Single-domain antibody to reduce the risk of rotavirus a infection

Abstract

A single-domain antibody (sdAb) which binds to head domain of VP6 derived from a human rotavirus A and has neutralization activity against different the rotavirus A types.

Description

[0001] P6791 PC00

[0002] 1

[0003] Single-domain antibody to reduce the risk of rotavirus A infection

[0004] Field of the invention

[0005] The present disclosure relates to a single-domain antibody (sdAb) which binds to head domain of VP6 derived from a human rotavirus A and has neutralization activity against different the rotavirus A types.

[0006] Background of the invention

[0007] As known in the art - a nanobody or single domain antibody (sdAb) refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.

[0008] Such single domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian host cell).

[0009] Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, single domain antibodies lack a light chain and so have just three complementary- determining region (CDR) loops (Gordon et al. (2023) A comparison of the binding sites of antibodies and single-domain antibodies. Front. Immunol. 14:1231623. doi: 10.3389 / fimmu.2023.1231623).

[0010] The Gordon et al. article reads on page 2, left column:

[0011] “SdAbs are approximately one tenth the mass of antibodies (~15 kDa). Given this smaller size, the structural diversity available to sdAbs is significantly reduced compared to Abs. However, sdAbs have been shown to achieve comparable binding specificities and affinities”.

[0012] Rotavirus (RV) is a non-enveloped, icosahedral virus of the Reoviridae family containing a genome of segments of double stranded RNA (dsRNA). It has been estimated that each year, rotavirus causes more than a 100 million episodes of gastroenteritis which results in more many deaths in children.

[0013] Rotaviruses are currently divided into seven serotypes (Rotavirus A-G). They exhibit broad genetic and antigenic diversity due to reassortment among rotavirus strains and the accumulation of point mutations in the surface protein genes. Group A rotaviruses are the major human pathogens and have been further categorized on the basis of the outer capsid proteins, VP4 (P-type) and VP7 (G-type), and the intermediate layer protein VP6.

[0014] The incidence and distribution of group A rotavirus genotypes varies between geographical areas during a rotavirus season, and from one season to the next.

[0015] (Aladin et al. (2012) In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments. PLoS ONE 7(3): e32949. doi: 10.1371 / journal. pone.0032949). P6791 PC00

[0016] 2

[0017] As known in the art - rotavirus A vaccines on the market may be said to only be able to protect infants and children from a relatively few rotavirus A genotypes, as they are generally eliciting antibodies targeting the capsid proteins VP7 and VP4 which are quite variable between different rotavirus A types.

[0018] W02009 / 016100A1 page 2, lines 4-5 reads: “VP6 is a highly conserved protein within all the Group A RV (>90% amino acid homology)” - i.e. intermediate layer protein VP6 is conserved across different Rotavirus A types.

[0019] W02009 / 016100A1 describes a monomeric single domain antibody (sdAb - “VHH”) termed 3B2 which binds to rotavirus A intermediate layer protein VP6 and has neutralizing activity against different rotavirus A strains / types - i.e. able to neutralize different rotavirus A types.

[0020] W02009 / 016100A1 page reads:

[0021] [p15, 110-11]: “The anti-VP6 VHH domains of the invention were capable of neutralising different RV strains in vitro."

[0022] [p17, 18-9]: “60% of the mice treated with monomeric VHH of the invention 3B2 were protected against RV-induced diarrhoea”

[0023] Aladin et al. (2012) describes a monomeric sdAbs (“VHH”) termed ARP1 and ARP3 which binds to rotavirus A intermediate layer protein VP6 and has neutralizing activity against different rotavirus A strains / types - see e.g. Abstract, Table 1 on page 4, and page 4, left column reading: “ARP1 and ARP3 recognised bands corresponding to those also recognised by the anti-VP6 monoclonal antibody”.

[0024] Summary of the invention

[0025] A problem to be solved by the present invention relates to the identification of a single domain antibody (sdAb) related construct / product that may be used to neutralize different rotavirus A types.

[0026] As discussed above - W02009 / 016100A1 describes sdAb termed 3B2 and Aladin et al. describes sdAbs termed ARP1 and ARP3.

[0027] Without being limited to theory - it is believed that these sdAbs may be seen as representing best known herein relevant prior art sdAbs - and based on data of Aladin et al. there is objectively no reason to believe that ARP3 is better than ARP1 .

[0028] Accordingly, in working Examples herein were 3B2 and ARP1 used as reference / benchmark sdAbs.

[0029] As discussed below - based on the Examples experimental data discussed herein - one may objectively say that a lead candidate M010_0004 sdAb of our invention is a better / improved sdAb over reference / benchmark ARP1 and 3B2 sdAbs.

[0030] Aladin et al. (2012) raised sdAb against full rotavirus strain as such (see Material and Methods - section P6791 PC00

[0031] 3

[0032] Antibody fragments) to identify sdAb termed “ARP1 ”; and

[0033] WQ2009 / 016100A1 raised sdAb against full length VP6 derived from a bovine RV Strain (see page 25, line 1) to identify sdAb termed “3B2”.

[0034] As discussed in further detail in Example 1 herein - the present inventors used a different strategy - i.e. raised sdAb against a head domain of VP6 (i.e. not full length VP6).

[0035] The used head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5.

[0036] The head domain of VP6 sequence may be seen as the structural most accessible part of VP6 in the RVA.

[0037] Accordingly, and without being limited by theory - an advantage of using the head domain of VP6 may be that the raised antibodies are more directed / focused to this accessible part of VP6 - which could explain the improvements of sdAb of the present invention as discussed herein.

[0038] Based om above discussed different strategy - the present inventors identified a lead candidate sdAb termed “M010_0004”.

[0039] As discussed in Example 3 and Figures 2+3 herein - sdAb M010_0004 of our invention has as good neutralization activity against different human RV types as prior art reference / benchmark ARP1 and 3B2 sdAbs - but for e.g. porcine RV, sdAb M010_0004 of our invention is significantly better / improved over reference / benchmark ARP1 and 3B2 sdAbs.

[0040] Further, as discussed in Example 4 and Figures 4 herein - sdAb M010_0004 of our invention has improved thermostability over reference / benchmark ARP1 and 3B2 sdAbs.

[0041] For the lead candidate sdAb termed “M010_0004” of our invention - the corresponding SEQ ID NO numbers are:

[0042] - M010_0004: CDR1 is SEQ ID NO: 1 , CDR2 is SEQ ID NO: 2 and CDR3 is SEQ ID NO: 3;

[0043] - M010_0004 full length is SEQ ID NO: 4.

[0044] Just as note, and as discussed above - single-domain antibodies (sdAbs) like “M010_0004” of our invention have three CDR loops - i.e. three CDR sequences.

[0045] Figures 5 herein shows a sequence alignment of M010_0004 of our invention and benchmark ARP1 and 3B2 sdAbs - where can be seen that e.g. the CDR sequences are different.

[0046] Example 5 herein provides a discussion of possible relatively minor changes in e.g. the M010_0004 CDR regions that one could make and probably / plausible maintain the herein relevant RV binding / neutralization positive characteristic of M010_0004 - the skilled person may use the new technical information herein to make relatively minor changes in e.g. the CDR regions, while maintaining herein relevant RV binding / neutralization positive characteristic. P6791 PC00

[0047] 4

[0048] Accordingly, a first aspect of the present invention relates to a single-domain antibody (sdAb) which binds to head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5 and has neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine), and wherein said single-domain antibody is comprising:

[0049] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and

[0050] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and

[0051] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered.

[0052] In working Example 1 herein is shown that lead candidate M010_0004 (SEQ ID NO: 4) binds to head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5.

[0053] It is routine work for the skilled person to measure binding affinity of an antibody to a target / antigen. In relation to the first aspect, the “binds to head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5” shall be done / assayed in accordance with Example 1 herein - and the binding affinity of the sdAb shall preferably at least be as good as for lead candidate M010_0004 (SEQ ID NO: 4).

[0054] In Example 3 is shown that M010_0004 has neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine) - which are all public available / known rotavirus A types (see e.g. table of Example 3 below).

[0055] It is routine work for the skilled person to measure neutralization activity against rotavirus A type of interest for an antibody of interest.

[0056] In relation to the first aspect, the “has neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine)” shall be measured in accordance with Example 3 herein - and the neutralization activity of the sdAb shall preferably at least be as good as for lead candidate M010_0004 (SEQ ID NO: 4). P6791 PC00

[0057] 5

[0058] A second aspect of the invention relates to a single-domain antibody (sdAb) of the first aspect and / or embodiment thereof for use in the reduction of risk, prevention or treatment of rotavirus A infection in a subject.

[0059] The term “treatment” encompasses both curative and ameliorative treatment. By ameliorative treatment is meant a treatment that results in the improvement of one or more symptoms of a herein relevant infection in a subject.

[0060] Embodiments of the present invention are described below, by way of examples only.

[0061] Drawing description

[0062] Figure 1 : Shows results of rotavirus A neutralization activity for the in the Example 1 identified sdAbs. See Example 2 herein for further details.

[0063] Figures 2+3: Shows that sdAb M010_0004 of our invention has as good neutralization activity against different human RV types as prior art reference / benchmark ARP1 and 3B2 sdAbs - but for e.g. porcine RV, sdAb M010_0004 of our invention is significantly better / improved over reference / benchmark ARP1 and 3B2 sdAbs. See Example 3 herein for further details.

[0064] Figure 4: Shows that sdAb M010_0004 of our invention has improved thermostability over reference / benchmark ARP1 and 3B2 sdAbs. See Example 4 herein for further details.

[0065] Figure 5: Figure 5 herein shows a seguence alignment of M010_0004 of our invention and benchmark ARP1 (SEQ ID NO: 6) and 3B2 (SEQ ID NO: 7) sdAbs - where can be seen that e.g. the CDR seguences are different. See Example 5 herein for further details.

[0066] Figure 6: Shows that also when expressed in Aspergillus - sdAb M010_0004 lead candidate of our invention had at least as good neutralization activity against human RV strain Wa as prior art reference / benchmark 3B2 sdAb. See Example 8 herein for further details.

[0067] Figure 7: Shows E. coll expressed M010_0004-dimer basically have the same neutralization activity of Aspergillus expressed M010_0004-dimer. The M010_0004-dimer had an improved neutralization activity as compared to the M010_0004 monomer. See Example 9 herein for further details.

[0068] Figure 8: Shows that M010_0004-dimer of our invention had an improved / stronger neutralization activity against human RV strain Wa as compared to benchmark 3B2-dimer. See Example 10 herein for further details.

[0069] Detailed description of the invention P6791 PC00

[0070] 6

[0071] First aspect - single-domain antibody (sdAb)

[0072] For the skilled person it is routine work to obtain a suitable single-domain antibody (sdAb) in relation to a suitable pathogen of interest - i.e. it is herein not considered necessary to discuss this in great details.

[0073] As discussed above, and as known in the art - a nanobody or single domain antibody (sdAb) refers to the smallest antigen binding fragment or single variable domain (“VHH”) derived from a naturally occurring heavy chain antibody.

[0074] Such single domain antibodies can be derived from antibodies raised in e.g. sharks or e.g. Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Single domain antibodies may also be synthetically produced, such as by recombinant expression in a suitable production host cell (e.g. a bacteria, a fungal or mammalian host cell).

[0075] As discussed above - single-domain antibodies (sdAbs) like “M010_0004” of our invention have three CDR loops - i.e. three CDR sequences.

[0076] The above discussed Gordon et al. article reads on page 2, left column:

[0077] “SdAbs are approximately one tenth the mass of antibodies (~15 kDa). Given this smaller size, the structural diversity available to sdAbs is significantly reduced compared to Abs. However, sdAbs have been shown to achieve comparable binding specificities and affinities”.

[0078] Preferably, the sdAb (as a monomer) has a size of from 10 to 20 kDa - more preferably from a size of from 11 to 18 kDa, such as from 12 to 17 kDa.

[0079] As known in the art - the average molecular weight of a single amino acid is about 110 Daltons (0.1 1 kDa).

[0080] Accordingly, in a preferred embodiment - the sdAb (as a monomer) has a size of from 90 to 182 amino acids - more preferably from a size of from 100 to 164 amino acids, such as from 109 to 155 amino acids.

[0081] In a preferred embodiment - in relation to the first aspect, the “binds to head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5” shall be done / assayed in accordance with Example 1 herein - and the binding affinity of the sdAb shall at least be as good as for lead candidate M010_0004 (SEQ ID NO: 4).

[0082] In a preferred embodiment - in relation to the first aspect, the “has neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine)” shall P6791 PC00

[0083] 7 be measured in accordance with Example 3 herein - and the neutralization activity of the sdAb shall at least be as good as for lead candidate M010_0004 (SEQ ID NO: 4).

[0084] Working Examples herein describes suitable methods for obtaining / producing examples of herein preferred / relevant sdAbs (such as e.g. “M010_0004”).

[0085] Based on the technical disclosure herein and the common general knowledge - it is routine work for the skilled person to obtain / produce a single-domain antibody (sdAb) of the first aspect and / or embodiment thereof of our invention.

[0086] Preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein said single-domain antibody is comprising:

[0087] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein 1 amino acid has been altered; and

[0088] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein 1 amino acid has been altered; and

[0089] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein 1 amino acid has been altered.

[0090] More preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein said single-domain antibody is comprising:

[0091] (i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and

[0092] (ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and

[0093] (iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3.

[0094] This embodiment is termed “embodiment CDR1-3 = SEQ ID NO: 1-3” herein.

[0095] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one preferred embodiment, the sequence variance is outside the CDRs.

[0096] Most preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4.

[0097] In one embodiment, the single-domain antibody of the present disclosure is a humanized version of the single-domain antibody of the first aspect and / or embodiment thereof.

[0098] It is routine work for the skilled person to a humanized version of an antibody of interest.

[0099] The present disclosure also relates to fusion proteins comprising at least one of the single-domain antibodies as described herein. Such fusion protein can be assembled by methods known to the person P6791 PC00

[0100] 8 skilled in the art (see e.g. WO2020 / 144164A1 (Bactolife)).

[0101] Preferably, the fusion protein is recombinantly designed by fusing gene sequences in vitro. In some embodiments, the fusion protein further comprises a linker connecting the sbAbs.

[0102] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is a fusion protein comprising a single-domain antibody as defined in the first aspect and / or embodiment thereof and one or more further single-domain antibodies as defined in the first aspect and / or embodiment thereof, and optionally one or more linkers, such as one or more GS linkers.

[0103] In a preferred embodiment the fusion protein comprises a linker connecting the sdAbs.

[0104] In one embodiment the linker is a GS linker, i.e. a linker which comprises or consists of glycine and serine residues. Such linkers are well known in the art (see e.g. WO2020 / 144164A1 (Bactolife)).

[0105] In one embodiment the linker is a GS linker of the structure (GxS)n, where x may be a number between 1 to 10, preferably 2 to 5, and n refers to a number of repeats of the GxS sequence, where n may be between 1 to 10, preferably 2 to 5.

[0106] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the fusion protein is a homodimer or a heterodimer - more preferably the fusion protein is a homodimer.

[0107] As discussed above - preferably, the sdAb (as a monomer) has a size of from 10 to 20 kDa.

[0108] As understood - if a sdAb (as a monomer) has a size of e.g. 15 kDa - then a homodimer thereof will have a size of around 30 kDa (depending on the size of a possible linker).

[0109] In a preferred embodiment, the fusion protein homodimer is comprising a single-domain antibody as defined in “embodiment CDR1-3 = SEQ ID NO: 1-3” discussed above and one more single-domain antibody as defined in “embodiment CDR1-3 = SEQ ID NO: 1-3” discussed above.

[0110] As discussed above - M010_0004 full length is shown in SEQ ID NO: 4 - which herein may be termed M010_0004 or M010_0004 monomer.

[0111] Preferably, the fusion protein homodimer is comprising a single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 4 and one more single-domain antibody comprising or consisting of the sequence as set forth in SEQ ID NO: 4 - wherein it is preferred that the fusion protein homodimer comprises a linker and the linker is a GS linker of the structure (GxS)n, where x may be a number between 1 to 10, preferably 2 to 5, and n refers to a number of repeats of the GxS P6791 PC00

[0112] 9 sequence, where n may be between 1 to 10, preferably 2 to 5.

[0113] As discussed in Examples below - M010_0004 homodimer (may herein be termed M010_0004-dimer) with a GS linker was made and the full length sequence is shown in SEQ ID NO: 9.

[0114] As discussed below - the result of Example 10 shows that M010_0004-dimer of our invention had an improved / stronger neutralization activity against human RV strain Wa as compared to benchmark 3B2- dimer.

[0115] In a preferred embodiment, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 9, or a sequence having at least 90% sequence identity thereto. In one embodiment, the sequence identity is at least 95%, such as at least 96%, 97%, 98% or 99%. In one preferred embodiment, the sequence variance is outside the CDRs.

[0116] Most preferably, the single-domain antibody of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 9.

[0117] The skilled person knows how to produce / obtain a herein relevant single-domain antibody of interest

[0118] - e.g. by recombinant expression in a suitable host cell.

[0119] It may be preferred that the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is a glycosylated single-domain antibody.

[0120] The skilled person knows how to produce / obtain a herein relevant glycosylated single-domain antibody

[0121] - e.g. by recombinant expression in a suitable eucaryotic production host cell (e.g. yeast, Aspergillus, etc.).

[0122] In one embodiment, the production host cell is a yeast, such as a yeast selected from the genus of Pichia (preferably Pichia pastoris), Hansenula or Saccharomyces.

[0123] As known in the art - Pichia may alternatively be named Komagataella and Pichia pastoris may alternatively be named Komagataella phaffii.

[0124] In one embodiment, the production host cell is a fungus selected from Aspergillus - such as e.g. Aspergillus oryzae or Aspergillus niger.

[0125] Preferably, the single-domain antibody (sdAb) of the first aspect and / or embodiment has an neutralization activity neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine) at least like the M010_0004 monomer sdAb of SEQ ID NO: 4 expressed in Pichia according to Example 1 herein, and wherein the neutralization activity is P6791 PC00

[0126] 10 measured according to Example 3.

[0127] Second aspect - use in the reduction of risk, prevention or treatment of rotavirus A infection in a subject.

[0128] As discussed above, a second aspect of the invention relates to a single-domain antibody (sdAb) of the first aspect and / or embodiment thereof for use in the reduction of risk, prevention or treatment of rotavirus A infection in a subject.

[0129] As discussed above, single-domain antibodies have been described in the art for use in the reduction of risk, prevention or treatment of rotavirus A infection in a subject (e.g. a human).

[0130] Based on the technical disclosure herein and the common general knowledge - it is routine work for the skilled person to identity a suitable preferred dose and / or preferred route of administration of a herein relevant sdAb to a subject (e.g. a human).

[0131] A subject may e.g. be an animal (a bovine or a pig) or a human - preferably, the subject is a human.

[0132] As discussed herein - for e.g. porcine RV, sdAb M010_0004 of our invention is significantly better / improved over reference / benchmark ARP1 and 3B2 sdAbs.

[0133] Accordingly, if subject is an animal - it is preferably a pig.

[0134] In one embodiment, the subject is a human child, such as a child of less than 10 years of age. In one embodiment the subject is a child of between 0 and 10 years of age (preferably between 0 and 7 years, more preferably 0 and 5 years), such as a child of between 1 and 7 years of age, such as a child of between 1 and 6 years of age, such as a child of between 1 and 5 years of age.

[0135] Preferably, the single-domain antibody for use of the first aspect and / or embodiment thereof is a sdAb, wherein the single-domain antibody is present in a dietary composition or a pharmaceutical composition.

[0136] According to the art - for pharmaceutical compositions, all components of the composition should be pharmaceutically acceptable. By “pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the sbAb. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)).

[0137] In one embodiment, the dietary composition comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fibers, and / or nutrients, such as dietary minerals.

[0138] In one embodiment, the dietary composition is an oral rehydration solution or yoghurt. P6791 PC00

[0139] 11

[0140] EXAMPLES

[0141] EXAMPLE 1 : Screening of single-domain antibodies

[0142] Material and methods

[0143] Single-domain antibodies were raised against a head domain of VP6 (i.e. not full length VP6) derived from a human RV.

[0144] The antigen for immunization, panning and screening is the head domain of VP6 shown in SEQ ID NO: 5. It was cloned into E. coli expression vector (pE_0090, C-terminal His Tag and pE_0093, C- terminal Avi-His Tag) and expressed in E. coli BL21 (DE3) with IPTG induction. Both His Tag and Avi- His Tag VP6 head domain proteins were purified from cell lysates using Ni-NTA resin (Thermofisher) followed by size exclusion chromatography HiLoad 16 / 600 Superdex 75 pg (Cytiva). The VP6 head domain Avi-His Tag protein was biotinylated in vitro using a BirA Biotin-Protein Ligase Kit (Avidity LLC) for phage display selection.

[0145] Two llamas were immunized with VP6 head domain His Tag. The amounts of antigen used per injection per animal were 100 pg with adjuvant Gerbu adjuvant P. The animals were subcutaneously injected 6 times, on weekly basis. Four and 8 days after last injection (4 d.p.i. & 8 d.p.i., respectively), each time about 100 ml anticoagulated blood was collected from each animal for the preparation of peripheral blood lymphocytes (PBLs). Individual VHH (Nanobody) libraries were constructed from each animal’s peripheral blood lymphocytes (PBLs). To this end, total RNA was prepared from PBLs (4 d.p.i. & 8 d.p.i.), pooled (1 :1 weight ratio) per animal, and used for cDNA synthesis. About 50 pg of each pool (4 d.p.i. + 8 d.p.i.) of total RNA from peripheral blood lymphocytes (PBLs) from each animal was used as template for first strand cDNA synthesis with an oligodT primer. The cDNA of single-domain antibodies was cloned into phagemid vector pMECS-GG at Sapl site. In pMECS-GG, the Nanobody sequence is followed by a linker, HA and His6 tags (AAAYPYDVPDYGSHHHHHH - SEQ ID NO: 8). Electro- competent E. coli TG1 cells were transformed with the recombinant pMECS-GG to obtain the singledomain antibody libraries.

[0146] The single-domain antibody libraries prepared from the two llamas were mixed and went through 4 rounds of panning. The panning experiments were performed with antigens in solution, at a final VP6 Avi-His Tag concentration of 50 nM for all panning rounds. In order to avoid enriching for phages displaying Nanobodies specific for Avi tag, an irrelevant protein fused to Avi tag was used as competitor at 10-fold molar excess, as compared to target antigen concentration. After each round of panning, TG1 cells were infected with part of the phage elute from each panning round and stored as glycerol stocks for DELFIA screening. In total, 190 clones (95 clones from panning round 2 and 95 clones from round 3) were randomly selected and analyzed by DELFIA assays (PerkinElmer) for the presence of VP6 specific single domain antibodies in their periplasmic extracts. The positive clones were sent for Sanger sequencing. The amnio acid sequences of positive clones were aligned and grouped according to CDR3 sequences. Representative sdAbs were cloned into a Pichia secretive expression vector P6791 PC00

[0147] 12

[0148] (pP_0126) in fusion with Histidine tag and FLAG tag on the C-terminal and expressed in Pichia Pastoris with glucose induction. All the sdAbs were purified from the culture medium by Ni-NTA resin (Thermofisher).

[0149] The binding affinities to VP6 of purified sdAbs were analyzed by ELISA. Serial dilutions of sdAbs were transferred to 96 well ELISA plates immobilized with VP6 His Tag protein. The binding affinities of sdAbs were detected by HRP conjugated anti-FLAG monoclonal antibodies M2 (Thermofisher).

[0150] The binding affinities to rotavirus of purified sdAbs were analyzed by ELISA. The ELISA plates were coated with rabbit polyclonal anti-rotavirus antibodies (ThermoFisher) and the rotavirus RRV (NCPV, 0904051v, G3P[3]) were captured. Serial dilutions of sdAbs were incubated with the captured virus and the binding affinities were detected by HRP conjugated anti-FLAG monoclonal antibodies M2 (ThermoFisher). The assays were developed using TMB substrate (ThermoFisher).

[0151] Results

[0152] Out of the screened 190 colonies, 170 colonies scored positive. Based on sequencing data of the positive colonies, 133 different Nanobodies were identified, belonging to 61 different CDR3 groups (B- cell lineages). The table below shows results of head domain of VP6 and rotavirus binding affinities for different identified sdAbs.

[0153] Table 1 Top VP6 sdAbs screened from the phage library

[0154] +++: ELISA EC50 is lower than 10 nM

[0155] ++: ELISA EC50 is between 10 and 100 nM

[0156] +: ELISA EC50 is higher than 100 nM

[0157] Conclusions

[0158] The result of this example shows that 12 sdAbs screened from the library had good VP6 binding affinity.

[0159] Four out of the 12 sdAb had good binding affinity to the rotavirus. P6791 PC00

[0160] 13

[0161] EXAMPLE 2: Screening of RVA neutralization activity of the Example 1 identified sdAbs

[0162] Material

[0163] The cell line used for infection MA-104 (ATCC, CRL-2378.1) is maintained in Minimum Essential Medium Eagle (MEM) supplemented with 10% fetal bovine albumin (FBS) ,1 % Non-essential amino acids (NEAA) and 100 U / ml Penicillin, and 100 mg / ml Streptomycin. The human rotavirus A strain Wa (ATCC, VR-2018, G1 P[8]) was propagated in MA-104 cells and harvested from the cell culture supernatant by centrifugation and filtration removing cells and cell debris.

[0164] The virus neutralization assay was performed as previous described with modifications (see Aladin et al. (2012)). MA-104 cells were seeded in 96 well plate and grown for 3 days until confluency. Rotavirus stocks were first activated by trypsin (5 pg / ml) at 37 °C for 1 h and diluted to 1000 FFU / ml in MEM w / o FBS. The sdAbs were diluted to 8 pg / ml Equal volumes of virus and sdAbs were mixed and incubated for one hour at 37 °C. The virus-sdAb mixtures were then plated onto MA-104 cells and incubated for one hour at 37 °C, before 50 ul of MEM w / FBS added to each well. The plate was then incubated in CO2 incubator at 37 °C for 16 h.

[0165] To detect the infections of rotavirus to MA-104 cells, cells were first fixed with ice-cold 100% Methanol. And then incubated with rabbit polyclonal anti-rotavirus antibodies (ThermoFisher) followed by incubation with goat anti-rabbit FITC conjugated antibodies (ThermoFisher). The fluorescence forming unites (FFUs) were counted under inverted microscope EVOS M5000 (ThermoFisher). The infection rates were calculated by dividing the FFUs of treatment groups by the FFUs of no treatment control.

[0166] Results

[0167] The virus neutralization screening of VP6 binding sdAbs showed that only one sdAb M010_0004 significantly reduced the infection rate of rotavirus strain Wa at 4 ug / ml concentration. The Figure 1 shows results of rotavirus A neutralization activity for the in the Example 1 identified sdAbs.

[0168] Conclusions

[0169] The result of this example shows that M010_0004 was the best of the different identified sdAbs - since it had best VP6 and rotavirus binding affinity (see Example 1) and had best neutralization activity against the tested RVAs - i.e. may be seen as our lead candidate herein.

[0170] EXAMPLE 3: M010_0004 lead candidate neutralization activity against different human RV - compare with benchmark ARP1 and 3B2 sdAbs

[0171] Material and methods

[0172] The rotavirus neutralization assays were perform on 5 different human rotavirus A strains, 3 human and 2 animal rotavirus A strains. (See table below).

[0173] RVA strain Host Genotype ATCC Cat. No.

[0174] " Wa Human G1 P[8] VR-2018 P6791 PC00

[0175] DS-1 Human G2P[4] VR-3391

[0176] Hu / Australia / 1-9-

[0177] Human NA VR-1546

[0178] 12 / 77 / S

[0179] B223 Bovine G10P

[0011] VR-1290

[0180] OSU Porcine G5P[7] VR-892

[0181] The RVA neutralization experiments were essentially done as in Example 2 above.

[0182] Results

[0183] As shown in Figures 2 and 3 herein - the sdAb M010_0004 lead candidate of our invention had as good neutralization activity against different human RV types as prior art reference / benchmark ARP1 and 3B2 sdAbs - but for e.g. porcine RV, sdAb M010_0004 of our invention was significantly better / improved over reference / benchmark ARP1 and 3B2 sdAbs.

[0184] Conclusions

[0185] The result of this example shows that sdAb M010_0004 lead candidate of our invention had as good neutralization activity against different human RV types as prior art reference / benchmark ARP1 and 3B2 sdAbs - but for e.g. porcine RV strain OSU, sdAb M010_0004 of our invention was significantly better / improved over reference / benchmark ARP1 and 3B2 sdAbs.

[0186] EXAMPLE 4: M010_0004 lead candidate improved thermostability over reference / benchmark ARP1 and 3B2 sdAbs

[0187] Material and methods

[0188] To study the thermal stability of lead candidate and benchmarks, Protein Thermal Shift™ assays were performed using Protein Thermal Shift Dye kit (4461146 Applied Biosystems™) and Quantstudio™ 6 pro real-time PCR instrument (Applied Biosystems™) according to the manufacture’s manual. The melting point determination was carried out by Protein Thermal Shift™ Software (Applied Biosystems™). The final assay volume was 20 pL and contained PBS buffer (pH 7.2), Protein Thermal Shift Dye (3X) and 10 ug purified sdAbs. A non-protein control was also performed. A continuously increased temperature range from 25.0 to 99 °C was scanned in a ramp increment of 0.05 °C per second. Scans were run in triplicates. The unfolding temperatures (Boltzmann Tm) of proteins were calculated from the inflection point of the melt curves by Protein Thermal Shift™ Software (Applied Biosystems™).

[0189] Results

[0190] As shown Figure 4 herein - the M010_0004 lead candidate of our invention had improved thermostability over reference / benchmark ARP1 and 3B2 sdAbs.

[0191] Conclusions P6791 PC00

[0192] 15

[0193] The result of this example shows that the M010_0004 lead candidate of our invention had improved thermostability over reference / benchmark ARP1 and 3B2 sdAbs.

[0194] EXAMPLE 5: M010_0004 lead candidate - benchmark ARP1 and 3B2 sdAbs - comparing sequences

[0195] Figure 5 herein shows a sequence alignment of M010_0004 of our invention and benchmark ARP1 and 3B2 sdAbs - where can be seen that e.g. the CDR sequences are different.

[0196] Below is provided a discussion of possible relatively minor changes in e.g. the M010_0004 CDR regions that one could make and probably / plausible maintain the herein relevant RV binding / neutralization positive characteristic of M010_0004 - the skilled person may use the new technical information herein to make relatively minor changes in e.g. the CDR regions, while maintaining herein relevant RV binding / neutralization positive characteristic.

[0197] The CDR1 regions of M010_0004 and the two benchmarks are highly conserved. The most dramatic difference is at position P27 and V33. Both of the two benchmarks have a positive charged Arginine at position 27 instead of Proline and ARP1 has a negatively charged Aspartic acid instead of hydrophobic Valine. One can expect that the mutations at P27 and V33 probably keep the binding affinity of M010_0004.

[0198] The CDR2 region of M010_0004 is highly homologous to benchmark 3B2, only differing at position S55 and P58. One can expect that mutations on S55 and P58 probably keep the binding affinity of M010_0004.

[0199] Among the VP6 binding sdAbs screened in Example 1 , the CDR3 of 2RTC_G11 is homologous to the best sdAb M010_0004. While 2RTC_G11 has a lower affinity to VP6 and rotavirus compared with M010_0004. Therefore, changing of the none conserved residues may still keep the binding and neutralization activities of M010_0004. For example, S104, and A109.

[0200] EXAMPLE 6: Antibody (e.g. M010_0004) expression in Aspergillus and E.Coli

[0201] Example 1 describes antibody expression / purification in Pichia.

[0202] M010_0004 of our invention and reference / benchmark ARP1 and 3B2 sdAbs of EXAMPLE 3 and 4 above were expressed in Pichia.

[0203] As discussed above, the skilled person knows how to produce / obtain a herein relevant single-domain antibody by recombinant expression in a suitable production host cell (e.g. Pichia, Aspergillus, E. coli, etc.).

[0204] Herein relevant single-domain antibodies (sdAbs) such as e.g. M010_0004 of our invention and reference / benchmark ARP1 and 3B2 sdAbs were expressed in Aspergillus oryzae essentially as described in WO2020 / 144164A1 (Bactolife). P6791 PC00

[0205] 16

[0206] Expression of herein relevant single-domain antibodies (sdAbs) in E. coli was essentially done according to Example 1 above for expression of the head domain of VP6 - i.e. the expression sdAbs in E. coli was essentially done as expression of the head domain of VP6 in E. coli.

[0207] EXAMPLE 7: Homodimer antibodies (e.g. M010_0004-dimer)

[0208] As discussed above - M010_0004 full length is shown in SEQ ID NO: 4 - which herein may be termed M010_0004 or M010_0004 monomer.

[0209] M010_0004 of our invention and reference / benchmark ARP1 and 3B2 sdAbs of EXAMPLE 3 and 4 above were monomers - or alternatively expressed monomeric single domain antibodies.

[0210] As discussed above - homodimer antibodies can routinely be assembled / made by methods known to the person skilled in the art based on e.g. use of a suitable GS linker (see e.g. WO2020 / 144164A1 (Bactolife) discussed above).

[0211] M010_0004 homodimer (may herein be termed M010_0004-dimer) with a GS linker was made and the full length sequence is shown in SEQ ID NO: 9.

[0212] A reference 3B2 homodimer (may herein be termed 3B2-dimer) with the same GS linker as for M010_0004-dimer of SEQ ID NO: 9 was also made.

[0213] EXAMPLE 8: M010_0004 lead candidate neutralization activity against human RV strain l / l / a - compared with benchmark 3B2 sdAb - both expressed in Aspergillus

[0214] M010_0004 and benchmark 3B2 (both monomers) were expressed in Aspergillus.

[0215] The rotavirus neutralization assays were essentially done as in Example 3 above with human rotavirus (RV) strain Wa.

[0216] As shown Figure 6 herein - the result of this example showed that also when expressed in Aspergillus - sdAb M010_0004 lead candidate of our invention had at least as good neutralization activity against human RV strain Wa as prior art reference / benchmark 3B2 sdAb.

[0217] EXAMPLE 9: M010_0004-dimer lead candidate neutralization activity against human RV strain l / l / a - compared with M010_0004 monomer

[0218] M010_0004 homodimer (may herein be termed M010_0004-dimer) with a GS linker and the full length sequence is shown in SEQ ID NO: 9 was expressed in Aspergillus and E. coli. P6791 PC00

[0219] 17

[0220] M010_0004 of SEQ ID NO: 4 (i.e. M010_0004 monomer) was expressed in Aspergillus.

[0221] The rotavirus neutralization assays were essentially done as in Example 3 above with human rotavirus (RV) strain Wa.

[0222] Results

[0223] As shown in Figures 7 herein - E. coll expressed M010_0004-dimer basically have the same neutralization activity of Aspergillus expressed M010_0004-dimer.

[0224] The M010_0004-dimer had an improved neutralization activity as compared to the M010_0004 monomer.

[0225] Figure 7 refers to concentration (ug / ml) - and as understood, the same amount in ug / ml ofthe monomer and the dimer implies the same amount of monomer units - i.e. a true comparative experiment.

[0226] EXAMPLE 10: M010_0004-dimer lead candidate neutralization activity against human RV strain l / l / a - compared with benchmark 3B2-dimer

[0227] M010_0004 homodimer (M010_0004-dimer) was expressed in E. coli and reference 3B2 homodimer (3B2-dimer) was expressed in Pichia.

[0228] The rotavirus neutralization assays were essentially done as in Example 3 above with human rotavirus (RV) strain Wa.

[0229] Results

[0230] As shown in Figures 8 herein - M010_0004-dimer had an improved / stronger neutralization activity against human RV strain Wa as compared to benchmark 3B2-dimer.

[0231] Conclusions

[0232] The result of this example shows that M010_0004-dimer of our invention had an improved / stronger neutralization activity against human RV strain Wa as compared to benchmark 3B2-dimer.

[0233] REFERENCE LIST

[0234] 1 : Gordon et al. (2023) A comparison of the binding sites of antibodies and single-domain antibodies. Front. Immunol. 14:1231623. doi: 10.3389 / fimmu.2023.1231623

[0235] 2: Aladin et al. (2012) In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments. PLoS ONE 7(3): e32949. doi: 10.1371 / journal, pone.0032949 P6791 PC00

[0236] 18

[0237] 3: W02009 / 016100A1

[0238] 4: WO2020 / 144164A1 (Bactolife)

Claims

P6791 PC0019CLAIMS1. A single-domain antibody (sdAb) which binds to head domain of VP6 derived from a human rotavirus A shown in SEQ ID NO: 5 and has neutralization activity against the rotavirus A types Wa (human), DS-1 (human), Hu (human), B223 (bovine), and OSU (porcine), and wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein one or more amino acids have been altered, with the proviso that no more than 2 amino acids have been so altered, for example wherein 1 amino acid have been so altered.

2. The single-domain antibody of claim 1 , wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 , or a variant thereof wherein 1 amino acid has been altered; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2, or a variant thereof wherein 1 amino acid has been altered; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3, or a variant thereof wherein 1 amino acid has been altered.

3. The single-domain antibody of claim 2, wherein said single-domain antibody is comprising:(i): a complementary-determining region 1 (CDR1) comprising or consisting of SEQ ID NO: 1 ; and(ii): a complementary-determining region 2 (CDR2) comprising or consisting of SEQ ID NO: 2; and(iii): a complementary-determining region 3 (CDR3) comprising or consisting of SEQ ID NO: 3.

4. The single-domain antibody of any of the preceding claims, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 90% sequence identity thereto.

5. The single-domain antibody of claim 4, wherein the single-domain antibody is a single-domain antibody of claim 3.P6791 PC00206. The single-domain antibody of claim 5, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 95% sequence identity thereto.

7. The single-domain antibody of claim 6, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4, or a sequence having at least 99% sequence identity thereto.

8. The single-domain antibody of claim 7, wherein the single-domain antibody is comprising or consisting of the sequence as set forth in SEQ ID NO: 4.

9. The single-domain antibody of any of the preceding claims, wherein the single-domain antibody is a fusion protein comprising a single-domain antibody as defined in any one of the preceding claims and one or more further single-domain antibodies as defined in any one of the preceding claims, and optionally one or more linkers, such as one or more GS linkers.

10. The single-domain antibody of claim 9, wherein the fusion protein is a homodimer or a heterodimer.

11. The single-domain antibody of claim 10, wherein the fusion protein is a homodimer.

12. The single-domain antibody of claim 11 , wherein the fusion protein homodimer is comprising a single-domain antibody as defined in claim 3 and one more single-domain antibody as defined in claim 3.

13. The single-domain antibody of any of the preceding claims, wherein the single-domain antibody has been obtained by recombinant expression in a suitable production host cell.

14. The single-domain antibody of claim 1 , wherein production host cell is an Aspergillus cell.

15. A single-domain antibody (sdAb) of any of the preceding claims for use in the reduction of risk, prevention or treatment of rotavirus A infection in a subject.

16. The single-domain antibody for use of claim 15, wherein the subject is a human.

17. The single-domain antibody for use of claim 16, wherein the human is a child.

18. The single-domain antibody for use of claim 17, wherein the child a child of less than 10 years of age.

19. The single-domain antibody for use of claim 15, wherein the subject is a pig.P6791 PC002120. The single-domain antibody for use of claim 19, wherein the single-domain antibody is a singledomain antibody of claim 3.

21. The single-domain antibody for use of claim 20, wherein the single-domain antibody is a singledomain antibody of claim 5.

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