or simultaneously with at least one additional active agent.

[0210] 77. The VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66, for use as described in embodiment 76, is administered in combination with an antibiotic against Staphylococcus aureus (e.g., vancomycin) and optionally in combination with other antibiotics against different bacteria.

[0211] 78. A pharmaceutical composition comprising, as an active agent, a VHH antibody according to any one of embodiments 1-63 or a kit according to any one of embodiments 64-66, and a pharmaceutically acceptable carrier.

[0212] 79. A nucleic acid molecule encoding a VHH antibody or a unit containing a VHH antibody according to any one of embodiments 1-63, preferably in an operational link with a heterologous expression regulatory sequence.

[0213] 80. A vector comprising the nucleic acid molecule as described in embodiment 79.

[0214] 81. Recombinant cells or non-human objects transformed or transfected with the nucleic acid molecule described in embodiment 79 or the vector described in embodiment 80.

[0215] 82. The cell or organism according to embodiment 81 is selected from bacteria (e.g., Escherichia coli, Bacillus sp.), single-celled eukaryotes (e.g., yeast such as Pichia pastoris, or Leishmania), insect cells, mammalian cells, or plant cells.

[0216] 83. A method for recombinantly generating VHH antibodies according to any one of embodiments 1-63, comprising culturing cells or organisms according to embodiment 81 or 82 in a suitable culture medium, and obtaining the VHH antibodies from said cells or organisms or from said culture medium.

[0217] 84. The method according to embodiment 83, comprising culturing yeast (e.g., Pichia pastoris) and obtaining the VHH antibody from the culture medium.

[0218] 85. Use of the VHH antibody according to any one of embodiments 1-63 or the kit according to any one of embodiments 64-66 for the detection of HLA in a sample.

[0219] 86. The use according to embodiment 85, wherein the sample is a biological sample, such as bodily fluids (e.g., saliva, sputum, lavage fluid, swabs, blood, serum or plasma), fecal sample, tissue sample or biopsy sample.

[0220] 87. The use according to embodiment 85 or 86, wherein the detection comprises the binding of at least two different VHH antibodies to HLA molecules.

[0221] 88. The use according to embodiment 87, wherein the detection comprises the binding of at least two different VHH antibodies (e.g., 2, 3 or 4 VHH antibodies) each targeting different epitopes to an HLA molecule.

[0222] 89. The use according to any one of embodiments 85-88, wherein the detection includes a sandwich assay, such as a sandwich ELISA.

[0223] 90. The use according to any one of embodiments 85-89, wherein the detection includes a prior HLA enrichment step.

[0224] 91. A method for the prevention, treatment and / or relief of symptoms caused by, associated with and / or accompanied by HLA-positive Staphylococcus aureus infection, comprising administering to a subject (particularly a human subject) an effective dose of a VHH antibody according to any one of embodiments 1-63, a kit according to any one of embodiments 64-66, or a pharmaceutical composition according to embodiment 78. Summary of the Invention

[0225] VHH antibody

[0226] This invention relates to VHH antibodies that recognize Staphylococcus aureus α-hemolysin (HLA).

[0227] The VHH antibody may be a monovalent heavy chain-only antibody comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain linked by a framework region, including but not limited to full-length VHH antibodies, such as natural VHH antibodies comprising framework regions derived from camels, as well as modified VHH antibodies comprising modified framework regions, VHH antibody fragments, and VHH antibody fusion proteins (e.g., fusion proteins with immunoglobulins or non-immunoglobulin peptides or polypeptides), provided that it exhibits the characteristics according to the invention.

[0228] The VHH antibody of the present invention can be glycosylated or non-glycosylated.

[0229] Several methods for determining the CDR sequence of a given antibody molecule are known in the prior art, but there is no standard, definitive method. The determination of the CDR sequence from the variable region of the antibody heavy chain can be performed according to any method known in the art, including but not limited to methods called Kabat, Chothia, and IMGT. The selected set of CDRs may include sequences identified by more than one method; for example, some CDR sequences can be determined using Kabat, and some can be determined using IMGT. According to some embodiments of the invention, the CDR sequence of the VHH variable region is determined using the Kabat method. CDRs can also be defined by performing multiple alignments (with many other VHH antibodies) to identify variable hotspots and associating them with standard VHH antibody structures. CDRs may also be defined by analyzing the structure of the VHH antibody and determining what constitutes a loop and what constitutes an antibody scaffold. In some cases, residues adjacent to the CDR are also variable and are therefore included in the CDR definition.

[0230] The present invention also relates to covalent or non-covalent conjugates of VHH antibody molecules with non-protein structures, such as labeling groups, trapping groups (e.g., solid-binding groups), or effector groups (e.g., toxins). For example, the heterologous portion may be derived from fluorescent groups, biotin, enzymes (e.g., peroxidases, phosphatases, or luciferases), haptens, affinity tags, or nucleic acids (e.g., oligonucleotides).

[0231] The VHH antibodies of the present invention are specifically monoclonal VHH antibodies characterized by a specific amino acid sequence. The VHH antibodies can be produced in prokaryotic host cells, yeast cells, or mammalian cells (e.g., CHO cells). In some embodiments, the VHH antibodies are non-glycosylated. In some embodiments, the VHH antibodies are glycosylated, wherein the carbohydrate structure may originate from a glycosylation site introduced into the VHH sequence and / or from a fusion partner.

[0232] The VHH antibody according to the invention is characterized by: (i) a CDR3 sequence, (ii) a combination of CDR1, CDR2, and CDR3 sequences, (iii) the complete VHH sequence, or (iv) competition with a specific reference antibody. Specific CDR and VHH sequences are provided in tables, figures, and sequence listings.

[0233] According to the present invention, sequences related to the above-described sequences are included. These related sequences are defined by having the lowest possible identity with a specifically indicated amino acid sequence (e.g., a CDR or VHH sequence). This identity is indicated over the full length of the respective reference sequence and can be determined using well-known algorithms such as BLAST.

[0234] In a particular embodiment, the associated CDR3 sequence has at least 80%, at least 90%, or at least 95% identity with a specifically designated CDR3 sequence, for example, by substitution of 1, 2, or 3 amino acids.

[0235] In a particular embodiment, the combination of the associated CDR1, CDR2, and CDR3 sequences has at least 80%, at least 90%, or at least 95% identity with the specifically indicated combination of the CDR1, CDR2, and CDR3 sequences, for example, 1, 2, 3, 4, 5, or 6 amino acids are replaced by different amino acid substitutions.

[0236] In a particular embodiment, the associated VHH sequence has at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with the VHH sequence, for example, substitutions of 1, 2, 3, 4, 5, or up to 20 amino acids.

[0237] Furthermore, this invention relates to VHH antibodies that compete with specific VHH antibodies disclosed herein for binding to Staphylococcus aureus HLA peptides. In some embodiments, the competitive VHH antibody binds to the same or overlapping epitopes on the Staphylococcus aureus HLA peptide. For example, this invention relates to VHH antibodies that compete with a reference antibody, such as VHH antibody Ma7A07 or Bm28C04 (which binds to epitope 1 on HLA), VHH antibody Ma7B01 or Ma7F02 (which binds to epitope 2 on HLA), VHH antibody Ma8A05 (which binds to epitope 4 on HLA), or VHH antibody Ma8D07 (which binds to epitope 3 on HLA). Competition can be determined by label-free biolayer interferometry, performed as a cross-competition or epitope binning assay, wherein a label-free detection system (e.g., from Sartorius) is used. (System), and follow the manufacturer's instructions.

[0238] Furthermore, the present invention relates to antibodies of a specific VHH antibody class characterized by: common amino acids or amino acid segments in the VHH sequence, and / or common amino acids or amino acid segments that interact with HLA. For each corresponding structure, the interacting amino acid residues are identified by determining the intermolecular contacts of the VHH antibody-HLA structure using the "Find Clashes / Contacts" tool from UCSF Chimera (Pettersen et al., 2004). Having ≤ The interatomic distance of the residues is shown Figure 7 middle.

[0239] In some embodiments, the VHH antibody is a member of VHH antibody category 1 (Ma7A07 category).

[0240] Category 1 VHH antibodies are characterized by their binding to HLA epitope 1. For example, in Figure 7 As shown, the Class 1 VHH antibody interacts with amino acid residues selected from the following on the HLA according to SEQ ID NO:107:

[0241] (i) Amino acid residues at positions 54-61, especially Y54, K56, E57, M60 and / or H61;

[0242] (ii) Amino acid residues at positions 86-96, especially T86, A88, Y91, R92, V93, Y94 and / or E96;

[0243] (iii) Amino acid residues at positions 233-239, especially A233, D234, D238, and P239; and

[0244] (iv) Amino acid residues at positions 279-302, especially R279, W300 and D302.

[0245] In some embodiments, category 1 VHH antibodies are characterized by having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6A Number:

[0246] - At position 55, A, V, or D;

[0247] -Y at position 59;

[0248] -I at position 60;

[0249] -D at position 62;

[0250] -K at position 65;

[0251] -T at position 69;

[0252] -W at position 103;

[0253] -R at position 104;

[0254] -W at position 105;

[0255] -V at position 106;

[0256] - P at position 107; and

[0257] -S at position 108.

[0258] In some embodiments, the VHH antibody is a member of VHH antibody category 2 (Ma7F02 category).

[0259] Category 2 VHH antibodies are characterized by their binding to HLA epitope 2. For example, in Figure 7 As shown, the category 2 VHH antibody interacts with amino acid residues selected from the following on the HLA according to SEQ ID NO:107:

[0260] (i) Amino acid residues at positions 38-44, especially T38, D39, I40, G41, S42, N43 and / or T44;

[0261] (ii) amino acid residues at positions 141-143, particularly T141 and / or T143; and

[0262] (iii) Amino acid residues at positions 162-169, especially I162, G163, N165, V166, S167, I168 and / or G169.

[0263] In some embodiments, category 2 VHH antibodies are characterized by having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6B Number:

[0264] - Y or H at position 32;

[0265] -F at position 47;

[0266] -N, T, or S at position 52;

[0267] - W or Y at position 59;

[0268] -Y at position 60;

[0269] -P at position 61;

[0270] -D at position 62;

[0271] -S or N at position 104;

[0272] -Y at position 105;

[0273] -W at position 106;

[0274] -W at position 109;

[0275] -H at position 111; and

[0276] -E at position 112.

[0277] In some embodiments, the VHH antibody is a member of VHH antibody category 3 (Ma8D07 category).

[0278] Category 3 VHH antibodies are characterized by their binding to HLA epitope 3. For example, in Figure 7 As shown, the category 3 VHH antibody interacts with amino acid residues selected from the following on the HLA according to SEQ ID NO:107:

[0279] (i) amino acid residues at positions 210-219, particularly R210, D211, S212, W213, P215 and / or N219; and

[0280] (ii) Amino acid residues at positions 285-297, especially H285, T287, S289, K292, T294, N295 and / or K297.

[0281] In some embodiments, category 3 VHH antibodies are characterized by having a VHH sequence comprising the following amino acid residues that interact with HLA, based on Figure 6C Number:

[0282] -Y at position 29;

[0283] -K at position 30;

[0284] -L at position 31;

[0285] -N at position 32;

[0286] -A at position 33;

[0287] -L at position 47;

[0288] -T at position 50;

[0289] -S at position 52;

[0290] -S at position 53;

[0291] -G at position 54;

[0292] -F at position 58;

[0293] -N at position 98;

[0294] -P at position 99;

[0295] -T at position 100;

[0296] -Y at position 101;

[0297] -N at position 102; and

[0298] -H at position 103.

[0299] In some embodiments, the VHH antibody is a member of VHH antibody category 4 (Ma8A05 category).

[0300] Category 4 VHH antibodies are characterized by their binding to HLA epitope 4. (As in...) Figure 7 As shown, the category 4 VHH antibody interacts with amino acid residues selected from the following on the HLA according to SEQ ID NO:107:

[0301] (i) amino acid residues at positions 204-208, particularly N204, W205, G206, and / or Y208; and

[0302] (ii) Amino acid residues at positions 213-227, especially W213, N214, V216, Q220, M223, K224, T225, R226 and / or N227.

[0303] In some embodiments, category 4 VHH antibodies are characterized by having a VHH sequence containing the following amino acid residues that interact with HLA, based on Figure 6D Number:

[0304] -A at position 53;

[0305] -S at position 57;

[0306] -D at position 58;

[0307] -I at position 60;

[0308] -T at position 61;

[0309] -Y at position 62;

[0310] -Y at position 63;

[0311] -S at position 64;

[0312] - D at position 65;

[0313] -A at position 103;

[0314] -F at position 104;

[0315] -D at position 105; and

[0316] -F at position 106.

[0317] In some embodiments, the VHH antibody is a member of VHH antibody class 5 (Bm28C04 class).

[0318] Category 5 VHH antibodies are characterized by their binding to HLA epitope 1. (As in...) Figure 7 As shown, the category 5 VHH antibody interacts with amino acid residues selected from the following on the HLA according to SEQ ID NO:107:

[0319] (i) Amino acid residues at positions 55-59, especially D55, K56, E57, N58 and / or G59;

[0320] (ii) amino acid residues at positions 88-97, particularly A88, G89, Q90, R92, V93, Y94, S95, E96 and / or E97; and

[0321] (iii) Amino acid residues at positions 240-302, especially K240, R277, R279, W300 and / or D302.

[0322] In some embodiments, category 5 VHH antibodies are characterized by having a VHH sequence containing the following amino acid residues that interact with HLA, based on Figure 6E Number:

[0323] -N at position 30;

[0324] -S at position 31;

[0325] -Y at position 32;

[0326] -Q at position 44;

[0327] -R at position 45;

[0328] -S at position 54;

[0329] -D at position 100;

[0330] -H at position 102;

[0331] -Y at position 104;

[0332] -A at position 105;

[0333] -F at position 106;

[0334] -G at position 107; and

[0335] - D at position 109.

[0336] In a particular embodiment, at least one amino acid in the reference sequence (including amino acids in the CDR1, CDR2, or CDR3 sequences and / or in the framework region) is replaced by another amino acid while maintaining the structural integrity and epitope binding of the VHH antibody. These exchanges can be conserved (i.e., using similar amino acids) or non-conserved.

[0337] In a further specific embodiment, at least one amino acid of the reference sequence (including amino acids in the CDR1, CDR2, or CDR3 sequences and / or in the framework region) is replaced by a conserved amino acid substitution, i.e., the amino acid is replaced by another amino acid with similar biochemical properties, such as an aliphatic amino acid (e.g., Gly, Ala, Val, Leu, or Ile) replaced by another aliphatic amino acid; a basic amino acid (e.g., His, Lys, or Arg) replaced by another basic amino acid or Met; an acidic amino acid or its amide (e.g., Asp, Glu, Asn, or Gln) replaced by another acidic amino acid or its amide; or an aromatic amino acid (e.g., Phe, Tyr, or Trp) replaced by another aromatic amino acid.

[0338] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:3 are replaced by another amino acid.

[0339] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7A08 having the VHH sequence shown in SEQ ID NO:7, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:7 are replaced by another amino acid.

[0340] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7B03 having the VHH sequence shown in SEQ ID NO:11, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:11 are replaced by another amino acid.

[0341] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7B12 having the VHH sequence shown in SEQ ID NO:15, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:15 are replaced by another amino acid.

[0342] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7B01 having the VHH sequence shown in SEQ ID NO:19, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:19 are replaced by another amino acid.

[0343] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7C02 having the VHH sequence shown in SEQ ID NO:23, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:23 are replaced by another amino acid.

[0344] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7E01 having the VHH sequence shown in SEQ ID NO:27, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:27 are replaced by another amino acid.

[0345] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7F02 having the VHH sequence shown in SEQ ID NO:31, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:31 are replaced by another amino acid.

[0346] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7D06 having the VHH sequence shown in SEQ ID NO:35, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:35 are replaced by another amino acid.

[0347] In a further specific embodiment, the VHH antibody is selected from: antibody Ma7C11 having the VHH sequence shown in SEQ ID NO:39, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:39 are replaced by another amino acid.

[0348] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8E03 having the VHH sequence shown in SEQ ID NO:43, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:43 are replaced by another amino acid.

[0349] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8G02 having the VHH sequence shown in SEQ ID NO:47, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:47 are replaced by another amino acid.

[0350] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8H03 having the VHH sequence shown in SEQ ID NO:51, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:51 are replaced by another amino acid.

[0351] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:55 are replaced by another amino acid.

[0352] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8D05 having the VHH sequence shown in SEQ ID NO:59, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:59 are replaced by another amino acid.

[0353] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:63 are replaced by another amino acid.

[0354] In a further specific embodiment, the VHH antibody is selected from: antibody Ma8D08 having the VHH sequence shown in SEQ ID NO:67, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:67 are replaced by another amino acid.

[0355] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28F09 having the VHH sequence shown in SEQ ID NO:71, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:71 are replaced by another amino acid.

[0356] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28H01 having the VHH sequence shown in SEQ ID NO:75, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:75 are replaced by another amino acid.

[0357] In a further specific embodiment, the VHH antibody is selected from: antibody Ma31A12 having the VHH sequence shown in SEQ ID NO:79, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:79 are replaced by another amino acid.

[0358] In a further specific embodiment, the VHH antibody is selected from: antibody Ma31B06 having the VHH sequence shown in SEQ ID NO:83, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:83 are replaced by another amino acid.

[0359] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28C04 having the VHH sequence shown in SEQ ID NO:87, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:87 are replaced by another amino acid.

[0360] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28H07 having the VHH sequence shown in SEQ ID NO:91, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:91 are replaced by another amino acid.

[0361] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28C09 having the VHH sequence shown in SEQ ID NO:95, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:95 are replaced by another amino acid.

[0362] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28H12 having the VHH sequence shown in SEQ ID NO:99, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:99 are replaced by another amino acid.

[0363] In a further specific embodiment, the VHH antibody is selected from: antibody Bm28A12 having the VHH sequence shown in SEQ ID NO:103, or a VHH antibody as a variant thereof. In some embodiments, amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO:103 are replaced by another amino acid.

[0364] Furthermore, the present invention relates to nucleic acid molecules (e.g., DNA molecules) encoding the VHH identified above, or vectors comprising the aforementioned nucleic acid molecules, said nucleic acid molecules being operatively linked to expression regulatory sequences (particularly, to heterologous expression control sequences). Furthermore, the present invention relates to cells comprising the nucleic acid molecules or vectors described above. Vectors for recombinantly generating VHH antibodies are well known in the art. In some embodiments, said vectors are extrachromosomal vectors; in other embodiments, said vectors are vectors for genome integration. The cells may be known host cells for generating antibodies or antibody fragments, such as prokaryotic cells (e.g., *Escherichia coli* or *Bacillus* species cells), yeast cells (particularly *Pichia* yeast cells), insect cells (e.g., Sf-9 cells), or mammalian cells (e.g., CHO cells), or plant cells. In some embodiments, said cells contain said nucleic acids or said vectors in an extrachromosomal manner. In other embodiments, said cells contain said nucleic acids or said vectors integrated into the genome, for example as an expression cassette for genome integration.

[0365] Another further aspect of the invention is a method for recombinantly generating VHH antibodies by growing the cells described above in a culture medium and obtaining the VHH antibodies from the cells or the culture medium. Suitable culture media and culture conditions are well known in the art.

[0366] Binding with Staphylococcus aureus HLA

[0367] The VHH antibody of this invention binds to Staphylococcus aureus HLA peptides. The inventors have identified VHH antibodies that bind with high affinity to different epitopes on Staphylococcus aureus HLA, as shown in Table 1 (see above).

[0368] In the context of this disclosure, the term "Staphylococcus aureus HLA" encompasses Staphylococcus aureus HLA from various strains, such as MRSA-resistant strains, such as USA300, USA400 (CC1), USA500 (CC8), or ST80. These and other suitable strains have been described, for example, by Diep et al. (2006); Cortes et al. (2017); Earls et al. (2019); Frisch et al. (2018); and Mairi et al. (2020), the contents of which are incorporated herein by reference.

[0369] However, it should be noted that the term "Staphylococcus aureus HLA" also covers naturally occurring variants and genetically modified constructs of Staphylococcus aureus HLA peptides, as described herein.

[0370] The inventors performed selection and crystallization experiments using the HLA shown in SEQ ID NO:1, and performed Octet assays using Cys-HLA, after biotinylation at the N-terminal ectocysteine ​​using Cys-HLA shown in SEQ ID NO:2.

[0371] In some embodiments, the VHH antibody of the present invention binds to Staphylococcus aureus HLA, wherein the dissociation constant K is used. D The binding affinity is expressed as approximately 1 nM or less, approximately 0.5 nM or less, or approximately 0.1 nM or less. Binding affinity can be determined using the peptides of SEQ ID NO:1 and SEQ ID NO:2 described above, as detailed in the examples herein.

[0372] HLA neutralization

[0373] The VHH antibody of the present invention is able to neutralize Staphylococcus aureus HLA, particularly by inhibiting the hemolysis of blood cells (e.g., erythrocytes), as shown in Table 1 (see above).

[0374] In some embodiments, the VHH antibody of the present invention alters the biological properties of HLA, particularly by inhibiting HLA receptor and / or membrane binding and / or heptamer pore formation.

[0375] In some embodiments, the VHH antibody (e.g., Ma8A05 or a competitive antibody) binds to the HLA protomer and blocks the assembly of heptamer pores. In further embodiments, the VHH antibody (e.g., Ma7A07 and Ma8D07, or a competitive VHH antibody) blocks subunit association. In further embodiments, the VHH antibody (e.g., Ma7F02 or a competitive VHH antibody) inhibits the conformational change that generates membrane-intercalating β-hairpins.

[0376] In some embodiments, the VHH antibody of the present invention neutralizes HLA at a concentration corresponding chemometrically to the concentration of HLA. The neutralizing potency can be determined as described in detail in the examples herein. In Example 3, in vitro erythrocyte lysis was observed at an HLA concentration of approximately 0.5-1 nM. This lysis can be neutralized with a VHH antibody concentration of approximately 1 nM.

[0377] stability

[0378] For the intended therapeutic application, anti-HLA antibodies should not only be highly potent in HLA neutralization, but they should also be developable as biological drugs. This includes being stable enough to survive long, large-scale manufacturing processes, as well as transportation and storage (ideally for several years in liquid formulations), without aggregation or loss of activity.

[0379] A good predictor of stability is thermal stability, which can be measured, for example, by thermal shift assay or, in particular, by differential scanning fluorimetry. The inventors of this invention have identified several thermally stable or ultrathermally stable VHH antibodies, as shown in Table 1 (see above).

[0380] In one particular embodiment, the present invention relates to VHH antibodies that are stable, particularly thermally stable or ultrathermally stable. Preferably, the 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, 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 (when measured under non-reducing conditions). The melting temperature and aggregation temperature are determined as described herein.

[0381] VHH antibody kit

[0382] In a further aspect, the present invention relates to kits comprising at least two, three, four, or more of the VHH antibodies described above. In such kits, individual VHH antibodies are present in a suitable molar ratio. Typically, the molar ratio is in the range of about 2:1 to about 1:2, particularly about 1.5:1 to about 1:1.5, and more particularly about 1:1. In some embodiments, the VHH antibody kit may comprise a single composition, wherein the VHH antibodies in the kit consist of a predetermined number of different types of VHH antibodies described above. The VHH antibody kit may comprise multiple compositions, each comprising a different type of VHH antibody described above. The kits of the present invention may not contain other VHH antibodies.

[0383] In a particular embodiment, the VHH antibody kit contains at least two (e.g., two, three, or four) complementary VHH antibodies, namely VHH antibodies targeting different epitopes of HLA, such as a VHH antibody targeting epitope 1, a VHH antibody targeting epitope 2, a VHH antibody targeting epitope 3, and / or a VHH antibody targeting epitope 4, as defined herein.

[0384] For example, the VHH antibody kit contains 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):

[0385] (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA;

[0386] (ii) VHH antibody, selected from 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, or VHH antibody competing with VHH antibody Ma7B01 and / or Ma7F02 for binding to HLA.

[0387] (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and

[0388] (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

[0389] The specially selected VHH antibody kits include:

[0390] - VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3

[0391] - VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO:19,

[0392] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0393] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0394] A further particularly preferred VHH antibody kit includes:

[0395] - VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3

[0396] - VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO:31,

[0397] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0398] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0399] A further particularly preferred VHH antibody kit includes:

[0400] - VHH antibody Ma8E03 having the VHH sequence shown in SEQ ID NO:43

[0401] - VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO:31,

[0402] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0403] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0404] A further particularly preferred VHH antibody kit includes:

[0405] - VHH antibody Ma8E03 having the VHH sequence shown in SEQ ID NO:43

[0406] - VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO:19,

[0407] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0408] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0409] A further particularly preferred VHH antibody kit includes:

[0410] - VHH antibody Bm28C04 having the VHH sequence shown in SEQ ID NO:87

[0411] - VHH antibody Ma7B01 having the VHH sequence shown in SEQ ID NO:19,

[0412] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0413] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0414] A further particularly preferred VHH antibody kit includes:

[0415] - VHH antibody Bm28C04 having the VHH sequence shown in SEQ ID NO:87

[0416] - VHH antibody Ma7F02 having the VHH sequence shown in SEQ ID NO:31,

[0417] - VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63,

[0418] And optionally, a VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55.

[0419] Different VHH antibody kits are useful for the therapeutic and diagnostic applications described in detail below.

[0420] Monovalent and multivalent VHH antibodies

[0421] In some embodiments, the VHH antibody of the present invention is monovalent, i.e., it has a single binding site for a Staphylococcus aureus polypeptide. In these embodiments, the VHH antibody may be present in this manner, or may be covalently or non-covalently attached to a heterologous portion, such as a peptide or non-peptide portion.

[0422] In further embodiments, the VHH antibody of the present invention is in the form of a polymer (e.g., a dimer, trimer, or tetramer). In these embodiments, several VHH antibody units may be partially covalently or non-covalently attached to each other via linkers and / or polymerization (e.g., dimerization, trimerization, or tetramerization). In these embodiments, the VHH antibody of the present invention may be a homomeric antibody or a heteromeric antibody.

[0423] The heteropolymeric VHH antibody comprises two or more (e.g., 2, 3, or 4) distinct VHH antibody units. These distinct VHH antibody units may all target the same Staphylococcus aureus polypeptide, particularly HLA, and more particularly, different epitopes on the HLA (e.g., as defined herein). Alternatively, the distinct VHH antibody units may target different Staphylococcus aureus polypeptides, for example, at least one VHH antibody unit may target HLA, and at least one VHH antibody unit may target one or more other Staphylococcus aureus polypeptides, such as Staphylococcus aureus virulence factors.

[0424] In some embodiments, the heteropolymeric VHH antibody comprises two or more different VHH antibody units, such as 2, 3 or 4 different VHH antibody units, which target different Staphylococcus aureus peptides or Staphylococcus aureus HLA, wherein at least one VHH antibody unit comprises the VHH antibody described herein.

[0425] In some embodiments, the heteropolymeric VHH antibody comprises at least two VHH antibody units against Staphylococcus aureus HLA selected from the following different VHH antibody classes (i), (ii), (iii), and (iv):

[0426] (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA;

[0427] (ii) VHH antibody, selected from 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, or a VHH antibody that competes with VHH antibody Ma7B01 for binding to HLA.

[0428] (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and

[0429] (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

[0430] In some embodiments, the heteropolymer VHH antibody comprises at least two VHH antibody units selected from different VHH antibodies belonging to at least two VHH antibody classes 1, 2, 3, 4 and 5 as defined herein.

[0431] In some embodiments, the VHH antibody may be covalently or non-covalently conjugated to a heterologous portion selected from labeling groups, capture groups, or effector groups, wherein the heterologous portion is particularly selected from fluorescent groups, biotin, enzymes (e.g., peroxidases, phosphatases, or luciferases), haptens, affinity tags, or nucleic acids (e.g., oligonucleotides). In further embodiments, the heterologous portion is selected from human serum albumin, albumin-binding moieties, or Fc fragments of immunoglobulin molecules (e.g., IgA, IgD, IgE, IgG, IgM, or their subtypes). In still further embodiments, the heterologous portion is selected from one or more non-peptide polymer portions, preferably hydrophilic polymer portions, such as polyethylene glycol (PEG).

[0432] In some embodiments, the VHH antibody is a homodimeric VHH antibody, wherein the VHH antibody unit is covalently attached to the dimerized portion, such as an immunoglobulin IgG Fc fragment.

[0433] In some embodiments, the VHH antibody is a heterodimeric VHH antibody, particularly 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.

[0434] Production of VHH antibodies

[0435] VHH antibodies (including monomeric and multimeric VHH antibodies) may be produced as described in WO 2022 / 023483 and WO 2022 / 023484 (the contents of which are incorporated herein by reference) or by other methods well known in the art.

[0436] VHH antibodies can be recombinantly generated in suitable host cells (e.g., in prokaryotic or eukaryotic host cells or host organisms). For this purpose, a nucleic acid molecule encoding the VHH antibody is introduced into the host cell or host organism and expressed therein. The nucleic acid molecule may encode a monomeric VHH antibody or a subunit of a multimeric VHH antibody.

[0437] In one particular embodiment, the VHH antibody is recombinantly generated in bacteria (e.g., *Escherichia coli* or *Bacillus* spp.). For example, expression in bacteria may involve cytoplasmic and / or periplasmic expression of the VHH antibody and purification of the VHH antibody from host cells, or secretory expression of the VHH antibody and purification of the VHH antibody from a culture medium. In some embodiments, the nucleic acid sequence encoding the VHH antibody is fused with at least one sequence that directs expression to the periplasm and / or to the culture medium.

[0438] In a further specific embodiment, the VHH antibody is recombinantly generated in eukaryotic host cells or host organisms, preferably in yeast, such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, or *Hansenula polymorpha*, or in animal cells, particularly insect cells (e.g., Sf-9 cells) or mammalian cells (e.g., human or hamster cells, such as HEK293F, CHO, or COS-7). For example, expression in eukaryotic host cells or host organisms (e.g., yeast) may involve cytoplasmic and / or periplasmic expression of the VHH antibody and purification of the VHH antibody from host cells, or preferably secretion of the VHH antibody from host cells and purification of the VHH antibody from a culture medium. In some embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence that directs expression into the culture medium.

[0439] VHH molecules Ma7A07, Ma7B01, Ma7F02, Ma8A05, Ma8D07, and Ma8H03 were converted to the VHH-IgG1-Fc pattern and transfected into CHO-DG44 cells using a transposase for integration into the active site, and selected for generating clonal libraries. When these libraries were compared with conventional IgG1 antibodies and other VHH-IgG1-Fc fusions in fed-batch fermentation, unexpectedly high yields of secreted products, approaching 14 g / L, were observed, up to 2–3 times higher. These unusually high titers can be attributed to the extremely low aggregation tendency and high stability of the VHHs disclosed herein.

[0440] Therapeutic applications

[0441] Another further aspect of the invention is the use of the VHH antibody described above in medicine, particularly for therapeutic and / or in vitro or in vivo diagnostic purposes. In some embodiments, the VHH antibody is used in human medicine.

[0442] The VHH antibody of the present invention is useful in the prevention, treatment, and / or alleviation of symptoms caused by, associated with, and / or accompanied by HLA-positive Staphylococcus aureus infection. In some embodiments, the Staphylococcus aureus is a drug-resistant Staphylococcus aureus.

[0443] Exemplary symptoms include, but are not limited to, skin infections, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis, and / or endocarditis.

[0444] In therapeutic applications, the VHH antibody is administered to the subject in need, specifically the human subject, in an effective dose. The dosage will depend on the specific reagent type (e.g., monovalent or multivalent VHH antibody), the disease type, and the route of administration.

[0445] Typically, the VHH antibody is administered as a pharmaceutical composition comprising an active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art.

[0446] Depending on the stage and severity of the illness, the pharmaceutical composition may be applied once or several times during the course of the illness. For example, it may be applied once or several times daily, once every two days, twice a week, or once a week for an appropriate period of time.

[0447] In some embodiments, the pharmaceutical composition is administered parenterally, such as by subcutaneous, intramuscular, or intravenous injection, or by infusion. In further embodiments, the pharmaceutical composition is administered topically, such as topically, orally, nasally (e.g., by spray), or intrapulmonaryly (e.g., by inhalation as an aerosol).

[0448] In some implementations, treatment of patients infected with HLA-positive Staphylococcus aureus involves systemic administration of one or more complementary neutralizing anti-HLA VHH antibodies, for example, via injection. In the case of pneumonia, inhalation may be the preferred route of administration. In both cases, treatment should reduce tissue damage caused by HLA and the virulence of the pathogen to allow the immune system to clear the infection.

[0449] Prophylactic use of anti-HLA VHH antibodies should be considered for the following subjects: those who have been colonized with Staphylococcus aureus or have been exposed to or may be exposed to the pathogen, and / or those who are immunocompromised, preparing for surgery, and / or have suffered injuries that may lead to infection.

[0450] Anti-HLA VHH antibodies will also be effective against highly antibiotic-resistant strains, and in the late stages of infection (e.g., in late-stage sepsis) (where antibiotic treatment alone is no longer effective in clearing the toxins).

[0451] The VHH antibody can be administered as a monotherapy alone, or as a combination therapy with other active agents, particularly with other agents useful in the prevention, treatment, and / or mitigation of symptoms caused and / or associated with infections of Staphylococcus aureus and / or optionally different bacteria (e.g., sequentially or simultaneously). In some embodiments, the VHH antibody is administered in combination with an antibiotic against Staphylococcus aureus (e.g., vancomycin), and optionally in combination with other antibiotics against different bacteria.

[0452] Diagnostic applications

[0453] Diagnostic applications include in vitro methods in which VHH antibodies or a kit of different VHH antibodies are used to detect HLA in samples, such as bodily fluids (e.g., saliva, sputum, bronchoalveolar lavage fluid, swabs, blood, serum, or plasma), stool samples, or tissue or biopsy samples.

[0454] Diagnostic applications further include in vivo methods in which VHH antibodies are used to detect HLA in subjects, particularly in human patients. For diagnostic applications, the VHH antibodies may carry a marker for direct detection, or be used in combination with a second detection reagent (e.g., biotin / streptoacidin, detection antibody, including conventional antibodies or VHH antibodies) for indirect detection, according to techniques established in the art.

[0455] This invention covers the use of anti-HLA VHH antibodies for the highly sensitive detection of toxins. The availability of four complementary epitope classes can be fully utilized for highly sensitive detection. In some embodiments, the detection pattern includes the binding of at least two different VHH antibodies (e.g., 2, 3, or 4 different VHH antibodies) to an HLA molecule, particularly the binding of at least two different VHH antibodies (e.g., 2, 3, or 4 VHH antibodies) each targeting a different epitope. In a particular embodiment, the detection includes using a kit of 2, 3, or 4 different VHH antibodies as described above.

[0456] In a particular embodiment, the detection includes a sandwich assay, such as a sandwich ELISA. In this type of assay, a first VHH antibody may be immobilized to a solid phase for capturing HLA from the sample, and a second VHH antibody may be used in a labeled form for the actual detection. A third and / or fourth VHH antibody may also be used for a prior enrichment step, for example by capture and proteolytic release methods (Frey and...). 2014b; Frey and (2014a; Vera Rodriguez et al., 2019). This allows for rapid and sensitive detection of HLA, which is crucial in life-threatening conditions.

[0457] The invention will be further described in more detail with reference to the following figures and embodiments. Attached Figure Description

[0458] Figure 1 Sequence alignment and highlighting of variable regions

[0459] Figure 1 The alignments of the VHH sequences from Table 2 are shown. Residues deviating from the common sequence are marked with a gray background. Three variable CDR regions are indicated.

[0460] Figure 2 Affinity measurement of VHH antibodies against HLA

[0461] Bio-Layer Interferometry (BLI) experiments were performed on the VHH antibody using a high-precision streptavidin sensor and biotinylated HLA on an Octet instrument. The black curve represents the fit using the mass transport model. Actual data points are shown in gray. K D This is derived from global fitting. See Example 1 for details.

[0462] Figure 3 Epitope framing of anti-HLA VHH antibodies and identification of four distinct HLA binding sites.

[0463] (A) Epitope framing and experiments were performed on the indicated VHH antibodies using biotinylated HLA in pairs. When HLA was saturated with Ma7A07, Ma8H03 could not bind further, 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.

[0464] (B) Representatives of four different epitopes VHH are sequentially bound to fixed HLA.

[0465] Figure 4 VHH antibodies inhibit HLA-induced hemolysis.

[0466] Red blood cells were aliquoted into 96-well plates and incubated with buffer, water, or HLA at 37°C for 3 hours, followed by incubation at 4°C for 1 hour. The plates were centrifuged to allow still intact cells to form a granular pellet, and the supernatant was analyzed on a plate reader for absorbance at 412 nm. Lysed cells released their cytoplasmic contents, including heme-bound hemoglobin, which has maximum absorbance at 412 nm. If instructed, HLA was pre-incubated with VHH antibody.

[0467] (A) Mouse red blood cells were used to test the antibodies shown;

[0468] (B) Human red blood cells were used to test the antibodies shown.

[0469] Figure 5 High-resolution crystal structure of the dimer VHH·HLA complex

[0470] HLA and VHH were generated in the cytoplasm of *E. coli* NEB Express or NEB Shuffle Express, respectively, and then purified by Ni chelation chromatography. A dimeric VHH·HLA complex was formed in solution by stoichiometric mixing of HLA and VHH at a ratio of 1:1.2, and excess VHH was removed by gel filtration. The homogeneous complex was crystallized, X-ray diffraction data were recorded on a Swiss Light Source synchrotron, and the structure was resolved by molecular substitution: regarding Ma7A07·HLA, The resolution and Rfree of 0.23; regarding Bm28C04·HLA, The resolution and 0.25 Rfree; regarding Ma7F02·HLA, The resolution and Rfree of 0.24; regarding Ma8A05·HLA, The resolution and Rfree of 0.26; regarding Ma7D07·HLA, The resolution and Rfree of 0.23.

[0471] Crystal structures of the Ma7A07·HLA(A), Bm28C04·HLA(B), Ma7F02·HLA(C), Ma8A05·HLA(D), and Ma8D07·HLA(E) complexes are presented in band form (AE). The conformationally important domains of HLA, namely the N-terminal stem and membrane-intercalating arms, are labeled.

[0472] F) The crystal structures shown in A, C, D, and E are superimposed by arranging them relative to HLA and depicted using surface representation. Note: The epitope frame is validated and experimentally verified by binding the four VHHs to different parts of the HLA molecule.

[0473] Computer simulation of docking of GH)Ma8A05 to membrane-bound HLA. Modeling was based on the alignment of HLA in D with individual HLA units in heptamer HLA structures (PDB ID 3M2L; Banerjee et al., 2010).

[0474] G) Ma8A05 bound to a single HLA is shown in a banded representation. For clarity, the other six HLA protomers are omitted. Note: Ma8A05 binds to epitopes in conformational domains distant from the HLA, thus enabling it to identify HLA in all its forms.

[0475] H) shows the model in G, with membrane-intercalating domains for all HLA protomers. The significant conflict with adjacent HLA protomers indicates that HLA bound to Ma8A05 cannot oligomerize into porous heptamers. This explains why this VHH neutralizes the toxic HLA effect. The structures of HLAs with Ma7A07, Ma8D07, or Ma7F02 are also incompatible with hemolysin that forms multimeric pores.

[0476] Figure 6: Sequence alignment of individual anti-HLA VHH classes, indicating residues that interact with HLA.

[0477] From Figure 5 The HLA-interacting residues of the VHH antibody were identified in the crystal structure shown or through homology-modelling based on the corresponding structures. The HLA-interacting residues are printed in bold.

[0478] (A) Ma7A07 category VHH antibody,

[0479] (B) Ma7F02 type VHH antibody,

[0480] (C)Ma8D07 type VHH antibody,

[0481] (D)Ma8A05 type VHH antibody,

[0482] (E)Bm28C04 category VHH antibody.

[0483] Figure 7 HLA sequence, which specifies the residues that interact with VHH.

[0484] Analysis in Figure 5The VHH-HLA structure is shown, and the HLA residues that interact with VHHs are identified. The numbers below the residues indicate which VHH they interact with: 1 represents Ma7A07, 2 represents Ma7F02, 3 represents Ma8D07, 4 represents Ma8A05, and 5 represents Bm28C04.

[0485] Figure 8 Thermostability of VHH Antibodies

[0486] The VHH antibody was subjected to differential scanning fluorometry (DSF), which utilizes the phenomenon that thermal defolding exposes aromatic / hydrophobic residues, which then bind to the added SYPRO orange dye and enhance their fluorescence. Two replicates of each sample were placed on a thermal cycler. The samples were incubated at 25°C for 5 minutes, and then the temperature was increased to 95°C in 1°C increments of 45 seconds. Fluorescence was measured at the end of each step.

[0487] Figure 9 Thermal stability of VHH as determined by BLI

[0488] The VHH antibody was incubated at room temperature or 95°C for 10 minutes and centrifuged at 20,000g for 20 minutes. The supernatant was diluted 50-fold (20 nM) and analyzed for HLA binding by BLI.

[0489] HLA sequence

[0490] HLA (SEQ ID NO:1) used in hemolysis assays and crystallization.

[0491] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTL KYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0492] Cys-HLA (SEQ ID NO:2) for Octet assay (after biotinylation at the N-terminal ectocysteine ​​residue)

[0493] SECGSSGADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRK ASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0494] HLA including a signal sequence for determining amino acid interactions (SEQ ID NO:104)

[0495] MKTRIVSSVTTTLLLGSILMNPVAGAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0496] VHH antibody sequence

[0497] >Ma7A07(SEQ ID NO:3)

[0498] QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSALSIYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAMDLWGKGTLVTVSS

[0499] >Ma7A08(SEQ ID NO:7)

[0500] QVQLVESGGGLVQPGGSLTLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSVLSVYIADSVKGRFTISRDDAKNTIYLHMNNLKPEDTGVYYCARGVDSWRWVPSAMDLWGKGTLVTVSS

[0501] >Ma7B03(SEQ ID NO:11)

[0502] QVQLVESGGGLVQPGGSLRLSCAASGFTLSNYYVGWFRQAPGKQREGVSSIGSSDLSIYIADSVKGRFTISRDNAKNTIYLHMNNLKPDDTGVYYCARGTDSWRWVPSAMDLWGKGTQVTVSS

[0503] >Ma7B12(SEQ ID NO:15)

[0504] QVQLVESGGGLVQPGGSLRLSCAASGFTLGDYTLGWFRQAPGKQREGVSSIASSALSIYIADSVKGRFTISRDNAKNTVYLHMNNLKPEDTGVYYCARGIDNWRWVPSAMDLWGKGTQVTVSS

[0505] >Ma7B01(SEQ ID NO:19)

[0506] QVQLVESGGGLVQAGDSLSLSCAASGRTFSNYAMGWFRQAPGKERHFVAVINQIGDSTWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWPHEYGYWGQGTQVTVSS

[0507] >Ma7C02(SEQ ID NO:23)

[0508] QVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAMGWFRQAAGKDRDFVAVITRSGDSTYYPDSTKGRFTISRDNAKNTMYLQMNSLKPEDTARYYCAATDPTNYWRIWEHEFDYWGQGTQVTVSS

[0509] >Ma7E01(SEQ ID NO:27)

[0510] QVQLVESGGGSVQAGDSLSLSCVASERTFSNYAMGWFRQAPGKERHFVAVINPIGEETWYPDFAKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAATDPRSYWRIWEHEFDYWGQGTQVTVSS

[0511] >Ma7F02(SEQ ID NO:31)

[0512] QVQLVESGGGLVQAGGSLRLSCTVSGSIDSLHTMGWFRQAPGKKRDFVAVVSWSGGNTYYPDYAKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAAESGSGNYWKIWEHEFDSWGQGTQVTVSS

[0513] >Ma7D06(SEQ ID NO:35)

[0514] QVQLVESGGGLVQAGGSLRLSCAASGSISGIDTMAWFRQIPGKDREFVAVIKWAAGSTWYPDFVKGRFTVSRDNAKNMVYLQMDSVKPEDTAIYYCAAAAGEKYYYRLFPYEYDYWGQGTQVTVSS

[0515] >Ma7C11(SEQ ID NO:39)

[0516] QVQLVESGGGLVRAGGSLRLSCAASGSISSVNGMGWFRQAPGKERLFVAQVSLLDSSTYYADSVKGRFTISRDDAKNTMYLQMNNVRPEETAVYYCAATKLRIGTAFSSEYDYWGQGTQVTVSS

[0517] >Ma8E03(SEQ ID NO:43)

[0518] QVQLVESGGGLVHPGGSLRLSCAASGSSLDHYVIGWFRQVPGKEREGVSCISRSGGSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLGWLEYDDWGQGTQVTVSS

[0519] >Ma8G02(SEQ ID NO:47)

[0520] QVQLVESGGGLVLPGGSLRLSCAASGSSLDHYVIGWFRQYPGKEREGVSCISRSGGSTNYADSVKGRFTVSRDNVKNMVYLQMNSLEPEDSAEYYCAAQRNLGNFCVLGWVEYDDWGQGTQVTVSS

[0521] >Ma8H03(SEQ ID NO:51)

[0522] QVQLVESGGGLVHPGGSLRLSCAASGSSLDHYVIGWFRQVPGKQREGISCISSGGSTNYADSVKGRFTVSRDNVKSTVYLQMNSLAPDDSAEYYCAAQRNLGNFCVLGWLEYDDWGQGTQVTVSS

[0523] >Ma8A05(SEQ ID NO:55)

[0524] QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0525] >Ma8D05(SEQ ID NO:59)

[0526] QVQLVESGGGLVQPGGSLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDTVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0527] >Ma8D07(SEQ ID NO:63)

[0528] QVQLVESGGGLVQPGGSLRLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGGSTFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQGTQVTVSS

[0529] >Ma8D08(SEQ ID NO:67)

[0530] QVQLVESGGGFVQAGGSLRLSCAASESIYKLNAMGWYRQAPGKELELVTTISSGGSTFYTDPVKGRFTISTDNAKNTVYLQMRSLKPEDTAMYYCAANPTYNHYSARGQGTQVTVSS

[0531] >Bm28F09(SEQ ID NO:71)

[0532] QVQLVESGGGLVEPGGSLTLSCAASGFTSKNYYIGWFRQAPGKYREGVASIGASDGSLYIADSVKGRFTISSDNAKNTVYLHPRNLKPEDSGVYYCATGRDSWRWVPSAMDYWGKGIQVTVSS

[0533] >Bm28H01(SEQ ID NO:75)

[0534] QVQLVESGGGLVQPGGSLTLSCAASGFTLGDRTLGWFRQVAGKQREGVASIASGVLSAYIADSVKGRFTISRDDAKNTIYLHMNNLKPDDTGVYYCARGVDSWRWVPSTMDLWGKGILVTVSS

[0535] >Ma31A12(SEQ ID NO:79)

[0536] QVQLVESGGGLVQPGGSLRLSCAASAASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEVDADYWGQGTQVTVSS

[0537] >Ma31B06(SEQ ID NO:83)

[0538] QVQLVQSGGGLVQPGGTLRLSCAASPASGFTLDLYTIAWFLQAPGKEREGVSAISLSDEITYYSDAVKGRFTISRDSAKNTVFLQMDRLKPEDTGVYYCAIGAFDFILKEADADYWGQGTQVTVSS

[0539] >Bm28C04(SEQ ID NO:87)

[0540] QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRSTGGTSYADSVKGRFTISYDNVQNTVDLQMNSLEPEDTAIYYCAGGDGHIYAFGYDYWGQGTQVTVSS

[0541] >Bm28H07(SEQ ID NO:91)

[0542] QVQLVESGGGSVQPGNSLRLSCKVSGRTFNSYALGWFRRRPGDQRDFVASIIRSTGGTSYADSVKGRFTISYDNVQNTVDLQMSSLEPEDTAIYYCAGGDGHIYAFGYDYWGQGTQVTVSS

[0543] >Bm28C09(SEQ ID NO:95)

[0544] QVQLVESGGSVVQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRSTGKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGYDYWGQGTQVTVSS

[0545] >Bm28H12(SEQ ID NO:99)

[0546] QVQLVESGGSVVQAGGSLRLSCTASGRNFNSYAMGWFRRPPGEQRDFIAAIIRSTGKTTYADSDSVKGRFTISKDSSGNTVYLQMNNLKPEDTAIYYCAGGDAGPYAFGYDYWGQGTQVTVSS

[0547] >Bm28A12(SEQ ID NO:103)

[0548] QVQLVESGGGLVQAGGSLRLSCVVSGRALSSYNLAWFRQSPGKEREWIATIMPSADKAHYPDFLEGRFTISGDNTENTLYLQMNSLKPEDTAIYYCAAREPSYYHALFEYEYNLWGQGTQVTVSS Examples

[0549] VHH antibodies that inhibit HLA activity were generated as follows: two alpacas were immunized three times at three-week intervals with a non-toxic HLA mutant (His61Thr), an immune library was prepared, and phage display was performed using HLA as bait.

[0550] 192 selected clones were sequenced and classified based on sequence similarity. Then, 17 representatives of all VHH classes were expressed and purified in *E. coli* (Ma7 and Ma8 series, see [link to specific sequences]). Figure 1 ).

[0551] For the generation of “second-generation” VHH antibodies, alpacas were immunized with HLA mutant proteins 16 months after the last immunization, followed by selection for immune library preparation and phage display, which generated additional VHH antibodies. These included previously isolated VHH variants (e.g., belonging to the Ma7A07 and Ma8A05 classes), and new VHH classes such as Bm28C04, Bm28C09, and Bm28A12.

[0552] VHH-HLA interactions were analyzed using biomembrane interferometry (BLI; Abdiche et al., 2008).

[0553] VHH antibodies belonging to the Ma8A05 and Ma8D07 classes bind to HLA with moderate affinity, for example, at 600 pM (for Ma8A05) and 800 pM (for Ma8D07). D Value (see) Figure 2 Other VHH antibodies showed significantly tight HLA binding (Table 1 and...). Figure 2 Ma7B01, Ma7F02 and Bm28C04 with K at ~50pM D The combination was carried out. For Ma7A07 and Ma8H03, no dissociation was detected, and K was estimated. D The value is ≤10pM.

[0554] To assess whether and how many complementary binding sites these VHHs possess on HLA molecules, frame-fit experiments were performed using BLI. In these experiments, one VHH antibody was bound to immobilized HLA, and the binding of another VHH was then monitored. Figure 3 An example is shown. When HLA was fully saturated with Ma7A07, Ma8H03 (the VHH with the highest affinity for HLA) could not bind further, while the binding of other tested VHHs was unaffected. By repeating this setting with all representative VHH class members, we found that the selected VHH antibody targeted four complementary epitopes (Table 1). Figure 3The results showed that up to four VHH antibodies can bind to HLA simultaneously if they are selected in such a way that each of them recognizes a different complementary epitope.

[0555] In subsequent experiments, the ability of these VHH antibodies to neutralize HLA was tested. Red blood cells (RBCs) are highly susceptible to HLA lytic activity. Furthermore, the high concentration of heme (as part of oxygen-carrying hemoglobin) in RBCs simplifies the detection of cell lysis. Therefore, we used RBCs to perform HLA-induced hemolysis experiments. We observed that 30 nM HLA resulted in complete cell lysis. When tested at a concentration of 100 nM, all anti-HLA VHH antibodies completely protected cells from hemolysis. Figure 4 ).

[0556] To understand the structural basis of VHH-dependent HLA inhibition, we planned to crystallize the HLA·VHH complex. We resolved the X-ray crystal structures of the dimer HLA complexes of VHH antibodies representing four different epitope binding agents: Ma7A07, Bm28C04, Ma7F02, Ma8A05, and Ma8D07. Figure 5 Crystallographic analysis confirmed that these VHHs have four distinct binding sites on HLA. Figure 5 The mechanism of action of Ma8A05 is also illustrated as an example: it binds to hemolysin promergams and blocks the assembly of heptamer pores through conflict with adjacent subunits. Further analysis revealed that other VHH antibodies either block subunit association (i.e., Ma7A07, Bm28C04, and Ma8D07) or block the prior conformational change that generates membrane-intercalating β-hairpins (i.e., Ma7F02).

[0557] Example 1: Binding of VHH antibody to HLA

[0558] Biofilm layer interferometry (BLI) experiments were performed at 25 °C using a high-precision streptavidin biosensor and an Octet RED96e instrument (ForteBio / Sartorius). The assay buffer consisted of phosphate-buffered saline (PBS) pH 7.4, 0.02% (w / v) Tween 20, and 0.1% (w / v) bovine serum albumin (BSA). HLA was expressed in an N-terminal ectocysteine ​​form and biotinylated via the attachment of a thiol-reactive maleimide-PEG11-biotin (Thermo Scientific, 21911).

[0559] Biotinylated HLA was immobilized on the biosensor until a wavelength migration / binding signal of 1 nm was achieved. The biosensor was immersed in wells containing 40, 20, 10, 5, 2.5, or 1.25 nM VHH for 10 minutes to associate, and then incubated with assay buffer for 30 minutes to dissociate. Data were subtracted from the reference, and the curves (grey) were fitted using a mass delivery model (Octet Data Analysis HT 12.0 software).

[0560] The results are shown in Table 1 and Figure 2 middle.

[0561] Example 2: Identification of complementary HLA epitopes

[0562] Epitope framing and experiments were performed using a similar setup to that described in Example 1. Biotinylated HLA was immobilized on the biosensor until a wavelength migration / 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 for 240 seconds with another VHH (VHH2) at a concentration of 100 nM. When binding of VHH2 was blocked in the presence of VHH1, they were considered to be the same epitope binders. By repeating this process for all VHH class representatives, we grouped the VHH antibodies into four different epitope binders.

[0563] The results are shown in Table 1 and Figure 3 In the middle, Ma7A07, Ma7F02, Ma8D07 and Ma8A05 can sequentially bind to fixed HLA.

[0564] Example 3: Red Blood Cell Hemolysis Assay

[0565] Red blood cell stock solutions were prepared fresh from blood samples taken from male CD1 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), with or without 100 nM VHH antibody. After incubation at room temperature for 30 minutes, this solution was added to 50 μL of 4 × 10⁻⁶ saturated sodium chloride solution in the wells of a standard 96-well plate. 8 One red blood cell.

[0566] The plates were incubated at 37°C with shaking at 180 rpm for 3 hours, followed by incubation at 4°C for 1 hour to complete cell lysis. Afterwards, the plates were centrifuged at 400×g and 4°C in a vibrating rotor for 10 minutes. 150 μL of the supernatant was transferred to another 96-well plate. Analysis was performed on a plate reader at 412 nm absorption.

[0567] The results show Figure 4In section A. Note: Diluting red blood cells in water resulted in complete lysis, and 30 nM HLA resulted in the same amount of cell lysis. All tested VHH antibodies strongly inhibited cell lysis and showed levels similar to those in the assay buffer.

[0568] For example, in the above text and Figure 4 The inhibitory activity was tested with second-generation VHH antibodies as described in section B, but using human erythrocytes. Since human erythrocytes are less susceptible to HLA, 250 nM HLA was used for cell lysis, and 1 μM VHH antibody was used, if indicated. Similarly, all tested VHH antibodies inhibited cell lysis.

[0569] Example 4: Measurement of the thermal stability of VHH antibodies

[0570] The VHH antibody was subjected to differential scanning fluorometry (DSF), which utilizes the phenomenon that thermal defolding exposes aromatic / hydrophobic residues, which then bind to the added SYPRO orange dye and enhance their 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 1x dye (diluted from 5000x stock solution; Life Technologies). Two replicates of each sample were pipetted to… In a 96-hole plate (Bio-Rad). The plate is placed using a transparent... The samples were sealed in 'B'Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and then placed on a CFX96 real-time system (C1000 thermal cycler, BioRad). The samples were incubated at 20°C for 5 minutes, then the temperature was increased to 95°C in 1°C increments every 45 seconds. Fluorescence was measured at the end of each step, using 532 nm excitation and a 555 nm long-pass filter. The melting temperature was defined as the inflection point of the first melting peak.

[0571] The results are shown in Table 1 and Figure 8 The VHH antibody Re9B09, generated in a reducing cytosol and therefore lacking disulfide bonds, dissolved at 35°C. Neutralizing VHH antibodies Ma8A05 and Ma7F02 similarly dissolved at 44°C and 62°C, respectively, while Ma7A07 and Ma8E03 showed only negligible dissolution and thus remained stable throughout the 95°C period.

[0572] Example 5: Testing VHH stability using BLI

[0573] BLI is highly sensitive to sample concentration and is therefore suitable for detecting any loss of active material in solution, including loss of active nanobodies due to instability. To determine whether VHHs that had been defused at 60–70 °C in DSF experiments retained their binding activity after heat treatment, these antibodies were incubated at 95 °C for 10 minutes (at a concentration of 1 μM), cooled, and then centrifuged to remove any aggregates that might have formed. When tested for HLA binding via BLI, heated and untreated samples of Ma31A12 and Bm28A12 showed no difference and behaved similarly to the thermally stable VHH Ma8E03. Figure 9 Therefore, after heat treatment, these nanobodies either partially unwind or robustly refold back to their native state. On the other hand, Ma8D07 exhibited a loss of activity, suggesting that they aggregated to some extent after heat treatment.

[0574] References

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[0586] Lodise, T. P., Graves, J., Evans, A., Graffunder, E., Helmecke, M., Lomaestro, B. M., & Stellrecht, K. (2008). Relationship between vancomycin MIC and failure among patients with methicillin-resistant Staphylococcus aureus bacteremia treated with vancomycin. Antimicrob Agents Chemother, 52(9), 3315 - 3320. https: / / doi.org / 10.1128 / AAC.00113-08

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Claims

1. VHH antibodies that recognize Staphylococcus aureus hemolysin (HLA), which: (i) Competing with the VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3 to bind to HLA; or (ii) Competing with 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 HLA binding; or (iii) Competing with the VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55 to bind to HLA; or (iv) Compete with the VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63 for binding to HLA.

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

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

4. VHH antibodies that recognize Staphylococcus aureus hemolysin (HLA) or Staphylococcus aureus HLA peptides, comprising: (1)： (a) The CDR3 sequence shown 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 that has at least 80%, at least 90%, or at least 95% identity with the CDR3 sequence of (a); (2)： (a) Combinations of the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 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) Combinations of CDR1, CDR2, and CDR3 sequences that have at least 80%, at least 90%, or at least 95% identity with the CDR1, CDR2, and CDR3 sequences of (a); (3)： (a) The VHH sequence shown 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) The VHH sequence of (a) has at least 80%, at least 90%, at least 95%, or at least 99% identity with the VHH sequence of (a).

5. The VHH antibody according to any one of the preceding claims, wherein the dissociation constant K is... D The indicated binding affinity to HLA is approximately 1 nM or less, approximately 100 pM or less, approximately 50 pM or less, approximately 20 pM or less, or approximately 10 pM or less.

6. The VHH antibody according to any one of the preceding claims, wherein HLA is neutralized.

7. The VHH antibody according to any one of the preceding claims is stable, particularly thermally stable or ultrathermally stable, and 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.

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

9. A heteropolymeric VHH antibody comprising two or more distinct VHH antibody units that recognize Staphylococcus aureus proteins, particularly HLA VHH antibody units, such as 2, 3 or 4 distinct VHH antibody units, wherein at least one VHH antibody unit comprises a VHH antibody according to any one of claims 1-8.

10. The heteropolymeric VHH antibody of claim 9, comprising at least two VHH antibody units selected from the following different VHH antibody classes (i), (ii), (iii), and (iv): (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA; (ii) VHH antibody, selected from 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, or a VHH antibody that competes with VHH antibody Ma7B01 for binding to HLA. (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

11. A kit of two or more different VHH antibodies that recognize HLA, comprising at least one VHH antibody according to any one of claims 1-10, wherein the kit particularly comprises at least two VHH antibodies selected from at least two different VHH antibody classes (i), (ii), (iii) and (iv) below: (i) VHH antibody, selected from VHH antibody Ma7A07 having the VHH sequence shown in SEQ ID NO:3, or VHH antibody that competes with VHH antibody Ma7A07 for binding to HLA; (ii) VHH antibody, selected from 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, or a VHH antibody that competes with VHH antibody Ma7B01 for binding to HLA. (iii) A VHH antibody selected from VHH antibody Ma8A05 having the VHH sequence shown in SEQ ID NO:55, or a VHH antibody that competes with VHH antibody Ma8A05 for binding to HLA; and (iv) VHH antibody, selected from VHH antibody Ma8D07 having the VHH sequence shown in SEQ ID NO:63, or VHH antibody that competes with VHH antibody Ma8D07 for binding to HLA.

12. The VHH antibody according to any one of claims 1-10 or the kit according to claim 11, for medical use, particularly for the prevention, treatment and / or mitigation of symptoms caused by, associated with and / or accompanied by HLA-positive Staphylococcus aureus infection, said symptoms particularly selected from skin infections, pneumonia, bacteremia, sepsis, meningitis, osteomyelitis and / or endocarditis.

13. The VHH antibody according to any one of claims 1-10 or the kit according to claim 11, for use in human subjects as described in claim 10.

14. The VHH antibody according to any one of claims 1-10 or the kit according to claim 11, for use according to any one of claims 12-13, administered as a monotherapy or administered as a combination therapy in sequence and / or simultaneously with at least one other active agent, for example in combination with an antibiotic against Staphylococcus aureus such as vancomycin, and optionally in combination with other antibiotics against different bacteria.

15. A pharmaceutical combination comprising, as an active agent, the VHH antibody according to any one of claims 1-10 or the kit according to claim 11, and a pharmaceutically acceptable carrier.

16. The use of the VHH antibody according to any one of claims 1-10 or the kit according to claim 11 for the detection of HLA in a sample, particularly in a biological sample.

17. The use according to claim 16, wherein the detection comprises the binding of at least two different VHH antibodies to an HLA molecule, particularly at least two different VHH antibodies, each targeting a different epitope, such as two, three, or four VHH antibodies, to an HLA molecule.

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