THERAPEUTIC AND DIAGNOSTIC VHH ANTIBODIES AGAINST SARS-COV-2 AND METHODS FOR THEIR ENHANCEMENT

Abstract

The present invention pertains in the fields of antibody technology, protein engineering, medicine, pharmacology, infection biology, virology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that prevent cell entry of and infection by SARS-CoV-2, a strategy for an enhanced block of the homotrimeric viral spike proteins by symmetry-matching VHH-fusions, implementations of this strategy, as well as VHH antibodies for sensitive detection of SARS-CoV2-infections.

Claims

1. A homotrimeric VHH antibody comprising 3 VHH antibody subunits recognizing the same target structure, wherein each VHH antibody subunit comprises a VHH antibody directly linked or linked via a spacer to a trimerization module.

2. The homotrimeric VHH antibody of claim 1, which comprises three identical VHH antibody subunits.

3. The homotrimeric VHH antibody of claim 1 wherein the VHH antibody is directed to a target structure having a threefold-rotationally symmetric subunit structure.

4. The homotrimeric VHH antibody of claim 1, wherein the VHH antibody is directed to a target structure comprising a homotrimeric protein or protein assembly.

5. The homotrimeric VHH antibody of claim 1, wherein the target structure is a viral protein or protein assembly, particularly a homo-trimeric viral protein or protein assembly, more particularly selected from a Coronavirus trimeric spike protein, an Orthomyxovirus trimeric HA protein, a Paramyxovirus trimeric fusion protein, a Dengue fever virus or Zikavirus trimeric or higher order protein assembly, a Herpesvirus trimeric gB fusion protein or a HIV trimeric gp120 protein.

6. The homotrimeric VHH antibody of claim 1, wherein the target structure is the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain including the RBD of wildtype SARS-CoV-2 and the RBD domain of a SARS-CoV-2 escape mutant including the British mutant (Alpha), the South African mutant (Beta), Brazilian mutant (Gamma), the Indian mutant (Delta), the Californian mutant (Epsilon) as well as mutants comprising at least one of the amino acid substitutions in any one of the above mutants.

7. The homotrimeric VHH antibody of claim 6, which is a neutralizing trimeric VHH antibody, e.g. a VHH antibody which has a lowest SARS-CoV-2 neutralizing concentration of about 100 pM or less, of about 25 pM or less, of about 17 pM or less, of about 10 pM or less, of about 5 pM or less or of about 1.7 pM or less.

8. The homotrimeric VHH antibody of claim 6, wherein the VHH antibody is selected from the VHH antibody comprising (a) a CDR3 sequence as shown in SEQ. ID NO: 20, 204, 208, 4, 8, 12, 16, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287, or 291, (b) a CDR3 sequence which has an identity of at least 80%, at least 90%, or at least 95% to a CDR3 sequence of (a), or (c) a VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

9. The homotrimeric VHH antibody of claim 6, wherein the VHH antibody is selected from the VHH antibody comprising (a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 18-20, 202-204, 206-208, 2-4, 6-8, 10-12, 14-16, 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, 102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-136, 138-140, 142-144, 146-148, 150-152, 154-156, 158-160, 162-164, 166-168, 170-172, 174-176, 178-180, 182-184, 186-188, 190-192, 194-196, 198-200, 225-227, 229-231, 233-235, 237-239, 241-243, 245-247, 249-251, 253-255, 257-259, 261-263, 265-267, 269-271, 273-275, 277-279, 281-283, 285-287, or 289-291, (b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90%, or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a), or (c) a VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

10. The homotrimeric VHH antibody of claim 6, wherein the VHH antibody is selected from the VHH antibody comprising (a) a VHH sequence as shown in SEQ ID NO. 17, 201, 205, 1, 5, 9, 13, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276, 280, 284 or 288, (b) a sequence which has an identity of at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% to a VHH sequence of (a), or (c) VHH antibody, which competes with a VHH antibody of (a) for the binding to the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

11. The homotrimeric VHH antibody of claim 6, wherein the VHH antibody is selected from: (a) the VHH antibody VHH-72 comprising an VHH amino acid sequence as shown in SEQ: ID NO. 217, or (b) a sequence which has an identity of at least 70%, at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).

12. The homotrimeric VHH antibody of claim 6, which is capable of virus neutralization, and which is particularly capable of neutralization of a SARS-CoV-2 escape mutant including the British mutant (Alpha), the South African mutant (Beta), Brazilian mutant (Gamma), the Indian mutant (Delta), the Californian mutant (Epsilon) as well as mutants comprising at least one of the amino acid substitutions in any one of the above mutants.

13. The homotrimeric VHH antibody of claim 1, which is stable.

14. The homotrimeric VHH antibody of claim 13, which has a melting temperature of at least about 65° C., of at least about 80° C., of at least 90° C. or of at least about 95° C. when measured under non-reducing conditions and/or under reducing conditions.

15. The homotrimeric VHH antibody of claim 1, wherein the target structure is different from spike protein S1 domain, particularly from the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain.

16. The homotrimeric VHH antibody of claim 1, wherein the target structure is a non-viral protein or protein assembly, particularly a member of the TNF Ligand superfamily or a member of the TNF Receptor superfamily.

17. The homotrimeric VHH antibody of claim 1, wherein each subunit comprises a VHH antibody linked via a spacer to a trimerization module.

18. The homotrimeric VHH antibody of claim 17, wherein the spacer has a length of about 5 to about 100 amino acids, particularly of about 10 to about 50 amino acids.

19. The homotrimeric VHH antibody of claim 17, wherein the spacer comprises amino acids selected from Gly, Ser, Ala, Thr, Pro, Asp and Glu, particularly in an amount of at least about 50%, about 60%, about 70%, about 80%, about 90% or 100%.

20. The homotrimeric VHH antibody of claim 17, wherein the spacer comprises amino acids selected from Gly, Glu, Ser and Pro particularly in an amount of at least about 50%, about 60%, about 70%, about 80%, about 90% or 100%.

21. The homotrimeric VHH antibody of claim 17, wherein the spacer comprises a succession of tripeptides, wherein the first and the second amino acid are selected from Gly, Ser, Ala, Thr, and Pro, and the third amino acid is selected from Asp and Glu.

22. The homotrimeric VHH antibody of claim 17, wherein the spacer is substantially free or free of amino acids selected from Asn, Lys, Arg, Trp, Tyr, Phe, Met, Leu, lie, Val, His and Cys.

23. The homotrimeric VHH antibody of claim 1, wherein the trimerization module is located N-terminal to the VHH-antibody.

24. The homotrimeric VHH antibody of claim 1, wherein the trimerization module is located C-terminal to the VHH-antibody.

25. The homotrimeric VHH antibody of claim 1, wherein the trimerization module is selected from: (a) a collagen trimerization domain, e.g. the NC1 domain of collagen XV, the NC1 domain of collagen XVIII or the NC1 domain of collagen X, or the lung surfactant protein D. (b) a trimerization domain which has an identity of at least 80%, particularly at least 90%, more particularly at least 95% to a trimerization domain of (a).

26. The homotrimeric VHH antibody of claim 1, wherein the trimerization module is the NC1 domain of collagen XVIII or a trimerization domain which has an identity of at least 80%, particularly at least 90%, more particularly at least 95% thereto.

27. The homotrimeric VHH antibody of claim 1, wherein the trimerization module is the NC1 domain of collagen XVIII or a trimerization domain which has an identity of at least 80%, particularly at least 90%, more particularly at least 95% thereto, located C-terminal to the VHH-antibody.

28. A subunit of a homotrimeric VHH antibody of claim 1.

29. A nucleic acid molecule encoding a homotrimeric VHH antibody according to claim 1 or a subunit thereof, preferably in operative linkage with a heterologous expression control sequence.

30. A vector comprising a nucleic acid molecule according to claim 29.

31. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule according to claim 29 or a vector comprising a nucleic acid molecule according to claim 29.

32. The cell or organism of claim 31, which is selected from a bacterium such as E. coli, Bacillus sp., a unicellular eukaryotic organism, e.g. yeast such as Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, or Leishmania, an insect cell, a mammalian cell or a plant cell.

33. The homotrimeric VHH antibody of claim 1, or a subunit thereof, which is non-glycosylated.

34. The homotrimeric VHH antibody of claim 1 or a subunit thereof, which is produced in a bacterium, e.g. E. coli.

35. The homotrimeric VHH antibody of claim 1 or a subunit thereof, which is produced in a yeast, e.g. Pichia pastoris.

36. The homotrimeric VHH antibody of claim 1 or a subunit thereof, which is conjugated to one or several polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG).

37. A method for preventing or treating a disorder caused by and/or associated with an infection with SARS-CoV-2 including an infection with a SARS-CoV-2 escape mutant including the British mutant (Alpha), the South African mutant (Beta), Brazilian mutant (Gamma), the Indian mutant (Delta), the Californian mutant (Epsilon) as well as mutants comprising at least one of the amino acid substitutions in any one of the above mutants, comprising administering a homotrimeric VHH antibody of claim 1 or a subunit thereof.

38. A method for recombinant production of a homotrimeric VHH antibody of claim 1, comprising cultivating a cell or an organism transformed or transfected with a nucleic acid molecule encoding a homotrimeric VHH antibody according to claim 1 or a subunit thereof, or a vector comprising a nucleic acid molecule encoding a homotrimeric VHH antibody according to claim 1 or a subunit thereof; in a suitable culture medium and obtaining the homotrimeric VHH antibody from the cell or organism or from the culture medium.

39. The method of claim 38, comprising cultivating a yeast such as Pichia pastoris Saccharomyces cerevisiae, or Hansenula polymorpha, and obtaining the homotrimeric VHH antibody from the medium.

40. A method for the recombinant production of a monomeric or multimeric VHH antibody, comprising: cultivating a yeast such as Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, comprising a nucleic acid molecule encoding the VHH antibody or a subunit thereof, in a suitable culture medium and obtaining the monomeric or multimeric, e.g. homotrimeric VHH antibody from the culture medium.

41. The method of claim 40 wherein the monomeric or multimeric VHH antibody is selected from a monomeric VHH antibody, or a homomultimeric, particularly a homotrimeric VHH antibody, more particularly a homotrimeric VHH antibody of claim 1.

42. The method of claim 40 wherein the yeast comprises a nucleic acid molecule encoding a polypeptide comprising (from N-terminus to C-terminus) (i) a cleavable co-translational signal sequence (pre-sequence), (ii) optionally a cleavable pro-sequence, which enhances secretion, and (iii) a VHH antibody sequence, particularly a fusion comprising the VHH antibody sequence, optionally a spacer, and a trimerization domain, e.g. the Collagen XVIII NC1 domain.

43. The method of claim 42, wherein the cleavable co-translational signal sequence (i) is selected from the Ost1 signal sequence of Saccharomyces cerevisiae (SEQ. ID NO: 219) or an Ost1 signal sequence from a related yeast species, e.g. from Pichia pastoris (SEQ. ID NO: 220), Schizosaccharomyces pombe (SEQ. ID NO: 221), or Candida albicans (SEQ. ID NO: 222) or a variant of those, e.g. having an amino acid identity of at least 80%, of at least 90% or of at least 95% thereto.

44. The method of claim 40, wherein the cleavable pro-sequence comprising a secretion signal sequence (ii) is selected from the propeptide of S. cerevisiae alpha-factor (SEQ. ID NO: 223) or a variant thereof, having an amino acid identity of at least 80%, of at least 90% or of at least 95%, or another Kex2-cleavable pro-peptide that promotes packaging into COPII vesicles and thus export from the ER.

45. A nucleic acid molecule encoding a polypeptide comprising (from N-terminus to C-terminus) (i) a cleavable co-translational signal sequence (pre-sequence), (ii) optionally a cleavable pro-sequence, which enhances secretion, and (iii) a VHH antibody sequence, e.g., a monomeric VHH antibody, or a subunit of a homomultimeric, e.g. homotrimeric VHH antibody, particularly a fusion comprising the VHH antibody sequence, optionally a spacer, and a trimerization domain, e.g. the Collagen XVIII NC1 domain.

46. A polypeptide encoded by the nucleic acid molecule of claim 45, which comprises (from N-terminus to C-terminus) (i) a cleavable co-translational signal sequence (pre-sequence), (ii) optionally a cleavable pro-sequence, which enhances secretion, and (iii) a VHH antibody sequence, e.g., a monomeric VHH antibody, or a subunit of a homomultimeric, e.g. homotrimeric VHH antibody, particularly a fusion comprising the VHH antibody sequence, optionally a spacer, and a trimerization domain, e.g. the Collagen XVIII NC1 domain.

Description

FIGURES

[0348] FIG. 1: Sequence alignment and highlighting of variable regions

[0349] FIG. 1 shows an alignment of VHH sequences from Table 1. Residues that deviate from the consensus are shown in colour. The three variable CDR regions are indicated.

[0350] FIG. 2: Staining of transfected cells transiently expressing the SARS-CoV-2 spike protein.

[0351] HeLa cells where transfected with a plasmid carrying the humanized coding sequence of the SARS-CoV-2 spike protein under the control of a CMV promoter. 36 hours post-transfection, cells were fixed for 5 minutes with 4% paraformaldehyde (PFA), permeabilized, blocked with 5% BSA, incubated with 30 nM VHH Re6D09 carrying two Alexa568 fluorophores per molecule, extensively washed and finally imaged with an LSM780 confocal laser scanning microscope using the 405 nm and 568 nm laser lines for excitation. Image shows the overlaid DAPI and 568 channels, detecting DNA and VHH antibody in blue and red, respectively. Note that the transfection efficiency was only ˜30%. The bright red signal corresponds to transfected cells, while non-transfected ones served as negative control.

[0352] FIG. 3: Fluorophore-labelled anti-SARS-CoV-2 VHH antibodies specifically detect the spike protein and assembling viruses in infected cells.

[0353] Vero E6 cells were infected by SARS-CoV-2 for three days, fixed for two hours with 4% paraformaldehyde, and stained as described above with 30 nM of the indicated Alexa488-labelled VHH antibodies. Imaging was with a standard epifluorescence microscope.

[0354] FIG. 4: SARS-CoV-2 neutralizing VHH antibodies.

[0355] At day 0, Vero E6 cells were inoculated with SARS-CoV-2, in the absence of VHHs or after a 60 min pre-incubation with the indicated VHH antibodies. Three days later, the virus-load increased ˜10 000-fold when the antibodies had been omitted (compare “inoculation” and “no VHH”). One VHH (Re7D02) had no effect. Two (Re7D05 and Re5C08) had a weak impact. Re6B06 inhibited in this experiment ˜300-fold. The other 18 VHH antibodies blocked viral infection completely. Quantitation of viral RNA in the supernatants of infected cells was by quantitative reverse transcription (RT) PCR as described previously (Stegmann et al., 2020). Note the Log 10-scale of the figure.

[0356] FIG. 5: VHH Re5D06 neutralizes SARS-CoV-2 with extreme potency.

[0357] Virus neutralization was performed as in FIG. 4. (A) Cells were fixed with PFA and stained with DAPI (to visualize cell nuclei), with a cocktail of Atto488-labelled VHHs recognizing the RBD (RBD epitopes) and Atto565-labelled VHHs recognizing an S1 epitope outside the RBD (S1ΔRBD epitope) in order to visualize newly synthesized Spike protein in successfully infected cells. Images show confocal sections. Cells that are positive in the Atto488 and Atto656 channels are infected. Note that infection was completely prevented when the virus was pre-incubated with VHH Re5D06 at a concentration of 50 pM. (B) A replicate experiment with identical outcome. Bars depict analysis by quantitative RT PCR, showing that 50 pM VHH Re5D606 prevented replication of the viral RNA completely.

[0358] FIG. 6: Hyper-thermostable anti-SARS-CoV-2 VHH antibodies.

[0359] Indicated VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposed aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Assays were performed in a volume of 20 μl, at 1 mg/ml VHH concentration in 50 mM Tris/HCl, 300 mM NaCl (pH 8.0 at 20° C.) and 1× dye (diluted from a 5000× stock; Life Technologies). Three replicates of each sample were pipetted in a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed onto a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak. Note that the super-neutralizing VHH Re5D06 melts already at 50° C., while the optimized Re5D06R13 version remained fully stable throughout 95° C., as did Re6H06 and Re6B06. Re5D06R13 and Re6B06 retained their hyper-thermostability even in the presence of disulphide-bond reducing DTT.

[0360] FIG. 7: Highly potent symmetry matching anti SARS-CoV-2 VHH antibodies.

[0361] (A) Scheme of a homotrimeric VHH fusion to match the C3 rotational symmetry of the Spike of SARS-CoV-2 and other viruses. (B) Comparison of neutralization potencies of ReB06 monomers and Re6B06-spacer-Collagen XVIII NC1 trimers. The experiment was performed analogously to FIG. 5; however, overlaid fluorescent channels in extended focus are shown. Note that the trimer neutralizes at a 30,000-fold lower VHH concentration than the monomer. (C) Comparison between a VHH-72 monomer and a VHH-72 trimer. Note that the trimer neutralizes at a 10000-fold lower VHH concentration than the corresponding monomer.

[0362] FIG. 8: Trimerization caused a strong avidity effect for the VHH-spike interaction

[0363] Hela cells were transfected as described in FIG. 2 to transiently express the SARS-Co-V2 spike protein. Following fixation, they were stained with Alexa488-labelled VHH Re6A11. In monomeric form, the staining was very weak even at 30 nM and with 2 fluorophores per VHH. In contrast, staining was strong with the trimerized version, even at a much lower concentration of 1 nM VHH and with only one fluorophore per VHH.

[0364] FIG. 9: Neutralization of SARS-CoV-2 B.1.351 by VHH antibodies.

[0365] (A) and (B): Neutralization of SARS-CoV-2 B.1.351 by the indicated VHH antibody constructs. The neutralization experiment was performed as described in FIG. 5, with the difference being that a mix of mutant-optimized anti-RBD/S1 VHH antibodies were used for immunofluorescence staining.

[0366]

TABLE-US-00005 VHH antibody sequences >Re5A08 GSQVQLVESGGGLVQAGGSLRLSCTASGHTFTANRMGWFRQAPGKEREFV AAINWGGDSTNYVDSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYFCAA RNHVTGEFDSWGQGTQVTVSSTS >Re5B06 GSQVQLVESGGGLVQPGGSLRLSCAASGSIRSIYATVWFRQAPGKEHEWV GSITSSNVTTYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTALYYCNVH FASEYSDYAQIQGQGTQVTVSSTS >Re5C01 GSQVQLVESGGGLVQAGGSLRLSCGVSGRTFSSYAMGWFRQAPGKEREFV ATISWSGGTTNYAHSVKGRFTISRDNAKNTVYLQMNSLKVEDTAVYYCYA VSSGSDYDGGMDYWGKGTQVTVSSTS >Re5C08 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNDYNMVWFRQAPGKEREFV AAIKWNGGNTSYADSVKGRFAVSRDNAKNTVYLQMNNLKHEDTAEYLCYT VGPEGDYWGQGTQVTVSSTS >Re5D06 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAIGWFRQAPGKEREGV SRIRSSDGSTNYADSVKGRFTMSRDNAKNTVYLQMNSLKPEDTAVYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5E03 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SCISNSDGSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAG GPQTYYSGSYYYTCAEGAMDYWGKGTLVTVSSTS >Re5E11 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SCISSSDGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT APLTYYSGSWYLTCNSDAMDYWGKGTLVTVSSTS >Re5F10 GSQVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEWV ATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCRRGQGTQVTVSSTS >Re5F11 GSQVQLVESGGALVQPGGSLRLSCATSGSISSYRMGWYRQGPGKQRELVA FITIGGITDYIDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNADP PLFNWGQGTQVTVSSTS >Re5G05 GSQVQLVESGGGLVQAGGSLRLSCAASGFTATSYAMGWYRQAPGKECEWV ATITSTGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCDWGQGTQVTVSSTS >Re6A11 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREFV AAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAA SDYGLPREDFLYDYWGQGTLVTVSSTS >Re6B02 GSQVQLVESGGALVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGV SRIRSSDGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re6B06 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTS >Re6B07 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SYIRSSDGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DEAYYSELGWESPWGWSYWGQGTRVTVSSTS >Re6D06 GSQVQLVESGGGLVQAGASLRLSCAASGRMFGVYRMGWFRQAPGKEREFV AGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RDPTTYEYDYWGQGTQVTVSSTS >Re6D08 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFHQAPGKEREFV ATINWSGDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA VVDPSPTYYSGKYYPPRVEYWGKGTQVTVSSTS >Re6D09 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNNYNMVWFRQAPGKEREFV AAINWNGGSTSYAASVKGRFAVSIDNAKNTLYLQMNNLKHEDTAEYLCYT VGPEGDYWGQGTQVTVSSTS >Re6E11 GSQVQLVESGGGLVQPGGSLRLSCAASGLTLDYYAIGWFRQAPGKEREGV SCISSRDGSTMYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA TPTTYYSGSYYYTCSPEGYDYWGQGTQVTVSSTS >Re6F06 GSQVQLVESGGGLVQAGGSLRLSCAASGFTFSNYAMGWYRQAPGKECEFV AVITITGSNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCESQGQGTRVTVSSTS >Re6G03 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSTYRMAWFRLAPGKEREFV AGINWSDGTTSYKDSVKGRFTISRDNAKNTVYLQMDSLKPEDTAVYYCNA HLSTGQEGPGEYFGMDYWGKGTQVTVSSTS >Re6H06 GSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGV SCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAV VPQTYYGGKYYSQCTANGMDYWGKGTLVTVSSTS >Re6H10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWYRQAPGKECEFV AVITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCRGGGQGTLVTVSSTS >Re7A01 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFV ATISFSGSTSYAGHVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCHAV TRASDQDGGMDYWGQGTQVTVSSTS >Re7B01 GSQVQLVESGGGLVQPGGSLRLSCGASGFTLDYYAIGWFRQAPGKEREGV SRIRSNDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re7D05 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFV ATISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNA VTHHSDQDGGMDYWGKGTLVTVSSTS >Re7E02 GSQVQLVESGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SYIRSSDGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DEAYYSELGWESPWGWSYWGQGTQVTVSSTS >Re7H02 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAENTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTS >Re8A03 GSQVQLVESGGGLVQPGGSLRLSCAASGRITGFNGMGWYRQTPGKQRELV ASITNGGITKYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYLCYFW RPEFPNLYWGQGTQVTVSSTS >Re8A06 GSQVQLVESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKERELV AAMGSSGWINYADSVKGRLTISRDNAKNTLYLQMNSLKPEDTAVYYCRGT GGVGPTSADYWGQGTQVTVSSTS >Re8C06 GSQVQLVESGGGLVQAGGSLRLSCAASGRTDTIYNMGWFRQAPGKEREFV AAISWSDGKTTFADSVKGRFTISRDNAKNTVYLQMNSLKPEDTANYYCAA KAFLVAGRSLEEYDYSGQGTQVTVSSTS >Re8E12 GSQVQLVESGGGSVQPGGSLRLSCKVSGFTSDVDLRNYLVSWNRQAPGKE RELVAAITPTVISGGNTNYADSVKGRFTISRDYSKSTVYLQMNSLNPEDT AVYYCKVGVYWGQGTQVTVSSTS >Re8F03 GSQVQLVESGGGLVQPGGSLTLSCKVSGLTSYVDLRNYLVSWYRQGPGKE RELVAAITPTAITGGSTNYADSVKGRFTISRDYSKSTVYLQMNSLNPEDT AVYSCKVGVYWGQGTQVTVSSTS >Re9B09 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT VPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTS >Re9B10 GSQVQLVESGGGLVQPGGSLRLSCAASGRMFGVYRMGWFRQAPGKEREFV AGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RDPTTYEYDYWGQGTQVTVSSTS >Re9C07 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFV AAITWNADTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA GGNHYYSRSYYSSLEYDHWGQGTQVTVSSTS >Re9C08 GSQVQLVESGGGLVQPGGSLRLSCAVSGNIFGITAWDWHRQAPGKQRELV AHITSRGDTYYLDSVKGRFAISRDHAKNTLSLQMNSLKPEDTAVYYCYLR TFGPPNDHWGQGTQVTVSSTS >Re9D02 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFV AAISWGGDTTYYADSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DRGLSYYYDRVTEYDYWGQGTQVTVSSTS >Re9G05 GSQVQLVESGGGLVQPGGSLRLSCAVSGNISSITAWDWHRQAPGKQRELV AHITSRGDTMYLDSVKGRFAISRDHAKNTLSLQMNSLKPEDTAVYYCYLR TFGPPYDYWGQGTQVTVSSTS >Re9G12 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFV AHISWSGDSTYYADSVKGRFTIFRDNAKNTAYLQMNSLKPEDTAVYYCAA DRGASYYYTWASEYNYWGQGTQVTVSSTS >Re9H01 GSQVQLVESGGGLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQRELV AFITSRGSTNYTDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAVYFCRGG YSDYDIYFGSWGQGTQVTVSSTS >Re10B02 GSQVQLVESGGGLVQPGGSLRLSCATSGSISSYRMGWYRQGPGKQRELVA FITIGGITDYIDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNADP PLFNWGQGTQVTVSSTS >Re10B10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRIRSSDGSTTYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re10F10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRIRNNDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re11C10 GSQVQLVESGGGSVEAGGSLRLSCAASGRTLDNYNAVWFRQAPGKEREFV AAINWNGSNTSYGNSVKGRFAVSRDNAKNTVYLQMNNLKHEDTAEYLCYT VGPEGDYWGQGTQVTVSSTS >Re11E11 GSQVQLVESGGGSVEAGGSLRLSCAASGRTFNNYNIVWFRQAPGKEREFV AAINWNGGSTSYANSVKGRFAVSRDNAKNTVYLQMNNLKHEDTAEYLCYT VGPEGDYWGQGTQVTVSSTS >Re11F07 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRQAPGKEREFV ATISWSGGSTSYARSVKGRFTISGDNAENTVYLQMNSLKPEDTAVYYCYA VSSGSDYDGGMDYWGKGTLVTVSSTS >Re11F11 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSNYHMSWYRQAPGKGRELV ADITSGGDYTHYADSVKGRFTVSRDNPKNTLYLQMNSLKPEDTAVYHCHV RIFGPGFPVDYRGQGTQVTVSSTS >Re11G09 GSQVQLVESGGGLVHTGGSLRLSCAASGSIFNIYRMAWYRQAPGKQREKV AIITTYGLTDYADSVKGRFTISRDNAKNTTYLQMNSLKPDDTAVYYCNTD PPDLGPGYWGQGTQVTVSSTS >Re11H04 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYTIAWFRQAPGKEREGV SCISGNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA DRGESYYPFRPSEYHYWGQGTQVTVSSTS >KG4B11 GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTS >Re5D06R11 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGV ARIRSNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTAVYYCAY GPLTKYGSEWYWPYEYDYWGQGTQVTVSSTS >Re5D06R13 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGV ARIRSNDGSVNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGSEWYWPYEYDYWGQGTQVTVSSTS >Re5D06R15 GSQVQLVESGGGLVQPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGV ARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5D06R23 GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGV ARIRNNDGSTDYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGSSWYWPYEYDYWGQGTQVTVSSTS >Re5D06R28 >GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREG VARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCA YGPLTKYGSSWHWPYEYDYWGQGTQVTVSSTS >Re5D06R28D GSQVQLVESGGGLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGV ARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGDEWHWPYEYDYWGQGTQVTVSSTS >Re5D06R15_3QE GSQVQLVESGGGLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGV ARIRNSDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGSSWYWPYEYDYWGEGTEVTVSSTS >Re5D06R28_3QE GSQVQLVESGGGLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGV ARWRNNDGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAY GPLTKYGSSWHWPYEYDYWGEGTEVTVSSTS >Re9F06 GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREFV AAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAA SDYGLPREDFLYDYWGQGTQVTVSSTS >Re9H03 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT VPGTYYSGNWYYTWHPKAVDYWGKGTLVTVSSTS >Re21B09 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKEREGV SCIRSSDGSTYYADSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCAT DGTFNPPCDDLYSWYFPERQGTQVTVSSTS >Re21D01 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEWV ATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCRRGQGTQVTVSSTS >Re21H01 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEWV ATITITGGSTNYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCNP DPGCRGGGQGTQVTVSSTS >Re22D04 GSQVQLVESGGGLVQTGGSLRLSCAASGRTFSDDAMGWFRQAPGKERDVV AALGWAGVSTYYADSVKGRFGISRDNAKNTVYLQMSSLKPEDTAVYYCAA APSVAHARLGEWAYWGKGTQVTVSSTS >Re22E05 GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT VPGTYYSGNWYYTWHPKAVDYWGKGTQVTVSSTS >Re25H10 GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGLEWV STISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATS TEPRTVVAGWGDYLGQGTQVTVSSTS >Re26D07 GSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGV SCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAV VPQTYYGGKYYSQCTANGMDYWGKGTQVTVSSTS >Re26E09 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEWV ATITITGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCRRGQGTRVTVSSTS >Re26E11 GSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEWV ATITITGGNTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNP DPGCRRGQGTQVTVSSTS >VHH-72 monomer GSGQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREF VATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCA AAGLGTVVSEWDYDYDYWGQGTQVTVSSGS

TABLE-US-00006 Sequences of heterodimeric VHH antibodies >Re9F06-SpacerA-Re9B09|pDG03599 (SEQ. ID NO: 292) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREFV AAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAA SDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGEGGEGSQVQLVESGGGL VQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTDYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWYYT WHPEAVDYWGKGTQVTVSSTS >Re9F06-SpacerA-Re5D06R28D|pDG03560 (SEQ. ID NO: 293) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREFV AAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAA SDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGEGGEGSQVQLVESGGGL VQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTNYA DSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGDEWHWP YEYDYWGQGTQVTVSSTS >Re22D04-SpacerA-Re5D06R28D|pD0G3661 (SEQ. ID NO: 294) GSQVQLVESGGGLVQTGGSLRLSCAASGRTFSDDAMGWFRQAPGKERDVV AALGWAGVSTYYADSVKGRFGISRDNAKNTVYLQMSSLKPEDTAVYYCAA APSVAHARLGEWAYWGKGTQVTVSSTSGEGEGGEGGEGSQVQLVESGGGL VQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTNYA DSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGDEWHWP YEYDYWGQGTQVTVSSTS >Re25H10-SpacerA-Re9B09|pDG03697 (SEQ. ID NO: 295) GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGLEWV STISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATS TEPRTVVAGWGDYLGQGTQVTVSSTSGEGEGGEGGEGSQVQLVESGGGLV QPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTDYAD SVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWYYTW HPEAVDYWGKGTQVTVSSTS >Re25H10-SpacerA-Re5D06R28D|pDG03798 (SEQ. ID NO: 296) GSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMSWARQAPGKGLEWV STISEDGSTYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCATS TEPRTVVAGWGDYLGQGTQVTVSSTSGEGEGGEGGEGSQVQLVESGGGLV QPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTNYAD SVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGDEWHWPY EYDYWGQGTQVTVSSTS >Pp086|Re9F06-SpacerB-Re5D06R28D|pDG03637 (SEQ. ID NO: 297) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVQPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTN YADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGDEWH WPYEYDYWGQGTQVTVSS >Pp087|Re9F06-SpacerC-Re6H06|pDG03625 (SEQ. ID NO: 298) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKER EGVSCTSSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY CAVVPQTYYGGKYYSQCTANGMDYWGKGTLVTVSS >Pp088|Re9F06-SpacerC-Re9B09|pDG03626 (SEQ. ID NO: 299) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGG SGEGSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY CATVPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSS >Pp089|Re9F06-SpacerB-Re6H06|pDG03627 (SEQ. ID NO: 300) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGVSCTSSSDGSTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAVVPQTYYGGKYY SQCTANGMDYWGKGTLVTVSS >Pp090|Re9F06-SpacerB-Re9B09|pDG03628 (SEQ. ID NO: 301) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTD YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWY YTWHPEAVDYWGKGTQVTVSS >Pp091|Re9F06-SpacerB-Re5D06R15_3QE|pDG03629 (SEQ. ID NO: 302) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGVARIRNSDGSTN YADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGSSWY WPYEYDYWGEGTEVTVSS >Pp092|Re9F06-SpacerB-Re5D06R28_3QE|pDG03630 (SEQ. ID NO: 303) EGSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREF VAAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAA ASDYGLPREDFLYDYWGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGG GLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTN YADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGSSWH WPYEYDYWGEGTEVTVSS >Pp093|Re21D01-SpacerB-Re5D06R15_3QE|pDG03663 (SEQ. ID NO: 304) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGGGLVEPGGSL RLSCAASGITLDYYAMGWFREAPGKEREGVARIRNSDGSTNYADSVKGRF TMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGSSWYWPYEYDYWG EGTEVTVSS >Pp094|Re21D01-SpacerB-Re5D06R28_3QE|pDG03664 (SEQ. ID NO: 305) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGGGLVEPGGSL RLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNNDGSTNYADSVKGRF TMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKYGSSWHWPYEYDYWG EGTEVTVSS >Pp095|Re21D01-SpacerC-Re5D06R15_3QE|pDG03665 (SEQ. ID NO: 306) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGGSGEGSQVQL VESGGGLVEPGGSLRLSCAASGITLDYYAMGWFREAPGKEREGVARIRNS DGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKY GSSWYWPYEYDYWGEGTEVTVSS >Pp096|Re21D01-SpacerC-Re5D06R28_3QE|pDG03666 (SEQ. ID NO: 307) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGGSGEGSQVQL VESGGGLVEPGGSLRLSCAISGSTLDYYAMGWFREAPGKEREGVARWRNN DGSTNYADSVKGRFTMSRDNAKDTYYLQMNSLEPEDTADYYCAYGPLTKY GSSWHWPYEYDYWGEGTEVTVSS >Pp097|Re21D01-SpacerB-Re6H06|pDG03667 (SEQ. ID NO: 308) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGGGLVQPGGSL RLSCAASGVTLDYYAIGWFRQAPGKEREGVSCTSSSDGSTYYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTGVYYCAVVPQTYYGGKYYSQCTANGMD YWGKGTLVTVSS >Pp098|Re21D01-SpacerB-Re9B09|pDG03668 (SEQ. ID NO: 309) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGEGSQVQLVESGGGLVQPGGSL RLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTDYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWYYTWHPEAVD YWGKGTQVTVSS >Pp099|Re21D01-SpacerC-Re6H06|pDG03669 (SEQ. ID NO: 310) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGGSGEGSQVQL VESGGGLVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGVSCTSSS DGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAVVPQTYY GGKYYSQCTANGMDYWGKGTLVTVSS >Pp100|Re21D01-SpacerC-Re9B09|pDG03670 (SEQ. ID NO: 311) EGSQVQLVESGGALVQPGGSLRLSCVASGFTFSSFAMGWYRQAPGKECEW VATITITGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCN PDPGCRRGQGTQVTVSSTSGEGEGGGEGGSGEGGSEGGEGGSGEGSQVQL VESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSS DGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYY SGNWYYTWHPEAVDYWGKGTQVTVSS

TABLE-US-00007 Sequences of subunits for trimeric VHH-spacer Collagen XVIII NC1 or collagen XV NC1 fusions expressed in E. coli >Re6B06 ColXVIII trimer (SEQ. ID NO: 211) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFSSAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADASDGSEGAS SGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEART PLPR >Re7H02 ColXVIII trimer (SEQ. ID NO: 212) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAENTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADASDGSEGAS SGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEART PLPR >KG4B11 ColXVIII trimer (SEQ. ID NO: 213) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADASDGSEGAS SGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEART PLPR >Re6D06 ColXVIII trimer (SEQ. ID NO: 214) GSQVQLVESGGGLVQAGASLRLSCAASGRMFGVYRMGWFRQAPGKEREFV AGISTSVGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA RDPTTYEYDYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADAS DGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRK VQLEARTPLPR >Re6A11/Re9F06 ColXVIII trimer (SEQ. ID NO: 215) GSQVQLVESGGGLVQAGGSLRLSCAASGRTFSNDALGWFRQAPRKEREFV AAINWNSGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCAAA SDYGLPREDFLYDYWGQGTLVTVSSTSEGSEGPESSDGSDSTDPGEQGEG ADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQN GFRKVQLEARTPLPR >Re5A08 ColXVIII trimer (SEQ. ID NO: 216) GSQVQLVESGGGLVQAGGSLRLSCTASGHTFTANRMGWFRQAPGKEREFV AAINWGGDSTNYVDSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYFCAA RNHVTGEFDSWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADAS DGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRK VQLEARTPLPR >VHH-72 ColXVIII trimer (SEQ. ID NO: 218) GSGQVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREF VATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCA AAGLGTVVSEWDYDYDYWGQGTQVTVSSGSGSEGPESSDGSDSTDPGEQG EGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRV QNGFRKVQLEARTPLPR >ColXV-Re9B09 ColXV trimer (SEQ. ID NO: 315) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQL GELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQLVESGG GLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTD YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWY YTWHPEAVDYWGKGTQVTVSSTS >Re9B09-ColXVIII trimer (SEQ. ID NO: 316) GSQVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGV SRISSSDGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAT VPGTYYSGNWYYTWHPEAVDYWGKGTQVTVSSTSEGSEGPESSDGSDSTD PGEQGEGADASDGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEE LYVRVQNGFRKVQLEARTPLPR >ColXV-Re9H01 trimer (SEQ. ID NO: 317) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQL GELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQLVESGG GLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQRELVAFITSRGSTNY TDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAVYFCRGGYSDYDIYFGSW GQGTQVTVSST >Re9H01-ColXVIII trimer (SEQ. ID NO: 318) GSQVQLVESGGGLVQAGDSLRLSCAASGNIFSINAMGWYRQAPGKQRELV AFITSRGSTNYTDSVKGRFTISRDTAKDTVYLQMNSLKPEDTAVYFCRGG YSDYDIYFGSWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADAS DGSEGASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRK VQLEARTPLPR >ColXV-Re7H02 trimer (SEQ. ID NO: 319) GSEGNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQL GELIPIPAGSEGPESSDGSDSTDPGEQGEGADASDGSEGGSQVQLVESGG GLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFVASVSWSGDSTN YADSVKGRFTISRDNAENTGYLQMNSLKPEDTAVYYCKRGPYWGQGTQVT VSSTS >KG4B11-ColXV trimer (SEQ. ID NO: 320) GSQVQLVESGGGLVQAGGSLRLSCAASGRAFESAPMSWFRQAPGKEREFV ASVSWSGDSTNYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKR GPYWGQGTQVTVSSTSEGSEGPESSDGSDSTDPGEQGEGADASDGSEGNL VTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPI PAD

TABLE-US-00008   Ost1 signal peptides >S. cerevisiae Ost1 signal peptide (SEQ. ID NO: 219) MRQVWFSWIVGLFLCFFNVSSAA >P. pastoris Ost1 signal peptide (SEQ. ID NO: 220) MKFISILFLLIGSVFG >S. pombe Ost1 signal peptide (SEQ. ID NO: 221) MLVLKLLLWSIISGLSLAE >C. albicans Ost1 signal peptide (SEQ. ID NO: 222) MWKFFITLGVIFSICSA

TABLE-US-00009 Propeptide sequence >S. cerevisiae propeptide alpha factor (SEQ. ID NO: 223) APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTT IASIAAKEEGVSLEKR

Examples

Example 1—Periplasmic Expression and Purification of VHH Antibody Monomers

[0367] VHH antibody Re6H06 was expressed with an N-terminal pelB signal sequence and a C-terminal His10 tag from a Kan-ColE1 plasmid harboring a T5/lac promoter in E. coli NEBExpress (New England Biolabs). A 125-ml pre-culture in Terrific Broth (TB) containing 50 μg/ml Kanamycin was grown overnight at 28° C. to early stationary phase. The culture was then diluted with fresh medium (500 ml, pre-warmed to 37° C.). After 30 minutes of growth at 37° C., protein expression was induced with 0.05 mM IPTG and growth was continued for 2 hours at 37° C., whereby the culture reached a final OD.sub.600 of ˜8. Bacteria were harvested by centrifugation and lysed by osmotic shock lysis: cell pellets were resuspended in 14 ml 130 mM Tris/HCl pH 8.0, 10 mM EDTA, and sucrose was immediately added to 20% (w/v). After gentle mixing at 23° C. for 30 min, four volumes of ice-cold water were added and mixing was continued at 4° C. for 30 min. 20 mM Tris/HCl pH 7.5, 50 mM NaCl and 20 mM imidazole were added to the cell suspension. Periplasmic extract was then recovered as the supernatant of two consecutive centrifugation steps at 4° C.: a low-speed spin at 4000×g (20 min, F13 rotor, Thermo Fisher Scientific) and a high-speed spin at 38000 rpm (˜1 hour, T647.5 rotor). The VHH antibody was purified at 4° C. via Ni.sup.2+ EDTA-amide chelate affinity chromatography (1 ml matrix). Beads were washed with ten column volumes of 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.2% (w/v) Triton X-100 and ten column volumes of buffer lacking detergent. After elution with 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 500 mM imidazole, the buffer was exchanged to 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 250 mM sucrose via a PD 10 desalting column (GE Healthcare). Aliquots were frozen in liquid nitrogen and stored at −80° C. This expression/purification strategy was used for all VHH antibodies containing four cysteines and thus two disulfide bonds (Re5E03, Re5E11, Re5G05, Re6E11, Re6F06, Re6G03, Re6H06, Re6H10) as well as for Re5F11.

Example 2—Cytoplasmic Expression and Purification of VHH Monomers

[0368] All other VHH antibodies comprising just two cysteines and thus a single disulfide bond were produced as His14-ScSUMO fusions by cytoplasmic expression in E. coli NEBExpress Shuffle, which allows forming disulfide bonds in the bacterial cytoplasm. In brief, 125 ml pre-cultures were grown overnight at 35° C. in TB+50 μg/ml kanamycin in 5 liter flasks to early stationary phase. They were then diluted with 250 ml fresh medium, shifted to 21° C. and induced for 5 hours with 0.08 mM IPTG. 5 mM EDTA was added, bacteria were pelleted, resuspended in 50 mM Tris/HCl pH 7.5, 20 mM imidazole/HCl pH 7.5, 300 mM NaCl, frozen in liquid nitrogen and lysed by thawing plus sonication. Insoluble material was removed by ultracentrifugation at 38000 rpm (˜1 hour, T647.5 rotor).

[0369] The supernatant was applied to a 1 ml Ni.sup.2+ EDTA-amide chelate column. The matrix was sequentially washed in resuspension buffer, resuspension buffer+0.2% TritonX100, resuspension buffer+700 mM NaCl, low salt buffer (resuspension buffer minus NaCl), and protease buffer (resuspension buffer with an imidazole concentration lowered to 10 mM and supplemented with 250 mM sucrose). The VHH antibody was finally eluted by cleaving the His14-SUMO-tag using 100 nM S. cerevisiae Ulp1 for 2 hours at room temperature. The eluted VHH antibodies were frozen in liquid nitrogen and stored an −80° C. until further use.

Example 3—Cytoplasmic Expression and Purification of VHH Trimers

[0370] The VHH trimers listed in Table 4 were expressed and purified as described for the VHH monomers, the only difference being that the VHH sequence was C-terminally extended by a 31 residues long spacer (in single letter code: EGSEGPESSDGSDSTDPGEQGEGADASDGSE) (SEQ. ID NO: 209) and by the 57 residues long human collagen XVIII NC trimerization domain (in single letter code: GASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEARTP LPR) (SEQ. ID NO: 210).

Example 4—Spike Protein-Staining in Transfected Cells

[0371] To prepare fluorophore-labelled probes, monomeric VHH antibodies were expressed with two additional cysteine residues, one at the N- and one at the C-terminus. These were then used for labelling with fluorophore-maleimides as described by Pleiner et al., 2015. The trimerized VHH antibody Re6A11 was labelled through a single N-terminal cysteine. Labelings were essentially quantitative as judged by ratiometric UV-VIS spectroscopy and electrophoretic size shifts.

[0372] HeLa cells were cultivated in DMEM+ 5% FCS, seeded in 10-well slides, and transiently transfected with a plasmid allowing expression of the SARS-CoV-2 spike protein from a CMV promoter and using the PolyJet transfection reagent according to the manufacturer's instruction (SignaGen). 2 days later, cells were fixed for 5 minutes with 4% paraformaldehyde (PFA) in PBS, washed in PBS, permeabilized with 0.5% saponin, blocked with 5% BSA in PBS, and incubated with gentle shaking for 60 min with fluorophore-labelled VHH antibodies (in PBS+ 1% BSA) at concentrations given in the figures. Following washes with PBS and counterstaining with DAPI, images were taken by confocal laser scanning microscopy.

Example 5—Spike Protein Detection in SARS-CoV-2 Infected Cells

[0373] Stainings were performed with Vero E6 cells infected for 2 days as described above. Fixation was with 4% paraformaldehyde (PFA) for 2 hours. This long period of fixation was due to safety reasons to make sure that no infectious material remained. As a consequence, only highly fixation-resistant epitopes remain visible—explaining the difference between the staining of transfected and virus-infected cells in Table 1. The fluorescence staining themselves were performed as described above. FIG. 3 shows epifluorescence images. The evaluations of Table 1 are based on confocal laser scans.

Example 6—Virus Infection and Neutralization Assays

[0374] Virus stocks were prepared as supernatants from Vero E6 cells infected with SARS-CoV-2. The virus stocks contained between 10.sup.11 and 10.sup.12 copies of the virus genome per ml, as determined by reverse transcription and quantitative PCR (qRT-PCR) according to a standard protocol (Corman et al., 2020). 2*10.sup.6 virus copies were diluted in 100 μl of cell culture media, DMEM/2% FBS, in the presence or absence of varying concentrations of the VHH antibodies under study. After 60 min incubation at 37° C., this was added to a monolayer of Vero E6 cells, i.e. 5000 cells in a well of a standard 96 well cell culture plate. After 48 or 72 hours of incubation, the supernatants of the cells were harvested. To quantify the amount of newly produced virus particles, the RNA from this material was isolated by the Trizol method, followed by qRT-PCR. Alternatively, cells were fixed as in Example 5, stained with a cocktail Atto488 labelled anti-RBD VHHs (10-15 nM each) and with 15 nM Atto565-labelled VHH (Re8H11) that recognizes an S1 SARS-CoV-2 epitope outside the RBD. Imaging was by confocal laser scanning or spinning disc fluorescence microscopy. Neutralization was considered as successful if no infected cells were detectable within the well.

Example 7—Producing Trimeric VHH Antibodies by Secretion from Pichia pastoris

[0375] The sequences of VHH trimers were cloned into a vector that was derived from pPICZα (Invitrogen/ThermoFisher Scientific, Cat. No. V19520) by replacing the signal peptide of the mating factor α precursor by the signal peptide of ScOst1p (Barrero et al., 2018) and replacing the zeocin resistance cassette by a coding sequence for the aminoglycoside 3′ phosphotransferase (UniProt ID: KKA1_ECOLX), conferring resistance to kanamycin and G418. The plasmids encoding VHH antibody trimers were transformed into wild-type P. pastoris cells using the protocol described by (Wu and Letchworth, 2004) with the following modifications: 2 μg of linearized vector DNA were used per transformation. After electroporation by pulse at 1.5 kV in a 2 mm electroporation cuvette, cells were allowed to recover in YPDS medium for 1 hour at 30° C. The selection was done directly for resistance to 1 mg/ml of G418 on YPDS plates for 60-80 hours.

[0376] Resulting clones were screened for expression of a desired protein by inoculating a single colony into BMMY medium in a well of a deep-well plate and incubating at 28° C. shaking at 500 rpm for 48 hours and analysing the resulting culture medium by SDS-PAGE. The best clones were used for medium scale expression by growing a culture of the desired strain in BMGY at 28° C. shaking at 120 rpm to mid-log phase, harvesting the cells and resuspending them in BMMY to induce expression under control of AOX1 promoter and incubating a culture at 28° C. shaking at 120 rpm. Additional methanol was supplied 24 hours post-induction to replenish consumed/evaporated methanol. After 48 hours post-induction, cells were removed from the culture by centrifugation at 3000 g for 10 minutes, then at 10000 g for 10 minutes. The final supernatant was filtered through a 0.2 μm filter before proceeding to VHH purification.

Example 8—Highly Potent Neutralizers of the South African B.1.351 SARS-CoV-2 Strain (Beta Variant)

[0377] In a further set of experiments, the inventors assessed the neutralization potency of their mutation-tolerant VHH antibodies, using the South African B.1.351 variant as a representative of the recently emerged mutant strains. As their microscopic assay relied on VHH antibody-based detection of newly made viral proteins, they had to ensure that the mutant spike also yields an unambiguous signal. This was achieved with a mix of VHH-antibodies against epitopes outside the RBD (anti-S1ΔRBD), against the non-mutated epitope 2 (Re7E02 or Re9C07), and by the mutation-tolerant main epitope-binder Re9H03.

[0378] By contrast, Re6D06, which fails in mutant RBD binding, also failed to stain mutant spikes of infected cells suggesting that combinations of mutation-sensitive and -tolerant VHH-antibodies can diagnose virus variants by simple staining procedures.

[0379] The inventors then compared two strategies for the actual neutralization of mutant B.1.351 (FIG. 9). They first tested the most promising VHH monomers and observed potent neutralization by Re5F10 (at 1.7 nM), Re6H06 (at 170 pM), Re9B09 (at 1.7 nM), and by the mutant-preferring Re9B09 class member Re9H03 (at 50-170 pM; FIG. 9A, Table 5). Even more potent B.1.351 neutralization was found for the heterodimers Re9F06-R28 (50 pM), Re9F06-Re9B09 (50 pM), and Re9F06-Re6H06 (17 pM; FIG. 9B), as well as for the analogous Re21D01-heterodimers with R28, Re9B09 and Re6H06, whereby VHH-antibody-connecting linkers of 14, 15, or 29 amino acids gave rather similar results (for additional data, see Table 5 for VHH monomers and Table 6 for VHH heterodimers).

TABLE-US-00010 TABLE 5 Lowest concentration VHH monomer SEQ ID neutralizing B.1.351 (nM) Re5F10 29 1.7-5 Re21D01 (Re5F10 class) 260 5 Re26E09 (Re5F10 class) 284 1.7-5 Re26E11 (Re5F10 class) 288 1.7-5 Re21H01 5 Re6H06 81 0.170 Re26D07 (Re6H06 class) 280 0.500 Re9B09 129   1.7 Re9H03 (Re9B09 class) 252   0.050-0.170

[0380] Neutralization of the South African B.1.351 SARS-CoV-2 variant by mutation-tolerant VHH antibodies.

TABLE-US-00011 TABLE 6 Lowest neutralizing concentration (pM) Beta Alpha South Wild UK African VHH1 Spacer VHH2 Expression type B.1.1.7 B.1.351 Re9F06 A Re9B09 E. coli 50-170 17-170 Re9F06 A Re5D06R28D E. coli 5-50  5-170 Re22D04 A Re5D06R28D E. coli 170 Re25H10 A Re9B09 E. coli 500  50-170 170 Re25H10 A Re5D06R28D E. coli 170  170 170 Re9F06 B Re5D06R28D Pichia  50 Re9F06 C Re6H06 Pichia 500   50 170 Re9F06 C Re9B09 Pichia 17-170 170 50-170 Re9F06 B Re6H06 Pichia 17-170  50 Re9F06 B Re9B09 Pichia 50-170 17-170 Re9F06 B Re5D06R15_3QE Pichia 5-50 50-170 Re9F06 B Re5D06R28_3QE Pichia 50-170 Re21D01 B Re5D06R15_3QE Pichia 50-170 Re21D01 B Re5D06R28_3QE Pichia 17-50  50-170 Re21D01 C Re5D06R15_3QE Pichia 50 170 Re21D01 C Re5D06R28_3QE Pichia 50  50 Re21D01 B Re6H06 Pichia 50 50-500 Re21D01 B Re9B09 Pichia 50 Re21D01 C Re6H06 Pichia 170  50-500 50-170 Re21D01 C Re9B09 Pichia 50-170 170

[0381] Neutralization of the South African B.1.351 SARS-CoV-2 variant by VHH-spacer-VHH tandem fusions. Spacer A: TSGEGEGGEGGEGS (SEQ. ID NO: 312); Spacer B: TSGEGEGGGEGGEGS (SEQ. ID NO: 313); Spacer C: TSGEGEGGGEGGSGEGGSEGGEGGSGEGS (SEQ. ID NO: 314).

Example 9: Binding of Mutation-Tolerant Monomeric VHHs and Heterodimeric VHHs to the RBD of the SARS-CoV-2Delta Variant

[0382] In further experiments, the inventors used Biolayer interferometry (BLI) to assess the binding strength of selected VHHs to the receptor-binding domain of the Delta variant of SARS-CoV-2, which carries the L452R and T478K mutations. For this, VHH constructs were labelled with Biotin-PEG.sub.11-Maleimide (#PEG1595; Iris Biotech) through N- and C-terminally introduced cysteines.

[0383] BLI experiments were performed using High Precision Streptavidin biosensors and an Octet RED96e instrument (ForteBio/Sartorius) at 25° C. with Phosphate-Buffered Saline (PBS) pH 7.4, 0.02% (w/v) Tween 20 and 0.1% (w/v) BSA as assay buffer.

[0384] The indicated VHHs were immobilized on sensors until a binding signal of 0.4 nm (for monomeric VHHs) or 0.75 nm (for heterodimeric VHHs) was reached. Subsequently, the biosensors were dipped into wells containing 3-fold dilutions (20, 6.66, and 2.22 nM) of the wild type RBD (Z03479, GenScript) or the B.1.617.2/Delta RBD (40592-V08H90, Sino Biologicals) for 450 s. RBD dissociation in assay buffer was followed for 900 s. Data were reference-subtracted and curves were fitted using a mass transport model (Octet Data Analysis HT 12.0 software). The dissociation constants (K.sub.Ds) are summarized in the Table shown below. They revealed very tight binding of the analyzed VHHs and heterodimeric VHHs.

TABLE-US-00012 TABLE 7 K.sub.D RBD K.sub.D RBD VHH monomer or VHH heterodimer SEQ ID Wild type Delta variant Re5F10 29 40 pM 40 pM Re6H06 81 ≤10 pM ≤10 pM Re9B09 129 ≤10 pM 100 pM Re5D06R15 224 ≤10 pM 30 pM Re5D06R28 232 ≤10 pM 80 pM Re9F06-spacerA-Re9B09 292 ≤10 pM ≤10 pM Re9F06-spacerA-Re5D06R28D 293 ≤10 pM ≤10 pM

Example 10: Neutralization of the SARS-CoV-2 Delta Variant by Mutation-Tolerant VHHs and VHH Tandems

[0385] The indicated VHH constructs were used in neutralization experiments of the Delta SARS-CoV-2 variant. Experiments were performed analogously to those described in FIGS. 5 and 9. Table 8 lists the lowest neutralizing concentrations as geometric means of three independent experiments.

TABLE-US-00013 TABLE 8 Lowest neutralizing SEQ concentration VHH monomer or VHH heterodimer ID Delta variant (nM) Re6H06 81 0.5 Re9B09 129 1.7 Re9F06-SpacerB-Re5D06R28D 297 0.05 Re9F06-SpacerB-Re5D06R15_3QE 302 0.09 Re9F06-SpacerB-Re5D06R28_3QE 303 0.09 Re21 D01-SpacerC-Re5D06R28_3QE 307 0.29 Re21D01-SpacerB-Re5D06R28_3QE 305 0.29 Re9F06-SpacerC-Re9B09 299 0.29 Re9F06-SpacerB-Re6H06 300 0.50 Re9F06-SpacerC-Re6H06 298 0.50 Re21D01-SpacerC-Re6H06 310 0.50

Example 11: The Trimerization Module of Collagen XVIII Allows for More Potent SARS-CoV-2 Neutralizing VHH Fusions than the Homologous NC1 Domain of Collagen XV

[0386] The indicated trimeric VHH fusions were expressed in E. coli, purified and tested in a SARS-CoV-2 neutralization assay as described in FIGS. 3 and 5.

[0387] The lowest neutralizing concentrations (referring to VHH moieties) are listed in Table 9. The collagen XVIII NC1 domain was used as a C-terminal fusion partner. The collagen XV NC1 domain was used an N-terminal fusion partner in Re9B09, Re9H01 und Re7H02 (Re6B06 class) fusions, and as a C-terminal fusion partner in the KG4B11 fusion.

[0388] The data shows that the C-terminal collagen XVIII fusion allows for greater potency than an collagen XV NC1 on either the N-terminus or the C-terminus.

TABLE-US-00014 TABLE 9 Collagen XV Collagen XVIII NC1 fusion NC1 fusion Lowest Lowest neutralizing neutralizing SEQ concentration SEQ concentration VHH trimer ID (pM) ID (pM) Re9B09 315 90 316 16 Re9H01 317 50 318 4 Re7H02 (Re6B06 class) 319 160 212 4 KG4B11 (Re6B06 class) 320 170 213 4

[0389] Further examples are detailed in the figures and figure legends.

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