WO2025125577A1 - Antibodies against influenza b virus - Google Patents

Abstract

The invention relates to antigen-binding polypeptides specifically binding neuraminidase and/or haemagglutinin protein from Influenza B virus resulting in broadly neutralizing IBV antigen-binding polypeptides or antibodies. More specifically the invention relates to immunoglobulin single variable domains specifically binding NA or HA, which upon fusion to bispecific antigen-binding proteins provide for a broader and improved neutralization capacity as compared to a combination of IBV NA- and HA- targeting ISVDs or antibodies. The invention further relates to the use of said antigen-binding proteins for medical purposes, more specifically in prophylactic or therapeutic treatment of a subject, even more specifically to prevent or treat IBV infections.

Description

ANTIBODIES AGAINST INFLUENZA B VIRUS

FIELD

The invention relates to antigen-binding polypeptides specifically binding neuraminidase and/or haemagglutinin protein from Influenza B virus resulting in broadly neutralizing IBV antigen-binding polypeptides or antibodies. More specifically the invention relates to immunoglobulin single variable domains specifically binding NA or HA, which upon fusion to bispecific antigen-binding proteins provide for a broader and improved neutralization capacity as compared to a combination of IBV NA- and HA- targeting ISVDs or antibodies. The invention further relates to the use of said antigen-binding proteins for medical purposes, more specifically in prophylactic ortherapeutictreatment of a subject, even more specifically to prevent or treat IBV infections.

INTRODUCTION

Influenza viruses cause annual human epidemics on a global scale. Whereas infections with influenza C viruses are typically milder, influenza A (IAV) and influenza B viruses (IBV) are more virulent and can cause severe acute respiratory infection. The influenza B virus contains a segmented, negative-stranded RNA genome that codes for eleven proteins of which four are membrane embedded: hemagglutinin (HA), neuraminidase (NA), matrix protein 2 (BM2), and NB (Koutsakos et al., 2015). To this day, IBV does not have an established animal reservoir, in contrast to IAV, and has a limited pandemic potential. Continuous drift however diverged IBVs in the 1980s in two antigenically distinct lineages that can be discriminated based on HA (Rota et al., 1990). The B/Yamagata/16/88-lineage and B/Victoria/2/87- lineage viruses have co-circulated globally ever since and made up roughly 23.4 % of human influenza cases between 2000 and 2018. The incidence of IBV varies widely depending on geography, influenza seasons and age group (Caini et al., 2019). Influenza B is thought to be dominant in about one in seven seasons as was the case for 2017 - 2018: Influenza B viruses predominated in most European countries and accounted for up to 40 % of infections in north America and up to 50 % in central Asia. Remarkably, the IBVs isolated were almost exclusively B/Yamagata-lineage (Caini et al., 2019; WHO, 2023). Almost no IBVs were detected in 2018-2019, while in the ensuing season of 2019-2020, IBV was again dominant in Europe. Up to date, almost no B/Yamagata viruses are detected (WHO, 2023). Looking overall at USA influenza seasons as of 2000, influenza was associated with substantial clinical and economic burden, and type B influenza illness was responsible for a considerable percentage of this burden, accounting for an average of 41 % of healthcare encounters and 37 % of healthcare costs (Yan et al., 2017). In children however IBV is often more lethal than IAV infection (Shang et al., 2010; Tran et al., 2016). During the flu season in 2010-2011 for example, influenza B was responsible for 38 % of deaths in the paediatric population, while only 26 % of circulating influenza viruses were attributed to IBV during this period (Shang et al., 2010).

To keep up with changing influenza epidemiology, WHO advices a yearly quadrivalent vaccine as prophylaxis. Different small molecule therapeutic antivirals were made available over the years, though influenza B virus is less susceptible than influenza A to marketed neuraminidase (NA) inhibitors oseltamivir, zanamivir, peramivir, and laninamivir, and resistance against these inhibitors is a problem. Influenza B viruses are susceptible to the polymerase acid inhibitor Baloxavir Marboxil (Xofluza™) with an IC₅o value that is approximately 5-fold higher than influenza A, and which is not approved under 12y (Noshi et al., 2018). So it is appealing to in addition have broad range antibody-based antivirals available for prophylactic treatments, especially for paediatric use.

In fact, pan-influenza neuraminidase monoclonal antibodies have been isolated inhibiting the neuraminidase activity via receptor mimicry and binding the cell surface expressed influenza B NAs broadly, with for example FNI9 and FNI17 antibodies having a binding affinity in the pM range (Momont et al. 2023). Furthermore, combinations with anti-HA stem antibodies have been suggested with the goal to provide broad-range long lasting antibody-based antivirals.

Hemagglutinin-targeting monoclonal antibodies have been reported, though none are in the clinic, as well as single domain antibodies targeting IBV HA were reported. For instance Laursen et al. (2018) describe SD83 as a HA stem-specific VHH and SD84 as HA head-specific VHH, together forming promising assets since SD83 neutralizes all tested IBV viruses with similar potency and a fusion as head- to-tail construct with SD84 further increased microneutralization breadth. Ramage et al. (2019) described IBV cross-neutralizing VHHs mostly targeting the HA stalk domain, besides few IBV crossneutralizing VHHs (Vic2a-20, Vic2b-3, and Yam2a-20) that target the HA head-domain.

By developing even more potent and clinically interesting broad-range IBV targeting antibodies, even more superior approaches to tackle influenza are in scope. One approach is to combine NA and HA targeting antibodies, which would further reduce potential escapes, and the combination of such antibodies has revealed beneficial outcome in that anti-NA mAbs increase Fc-mediated protection by adding anti-HA stem mAbs (Zhang, et al. 2022). Also Zhai et al. (2022) reported that a combination of mAbs against IBV HA and NA active site, covering different lineages, resulted in more effective antibodies with enhanced ADCC. Though whether such a combined approach may be further improved through the application of novel bispecific antibody-based biologicals is not clear, since for instance in WO2022/109317A1 (Vir Biotechnology Inc. and Humabs Biomed), the bispecific antibodies including an IAV/IBV NA-specific monoclonal antibody FN 117 fused to the N-terminus of influenza hemagglutinin (HA)-specific FM08: FNI17-L-FIVI08-DVDIgGl-LS seems to have a lower potency as compared to the HA- specific FM08. An observation that also translates in vivo, since the bispecific FNI17_FM08-DVD-LS antibody also protects BALB/c mice less well against body weight loss following challenge with an H1N1 influenza virus compared to FNI17 mono-specific IgGl. However there would be an advantage of providing single-chain antibodies with a broader, hence multispecific, neutralizing effect on influenza, and to date it is not yet clear what approach may lead to improved Influenza antibodies for the clinic.

SUMMARY OF THE INVENTION

The invention is based on the identification of antigen-binding polypeptides, specifically immunoglobulin single variable domains (ISVDs), even more specifically VHH or nanobodies®, which were specifically raised against a broad panel of Influenza B viral (IBV) antigens, against the IBV hemagglutinin (HA) or neuraminidase (NA), as to provide for broadly neutralizing ISVDs. More specifically, the present invention relates to the finding of VHH families specifically binding IBV HA, which are VHH families that compete for the same HA binding site, providing for a novel epitope, as derived from the structurally resolved humanized VHH69 in complex with IBV HA protein, at a unique quaternary epitope. The ISVDs were also shown to in vitro microneutralize (a diverse set of) IBVs through inhibition of membrane fusion. The invention further relates to the finding of VHH families specifically binding IBV NA, which neutralize IBV through NA active site inhibition.

Bivalent ISVDs, as head-to-tail or Fc fusions, as well as tetravalent antibodies comprising said ISVDs potently neutralize a broad range of IBV strains of both lineages. Finally, the invention provides for bispecific antigen-binding molecules wherein said ISVDs targeting NA and HA are fused, and for which in vivo potency was shown to prophylactically protect mice upon against IBV. Moreover, as compared to an equimolar mix combining single targeting NA- and HA-specific ISVD-based constructs, the bispecific single chain molecules or ISVD-based antibodies improved neutralization in a synergistic manner.

In the present application, broad-range IBV neutralizing bispecific ISVD-based proteins or antibodies targeting HA and NA, and which are exclusive in IBV-specificity since IAV NA or HA is not affected with these ISVDs, have been disclosed for the first time, with a superior effect as compared to applying a composition that combines monoclonal or single domain antibodies targeting each of said IBV HA or NA proteins. Moreover, said monovalent HA- and NA-specific VHHs are superior in neutralization potential over the existing binders, due to their unique epitope.

Overall, the present invention provide for single chain engineering of antibody formats with VHHs holding promise as superior candidates for the development of broad-spectrum prophylactics or therapeutics against IBV virus infections with enhanced neutralization by hitting multiple epitopes simultaneously.

In a first aspect, the invention relates to an antigen-binding polypeptide which binding Influenza B virus (IBV), wherein said antigen-binding polypeptide comprises or consists of an immunoglobulin single variable domain (ISVD) specifically binding IBV neuraminidase (NA) protein and an ISVD specifically binding IBV haemagglutinin (HA) protein. More specifically, said antigen-binding polypeptide specifically binds an Influenza B virion through the interaction between said NA-specific ISVD and HA- specific ISVD and the surface viral NA and HA proteins. More specifically, said antigen-binding protein comprises an ISVD specifically binding IBV NA and comprises an ISVD specifically binding HA which are fused or linked directly or via a linker, or via an Fc tail or antibody forming moiety, resulting in a bispecific antigen-binding polypeptide specifically binding IBV NA and HA proteins. In one embodiment, the bispecific antigen-binding protein described herein neutralizes Influenza B Virus from both B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage. In another embodiment said bispecific antigen-binding polypeptide comprises a NA-specific ISVD which binds the NA catalytic region, and/or comprises an HA-specific ISVD that binds to the stalk region of the IBV HA protein.

A further specific embodiment relates to said bispecific antigen-binding polypeptide which comprises an NA-specific ISVD binding an epitope comprising at least residues E275, E276, R292, S345, G347, E373, R374, and W408 of the NA protein as present in the reference sequence of B/Washington/02/2019 NA (see Table 2), and/or comprises and HA-specific ISVD that binds an epitope comprising at least residues 38, 80, 317, 327, and 403, more specifically further comprising residue 308, 309, and 434, even more specifically comprising residues 47 and 324, as present in the PDB HA sequence (see Table 1).

A second aspect of the invention relates to an antigen-binding protein specifically binding Influenza B virus (IBV) which (micro)neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage, which comprises ISVD specifically binding NA. In a more specific embodiment said antigen-binding protein specifically binding Influenza B virus (IBV) is a monovalent or multivalent antigen-binding protein that comprises said NA-specific ISVD. In a more specific embodiment, said antigen-binding protein consists of an ISVD specifically binding IBV NA protein, preferably as present on the surface of an IBV virion.

A further specific embodiment relates to said antigen-binding protein specifically binding IBV NA and capable of (micro)neutralizing IBV, comprising an NA-specific ISVD specifically binding the NA active site at least at residues E275, E276, R292, S345, G347, E373, R374, and W408 of the NA protein as present in the reference sequence of B/Washington/02/2019 NA (see Table 2). A further specific embodiment relates to said antigen-binding protein specifically binding IBV NA and capable of (micro)neutralizing IBV, comprising an NA-specific ISVD composed of the formula of FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4, and which comprises the complementarity determining regions (CDRs) as presented in any of SEQ ID NOs: 1-3, representing VHH504, 525 and 508, respectively, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia, or comprising said ISVD comprising a sequence wherein

- CDR1 is SEQ ID NO: 19, CDR2 is SEQ ID NO: 20, and CDR3 is SEQ ID NO: 21;

- CDR1 is SEQ ID NO: 22, CDR2 is SEQ ID NO: 23, and CDR3 is SEQ ID NO: 24.

A further specific embodiment relates to the NA-specific antigen-binding protein wherein said ISVD comprises a sequence selected from the group of sequences of SEQ ID NO: 1-3, corresponding to the sequence of VHH504, 525 and 508 respectively, or a humanized variant thereof as present in SEQ ID NOs:7-9, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the CDRs remain identical, or a humanized variant of any one thereof as known by the skilled person.

A third aspect of the present invention relates to an antigen-binding protein specifically binding Influenza B virus (IBV), which (micro)neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87- lineage, and which comprises an ISVD specifically binding HA, wherein the binding site comprises at least residues 38, 80, 317, 327, and 403, more specifically further comprises residues 308, 309, and 434, even more specifically comprises residues 47 and 324, as present in the PDB HA sequence (see Table 1).

A further specific embodiment relates to an antigen-binding protein specifically binding Influenza B virus (IBV), which (micro)neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage, and which comprises an ISVD specifically binding HA wherein said ISVD is composed of the formula of FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4 and comprises the CDRs as present in any of SEQ ID NOs: 4-6, corresponding to the sequences of VHH69, VHH75 and VHH72, and wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia.

Alternatively, said HA-specific antigen-binding protein specifically binding and (micro)neutralizing Influenza B virus (IBV), comprising an ISVD comprising the CDRs as present or comprising an ISVD comprising a sequence wherein:

- CDR1 is SEQ ID NO: 13, CDR2 is SEQ ID NO: 14, and CDR3 is SEQ ID NO: 15;

- CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 18. A further specific embodiment relates to the HA-specific antigen-binding protein wherein said ISVD comprises a sequence wherein:

- CDR1 is SEQ ID NO: 13, CDR2 is SEQ ID NO: 14, and CDR3 is SEQ ID NO: 15;

- CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 17, and CDR3 is SEQ ID NO: 18; and/or

FR2 residues 44 and 64 (according to Kabat annotation) are a Glutamic acid residue.

A further specific embodiment relates to the HA-specific antigen-binding protein wherein said ISVD comprises a sequence selected from the group of sequences of SEQ ID NO: 4-6, corresponding to the sequence of VHH69, 75 and 72 respectively, or a humanized variant thereof as present in SEQ ID NQs:10-12, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the CDRs remain identical, or a humanized variant of any one thereof as known by the skilled person.

A further aspect of the invention relates to the bispecific antigen-binding protein described herein, comprising said NA-specific ISVD of the second aspect of the invention and the HA-specific ISVD of the third aspect of the invention. In a further embodiment said bispecific antigen-binding protein comprises said NA- and HA-specific ISVDs fused or linked directly, as a genetic fusion, or via a linker, or as part of an ISVD-FC fusion or part of a bispecific antibody format.

The invention likewise relates to any of the above-described antigen-binding polypeptides comprising NA- and/or HA-specific ISVD(s) provided as (pharmaceutical) compositions, their encoding nucleic acids, and/or recombinant vectors, and derived products thereof such as multivalent formats, conjugates, or labelled polypeptides. The invention likewise relates to any of the above-described antigen-binding polypeptides comprising NA- and/or HA-specific ISVD(s), (pharmaceutical) compositions, nucleic acids and/or recombinant vectors, for use as a medicament, preferably for use in prophylactic or therapeutic treatment of a subject, wherein the subject preferably is a human. The invention likewise relates to any above-described antigen-binding polypeptides comprising NA- and/or HA-specific ISVD(s), (pharmaceutical) compositions, nucleic acids and/or recombinant vectors, for use in the prevention or treatment of an Influenza virus infection, more specifically an Influenza B virus infection.

DESCRIPTION OF THE FIGURES

Figure 1. Seroconversion monitoring of llama 178. Llama 178 was immunized biweekly with 10 phylogenetically distinct IBV HAs by DNA immunization. Serum was sampled at day 0 post immunization (pre-immune serum). Plasma was sampled at day 46, 50, 88 and 92 post immunization. Two phage display libraries were constructed; Library A from plasma sampled at day 46 and 50, library B from plasma sampled at day 88 and 92. Seroconversion was monitored by ELISA with recombinantly coated influenza B HAs. Importantly, none of the coated HAs were included in the immunization regimen. BSA was coated as a negative control. Samples were diluted 1/3 starting from an initial 1/100 dilution. Irrelevant control plasma were included as negative control plasma. The ELISA was read out using a anti-camelid VHH HRP-coupled monoclonal antibody (Genscript). Absorbances were normalized and the mean values of two independent experiments are shown.

Figure 2. Seroconversion monitoring of llama 179. Llama 179 was immunized biweekly with 10 phylogenetically distinct IBV NAs by DNA immunization. Serum was sampled at day 0 post immunization (pre-immune serum). Plasma was sampled at day 46, 50, 88 and 92 post immunization. Two phage display libraries were constructed; Library A from plasma sampled at day 46 and 50, library B from plasma sampled at day 88 and 92. Seroconversion was monitored in a fetuin-based ELLA assay. Viruses (B/Lee/1940, B/Washington/02/2019 or B/Phuket/3073/2013) were incubated with 3-fold dilutions of plasma, starting from a 1/100 initial dilution. Pre-immune serum was included as a control. The amount of desialylated fetuin was measured with a HRP-coupled peanut agglutinin (Sigma-Aldrich). The mean values of four technical replicates are shown.

Figure 3. Timeline of llama immunization. Two llamas were immunized with a mix of influenza B HA expressing plasmids (llama 178) or a mix of influenza B NA expressing plasmids (llama 179). Numbers in circles represent days since first immunization. Plasma was sampled at day 46, 50, 88 and 92 and two libraries were constructed for each llama: library A at day 50 and library B at day 92.

Figure 4. Screening of anti-HA VHH PE extracts from library 178B. PE extracts of 90 individual clones were mixed with 100 TCID50 virus and incubated on MDCK cells for 3 days. The NP-protein of infected cells was detected using a mouse anti-influenza B NP monoclonal antibody (Thermo Scientific) and antimouse IgG-HRP coupled monoclonal antibody (GE healthcare). Absorbance values are given on the X- axis and individual clone numbers are denoted on the Y-axis. Clones are numbered Cl - C90 with VHH- 69 being C9 and VHH-75 being C31. Viruses used are B/Lee/1940, B/Harbin/07/94, B/HongKong/05/1972, B/Phuket/3073/2013 and B/Washington/02/2019.

Figure 5. Screening of anti-NA VHH PE extracts from library 179B. VHH PE extracts of 216 clones were incubated with an EC70 amount of virus on fetuin-coated plates. The amount of desialylated fetuin was measured using an HRP coupled peanut agglutinin (Sigma-Aldrich). Absorbance values are given on the X-axis and individual clone numbers are denoted on the Y-axis. Clones are numbered Cl - C216 with VHH-504 being C12 and VHH-525 being C110. Viruses used are B/Lee/1940, B/Washington/02/2019 and B/Phuket/3073/2013. Figure 6. Humanization of VHH-69 and VHH-75. A) Original and humanized sequences of VHH-69 and VHH-75 with Kabat numbering. CDRs are boxed and annotated according to Kabat. Glutamic acid at position 64 is colored in red. B) Binding to recombinant influenza B HA in ELISA. mAb mouse anti-his tag (MCA1396, biorad) was used to detect the HIS tagged VHHs. An anti-mouse IgG HRP coupled mAb (GE healthcare, NA931V) was used for read out in combination with TMB substrate. As control a anti- GFP VHH. Means of two independent experiments are plotted.

Figure 7. Mechanism of action for hVHH-69 and hVHH-75. A) hVHH-69 and hVHH-75 pre-incubated with recombinant soluble HA/B/Washington/02/2019 or HA/B/Utah/09/2014. Recombinant VHH at an 8-fold molar excess does not inhibit proteolytic cleavage of HA. The HA was analyzed by reducing SDS- PAGE and representative images of two independent experiments are shown. B) hVHH-69 protects HA/B/Utah/09/2014 and HA/B/Lee/1940 completely, while HA/B/Washington/02/2019 only partially, from the pH-induced protease sensitivity associated with membrane fusion. hVHH-75 protects all tested HAs except HA/B/Utah/09/2014 from pH-induced protease sensitivity. Exposure to acidic pH (pH 5,5) converts the HAs to the post-fusion conformational state, rendering them sensitive to trypsin digestion. Stabilization of the HA in the pre-fusion form by the hVHHs makes for a protease resistant HA that can be observed as HAO on non-reducing SDS page. (n= 2).

Figure 8. Polykaryon assay with HeLa cells expressing B/HA/Colorado/06/2017 or B/HA/Wisconsin/01/2010. hVHHs were pre-incubated in three concentrations: 5 pg/ml, 0,5 pg/ml and 0,1 pg/ml. The control (VHH-a-GFP) was used at 5 pg/ml. HAs were untreated (precursor HA, HAO), treated with TPCK-treated trypsin to activate the HA (HA1+HA2) or activated and in low pH (postfusion). The experiment was conducted four times (n=4). Each time four technical replicates were counted under the microscope (20xl0x). Mean values of the four technical replicates were used to analyze the variance. An ANOVA test was conducted assuming gaussian distribution of residuals and equal standard deviations. As post hoc analysis, the DUNNETT test was used where everything was compared to the control group. Multiplicity adjusted P-values were used to calculate the significance of the values. Significant P-value corrected for multiple testing: * P < 0,0332, ** p < 0,0021, *** P < 0,0002, **** p < 0,0001 .

Figure 9. Humanization of VHH-504 VHH-525. A) Original and humanized sequences of VHH-504 VHH- 525 with Kabat numbering. CDRs are boxed and annotated according to Kabat. B) Humanization does not impact potency on the ELLA assay for hVHH-504 and hVHH-525. Original VHHs (n=4), humanized VHHs (n=4) are tested in ELLA assay. Three influenza B viruses are used: B/Phuket/3073/2013, B/Washington/02/2019 and B/Lee/1940. IC₅o values are reported in pg/ml. Figure 10. Characterization of hVHH 504 and hVHH 525. A) 50 ng of recombinant enzymatically active NA was incubated with 160-fold molar excess of hVHH or 288-fold molar excess of zanamivir. The fluorescent signal was measured after incubation with 4-methylumbelli-feryl N-acetyl-a-D-neuraminic acid (Sigma-Aldrich). Relative percent inhibition per treatment is depicted compared to PBS. Mean with 95% Cl is given for 4 biologic replicates. B) B/Washington/02/2019 virus was incubated for 2 hrs on MDCK cells before VHHs or Zanamivir was added. Concentrations are in ng/pL for the hVHHs and in pM for zanamivir. Detection of HAO was done with a rabbit polyclonal antibody directed to influenza B HA (Sino Biological) and a goat anti-Rabbit IgG-HRP polyclonal antibody (Southern Biotech). Representative image of one experiment is shown, in total n=2. C) EC₇o amounts of virus (right panel) or recombinant NA (left panel) were mixed with VHHs and transferred to fetuin coated plates. After 18 hrs, the amount of desialylated fetuin was measured with horseradish peroxidase coupled peanut agglutinin (Sigma- Aldrich) by colorimetry. Y: yamagata-lineage virus (B/Phuket/3073/2013), V: Victoria-lineage virus (B/Washington/02/2019), A: ancestral-lineage virus (B/Lee/1940). EC₅o values are reported in pg/ml and representative of three independent experiments (on recombinant NA) or two independent experiments (on virus).

Figure 11. Microneutralization data of multivalent VHHs. Purified VHH was mixed with lOOTCIDso virus and incubated on MDCK cells for 3 days. IBV NP was detected with a mouse monoclonal (Thermo Scientific) and an anti-mouse IgG-HRP antibody to incubate (GE healthcare). Reported values are average IC50 in nM for three independent experiment (n=3). A) monovalent and homobivalent head-to- tail hVHHs B) Heterobivalent hVHH head-to-tail and equimolar mixes of monovalent constructs.

Figure 12. Microneutralization data comparing WT and NA mutation E117A. NA mutation E117A reduces susceptibility for NAIs (peramivir, zanamivir and laninamivir), while increasing susceptibility for hVHH 504 and hVHH 525. An anti-GFP VHH and hVHH 69 K65E were used as controls. Data represents three independent experiments, performed with technical duplicates. Means with SD are reported and an Anova was performed to assess the difference between WT and E117A samples. A follow-up Sidak test was used for multiple comparison correction. ** <0,0021, **** <0,0001.

Figure 13. Comparison of heterobivalent head-to-tail fusions with representative equimolar mixes. Fold difference is indicated in gradients at the right. An improvement with fold difference >2 as compared to an equimolar cocktail was observed in 55/64 combinations (85 %). An improvement with fold difference >10 was obtained in 20/64 combinations (31 %) tested.

Figure 14. Microneutralization data of purified VHH-Fc antibodies. Protein was mixed with 100 TCID50 virus and incubated on MDCK cells for 3 days. IBV NP was detected with a mouse monoclonal (MA1- 80712 monoclonal, Thermo Scientific) and an anti-mouse IgG-HRP antibody to incubate (NA931V, GE healthcare. Reported values are average IC₅o in nM for three independent experiment (n=3).

Figure 15. Prophylactic intraperitoneal or intranasal treatment with hVHH69-Fc and hVHH525-Fc protects mice against lethal IBV infection. Mice were treated with hVHH69-Fc, hVHH525-Fc, or an equimolar mix of hVHH69-Fc + hVHH525-Fc (n=8, split over 2 independent experiments). Differences in body weight between two groups were tested by two-way ANOVA with Sidak's multiple comparison. P <0.0001 for all treatments compared to control. Differences in survival were tested with a log-rank (Mantel-Cox) in graphpad prism. P <0.0001 for all treatments compared to control. A) Intranasal administration of 0,5 mg/kg of treatment was done 4 hours before viral challenge. B) Intraperitoneal administration of 5 mg/kg was done 24 hrs before viral challenge. Lethal challenge was done with 2LD₅o mouse adapted B/Washington/02/2019 virus. PBS intranasally dosed was included as a control group for both IP and IN experiment, since the challenge was done simultaneously.

Figure 16. Prophylactic intraperitoneal treatment with hVHH69-Fc, hVHH525-Fc or a combination protects mice against lethal IBV infection. Mice were treated IP with 1 mg/kg, 0,1 mg/kg or 0,01 mg/kg (n=4, except for the CR9114-Fc group). Differences in body weight between two groups were tested by two-way ANOVA with Sidak's multiple comparison. P <0.0001 for all treatments compared to control. Differences in survival were tested with a log-rank (Mantel-Cox) in graphpad prism. P <0.0001 for all treatments compared to control. Lethal challenge was done with 2LD₅o mouse adapted B/Washington/02/2019 virus.

Figure 17. Broad binding capacity of anti-HA hVHH69-Fc and hVHH75-Fc at 10 mg/ml, 2 independent repeats. HEK cells were transfected with different IBV HA expression plasmids or an irrelevant control plasmid (expressing influenza B NA) together with a GFP- expressing plasmid. 24 hrs post infection, cells were fixed and live cells were labelled with hVHH69-Fc, hVHH75-Fc or synagis. The median fluorescent signal of the GFP+ population was compared to the GFP- and the ratio was plotted on the graphs. The graphs represent the mean of two independent experiments with SD. All signals were statistically different (P < 0,5) compared to the cells transfected with irrelevant control plasmid, except for the synagis labelled cells. In green: B/Victoria-lineage. In red: B/Yamagata-lineage.

Figure 18. Amino acid sequence of NA- and HA-specific VHHs with CDR annotations. Kabat numbering of the amino acids is shown, and CDRs are displayed as annotated by MacCallum, AbM, Chothia, Kabat, and IMGT. The bottom line indicates the potential amino acid substitutions that can be made to produce alternative humanized variants of any of the NA- or HA-specific VHHs as described in the application, wherein any combination of one or more of said alternative substitutions presented herein is disclosed based on any of the original and/or humanized VHH sequences described in the present application. X represent any possible amino acid identity as conventionally known in the art.

Figure 19. Structural representation of binding region of hVHH69 and recombinantly produced HA/B/Washington/02/2019 protein forming a complex. A) Amino acid residues part of the binding region including the paratope of hVHH69 are shown in stick representation, as present on the CDR loops, and B) visual indication of the epitope region where the hVHH69 is bound to the HA protein, with the numbers corresponding to the amino acid positions of the HA protein being part of said epitope.

Figure 20. hVHH-69 binds a conserved quaternary epitope on the stem of IBV HA. Side view (a) or bottom view (c) of a cryo-EM 3D reconstruction of hVHH-69 in complex with B/Washington/02/2019 HA. Side (b) or bottom (d) cartoon view of a structural model built in the corresponding cryo-EM map. Two hVHH-69s are coloured in dark grey, one hVHH-69 is coloured in red. Two HA protomers are coloured in light grey, one is coloured in blue, e, f, Close-up of the interaction interface between hVHH- 69 and HA derived from B/Washington/02/2019 as determined by cryo-EM. Selected residues are shown as sticks, with polar contacts shown as black dotted lines.

Figure 21. Epitope comparison of hVHH-69. Epitopes of single-domain antibodies (SD83 and hVHH-69) and human broadly binding/neutralizing influenza B HA-stem antibodies CR9114, 5A7 and CR8071 are shown on B/Brisbane/60/2008 HA (PDB: 4FQM). HA1 epitope residues are coloured in red and HA2 epitope residues in blue. Lighter shades of red and blue were used when the epitope comprised multiple protomers (hVHH-69 and 5A7). One protomer of the trimeric HA complex is coloured in dark grey with other two protomers coloured in light grey.

Figure 22. Flow cytometry binding assay of hVHH-69 to cells expressing alanine mutant HA variants in the epitope region. Median fluorescent intensity (MFI) ratio of B/Washington/02/2019 HA transfected (GFP⁺) and non-transfected cells (GFP ). Binding curves of hVHH-69 were fitted through datapoints that represent mean values (n=3). IC₅o values are expressed in pg/mL. Identical data points of wild type (WT) B/Washington/02/2019 HA were plotted in both graphs in red. The binding of control VHH (ctrl) was evaluated at a fixed concentration of 10 pg/mL.

Figure 23. hVHH-525 binds a conserved active site epitope on IBV NA. Side view (a) and top view (c) of a cryo-EM 3D reconstruction of hVHH-525 in complex with B/Washington/02/2019 NA. Side (b) or top (d) cartoon view of a structural model built in the corresponding cryo-EM map. One NA protomer is coloured in blue, the other three protomers in grey. One hVHH-525 molecule bound to NA is shown in red. e, g, Close-up of the interaction interface. Selected residues are shown as sticks, with polar contacts (black), cation-n interactions (green) and additional contacts closer than 4 A (purple) are shown as dotted lines, f, Close-up of the modelled CDR3 loop and its fit in the cryo-EM map. h, Median fluorescent intensity (MFI) ratio of B/Washington/02/2019 NA expressing (GFP⁺) cells and nonexpressing cells (GPF ). Binding curves of hVHH-525 were fitted through datapoints that represent mean values (n=3). Wild type (WT) B/Washington/02/2019 NA is plotted in red and the binding of a control VHH (ctrl) was determined at a fixed concentration of 10 pg/mL. i, Epitope footprint of hVHH-525 on NA derived from B/Washington/02/2019 with IBV conservation colouring according to key. The contact residues were determined by PISA analysis.

Figure 24. Competition assays using the VHH-Fc constructs for binding to NA of HA in bio-layer interferometry, a) VHH525-Fc competes with VHH504-Fc and peramivir for binding to recombinant B/Washington/02/2019 NA; b) VHH69-FC competes with VHH75-FC but not with CR9114 for binding to HA, and similarly VHH75-FC competes with VHH69-FC but not with CR9114 for HA binding.

NA /HA recombinant proteins were immobilized on SA biosensors (400 s) and saturated with 504-Fc , 525-Fc, VHH69-FC, VHH75-Fc, or CR9114 antibodies (200 nM) or Peramivir (15 nM). Grey shades indicate wash steps with assay buffer. A representative experiment of two independent experiments is shown.

Figure 25. Polyreactivity assay. Several of the VHH-Fc construct were tested for unspecific binding to HEK293 cells: 69-Fc, 75-Fc, 504-Fc, and 525-Fc, which even at high concentration (100 pg/mL) did not indicate to bind to living (HEK293) cells, as for the controls Synagis (clinically approved monoclonal antibody), CR9114 (anti-HA stem monoclonal antibody).

Figure 26. IC₅o (nM) on microneutralization of B/Phuket/3073/2013 and B/Washington/02/2019 virus. Further antibody formats such as tetravalent VHH-Fc-VHH type of fusions provides lower IC50 values as compared to bispecific head-to-tail Fc fusions of combined mixtures of monovalent Fc fusions.

Figure 27. VHH69-Fc and VHH75-Fc are specific and exclusively binding to Influenza B virus HA. The binding of VHH69-Fc and VHH75-Fc to recombinant HA was evaluated in an ELISA. Recombinant HA proteins derived from A/Victoria/03/1972 (H3), A/New Caledonia/20/1999 (Hl), and B/Washington/02/2019, were coated in wells of a 96-well plate at a concentration of 2 ng/pL in a volume of 50 pL/well. Wells were washed with PBS supplemented with 0.05 % Tween20 and blocked with 3 % BSA. Antibodies were incubated in a 3-fold serial dilution range starting at a concentration of 30 pg/mL in 1.5 % BSA in PBS and incubated at room temperature for 1 hour. CR9114, a human monoclonal antibody that binds both IBV and IAV HA (Dreyfus et al., 2012), was included as a positive control. FNI9, a human monoclonal antibody specific to IBV and IAV NA, was included as a negative control (Momont, et al., 2023). Bound antibodies were detected with a rabbit anti-human IgG peroxidase linked polyclonal serum (Sigma, A8792). The peroxidase activity was detected using a 3, 3', 5, 5' tetramethylbenzidine solution (TMB, BD Pharmingen, 555214). The colorimetric reaction was stopped by the addition of 50 pL of IM H2SO4. The absorbance at 450 nm R6, 3, C2 (HA band)was measured with an Infinite pro 200 Tecan reader.

Figure 28. VHH504-Fcs and VHH525-Fcs inhibit neuraminidase (NA) activity of IBV NA but not of A/H1N1 Nl. NA activity was assessed using the (2'-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid) (MUNANA) substrate. Two soluble enzymatically active recombinant NAs derived from B/Washington/02/2019 (IBV) and A/HINlpdm NA were used as target enzymes. A VHH-Fc fusion construct targeting the SARS-CoV-2 spike protein was used as a negative control (Ctrl). FNI9 is a human monoclonal antibody that can inhibit the enzymatic activity of influenza A and B virus NA (Momont et al. ,2023) and was produced in house. The assay was conducted in PBS supplemented with 10 mM CaCI₂. Recombinant NA (2 ng/pL) was incubated with either 5 ng/pL of FNI9 antibody or VHH-Fcs, or 30 pM of zanamivir in a total volume of 40 pL, for 30 minutes at room temperature. Relative fluorescent units (RFU) were recorded with a Infinite Pro reader (Tecan) over 60 minutes following the addition of 2'-(4- Methylumbelliferyl)-a-D-N-acetylneuraminic acid at a final concentration of 1 mM.

Figure 29. Bispecific constructs can bind recombinant IBV HA and NA simultaneously, a) ELISA performed on coated recombinant B/Washington/02/2019 derived NA. Head-to-tail fusions (69-525, 525-69, 69-69, or 525-525) or head-to-tail fused to a human IgGl Fc domain (69-525-Fc, 525-69-Fc, 69- 69-Fc, or 525-525-Fc; resulting in tetravalent antibodies) were used to capture strepll-tagged B/Washington/02/2019 HA. The strepll-tag was detected with a strepll-tag specific monoclonal antibody. Monospecific constructs (525-525, 525-525-Fc, 69-69, and 69-69-Fc) cannot bind HA and NA simultaneously. Bispecific constructs (69-525, 69-525-Fc, 525-69, and 525-69-Fc) can bind inter-protein, although bivalent 69-525 does so with a lower potency compared to bivalent 525-69 or to the tetravalent Fc-fusion antibodies. Mean with SEM are given for 2 independent experiments. The cartoon (upper left corner) visualizes the experiment using a bispecific single domain antibody (SdAb). b) HA derived from B/Washington/02/2019 was immobilized (HA binding) on biosensors and saturated (SdAb binding) with head-to-tail SdAbs (525-69, 69-525, 69-69, or 525-525). Sensors were transferred (NA binding) to wells containing NA derived from B/Washington/02/2019 and binding was observed for bispecific constructs. Representative experiment of 2 independent experiments is shown. The cartoon (top) visualizes the different steps using a bispecific SdAb construct.

Figure 30. Monospecific bivalent VHH69-Fc and VHH525-Fc, as well as tetravalent bispecific VHH69- 525-Fc and VHH525-69-Fc, can induce Fey receptor-mediated effector functions. MDCK cells infected with influenza B/Washington/02/2019 virus were used as target cells for measuring VHH-Fc-mediated effector functions with a human FcyRllla-expressing Jurkat reporter cell line. VHH-Fc (LALAPG) or monoclonal antibody CR9114 (LALAPG) were used at the indicated concentrations. Monospecific bivalent (a, b) or bispecific (c, d) VHH-Fc constructs were tested. Relative luminescence units (RLU) represent the luciferase activity of the reporter cells incubated with infected MDCK cells, subtracted with the luciferase activity of the reporter cells incubated with non-infected MDCK cells, incubated with the indicated VHH-Fc constructs or CR9114. b, d The statistical differences between the areas under the curve (AUC) of the graphs in a and c were determined by one-way ANOVA with Sidak correction: **** p < 0.0001. Data represent the compiled data from 2 independent experiments, with two technical repeats each. Error bars represent SD.

DETAILED DESCRIPTION

Definitions

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).

'Nucleotide sequence', "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and singlestranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" or "construct sequence(s)" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. "Coding sequence" or a "nucleic acid molecule encoding" is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. An "expression vector" comprises an expression cassette which in turn comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell.

The terms "protein", or "polypeptide", are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for instance after tryptic digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation, and also myristoylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). A "protein domain" is a distinct functional and/or structural unit in a protein. Usually a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.

By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., an antibody or VHH as identified and disclosed herein which has been removed from the molecules present in the sample or mixture, such as a production host, that are adjacent to said polypeptide. An isolated protein or peptide can be generated by amino acid chemical synthesis or can be generated by recombinant production or by purification from a complex sample.

The term "linked to", or "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "genetic fusion", e.g., by recombinant DNA technology, as well as to "chemical and/or enzymatic conjugation" resulting in a stable covalent link.

"Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the percentage of identity is calculated over a window of the full-length sequence referred to. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity, which is hereby defined as a 'functional variant'. A 'functional variant' thus also refers to variants comprising one or more substitutions or mutations, resulting in a homologue, preferably of at least 70 %, at least 80%, or at least 90 % , or even 95 % amino acid identity, wherein the functionality is retained or at least similar as compared to the wild type protein or reference protein.

The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified", "mutant", "engineered" or "variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wild- type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. A 'functional variant' thus also refers to 'variants' as compared to the wild type, though with the limitation that such a functional variant has retained function and properties relevant for its function. With 'function' in view of the present disclosure is referred for instance to the function of the VHH or VHH-based product, which preferably is the specificity for NA and/or HA binding, as well as the neutralizing capacity of IBV or Influenza virus.

"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. In the present application, with specific binding of HA or NA proteins as present in Influenza B virus, this means that those protein targets are bound by said VHHs with high affinity. Thought this does not exclude that other HA or NA protein, such as those with Influenza A origin may be bound by said VHHs. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding.

The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favourably associates with another chemical entity or binding domain, such as a compound, proteins, peptide, antibody or Nb, among others. For antibody-related molecules, the term "epitope" or "conformational epitope" is also used interchangeably herein. The IBV HA and/or NA proteins herein described comprise a binding pocket or binding site which includes, but is not limited to an ISVD binding site. The term "part of a binding pocket/site" or "partial epitope" refers to less than all of the amino acid residues that define the binding pocket, binding site or epitope. For example, the atomic coordinates of residues that constitute part of a binding pocket may be specific for defining the chemical environment of the binding pocket, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or noncontiguous in primary sequence.

The term "epitope" refers to a region of a molecule or molecular complex, also called the antigen in particular cases, that, as a result of its shape and charge, favourably associates with another chemical entity or binding domain, such as a compound, proteins, peptide, antibody or ISVD or Nb, among others. An "epitope", as used herein, thus refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule, such as NA or HA. Said epitope may comprise at least one amino acid that is essential for binding the binding agent, though preferably comprise at least 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance, cryo-EM, or other structural analyses, including at least the ones exemplified herein to determine the HA and NA conformational epitopes respectively.

The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. "Antibodies" can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "active antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, defined by 6 CDRs. Non-limiting examples of active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. The term "antibody fragment" and "active antibody fragment" or "functional variant" as used herein refers to a protein comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding IBV NA or HA. Antibodies are typically tetramers of immunoglobulin molecules. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VLwill contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, binds to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. An "immunoglobulin single variable domain (ISVD)" as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An "immunoglobulin domain" of this invention refers to "immunoglobulin single variable domains" (abbreviated as "ISVD"), equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR's. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL- sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH- sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH- sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody , reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. For numbering of the amino acid residues of an IVD different numbering schemes can be applied. For example, numbering can be performed according to the AHo numbering scheme for all heavy (VH) and light chain variable domains (VL) given by Honegger, A. and Pluckthun, A. (J. Mol. Biol. 309, 2001), as applied to VHH domains from camelids. Alternative methods for numbering the amino acid residues of VH domains, which can also be applied in an analogous manner to VHH domains, are known in the art. For example, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from camelids in the article of Riechmann, L. and Muyldermans, S., 231(1-2), J Immunol Methods. 1999. It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein. Determination of CDR regions may also be done according to different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745). Or alternatively the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22)). Those annotations exist for numbering amino acids in immunoglobulin protein sequences, though in the present application solely the Kabat numbering is used, or the specific SEQ. ID numbering, as indicated. Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any immunoglobulin protein sequences without undue burden. These annotations differ slightly, but each intend to comprise the regions of the loops involved in binding the target.

Immunoglobulin single variable domains (ISVDs) such as Domain antibodies and Nanobody® (including VHH domains) can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody® (including VHH domains) may be immunoglobulin single variable domains in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that corresponds to a humanizing substitution. Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody® (including VHH domains) may be partially humanized or fully humanized.

Humanized immunoglobulin single variable domains, in particular Nanobody®, may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. By humanized is meant mutated so that immunogenicity upon administration in human patients is minor or non-existent. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence and/or VHH still retains the favourable properties of the VHH, such as the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee. A human consensus sequence can be used as target sequence for humanization, but also other means are known in the art. One alternative includes a method wherein the skilled person aligns a number of human germline alleles, such as for instance but not limited to the alignment of IGHV3 alleles, to use said alignment for identification of residues suitable for humanization in the target sequence. Also a subset of human germline alleles most homologous to the target sequence may be aligned as starting point to identify suitable humanisation residues. Alternatively, the VHH is analyzed to identify its closest homologue in the human alleles and used for humanisation construct design. A humanisation technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is known from literature, are from known humanization efforts, as well as from human consensus sequences compared to the natural VHH sequences, or the human alleles most similar to the VHH sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5-A-8 of WO 08/020079, some amino acid residues in the framework regions are more conserved between human and Camelidae than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single domain antibodies contain the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by other substitutions, such as at position 103 that substitutes the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanisation. Indeed, some Camelidae VHH sequences display a high sequence homology to human VH framework regions and therefore said VHH might be administered to patients directly without expectation of an immune response therefrom, and without the additional burden of humanization.

Suitable mutations, in particular substitutions, can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (reference is made for example to WO 2012/175741 and WO2015/173325), for example at at least one of the positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined below) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequence, VHH or polypeptide of the invention, such deletions and/or substitutions may also be designed in such a way that one or more sites for posttranslational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation.

In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see W02008/020079 Table A-03). Another example of humanization includes substitution of residues in FR 1, such as position 1, 5, 11, 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 10 103, 104, 108 and/or 111 (see W02008/020079 Tables A-05 -A08; all numbering according to the Kabat). Humanization typically only concerns substitutions in the FR and not in the CDRs, as this could/would impact binding affinity to the target and/or potency.

'Antigen-binding proteins' , 'antigen-binding polypeptides' or 'antigen-binding domains' as described herein provide for polypeptides preferably provided as a single chain molecule or present as covalently linked polypeptide chains such as in the format of an antibody. The antigen-binding property of said polypeptide or protein refers to its characteristics of begin capable to specifically bind one or more antigens, and thus refers to for instance those antigen-binding domains that are derived from an antibody as described herein, or are derived from alternative antigen-binding proteins with a different fold, so non-immunoglobulin binding proteins such as but not restricted to avimers, DARPins, alphabodies, affitins, nanofitins, anticalins, monobodies and lipocalins. The term 'antibody' or 'Fc-fusion' or 'ISVD-Fc fusion' or 'VHH-Fc fusion' as used herein further refers to the genetic linking or fusion of antigen-binding fragments or antigen-binding domains with an Fc constant domain as to obtain dimers forming an antibody structure when expressed in a recombinant host. In particular, antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N-terminus of an Fc domain, preferably via a linker or hinge region. Alternatively, antibody fragments, or single domain antibodies such as ISVDs, may be fused at the N-terminus to the C-terminal end of an Fc domain, preferably via a linker or hinge region. Said single domain antibody or ISVD fused to said Fc may comprise one or more VHHs or Nbs, as described herein.

"Fc domains" or "Fc-regions" or "Fc-tails", as interchangeably used herein, and refer to the single Fc chain and/or the dimeric Fc domain of an Fc-containing proteins. Specifically in antibodies, said Fc domain is thus responsible for antibody function, and 'antibody Fc engineering' stands for engineering functions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life. Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post- translational modifications, dimerization behavior, effector function, serum half life, among others. To indicate the variations present in Fc domains based on the sequence of naturally occurring IgGs, conventional antibody numbering annotations are known in the art, such as for instance IMGT numbering (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)), or preferably used herein EU numbering (Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.;63:78-85).

As used herein, a "therapeutically active agent" or "therapeutically active composition" means any molecule or composition of molecules that has or may have a therapeutic effect (i.e. curative or prophylactic effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. The binding agent or the composition, or pharmaceutical composition (described below), of the invention may act as a therapeutically active agent, when beneficial in treating patients infected with or at risk of infection with Influenza virus, specifically Influenza B virus. The therapeutically active antigen-binding protein or therapeutically active or pharmaceutical composition may include an antigen-binding protein comprising an ISVD specifically binding the IBV NA and/or HA proteins and/or may contain or be coupled to additional "functional groups", interchangeably called "functional moieties" herein, which are advantageous when administered to a subject. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the art as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments, for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to the ISVD, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a immunoglobulin single variable domain of the invention, a immunoglobulin single variable domain of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of an ISVD or active antibody fragment of the invention, all using techniques of protein engineering known per se to the skilled person. Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the antibody or active antibody fragment. Another technique for increasing the half-life of a binding domain may comprise the engineering into bifunctional or bispecific domains (for example, one ISVD or active antibody fragment against the IBV HA or NA antigen and one against a serum protein such as albumin aiding in prolonging half-life) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin).

As used herein, the terms "determining," "measuring," "assessing,", "identifying", "screening", "addressing", "testing", and "assaying" are used interchangeably and include both quantitative and qualitative determinations. "Similar" as used herein, is interchangeable for alike, analogous, comparable, corresponding, and -like or alike, and is meant to have the same or common characteristics, and/or in a quantifiable manner to show comparable results i.e. with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less.

The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, or is expected to be at high risk of developing a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication or a treatment designed and used to prevent a disease from occurring, herein referred to as "prevention".

A "composition" relates to a combination of one or more active molecules, and may further include buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance. Suitable conditions as used herein could also refer to suitable binding conditions, for instance when antigen-binding protein comprising ISVDs are aimed to bind IBV NA and/or HA surface antigens.

A "pharmaceutical composition" is a therapeutically active composition comprising the one or more antigen-binding agents or therapeutically active agents or therapeutically active compositions and optionally comprising a carrier, diluent or excipient. A "carrier", or "adjuvant", in particular a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, or preservatives may be included in such vehicles. A pharmaceutically effective amount of polypeptides, or conjugates of the invention and a pharmaceutically acceptable carrier is preferably that amount which produces a result or exerts an influence on the particular condition being treated. For therapy, the pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, and the like. Still other techniques of formulation as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be taken into account by the clinician. The pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012). The dosage and concentration of the carrier, excipient and stabilizer should be safe to the subject (human, mice and other mammals), including buffers such as phosphate, citrate, and other organic acid; antioxidant such as vitamin C, small polypeptide, protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as PVP, amino acid such as amino acetate, glutamate, asparagine, arginine, lysine; glycose, disaccharide, and other carbohydrate such as glucose, mannose or dextrin, chelate agent such as EDTA, sugar alcohols such as mannitol, sorbitol; counterions such as Na+, and /or surfactant such as TWEEN™, PLURONICS™ or PEG and the like. Detailed description

The present invention relates to the targeted selection of single domain antibodies, in particular ISVDs, even more in particular nanobodies, with a broad range neutralization potential in Influenza B virus strains as to prevent and overcome resistance and escapes for these antivirals upon future Influenza seasons. This unique approach has resulted in the isolation of four immunoglobulin single variable domain (ISVD) families, with as most potent representative IBV hemagglutinin (HA) -specific ISVDs VHH69 and VHH75, and as most potent representative IBV neuraminidase (NA) -specific ISVDs VHH504 and VHH525. These were selected from llamas immunized with a pDNA cocktail expressing 10 different IBV HAs or NAs, according to an approach called epitope dilution phenomenon (EDP). The rationale for choosing this immunization strategy is based on the fact that an antigen cocktail focuses humoral response on epitopes that are common, and thus conserved. Similarly to a vaccination strategy used previously to broaden the humoral response for the Plasmodium falciparum AMA-1 protein (Kusi et al., 2009), by diluting out the antigen, or epitope dilution, the goal was to find broadly conserved epitopes for HA and NA Influenza B proteins, since this approach seems especially well-suited for targeting Influenza B since in general diversity is less as compared to IAV (Hernandez-Davies, et al., 2021; Zhuang et al., 2019). However, so far, it was not clear from the literature how many antigens are required to adequately cover all influenza B HA's /NA's and what diversity is needed (Carter et al., 2013; Prabakaran et al., 2014; Schwartzman et al., 2015), as circulating strains of influenza B viruses derive from two distinct lineages.

The immunization was based on the choice of ten different IBV strains in view of their the antigenic phylogeny, with five belonging to the Victoria-lineage, and fiver to the Yamagata-lineage. Together they span over 20 years of antigenic drift. Immunization with ten different DNA encoded IBV hemagglutinin or neuraminidase proteins yielded broad seroconversion in the respective llamas.

The present invention relates thus to the selection of superior VHH families targeting IBV NA or HA surface antigens in a unique manner, which is clear from the structural epitopes that have been identified herein, in view of developing antigen-binding agents which are capable of broadly neutralizing IBV strains through targeting conserved epitopes to avoid resistance and escapes in future flu seasons. Hence the antigen-binding agents or proteins of the invention may be applied in several ways. Moreover, due to their high potency as monovalent NA and/or HA binders, these VHHs outperform currently available antibodies or single domain antibodies which is beneficial to apply such small monovalent building blocks in multispecific antibody assets targeting a broad range of IBV strains and even to combine them in a superior manner with assets targeting different viruses (e.g. IAV strains or other viral targets). In a first aspect, the invention provides for the broadest possible coverage of antigen-binding proteinbased antivirals by combining the ISVDs against both targets, HA and NA, into a bi- or multi-specific antigen-binding protein as to potently neutralize Influenza B virus, and for use in treatment or prevention of Influenza infection in a subject. Several approaches have been reported wherein antivirals against both targets are combined or wherein a combination is anticipated as to improve the efficacy. However, this is the first time that a single chain biological with a bispecific targeting mode on IBV is reported, more specifically targeting HA and NA, and remarkably revealing a synergistic improvement in potency when comparing the bispecific antiviral with a composition using an equimolar mixture of the single targeting antigen-binding proteins. Moreover, such bi- or multispecific binders are advantageous when a simultaneous binding or targeting of NA and HA is desired upon administration of the binding agent. The proposed antigen-binding protein or antibody targeting IBV by comprising an NA-specific ISVD and an HA-specific ISVD thus provides for a next generation developable clinical asset in treatment and prevention of Influenza.

In one embodiment, said bispecific antigen-binding protein or antibody comprising said HA-specific ISVD and NA-specific ISVD neutralizes IBV strains classified under both lineages: B/Yamagata/16/88- lineage and B/Victoria/2/87-lineage. As used herein, a "neutralizing" antigen-binding protein or binding agent refers to an agent that binds one or more target proteins on a virion, specifically as described herein the antigen-binding proteins specifically bind IBV HA and/or NA protein, wherein upon said binding the agent inhibits or reduces at least one biological activity of the virion (e.g. entry of the IBV- virion into a host cell), thereby having the capacity to be "neutralizing" to the extent that such viral activity is affected, optimally thereby preventing or reducing viral infection of the host. With inhibiting or reducing the biological activity as to obtain a neutralizing effect, it is meant herein that the agent is inhibits, reduces or alters the activity by showing an extent of at least 10 % lower activity as compared to a virion or target protein in the absence of the binding agent, or in the presence of a control agent, or preferably of at least 20 % lower activity, or at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % lower activity, or undetectable levels of activity, as compared to a control or vehicle.

In a specific embodiment described herein, the NA-specific ISVD as part of the antigen-binding proteins described herein, targets the NA protein by binding to the catalytic or active site of said neuraminidase protein, thereby inhibiting its neuraminidase activity. The active site inhibition of the NA-specific ISVDs of the present invention further envisages that said ISVD upon binding IBV NA competes with or even blocks the binding of known antivirals that bind to the active site, such as zanamivir and peramivir, among others. The term "competing agent" or "antigen-binding protein or ISVD competing for" or "ISVD competing with" herein thus refers to said antigen-binding protein which specifically binds to an antigen whereby said binding resulting in an inhibition or blocking of another binding agent (e.g. an active site NA inhibitor) to that same antigen or target, often in such a way that binding of one inhibits or blocks the binding of the other one, e.g. by steric hindrance. Competition may be measured by methods known in the art, such as an AlphaLISA, and as further exemplified herein. With 'competing' is meant that the binding of one NA binder, in particular active site or orthosteric binding agent, as described herein is reduced with at least 30 %, or at least 50 %, or preferably at least 80 % in strength in the presence of said competing binding agent.

In another specific embodiment, the HA-specific ISVD as part of the antigen-binding proteins described herein, targets the HA protein by binding to a conserved region of said hemagglutinin protein, in particular to the stalk or stem region, which is closer to the membrane and less prone to mutations. Indeed also for IBV HA, the diversity is mainly to be found in the immunogenic head domain of the trimer, while big patches of conserved residues make up the stalk of the IBV trimer. In a specific embodiment, the HA-specific ISVDs comprised in the antigen-binding proteins of the present invention thus bind to an epitope that , at least partially, is a region present in the stalk or stem of the HA trimer, and preferably binds more than one protomer of said trimer, thereby increasing its neutralization potential. In a particular embodiment, said HA-specific ISVDs comprised in the antigen-binding proteins of the present invention neutralize the IBV virions by inhibiting the fusion process.

In particular, from the structural data obtained on the complex of hVHH-69 with IBV HA, it was shown that the HA-binding VHH binds two HA protomers, with contact residues on both HA1 and HA2, essentially holding the trimer together and thereby preventing or strongly hindering HA-mediated membrane fusion. In contrast, most of the described broad IBV HA-binding antibodies bind only one protomer. Human monoclonal antibody CR8071, for example, binds the vestigial esterase domain in the HA-head and can microneutralize B/Yamagata more potently than B/Victoria-lineage viruses. CR9114 can bind IBV HA broadly, but does not microneutralize IBV in vitro. The epitope of the IBV HA stem-specific SdAb SD83 shares conserved contact residues with hVHH-69 in the fusion subdomain, but contact residues of SD83 are also limited to one protomer. The dominance of strategies targeting a single protomer of HA in the current state of the art is thus different to the HA-specific VHHs obtained in the approach used herein.

The first aspect of the invention thus covers all embodiment wherein the antigen-binding protein or polypeptide or antibody as described herein is a multispecific binding agent, in particular with specificity at least for targeting IBV NA and HA surface antigens. Said multispecific agents moreover exclusively bind to IBV HA and NA antigen in the sense that they do not bind or neutralize IAV virions. The binding moieties, in particular the ISVDs, within said multispecific protein may be directly linked, or fused by a linker or spacer. The composition or antigen-binding proteins as described herein may appear in a "multispecific" form and thus be formed by bonding, chemically or by recombinant DNA techniques, together two or more identical or different binding agents. Said multispecific forms may be formed by connecting the building blocks, in particular the ISVDs, directly or via a linker, or through fusing the building block(s) with an Fc domain encoding sequence. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, and so on. The immunoglobulin single variable domains comprised within a multispecific construct may be identical, but at least one ISVD should be different, preferably binding to a different binding site or different antigen. In another particular embodiment, the antigen-binding proteins or antibodies of the invention are in a "multispecific" form and are formed by bonding together two or more building blocks or agents, of which at least one binds to one epitope or binding site on IBV HA, and at least one binds to another epitope or binding site on IBV HA. Alternatively, a "multispecific" form is formed by bonding together two or more building blocks or agents, of which at least one binds to one epitope or binding site on IBV NA, and at least one binds to another epitope or binding site on IBV NA, with at least one of said building blocks preferably binding to the active site of NA. Furthermore, a "multispecific" form may be formed by bonding together two or more building blocks or agents, of which at least one binds to one epitope or binding site on IBV HA, and at least one binds to one epitope or binding site on IBV NA, preferably the active site of NA. Optionally, said multispecific antigen-binding protein comprises a building block which binds to a further target or alternative molecule, such as for instance but not limited to, HA or NA derived from a Influenza A, or for example a building block as a half-life extension against Serum Albumin or SpA, or another target. So a multispecific fusion or protein or antibody as provided herein is capable of specifically binding two or more epitopes or targets, thus comprising binders with a different specificity.

Multivalent or multi-specific ISVDs of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for the desired IBV interaction, and/or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multispecific immunoglobulin single variable domains. Upon binding IBV, through HA and/or NA, said multi-specific or multivalent binding agent may have an additive or synergistic impact on the binding and/or therapeutic effect on IBV, such as an increase in its potency for blocking or inhibiting viral entry. In another embodiment, the invention provides a polypeptide comprising any of the immunoglobulin single variable domains according to the invention, either in a monovalent, multivalent or multispecific form. Thus, polypeptides comprising monovalent, multivalent or multispecific nanobodies are included here as non-limiting examples. The multivalent or multispecific binders or building blocks may be fused directly or fused by a suitable linker, as to allow that the at least two binding sites can be reached or bound simultaneously by the multivalent or multispecific agent.

Alternatively, at least one ISVD as described herein may be fused at its C-terminus to an Fc domain, for instance an Fc-tail of an Ig, resulting in an antigen-binding protein of bivalent format wherein two of said VHH-lg Fes, or humanized forms thereof, form a heavy chain only-antibody-type molecule through disulfide bridges in the hinge region of the Fc part, called "Fc fusion" herein. In a specific embodiment, the multivalent or multispecific agent as described herein is an Fc fusion or an antibody. Another embodiment relates to an antigen-binding protein or antibody comprising a humanized ISVD specifically binding HA and/or a humanized ISVD sequence specifically binding NA, as described herein, comprised in a multivalent or multispecific agent, which may be provided as a humanized ISVD-IgG or ISVD-Fc fusion, and which may further include but is not limited to the use of IgG humanization variants known in the art, such as C-terminal deletion of Lysine, alteration or truncation in the hinge region, LALA or LALAPG mutations as described, among other substitutions in the IgG sequence. In an alternative setup, the "Fc fusion" is designed by linking the C-terminus of such a bivalent or bispecific binder fused by a linker to an Fc domain, which then upon expression in a host forms a multivalent or multispecific-antibody-type molecule through disulfide bridges in the hinge region of the Fc part.

Further multivalent or multispecific agents are also comprised herein, wherein said ISVDs comprised in said multispecific or multivalent agent comprises any one of said humanized variants of the ISVDs disclosed herein, and/or is present in a multispecific or multivalent antibody format known in the art, such as any type of Fc (native or variant IgG, or in particular IgGl Fes), or in head-to-tail multivalent or multispecific format, or a combination of head-to-tail and Fc fusion, and/or N- and C-terminally fused ISVDs to an Fc tail; and/or a knob-into-hole Fc fusion format, among others.

As supported by the examples herein, the formats that are described herein may envisage thus monovalent ISVDs (VHHx), monospecific Fc fusions (VHHx-Fc) which will form a bivalent antibody upon recombinant expression, bivalent head-to-tail ISVD fusions (VHHx-VHHx) or bispecific head-to-tail ISVD fusions (VHHx-VHHy) or Fc fusions of any thereof (VHHx-VHHx-Fc or VHHx-VHHy-Fc) which will result in monospecific or bispecific tetravalent antibodies upon recombinant expression, respectively. The invention also envisages alternative antibody formats, exemplified in a non-limiting way herein for instance as monospecific or bispecific fusions to an N- and C-term of the Fc tail (VHHX-Fc-VHHx or VHHx- Fc-VHHy, resp), which upon recombinant expression in a cell also result in antibodies of tetravalent format.

In another aspect the antigen-binding protein is a multispecific protein comprising an ISVD specifically binding NA, and an ISVD specifically binding HA, wherein said ISVD specifically bind a conformational epitope on IBV NA and HA, respectively, as determined herein for VHH525 (or hVHH525) and for VHH69 (or hVHH69), respectively, or an ISVD competing for binding to IBV NA and HA with VHH525 and VHH69, respectively. More specifically, a multispecific agent is envisaged comprising an ISVD targeting the IBV NA epitope as provided in Example 9, and comprising an ISVD targeting the IBV epitope as provided in Example 3.

A further aspect relates to an antigen-binding protein comprising an ISVD specifically binding NA which neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage, preferably wherein said ISVD specifically binds the epitope of NA as determined for hVHH525 in Example 9. A further aspect relates to an antigen-binding protein comprising an ISVD specifically binding HA which neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage, preferably wherein said ISVD specifically binds the epitope of HA as determined for hVHH69 in Example 3.

In a further aspect of the invention, the antigen-binding protein may be a multispecific, preferably at least bispecific protein, or may be a monovalent or multivalent antigen-binding protein, as further described herein, wherein said protein, antibody or agent comprises at least one or more of the following ISVDs: an ISVD which specifically binds the IBV NA protein capable of neutralizing IBV virions, preferably of both lineages B/Yamagata/16/88 and B/Victoria/2/87; an ISVD which specifically binds the IBV NA protein through binding of the CDRs as depicted in any one of SEQ ID NO:l-3, more specifically wherein said CDR1, CDR2, and CDR3 are defined by anyone of the annotations known in the art, and as provided for VHH504 and VHH525 in Figure 18 further herein, specifically according to MacCallum, Kabat, Chothia, IMGT or AbM annotation; an ISVD which specifically binds the IBV NA protein through binding of the CDRs, wherein CDR1 is annotated according to AbM annotation and CDR2 and 3 are annotated according to Kabat annotation:

CDR1 comprises SEQ ID NO: 19, CDR2 comprises SEQ ID NO: 20, and CDR3 comprises SEQ ID NO: 21;

CDR1 comprises SEQ ID NO: 22, CDR2 comprises SEQ ID NO: 23, and CDR3 comprises SEQ ID NO: 24; an ISVD which specifically binds the IBV NA protein comprising a sequence selected from the group of sequences of SEQ ID NO: 1-3, representing VHH504, 525 and 508, resp. or a functional variant of any one thereof with at least 80%, at least 90 %, or at least 95% amino acid identity thereof, calculated over the full length of the ISVD sequence wherein the CDR remain identical to the CDRs as described above, and the variations are located in one or more framework residues; an ISVD which specifically binds the IBV NA protein comprising a humanized sequence variant said VHH504, 525 or 508 ISVDs, wherein the sequence of said humanized variant is selected from the group of sequences of SEQ ID NO: 7-9, or an alternative humanized variant of any one SEQ ID NOs: 1-3, as for instance provided in the alternative substitutions and combinations disclosed in Figure 18; and/or an ISVD which specifically binds the IBV HA protein capable of neutralizing IBV virions, preferably of both lineages B/Yamagata/16/88 and B/Victoria/2/87; an ISVD which specifically binds the IBV HA protein through binding of the CDRs as depicted in any one of SEQ ID NO:4-6, more specifically wherein said CDR1, CDR2, and CDR3 are defined by anyone of the annotations known in the art, and as provided for VHH69 and VHH75 in Figure 18 further herein, specifically according to MacCallum, Kabat, Chothia, IMGT or AbM annotation; an ISVD which specifically binds the IBV HA protein through binding of the CDRs, wherein CDR1 is annotated according to AbM annotation and CDR2 and 3 are annotated according to Kabat annotation, as this annotation for the binding site became clear based on the structural insights obtained for the complex of one VHH family, so wherein :

CDR1 comprises SEQ ID NO: 13, CDR2 comprises SEQ ID NO: 14, and CDR3 comprises SEQ ID NO: 15;

CDR1 comprises SEQ ID NO: 16, CDR2 comprises SEQ ID NO: 17, and CDR3 comprises SEQ ID NO: 18; an ISVD which specifically binds the IBV HA protein through binding of the CDRs, wherein CDR1 is annotated according to AbM annotation and CDR2 and 3 are annotated according to Kabat annotation, as this annotation for the binding site became clear based on the structural insights obtained for the complex of one VHH family, so wherein :

CDR1 comprises SEQ ID NO: 13, CDR2 comprises SEQ ID NO: 14, and CDR3 comprises SEQ ID NO: 15;

CDR1 comprises SEQ ID NO: 16, CDR2 comprises SEQ ID NO: 17, and CDR3 comprises SEQ ID NO: 18; and/or wherein the FR2 residues 44 and 64 (according to Kabat numbering) are an Glu € residue contributing to the HA binding; an ISVD which specifically binds the IBV HA protein comprising a sequence selected from the group of sequences of SEQ ID NO: 4-6, representing VHH69, 75 and 72, resp. or a functional variant of any one thereof with at least 80%, at least 90 %, or at least 95% amino acid identity thereof, calculated over the full length of the ISVD sequence wherein the CDR remain identical to the CDRs as described above, and the variations are located in one or more framework residues; an ISVD which specifically binds the IBV HA protein comprising a humanized sequence variant said VHH69, 75 or 72 ISVDs, wherein the sequence of said humanized variant is selected from the group of sequences of SEQ ID NO: 10-12, or an alternative humanized variant of any one SEQ ID NOs: 4- 6, as for instance provided in the alternative substitutions and combinations disclosed in Figure 18.

The CDR region annotation for each VHH sequence described herein slightly differ depending on the cited reference, as also cited and incorporated herein above, but have been shown at least for HA- specific binding to match the AbM and Kabat annotation, though the loop regions of the ISVD form the CDRs and depending in the target protein may vary in their interaction positions.

VHHs or Nbs are often classified in different sequences families or even superfamilies, as to cluster the clonally related sequences derived from the same progenitor during B cell maturation (Deschaght et al. 2017. Front Immunol. 10; 8 :420). This classification is often based on the CDR sequence of the Nbs, and wherein for instance each Nb family is defined as a cluster of (clonally) related sequences with a sequence identity threshold of the CDR3 region. Within a single VHH family defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80 % identity, or at least 85 % identity, or at least 90 % identity in the CDR3 sequence, resulting in Nbs of the same family binding to the same binding site, having the same effect or functional impact.

Humanized and/or functional variants are obtained as described herein, and are based on primary sequence alignment with the human IGHv3 coding sequence, to substitute one or more key residues of the alpaca-derived framework regions of the VHHs, followed by biophysical analysis of the resulting VHHs after recombinant production. Specifically, the stability and neutralizing properties of the resulting VHHs are analysed. Moreover, said original and/or humanized variant sequence can be fused directly or via a linker, as to provide for (humanized) bivalent or bispecific or multispecific VHH variants, as tandem repeats, or head-to-tail fusion, as interchangeably used herein. Alternatively, they may be additionally fused to an Fc tail, more specifically a human IgGl Fc.

Examples of the possible humanization variants of the monovalent VHHs or ISVDs have been disclosed in a non-limiting manner herein, wherein said humanized variant of the ISVD of said antigen-binding protein comprises: at least one of the following substitutions (according to Kabat numbering): 1D/E, 5V, 14P, 16G, 46E, 71R, 73X, preferably 73N or D, 74X, preferably A or I, 75K, 76N, 77T, 81N, 83R, 91Y, and/or 108L.

In further aspects of the invention, the antigen-binding protein(s) as described herein may be labelled, tagged or conjugated. More specifically, a detection label may be useful for localizing, visualizing, and quantitating a binding or recognition event, hence also for in vivo imaging or for diagnostic purposes. The labelled antigen-binding proteins as described herein can detect NA and/or HA present on the surface of an influenza B virion. Another use for detectably labelled antigen-binding proteins of the invention is a method of bead-based immunocapture comprising conjugating a bead with a fluorescent labelled binding agent and detecting a fluorescence signal upon binding of a ligand. Similar binding detection methodologies utilize the surface plasmon resonance (SPR) effect to measure and detect antigen-binding protein/antigen interactions.

The term detectable label or tag, as used herein, refers to detectable labels or tags allowing the detection and/or quantification of the HA and/or NA-specific antigen-binding polypeptide of antibody as described herein, and is meant to include any labels/tags known in the art for these purposes. Particularly preferred, but not limiting, are affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin and cyanine); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease or glucose oxidase); radioisotopes. Also included are combinations of any of the foregoing labels or tags. Technologies for generating labelled polypeptides and proteins are well known in the art. An antigen-binding protein comprising a HA- and/or NA-specific ISVD of the invention, coupled to, or further comprising, a label or tag allows for instance immune-based detection. Immune-based detection is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as described above. See, for example, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In the case where multiple antibodies are reacted with a single array, each antibody can be labelled with a distinct label or tag for simultaneous detection. Yet another embodiment may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, or tags, depending on the intended use of the labelled or tagged IBV-specific binding agent of the present invention. Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. Such labelled antigen-binding agents, such as HA- and/or NA-specific ISVDs or Nanobodies for the detection of IBV, as described herein may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes, depending on the choice of the specific label.

The labelled or tagged binding agents as described herein may also be used as an affinity purification agent. In this process, the labelled agent or antigen-binding protein is immobilized on a solid surface, such as a Sephadex, Sepharose or other polymeric resin, or filter paper, or a cartridge, using methods well known in the art. The immobilized binding agent is subsequently contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the antigen to be purified, which is bound to the immobilized binding agent. Finally, the support is washed with another suitable solvent, which is capable to outcompete the binding.

In a further aspect of the invention, the antigen-binding protein or antibody or multi/bispecific fusion protein as described herein may be conjugated to a further functional moiety, such as a therapeutic or half-life extension moiety, or to a cell-penetrant carrier. More specifically, the antigen-binding proteins as described herein may as fusion be further coupled or operably linked to further binding moieties, which may be additional ISVDs, or antigen-binding domains specific for a target protein, preferably a target present on the cell surface or extracellularly, or to extend the half-life (e.g. serum albumin specific binders), or alternative compounds that are providing a function. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Another technique for increasing the half-life of a binding agent may comprise the engineering into bifunctional or bispecific domains (for example, one or more ISVDs or active antibody fragments against IBV coupled to one ISVD or active antibody fragment against serum albumin or pulmonary surfactant protein A (Spa) aiding in prolonging half-life)) or into fusions of antibody fragments, in particular immunoglobulin single variable domains, with peptides (for example, a peptide against a serum protein such as albumin). The coupling to additional moieties will result in multispecific antigen-binding protein agents, as further disclosed herein. In a final aspect of the invention, the antigen-binding protein as described herein which specifically binds IBV HA and/or NA, and has neutralization activity against Influenza, in particular Influenza B virus, is used as a medicament, or for prevention or treatment of viral infection, specifically influenza viral infection, more specifically Influenza B viral infection. "Influenza (B) infection" refers to the invasion of a living subject's tissue by IBV, multiplication of influenza B virus, and/or the reaction of host tissues to the infectious influenza B virions. IBVs are etiological agents of contagious respiratory illness that infect the nose, throat, and lungs, and can cause mild to severe illness, and at times can lead to death.

So in one embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used as a medicine, in terms of prophylaxis or in therapy.

In a preferred embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or resulting antibodies, or the pharmaceutical composition comprising any of the preceding, may be used in prevention or treatment of an influenza infection in a subject, preferably a human.

In another embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used in prevention or treatment of an influenza B infection in a subject, preferably a human.

In another embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used for diagnosing Influenza, specifically Influenza B.

In another embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used for in vivo imaging of IBV infection in a subject, preferably in a human subject.

In another embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used to treat an Influenza infection, specifically to treat Influenza B infection of a subject, preferably a human subject.

In another embodiment, the antigen-binding proteins or antibodies described herein, comprising HA- and /or NA-specific ISVD(s) and capable of neutralizing IBV, or the nucleic acid molecule encoding any of said antigen-binding proteins or antibodies, or the pharmaceutical composition comprising any of the preceding, may be used for treatment of l( B) V infection by intranasal administration.

Indeed, it has been observed herein that although intranasal delivery of molecules is often complex due to stability of the biomolecules, the ISVD-based agents provide for a stability capable to resist degradation and to overcome shear forces often observed with nebulization, resulting in a higher potency as for instance compared to intraperitoneal administration. The latter also allows to lower the dosing significantly. The possibility for intranasal administration makes it possible to deliver therapeutics to the forefront of influenza virus infection and for acting promptly to neutralize any (starting) influenza virus infection.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for methods, samples and products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

EXAMPLES

Example 1. Isolation of HA- and NA-specific VHHs with broad influenza B virus-neutralizing activity.

The aim of this study was to obtain VHHs with broad Influenza virus B (IBV)-neutralizing activity that target conserved epitopes in either IBV HA or NA. Two immune libraries were generated from llamas for which we aimed to broaden the B-cell immune-repertoire by applying a DNA immunization strategy using a diverse set of IBV-HA or -NA expression plasmids, thereby diluting out the antigen, an approach called Epitope Dilution Phenomenon. Therefore, we selected 10 phylogenetically distinct IBV HAs and 10 phylogenetically distinct IBV NAs, each time consisting of 5 B/Yamagata-lineage derived components and 5 B/Victoria-lineage derived components from WHO proposed vaccine viruses (with the exception of B/Memphis/12/97) of the past 40 years. One llama was immunized with a cocktail of 10 HA- expressing vectors (llama 178) and a second llama was immunized with a cocktail of 10 NA-expressing plasmids (llama 179). Both llamas seroconverted as evidenced by HA-binding in ELISA and NA-inhibition activity of immune plasma (Figure 1 and 2, respectively). Interestingly, NA-inhibitory (NAI) activity in plasma from the NA-immunized llama increased roughly 10-fold after the 6^th immunization compared to the 4^th immunization. Peripheral blood mononuclear cells were isolated to construct the VHH phagedisplay libraries from which the presented VHHs with broad IBV inhibitory activity were isolated (Figure

3).

Example 2. Broad IBV-HA binding VHHs that block the influenza B fusion.

Phage-display Library 178B was panned consecutively on B/Phuket/3073/2013 (B/Yamagata-lineage) and B/Washington/02/2019 (B/Victoria-lineage) infected MDCK cells. Periplasmic extracts from ninety VHH clones were prepared and screened for neutralizing activity against a broad panel of IBVs (Figure

4). PE extracts from clone C9 (renamed VHH69- SEQ ID NO:4) and C31 (renamed VHH75- SEQ ID NO:5) were found to have broad IBV-neutralizing activity and unique CDR3 sequences (See Table 3).

Next, we humanized VHH69 and VHH75 to minimize differences between the selected VHHs and the hlGHv3 germline sequence (Figure 6A). The N-terminal glutamine was replaced by a glutamic acid codon to avoid N-terminal pyroglutamate formation. CDR regions remained unaltered except for CDR2 in VHH69: glutamic acid at position 64 was changed to lysine (E64K) to correct the electrostatic charge. However, since this mutation proved detrimental for recombinant HA/B/Washington/02/2019 binding (Figure 6B), this substitution was not included in the final humanized sequence of hVHH-69 (SEQ ID NO:10).

HA mediates attachment of the virus to the host cell and subsequent fusion of the viral and host cell membrane at acidic pH in the endosomes. Mechanistically this membrane fusion can be inhibited by antibodies either by inhibition of proteolytic maturation of HAO into HA1 and HA2 or interference with the subsequent low pH-dependent conformational change. To unravel the mode of action of hVHH-69 and hVHH75, HAO cleavage after incubation with TPCK-treated trypsin was examined with SDS-page. Neither hVHH69 nor hVHH-75 could prevent proteolytic maturation, suggesting that their epitope in HAO does not include the fusion loop of HAO (Figure 7A). Upon acidification, the proteolytically activated HA undergoes a pH triggered conformational change to fuse the viral and host cell membrane. This conformational change renders recombinant HA sensitive to protease cleavage. In a protease sensitivity assay, hVHH69 protects HA/B/Utah/09/2014 and HA/B/Lee/1940 completely, while HA/B/Washington/02/2019 only partially, from the pH-induced protease sensitivity associated with membrane fusion. hVHH75 protects all tested HAs except HA/B/Utah/09/2014 from pH-induced protease sensitivity (Figure 7B). To confirm the inhibition of low-pH HA rearrangement, an assay was performed where the amount of polykaryons is directly related to the HA fusion activity. Both hVHH69 and hVHH75 are able to inhibit polykaryon formation for HA/B/Colorado/06/2017. hVHH75 is not able to inhibit polykaryon formation for HA/B/Wisconsin/01/2010, in contrast to hVHH69 (Figure 8). On both HAs, polykaryon formation can be inhibited in a dose dependent manner starting from precursor HAO or activated (cleaved) HA, indicating the binding epitope is not disturbed by cleavage of HA (Figure 8).

Example 3. Structure, epitope and paratope of hVHH69 in complex with HA/B/Washington/02/2019.

As to resolve the binding site of the hVHH69 Nb, we resolved a 2.97 Angstrom structure of hVHH-69 in complex with HA/B/Washington/02/2019 (recombinantly produced as present in SEQ. ID NO:25).

The HA epitope and hVHH69 paratope were analysed using the PISA algorithm on said structure of the complex of hVHH69 with HA/B/Washington/02/2019 according to Krissinel and Henrick (2007).

Figure 19A shows the VHH structure indicating in sticky representation the side chains of that residues that are part of the paratope. These include the following amino acid residues (according to Kabat numbering): G26, V28, D29, S30 as part of CDR1 (wherein CDR1 is annotated according to AbM); K52, S54, S56, 158, Y59, D61, E64 as part of CDR2 (annotated according to Kabat), and Q96, W97, Y98, S99, G100, GlOOa, YlOOb, YlOOc, GlOOd, D101, and Y102 as part of CDR3 (annotated according to Kabat).

Based on the structure, the epitope was found to involve the residues of the HA protein as shown in table 1, as being in contact with the VHH CDR loops (Figure 19B). More specifically, amino acid residues present in the HA1 and HA2 are found to be in contact with hVHH69, revealing a novel epitope as compared to known antibodies targeting IBV HA (e.g. CR9114 and 5A7).

Indeed, hVHH-69 binds a unique quaternary epitope below the HA-head domain split between two adjacent HA protomers, covering ~ 860 A² of buried surface area (Figure 20). On one side, a set of three tyrosine residues, Y98, YlOOb, and YlOOc of the CDR3 loop of hVHH-69, interact with HA residues D434, E403, and T308/G309 respectively via hydrogen bonds (Y98 and YlOOb) and van der Waals interactions (YlOOc) (Figure 20e). The interaction with HA E403 is additionally stabilized via a salt bridge with K52 of hVHH-69 CDR2 at the tip of strand P4, while residues E44 and E64 in hVHH-69 FR2 and FR3 interact with K38 and K327 respectively (Figure 20e). On the other side of the interaction epitope, W97 of the hVHH-69 CDR3 loop locks into a hydrophobic pocket formed by HA residues L44, T47, V81, 1315 and G316, while salt bridges/hydrogen bonds are formed between hVHH-69 residues D101/Y102 and R80 of HA, hVHH-69 S30 and HA N317, and CDR3 loop Q96 with the backbone nitrogen of HA residue T47 (Figure 2f). The HA epitope involves contact residues that are highly conserved amongst influenza B viruses (Figure 19B). For example, the amino acid at position 80 is always positively charged, i.e. R80 for B/Victoria-lineage HAs and K80 for B/Yamagata-lineage HAs. The amino acid residue at position 81 is always hydrophobic, varying between V81 and A81 for B/Victoria-lineage HA and B/Yamagata-lineage HA respectively, thus shaping the hydrophobic pocket for hVHH-69 W97. Uniquely, hVHH-69 binds the HA stalk of two adjacent protomers (Figure 21). We calculated the free energy contribution per amino residue and identified hotspots ( | AG | > 0.5) on both adjacent HA protomers (Figure 19B). By holding HA protomers together, and simultaneously binding both HA1 and HA2, hVHH-69 is poised to prevent the transition to the post-fusion HA state. We validated our structural model by interrogating the binding of hVHH-69 to cells expressing mutant HA via flow cytometry (Figure 22). Alanine mutants of key interaction residues R80, E403, K327, K38, and N317 altered binding of hVHH- 69 and increased IC₅o values, while such substitutions at T47 and T324 did not (Figure 22).

Amino acid residues contacted by hVHH-69 in HA derived from B/Washington/02/2019 are numbered after alignment with a reference sequence of B/Washington/02/2019 HA, or after alignment with the PDB sequence (9FM1). The percentage abundance of the most prevalent amino acid(s) in the column "amongst all IBV HA" is based on 3837 full-length and non-redundant influenza B HA sequences from the Influenza Virus Resource at the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/genomes/FLU). The percentage abundance per amino acid in the column "amongst B/Victoria-lineage HA" and " amongst B/Yamagata-lineage HA" was calculated after submitting the 3837 sequences to GISAID annotation and retaining respectively 1103 B/Victoria-lineage and 878 B/Yamagata-lineage HAs.

Since the PDB sequence is different in its starting point and misses certain loop regions (according to the structural data), and the reference sequence may slightly differ depending on the inclusion of the N-terminal signal peptide etc., for clarity, the amino acid residue positions comprised in the conformational epitope are indicated (bold underlined) in the protein sequence of B/Washington/02/2019 HA as provided in SEQ ID N0:31 here below:

SEQ ID No: 31: Influenza B/Washington/02/2019 Hemagglutinin

MPMGSLQPLATLYLLGMLVASVLGDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGIETRGKLC PKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIM HDRTKIRQLPNLLRGYEHVRLSTHNVINAEDAPGR PYEIGTSGSCPNITNGNGFFATMAWAVPKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQK FTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIKGSLPLI GEADCLHEKYGGLNKSKPYYTGEHAKA1GNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGM IA GWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIE LAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASL NDDGLDNHTISGKRM KQIEDKIEEIESKQKKIENEIARIKKLIGERSAWSHPQFEK

Example 4. Broad influenza B NA-binding VHHs that occupy the catalytic site.

Enrichment of phage-displayed VHHs with broad IBV NA-binding activity from Lib 179B was performed by 3 consecutive panning rounds on purified, tetrameric, enzymatically active recombinant NA derived from B/Utah/09/2014, B/Washington/02/2019 and B/Lee/1940. PE extracts were prepared from 216 individual clones and were screened in an ELLA-assay. 54 VHH candidates had a broad neuraminidase inhibitory activity (Figure 5). VHHs derived from 21 clones were produced in Pichia pastoris and bench- top purified with NTA beads. Two VHHs with similar CDR3 sequences were broadly neutralizing 100 TCID50 of B/Phuket/3073/2013, B/Washington/02/2019 and B/Lee/1940 virus: VHH504 (SEQ ID NO:1) and VHH525 (SEQ ID NO:2). We humanized VHH504 and VHH525 similarly as before (SEQ ID NO:7 and 8, resp.) and none lost potency on the ELLA assay when humanized on all three tested viruses compared to their original sequences (Figure 9).

Release of newly formed progeny virus from infected cells and elimination of decoy receptors are all supported by the sialidase activity of neuraminidase. Blocking the enzymatic pocket is accepted as the mechanism of action for NAIs (like zanamivir) and this can be examined with the small MUNANA substrate. If the activity is inhibited, most likely the active pocket itself is occupied. Both hVHHs were shown to significantly inhibit NA enzymatic activity with the MUNANA substrate on NA/B/Washington/02/2019 and NA/B/Utah/09/2014. Remarkably, no clear reduction in sialidase activity is seen on the NA/B/Lee/1940 (Figure 10A). To assess the biophysical effects of hVHH-504 and hVHH525 treatment, the egress of virus from infected cells was studied. Immunoblots of HAO detected in the supernatant and lysate of infected MDCK cells show that both hVHH504 and hVHH525 are able to inhibit the egression of the virus from infected cells and have a mechanism of action comparable to zanamivir (Figure 10B).

Example 5. Bivalent and bispecific VHHs as NA inhibitors analyzed in the ELLA assay.

In an effort to increase potency, bivalent constructs were made, expressed in Pichia pastoris and His6- tag purified. To assess the best combinations and intern positions of NA targeting, head-to-tail VHHs of NA-specific hVHH504 and hVHH525, and/or HA-specific hVHH69 and hVHH75, in both directions, N- or C -terminally, were assessed using ELLA experiments on recombinant NA protein and on virus (Figure IOC). On recombinant NA/B/Lee/1940 protein, neither homobivalent or heterobivalent formatting increased the potency. Both VHHs hVHH504 and hVHH525 were potently inhibiting the tested NA/B/Washington/02/2019. Heterobivalent formatting with hVHH504 or hVHH525 N-terminal in combination with hVHH69 C-terminal increased the potency over 10-fold. On the NA/B/Phuket/3073/2013, formatting of hVHH504 or hVHH525 did not increase potency more than 10- fold and overall hVHH525 seemed to be more potent in inhibiting recombinant NA/B/Phuket/3073/2013. On B/Lee/1940 virus, clear improvements were seen with both hVHH504 and hVHH525 as homobivalent constructs. hVHH504 N-terminally combined with either hVHH69 or hVHH75 gave an equal improvement in potency as homobivalency, which is remarkable. For hVHH 525, all tested bivalent/specific combinations improved potency. On the B/Washington/02/2019, no clear improvements were seen, possibly due to the already low IC50s. Remarkably, hVHH504 C-terminal is less well tolerated, as it resulted in higher IC50s. The B/Phuket/3073/2013 tested with hVHH504 N- terminal coupled to C-terminal hVHH69 gave clear improvements. This was not seen when hVHH525 was used instead of hVHH504. Overall potent inhibition was observed using the ELLA assay, both on recombinant NA and on virus, with both hVHH504 and hVHH525. Remarkably, despite hVHH525 being less potent on recombinant NA/B/LEE/1940, an increase in potency was observed for heterobivalent combinations tested on virus. Coupling of anti-HA molecules may increase the potency of the anti-NA hVHHs. A similar feature was observed for hVHH504 on the B/Phuket/3073/2013 virus (Figure IOC).

Example 6. Microneutralization potential of IBV HA and NA targeting VHHs.

To assess the in vitro antiviral potency of the hVHHs, the monovalent and bivalent head-to-tail hVHH constructs were used for microneutralization tests with 100 TCID50 of a broad panel of representative IBV viruses. The anti-HA VHHs, hVHH69 and hVHH75, but none of the anti-NA hVHHs, hVHH504 and hVHH525, microneutralized the ancestral IBV B/Lee/1940 and B/Hong Kong/5/1972 (Figure 11A). Homobivalent combination of anti-NA hVHHs seems to increase potency for B/Lee/1940 but not for B/HongKong/5/1972.

Testing of the hVHHs on B/Yamagata-lineage B/Harbin/07/94 and B/Phuket/3073/2013 often resulted in similar IC50S, except for hVHH75. Remarkably, none of the hVHHs was able to microneutralize the B/Yamagata-lineage B/Florida/4/2006 strain. hVHH75 microneutralizes the B/Victoria-lineage broadly and potently, while hVHH69 and hVHH504 seem to cope with the more recent B/Washington/02/2019 but not with older strains. hVHH525 microneutralizes all tested B/Victoria-lineage viruses with performance being highest against the most recent B/Washington/02/2019 strain (Figure 11A).

Bivalent formatting for hVHH-525-525 is able to microneutralize the B/Florida/4/2006 virus, while this is not the case for hVHH-504-504. hVHH-504-504 increases potency for B/Malaysia/2506/2004 compared to monovalent VHH-504. Also hVHH-525-525 increases potency for B/Brisbane/60/2008 compared to monovalent VHH-525 (Figure 11A).

Head-to-tail bivalent fusions of hVHH-69 and hVHH-75 are all able to microneutralize all tested viruses, with increased potency (Figure 11A). This is in sharp contrast to the monovalent VHHs that sometimes did not neutralize certain viruses.

Furthermore, also single molecule head-to-tail heterobivalent or bispecific VHHs, combining the hVHH69 or hVHH75 with hVHH504 or hVHH525, are able to microneutralize, thus combining anti-HA and anti-NA activity (Figure 11B). Remarkably, these bispecific molecules show an improved potency over the treatment using a combination of the single monovalent VHHs for a number of viruses (Figure 11B).

Compared to equimolar mixes of single monovalent molecules, the bispecific molecules showed a synergistic improvement (fold difference >2) in 55 out of 64 combinations tested (85 %), and even more a synergistic improvement with a fold difference >10 was seen in 20/64 cases (31 %) (Figure 13). In addition, it was shown that hVHH525 and hVHH504 are able to microneutralize a virus with E117A mutation in the NA segment, even with increased potency as compared to WT virus, whereas the E117A mutation significantly reduced susceptibility for peramivir and zanamivir (Figure 12).

Example 7. Microneutralization potential of IBV HA and NA targeting VHH-Fc fusions.

To target both HA and NA with the a bispecific antibody-type molecule, we tested mammalian expressed Fc-fusion constructs in a microneutralization assay (Figure 14). Importantly, no exogenous TPCK treated trypsin was added in this assay since this can influence VHH-Fc stability. Without exogenous TPCK treated trypsin added, ancestral lineages (B/Lee/1940 and B/HongKong/5/1972) did not grow and could not be tested in this assay. All tested VHH-Fc constructs microneutralize with IC50S lower than 100 nM. To assess synergy of the bispecific antibodies (hVHH69-hVHH52-Fc and hVHH525- hVHH69-Fc), an equimolar mix of the monovalent Fc fusions hVHH69-Fc and hVHH525-Fc was included. hVHH69-hVHH525-Fc treatment resulted in synergy, however minimal, on B/Harbin/07/94, B/Brisbane/60/2008, and B/Washington/02/2019 virus as compared to the equimolar mix. Bispecific hVHH525-hVHH69-Fc showed synergy on all viruses except B/Brisbane/60/2008 as compared to the equimolar mix treatment. Both tetravalent monospecific hVHH525-hVHH525-Fc and hVHH69-hVHH69- Fc increased potency compared to monovalent constructs and overall hVHH525-hVHH525-Fc was the most potent construct for all tested influenza B viruses of both B/Yamagata- and B/Victoria-lineages (Figure 14).

In BLI, the NAI peramivir, hvHH504-Fc and hVHH525-Fc do compete for binding the recombinant NA, again indicating the active site epitope is similar in the Fc fusion constructs (Figure 24a). For the anti- HA hVHH69-Fc and hVHH75-Fc, competition for binding to recombinant HA was also confirmed by BLI, as well as the non-competitive binding of HA to CR9114 (Figure 24b). Moreover, the anti-HA hVHH69- Fc and hVHH75-Fc broad range Influenza B virus binding capacity was also confirmed for these constructs as shown in the results in Figure 17.

Finally, further antibody constructs were produced as a tetravalent format in which the NA and HA specific VHHs are combined as bispecific VHH(NA)-Fc-VHH(HA) construct, or vice versa VHH(HA)-Fc- VHH(NA) in which a first VHH is fused to the N-terminus of the Fc via a linker and hinge and were a second (different) VHH, is fused to the C-terminus of a human Fc via a linker. A first set of constructs was designed to fuse VHH69 and VHH525under such bispecific Fc antibody format and after production these were analyzed for their neutralization potency as compared to monospecific Fc fusions (69-Fc or 525-Fc), bispecific head-to-tail Fc fusions (69-525-Fc and 525-69-Fc), or to the equimolar mixture of the monospecific Fc fusions. The IC₅o (nM) values on microneutralization of B/Phuket/3073/2013 and B/Washington/02/2019 virus was tested and indicates that such antibody formats may provide for improved potencies over certain head-to-tail bispecific fusions, suggesting the steric impact may be different in view of otherwise more limiting bispecific fusion format options.

Indeed, as shown in Figure 26, the IC₅o (nM) on microneutralization of B/Phuket/3073/2013 and B/Washington/02/2019 virus for the tetravalent bispecific VHH69-Fc-VHH525 performs ten-fold better as compared to the bispecific head-to-tail VHH69-VHH525-Fc format, and the bispecific VHH525-Fc- VHH69 performs 3 times better as compared to the head-to-tail VHH525-VHH69-Fc format. In fact, especially for 69-525-Fc where microneutralization potency is limited due to steric hindrance of C- terminal VHH525, a VHH-Fc-VHH format is able to increase potency, thereby indicating that the combined use of both NA and HA-specific VHHs within a single chain molecule provides broadly neutralizing highly potent influenza B specific antibodies.

In addition, for the bispecific head-to-tail (69-525, 525-69, 69-69, or 525-525) and bispecific head-to- tail fused to a human IgGl Fc domain (69-525-Fc, 525-69-Fc, 69-69-Fc, or 525-525-Fc) simultaneous binding to IBV HA and NA recombinant proteins was confirmed in 2 independent experiments (Figure 29). Bispecific constructs bind inter-protein, with the tetravalent VHH-Fc antibodies and the bivalent 525-69 head-to-tail fusion having the highest potency. As a negative control, the corresponding monospecific constructs were used.

Example 8. In vivo protection.

Next, we evaluated the in vivo efficacy of the humanized hVHH69-Fc and hVHH525-Fc and compared the prophylactic efficacy in DBA/2J mice challenged with 2 LD₅o mouse adapted B/Washington/02/2019 virus. Mice were treated either intraperitoneally (IP) 24 hours before challenge or intranasally (IN) 4 hours before challenge to evaluate the efficacy of systemically or locally administered VHH-Fc, respectively. 0.5 mg/kg IN or 5 mg/kg IP prophylactic administration of hVHH69-Fc, hVHH525-Fc or an equimolar mix (hVHH69-Fc + hVHH525-Fc) completely protected mice from morbidity and mortality imposed by the mouse adapted B/Washington/02/2019 virus (Figure 15, A and B).

To evaluate the prophylactic effects of VHH-Fc fusions that combined target both HA and NA, dosages of lmg/kg, 0.1 mg/kg or 0.01 mg/kg were administered IP 24 hours before challenge. At a dose of only 1 mg/kg, all tested VHH-Fc fusions protected mice completely against mortality (Figure 16). For hVHH525-Fc, exceptionally, this dose could even be reduced to 0.1 mg/kg. In this prophylactic setting, the synergistic effect as observed previously was not observed for the bispecific hVHH69-hVHH525-Fc and hVHH525-hVHH69-Fc as compared to the equimolar mix (hVHH69-Fc + hVHH525-Fc). CR9114-Fc did not completely protect mice even at the highest dose of 2.5 mg/kg (Figure 16). Remarkably, hVHH525-hVHH69-Fc and hVHH69-Fc+hVHH525-Fc protected better at 0.1 mg/kg compared to hVHH69-hVHH525-Fc against morbidity in this experimental setting. Example 9. Structure of hVHH-525 in complex with IBV NA

To structurally characterize the enzymatic inhibition of NA by hVHH-525, we determined a 3.6 A cryo- EM map of hVHH-525 in complex with NA derived from B/Washington/02/2019 (Figure 23a-d). A data processing strategy combining symmetry expansion, focused 3D classification without alignment, signal subtraction and localized refinement led to an interpretable map for an NA tetramer bound by one hVHH-525 molecule at medium resolution, but with a clearly resolved interaction epitope (Figure 23e- g). hVHH-525 binds the active site perpendicular to the tetramer NA plane, with the majority of interactions mediated by its CDR3 loop. R374 of NA forms the epicenter of the interaction interface, and is sandwiched between NA W408 and hVHH-525 CDR3 loop YlOOa thereby forming a prominent cation-n interaction network. Y98 of the hVHH-525 CDR3 loop sits atop of R374 making van der Waals interactions, while YlOOa makes hydrogen bonds with the backbone N and O atoms of neighbouring G347 (Figure 23e). Additional hydrogen bonds are formed by hVHH-525 D27 and NA S345, CDR2 loop T53 and NA E373, CDR3 loop D100 and NA R292 and a salt bridge between CDR3 loop RIOOb and E275 (Figure 23f, g). To validate the structural model of the NA:hVHH-525 complex, NA residues comprising the structural epitope were mutated to alanine (R374A, R292A, E275A, G347A, and E276A), all of which showed decreased hVHH-525 binding as determined by flow cytometry using cells expressing the mutant NA variants (Figure 23h). The NA epitope bound by hVHH-525 covers 593 A² buried surface area and is highly conserved amongst influenza B NAs (Figure 23i) (Table 2).

Table 2. Amino acid conservation of residues in B/Washington/02/2019 NA contacted by hVHH-525.

This table is based on 4651 full-length and non-redundant influenza B NA sequences from the Influenza

Virus Resource at the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/genomes/FLU) after alignment with a reference sequence of B/Washington/02/2019 NA.

Since the PDB sequence is different in its starting point and misses certain loop regions (according to the structural data), and the reference sequence may slightly differ depending on the inclusion of the N-terminal signal peptide etc., for clarity, the amino acid residue positions comprised in the conformational epitope are indicated (bold underlined) in the protein sequence of

B/Washington/02/2019 NA as provided in SEQ. ID NO:32 here below:

SEQ ID No: 32: Influenza B/Washington/02/2019 Neuraminidase

MPMGSLQPLATLYLLGMLVASVLSAWSHPQFEKGSGIINETADDIVYRLTVIIDDRYESLKNLITLRADRLEIVIIINDNV

STILASIGSGVTLLLPEPEWTYPRLSCPGSTFQKALLISPHRFGETKGNSAPLIIREPFVACGPNECKHFALTHYAAQPG GYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHS YANNILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRIIKEIFPTGRVKHTEECTCGFASNKTIECACRDNRY TAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPCESDGDKGSGGIKGGFVHQRMKSKIGRWYSRTMSKT ERMGMGLYVKYGGDPWADSDALTFSGVMVSMKEPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKETWHSAATAI YCLMGSGQLLWDTVTGVDMAL

Example 10. Polyreactivity assay.

Polyreactive antibodies bind with low affinity to multiple antigens via various noncovalent interactions such as hydrophobic or charge interactions, such as interactions of basic arginine residues in antibody CDRs with acidic phosphate backbone of DNA, resulting in aspecific interactions, binding a variety of self and foreign antigens. Polyreactivity seems to augment in antibodies targeting antigen binding sites which are poorly accessible or buried, as is typically observed for HA stalk binding antibodies. Indeed, antibodies with epitopes located on HA-stalk regions were shown to have a tendency towards higher polyreactivity, pointing to polyreactivity as being an innate feature for such antibodies capable of binding broadly protective epitopes on the influenza HA stalk (Andrews et al., 2015).

Development of novel prophylactic or therapeutic antibodies targeting the HA stalk requires to take into account polyreactivity as it is not desired and has to be overcome, so our VHHs have been tested on HEK293 cells for their polyreactivity or aspecific binding. As shown in Figure 25, the IBV NA- or IBV HA-specific VHH-Fc constructs described herein were tested for unspecific binding to HEK293 cells (hVHH69-Fc, hVHH75-Fc, hVHH504-Fc, and hVHH525-Fc), revealing that even at high concentration (100 pg/mL) no binding to living (HEK293) cells was observed. Comparison was made with the controls Synagis (clinically approved monoclonal antibody) and CR9114 (anti-HA stem monoclonal antibody). In conclusion, all tested VHH-Fcs are less polyreactive than Synagis (which is commercially approved), and favouring these novel candidates towards clinical development.

Example 11. The VHH-Fc antibodies act on Influenza B virus but not on Influenza A virus.

As described in Example 3, the HA epitope of VHH69 is broadly conserved among the Influenza B virus lineages. Though, the binding of both VHH69 and VHH75, which competes for the VHH69 epitope, could not be confirmed on Influenza A HA protein derived from A/Victoria/03/1972 (H3), A/New Caledonia/20/1999 (Hl), and B/Washington/02/2019, as shown in Figure 1 for the VHH69-FC and VHH75-Fc fusion antibodies.

Inhibition of the neuraminidase (NA) activity for IBV and IAV NA was also analyzed for the VHH-Fcs binding to NA, and confirmed that mainly IBV NA activity was blocked by the VHH525-Fc and VHH504- Fc, but not significantly inhibiting the IAV NA activity (Figure 28). Overall, these data confirm that the VHH building blocks and derived fusions and antibodies thereof specifically act on IBV and not on IAV.

Example 12. The HA- and NA-specific VHH-Fc constructs can induce Fey receptor-mediated effector functions, which may boost their effectiveness in vivo. Antibody-dependent cellular cytotoxicity (ADCC) is induced through the Fey receptor binding of the human IgGl Fc tails used in the VHH-Fc fusions herein.

Indeed, Figure 30 demonstrates that monospecific bivalent VHH69-Fc and VHH525-Fc, as well as tetravalent bispecific VHH69-525-Fc and VHH525-69-Fc, can induce Fey receptor-mediated effector functions, whereas the effector-dead negative controls with an Fc containing the LALAPG mutations, did not show an effect on the MDCK cells infected with influenza B/Washington/02/2019 virus.

Materials and Methods

Selection of HAs and NAs to be used for llama immunization. HAs and NAs were selected based on their phylogeny to obtain a maximal antigenic diversity. Only WHO proposed vaccine components were used, except for B/Memphis/12/97. Amino acid sequences of the selected HAs and NAs were obtained through the NCBI viral genomes resource database using the corresponding Genbank ID (Table 3)(Bao et al., 2008). Sequence integrity was checked after alignment to a fully annotated B/Victoria-lineage hemagglutinin (https://www.uniprot.org/uniprot/P22092) or B/Yamagata-lineage neuraminidase (https://www.uniprot.org/uniprot/Q90021).

Table 3. Genbank ID's for HA and NA

The complete ectodomain sequences were codon optimized for mammalian expression and the Kozak consensus sequence was added to the start codon. No tags were added in the design of the transcribed construct. The 5' and 3' ends were optimized for cloning in vector PCAXL using Gibson assembly (New England Biolabs). The plasmids were propagated in E. Coli DH5A and LPS-free purified (Qiagen). The pDNA was batch-frozen and a sample was taken in order to confirm the quality (data not shown).

Llama immunization and seroconversion. Two male llamas were immunized intradermally four times bi-weekly, followed by electroporation, each time with 2 mg of a pool of recombinant pCAXL vector encoding either 10 different hemagglutinins or 10 different neuraminidases from influenza B. Immunizations and handling of the llama were performed according to directive 2010/63/EU of the European Parliament for the protection of animals used for scientific purposes and approved by the Ethical Committee for Animal Experiments of Lamaste BVBA, Vrije Universiteit Brussel (Licence: LA1700601).

Seroconversion of llama 178 was monitored with a recombinant HA ELISA as described below. None of the three tested HA-antigens were included in the immunization regime of llama 178. BSA was coated as a control and pre-immune serum was used as negative control. Sera dilutions started at 10^-2 and were done 3-fold until IO^-7. The secondary antibody was a rabbit anti-camelid VHH HRP-coupled monoclonal antibody (Genscript). The assay was performed in duplicate. Curves were fitted with GraphPad Prism software (GraphPad Software).

Seroconversion of llama 179 was monitored with a fetuin-based ELLA assay as described below. Serial dilutions of plasma were prepared starting at 10^-2 and were done in 3-fold to IO^-7. Viruses used are B/Lee/1940 (Ancestral-lineage), B/Washington/02/2019 (Victoria-lineage) and B/Phuket/3073/2013 (Yamagata-lineage). None of the three viruses' NA was included in the immunization regime for llama 179. Four technical replicates were done for this assay. Curves were fitted with GraphPad Prism software (GraphPad Software). Library generation. One hundred ml anticoagulated blood was collected from the animals for the preparation of peripheral blood lymphocytes (PBLs). The total RNA prepared from PBLs was then pooled in a 1:1 ratio and used for cDNA synthesis. About 50 pg of total RNA was used as template for first strand cDNA synthesis with oligo-dT primers. Using this cDNA, the VHH encoding sequences were amplified by PCR and cloned in the phagemid vector pMECS-GG. pMECS-GG transfected E.coliTGl cells were infected with VCS M13 helper phages to obtain a library of VHH-presenting phages; Lib 178 and Lib 179 for HA and NA respectively. Two libraries were constructed for each llama: library A from plasma sampled at day 50 and library B from plasma sampled at day 92. Since the B libraries were more matured, only the B libraries were biopanned to isolate interesting phages.

Isolation of HA and NA VHH phages. Lib 178B was panned in two consecutive rounds on MDCK infected cells. Confluent T175 falcons of MDCK cells were infected with IBV virus at a MOI of 1 in DMEM supplemented with TPCK-treated trypsin (2 pg/ml Sigma-Aldrich). In the first round B/Phuket/3073/2013 was used and in the second round of panning B/Washington/02/2019 was used. A falcon with non-infected MDCK cells served as a negative control. After 12 hours of infection, cells were detached using trypsin, washed and fixated in a 15 ml tube with 2% PFA. After subsequent washing and blocking (in 4% milk for the first panning round and 4% BSA for the second panning round) of the cells, phages were added. Phages were first adsorbed to the non-infected cells to eliminate nonspecific interactions with the fixed MDCK cells. lxlO¹¹ phages were incubated for 1 hr at room temperature. After extensive washing with PBS + 0.05% Tween 20 (10 times, 15 times and 20 times respectively for panning round 1, 2 and 3) retained phages were eluted with a TEA-solution of pH 10 (14% triethylamine (Sigma)) and the pH was neutralized with IM Tris-HCI (pH 8). Phages were amplified in exponentially growing TGI E. Coli cells, infected with VCS M13 helper phages and subsequently purified using PEG 8000/NaCI precipitation for the next round of selection. A dilution of phages was used to infect TGI cells, after which the bacteria were plated on LB agar plates supplemented with 100 pg/ml ampicillin and 1% glucose to select single colonies for PE extracts. A total of 90 individual VHH-expressing clones were selected to be screened with PE extracts.

Lib 179B was panned in three consecutive rounds, each time with 100 ng of recombinant NA in one well of a MaxiSorb 96-well plate (Nunc, Thermo Scientific) and an uncoated control well. Three different panning strategies were followed in parallel where each time three different recombinantly produced NAs were coated and panned in a different succession. The three NAs used are NA/B/Lee/1940, NA/B/Utah/09/2014, NA/B/Washington/02/2019. The blocking buffer varied between 4% milk, 4% BSA or Seablock blocking buffer (Thermo Scientific). Panning was performed as described above. From each panning strategy, 72 VHH-expressing clones were selected to screen in a fetuin-based ELLA assay. Periplasmic extracts to screen the VHH expressing phages. To prepare VHHs from the bacterial periplasm, single colonies were grown in 2 ml terrific broth (TB) medium supplemented with ampicillin (100 pg/ml) at 37°C in 24-well deep well plates. After 6 hours, cultures were spiked with Isopropyl P-D- 1-thiogalactopyranoside (IPTG; ImM) and incubated overnight at 28°C to start the production of VHH. The next day, bacterial cells were pelleted, resuspended in 200 pl TES buffer (0.2 M Tris-HCI pH 8, 0.5 mM EDTA, 0.5 M sucrose) and incubated at 4 °C for 30 min. Next 300 pl of 4 times diluted TES was added and incubated for 1 h at 4 °C. The plates were centrifuged and the supernatant was collected and is the periplasmic extract (PE). The PE extract was diluted 1/10 in PBS for experiments. PE extracts from Lib 178B were used in a microneutralization assay as described below. PE extracts from Lib 179B were used in an fetuin-based ELLA assay, as described below.

Recombinant protein expression of HA and NA. Ectodomain encoding information for hemagglutinin was retrieved from the NCBI database with accession numbers MK676294.1, KU592766.1 and K00423.1 for respectively B/Washington/02/2019, B/Utah/09/2014 and B/Lee/1940. Gblocks (IDT genomics) were cloned under transcriptional control of the CMV promotor in the pCDNA3.4 plasmid (thermo fisher). Fragments were codon optimized for mammalian expression (IDT genomics) and completed with a CD5-secretion signal, a trimerizing GCN4 derived zipper domain and C-terminal strep-tag. Neuraminidase head encoding information was retrieved from the NCBI database with accession numbers QBP37483.1, AMB72005.1 and AAA43749.1 for respectively B/Washington/02/2019, B/Utah/09/2014 and B/Lee/1940. Fragments were codon optimized for mammalian expression and ordered as Gblocks (IDT genomics), including an N-terminal CD5-secretion signal, C-terminal strep-tag and tetramerizing tetrabrachion stabilization domain. Cloning was done using a double digest with BspEI (New England Biolabs) and Xbal (New England Biolabs) in the PEF vector. The NA/B/Washington/02/2019 ectodomain had 2 internal BspEI sites, which were silently mutated. Ligation was performed with T4 ligase (Promega) according to standard protocol. Plasmids were transformed in DH5a E.coli using standardized heat shock protocol and pDNA was isolated (NucleoBond Xtra Midi EF, Macherey-Nagel). The Expi293 transient expression system (Gibco) was used for rapid and high-yield production with ensured native glycosylation patterns. HEK Suspension (HEK S) cells were maintained and cultured according to standard cell culture protocols using a 1:1 mixture of ExCell-293 (Sigma Aldrich) and Freestyle-293 (Thermo Scientific) medium. Cells were transfected using polyethylenimine (Polyethylenimine, linear, 25000 MW, polysciences europe) and cell-viability was frequently monitored. LPS-free purification was done using an AKTA system (cytiva), coupled to a StrepTrap-HP column (Cytiva). Elution was performed with 2.5 mM D-desbiothin (Sigma Aldrich). Relevant fractions were pooled and loaded on a Hiload 16/600 superdex column (Cytiva). Upconcentration was done if necessary (Sartorius vivaspin 20, 5000 MWCO). Concentrations were measured on a spectrophotometer and protein purity was checked on SDS page with Coomassie staining (data not shown).

Expression of VHHs in Pichia pastoris. Original VHH (oVHH) sequences were PCR amplified from the respective pMECS-GG vectors using following forward and reverse primers (SEQ ID NO:27-28, resp.) and digested with Xhol (New England Biolabs) and Spel (New England Biolabs) before ligation into a Xhol/Spel-digested pKai61 backbone. OVHHs were cloned in frame with a modified version of the S. Cerevisiae alfa-mating factor signal sequence and C-terminal Hisg-tag. OVHH sequences were expressed under the control of the methanol inducible AOX1 promotor (Schoonooghe et al. 2009). Vectors were linearized using the Pmel (Promega) before transformation to electrocompetent GS115 Pichia pastoris (Gene Pulser, Biorad). Electroporation settings were: voltage 1.5 kV, Capacitance 25 pFD, Extended capacitance 125 pFD and Resistance 200 O. After electroporation the cells were plated on zeocine- selective (100 pg/ml, InvivoGen) YPD plates (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar) and incubated 3 days at 30°C.

Monovalent and bivalent humanized VHHs (hVHHs) expression plasmids were assembled using an adapted version of the Yeast Modular Cloning toolkit on the basis of Golden Gate assembly (reference M. E. Lee, W. C. DeLoache, B. Cervantes, J. E. Dueber, A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth. Biol.4, 975-986 (2015)). Briefly, VHH coding sequences were ordered as codon optimized Gblocks (IDT Genomics) for yeast expression. The assembled MOCLO expression vector contained the pAOXl promotor, S. Cerevisiae alfa-mating factor minus EA-repeats, VHH(s), (G₄S)₄ linker, C-terminal Hisg-tag as well as resistance markers for bacterial (ampicilline) and yeast (zeocine) selection.

Expression of VHHs by transformed P. Pastoris clones was done by picking single clones and inoculation of 50 ml of YPNG (1% yeast extract, 2% peptone, 100 mM KH2PO4/K2HPO4, 1.34% YNB, 1% glycerol) medium and incubation while shaking at 28°C for 24 h. After 24 hours, the YPNG medium was replaced by YPNM (same composition but with 1% methanol replacing the 1% glycerol) to induce VHH expression. To boost VHH production for 48 hours, 500 pl methanol was added to the cultures at 16, 24 and 40 h. After 48 h, the yeast cells were pelleted and the supernatant was retained and VHHs were extracted using Ni-NTA Beads (Ni Sepharose High Performance; GE healthcare) according to standard protocol in combination with spin columns (Pierce; Thermo Fisher Scientific). The eluate was concentrated with the 0.5 ml Amicon ultra columns (cutoff 3kDa) (Millipore). Buffers were exchanged by passing 400 pl PBS though the filter 3 times.

ELISA. MaxiSorb 96-well plates (Nunc, Thermo Scientific) were coated overnight with 100 ng of in-house produced recombinant hemagglutinin or bovine serum albumin (Sigma-Aldrich). Plates were blocked with 3% milk. Purified Pichia pastoris produced VHHs or llama plasma was incubated for 1 hr at room temperature. Plates were washed with PBS. The secondary antibody was HRP-coupled and the readout was performed with 50 pl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD OptEIA) and the reaction was stopped by addition of 50 pl of IM H2SO4. Absorbance at 450 nM was measured with an iMark Microplate Absorbance Reader (Bio Rad).

ELLA assay. MaxiSorp 96-well plates (Nunc, Thermo Scientific) were coated with fetuin (25 pg/ml; Sigma-Aldrich) overnight at 4°C. The amount of virus or recombinant NA to reach EC70 was titrated in a separate experiment. Virus or recombinant NA were mixed with Pichia pastoris purified VHH, plasma or VHH PE extract in MES-buffer (Thermo Scientific) supplemented with 20 mM CaCL, 1% BSA (Sigma- Aldrich) and 0.5% Tween 20 (Sigma-Aldrich). After one hour of pre-incubation, the mixture was transferred to the coated fetuin plates, from which the excess fetuin was washed away, and incubated for 18 hours at 37°C. The amount of desialylated fetuin was measured by colorimetry. Plates were washed three times with PBS supplemented with 0.1% Tween 20 and incubated for one hour with PNA- HRP (Sigma-Aldrich)(2.5 pg/ml). Absorbance at 450 nm was measured with an iMark Microplate Absorbance Reader (Bio Rad) after addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD OptEIA) and neutralization with IM H2SO4.

MUNANA. 50 ng recombinant stabilized influenza B neuraminidase was mixed with 500 ng hVHH, 20 ng NAI or equal volume PBS and incubated for 30 minutes at 21°C. All dilutions were done in PBS (thermo fisher) supplemented with CaCL at a final concentration of 400 mM. 2'-(4-Methylumbelliferyl)- a-D-N-acetylneuraminic acid (= MUNANA substrate) was added to the mixtures at a final concentration of 1 mM. (merck, catalog nr: M8639-25mg) and the mixture was incubated for 1 hr at 37°C before measuring the fluorescence absorbance at 460 nm after excitation at 360 nm. A background sample (containg only PBS+MUNANA) was substracted as a reference. All percentages are relative to NA + PBS. Mean + 95% Cl is given of 4 biologic replicates.

Egress inhibition assay. MDCK cells were seeded for confluency in 96-well plates (Nunc, Thermo Scientific) in complete DMEM (DMEM + 10% FCS + 1% Penicillin-Streptomycin + 1% NEAA) (Gibco). The amount of B/Washington/02/2019 virus to achieve 90-100% infection was titrated in a separate experiment. 10⁶ pfu/well of B/Washington/02/2019 was added to the cells and incubated at 37°C, 5% CO2 for two hours plain DMEM. After two hours, the supernatant was removed, and non-infected virus particles were washed away (3x). DMEM containing a serial dilution of nanobodies or a serial dilution of zanamivir (Sigma-Aldrich) was incubated on the cells for 18 hours at 37°C.

HA protease sensitivity assay. In-house produced recombinant HA (2pg) was proteolytically cleaved by TPCK-treated trypsin (Sigma Aldrich) in a molar ratio of 100:1 (HA:TPCK treated trypsin), followed by inactivation with an excess of trypsin soy bean inhibitor (Sigma Aldrich). hVHH were added in a 3-fold molar excess and incubated for 30 minutes. If no VHH was added, an equal amount of PBS was added. The pH was adjusted with sodium acetate buffer pH 5,5 (Chemlab) and neutralized after exactly 5 minutes of room temperature incubation with an equal volume of IM pH8 Tris-HCI buffer. In the control reactions without acidification, the acid and neutralization buffers were mixed in advance. TPCK- treated trypsin was added to the reaction mixture in a molar ratio of 20:1 (TPCK treated trypsin:HA) and incubated for 1 hr at 37°C. The reaction was stopped by adding non-reducing laemli buffer and boiling for 5 minutes at 95°C. Samples were run on precast SDS-page gels (Biorad) and stained with Coomassie. All blue protein ladder was used as marker and a representative of two independent experiments is shown.

HA cleavage assay. In-house recombinant HA (2pg) was incubated with an 8-fold molar excess VHH or mAb for 30 min. The HAO is proteolytically activated byTPCK-treated trypsin at 37°C for 15 min. Samples were run on precast SDS-page gels (Biorad) and stained with Coomassie. All blue protein ladder was used as marker and a representative of two independent experiments is shown.

Polykaryon assay. The polykaryon assay protocol was taken and adapted from (Vanderlinden et al, 2010). The protein sequences of HA B/Wisconsin/01/2010 and HA B/Colorado/06/2017 were obtained from the NCBI database (respectively with entry AFH57953.1 and ARQ85589.1) and cloned into the pCAXL vector. HeLa cells were seeded into 12-well plates and transfected at 90% confluency using polyethylenimine (PEI, linear, molecular weight 25.000, Polysciences.com) in serum-free medium. Two days later, the cells were washed and the membrane expressed HAs were proteolytically activated by incubation with TPCK-treated trypsin (100 nM in DMEM)(Sigma-Aldrich) for 15 min at 37°C. After two rinses with PBS, the cells were incubated for 15 minutes with acidic buffer (PBS adapted to pH 5.1 with 0,lM citric acid (Sigma-Aldrich)) and then washed and incubated for 3 hours at 37°C, 5% CO2 in complete medium. To determine the optimal pH of the acidic buffer, the experiment was performed in the absence of hVHHs, and the pH of the acidic buffer was varied in increments of 0.1 pH (range pH 4.9- 6.0). The effect of the hVHH69 K64E and hVHH 75 was surveyed at different timepoints in the polykaryon assay. hVHHs were added either before TPCK-treated trypsin treatment, either between TPCK-treated trypsin treatment and acidification or after the acidification. hVHH 69 K64E and hVHH75 were tested in three different concentrations (5 pg/mL, 0,5 pg/mL and 0,1 pg/mL) and the control (an anti-GFP VHH) was tested at 5 pg/mL. HeLa cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 (0,2% in PBS)(Sigma-Aldrich). The nuclei were stained with (4',6- Diamidino-2-Phenylindole, Invitrogen TM) and syncytium formation was counted using fluorescent microscopy (Olympus CKX53 with pE-300 C00ILED and ToupCAM E3ISPM). The number of polykaryons (containing five or more nuclei) were counted in four randomly selected microscopic fields (20x lOx). The experiment was conducted four times.

Microneutralization. MDCK cells were seeded in 96-well plates (Nunc, Thermo Scientific) in complete DMEM (Gibco) for confluency on the day of the experiment. TCID50 titers were determined in a separate experiment. One hundred TCID50 of panel of influenza B viruses was mixed with a dilution series of VHH PE extract, purified VHH, VHH-Fc or controls in DMEM supplemented with TPCK-treated trypsin (2 pg/ml)(Sigma-Aldrich). After one hour, 100 pl of the VHH-virus mixture was transferred to washed MDCK cells and incubated for 3 days at 37°C and 5% CO2. The readout was performed after fixation of the cells with 4% PFA and permeabilization with 0,5% triton-XlOO (Sigma-Aldrich). The primary antibody was a mouse monoclonal antibody directed against IBV NP (MAI-80712, Thermo Scientific). Three wash steps with PBS were performed before allowing the anti-mouse IgG-HRP antibody to incubate (NA931V, GE healthcare). After washing, 50 pl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD OptEIA) was added and the reaction was stopped by addition of 50 pl of IM H2SO4. Absorbance at 450 nM was measured with an iMark Microplate Absorbance Reader (Bio Rad). Reported values are average IC50 in pg/ml for three independent experiment (n=3)

Mammalian expression of VHH-Fc fusions. Humanized VHHs were fused to a human immunoglobulin G1 domain (IgGl) with a (G₄S)2 linker and the IgGl hinge region. The cysteine at position 230 was mutated to a serine to avoid reactive cysteines and the last lysine was removed. Coding sequences for the Saccharomyces cerevisiae a-mating factor minus EA-repeats were fused N-terminally as secretion signal and cloned in the pcDNA TOPO 3.4 vector following standard protocol (Thermo Scientific). VHH- Fc constructs were produced in human embryonic kidney cells (HEK) 293S cells by transient transfection of the respective expression plasmids with polyethylenimine (Polyethylenimine, linear, 25000 MW, Polysciences europe). Cell viability was monitored daily and the culture medium was harvested once viability reached 75% living cells. VHH-Fc constructs were purified from the clarified culture medium using a MabSelect SuRe column (GE Healthcare). After a wash step with Mcllvaine buffer (pH 7.2), bound proteins were eluted using Mcllvaine buffer pH 3. The eluted protein containing fractions were neutralized with a saturated NasPC buffer. The fractions of interest were pooled and loaded onto a HiPrep Desalting column (Cytiva) for buffer exchange into storage buffer (PBS) and frozen at -80°C until use. Open reading frames (ORFs) corresponding to the light and heavy chains of the CR9114 monoclonal antibody were ordered synthetically at IDT. A similar cloning, expression and purification strategy was followed as for VHH-Fc.

Cells and viruses. Viruses used were B/Lee/1940, B/HongKong/5/1972, B/Harbin/7/94, B/florida/4/2006, B/Phuket/3073/2013, B/Malaysia/2506/2004, B/Brisbane/60/2008, and B/Washington/02/2019. Virus stocks were prepared on Madin-Darby Canine kidney (MDCK) cells, in the presence of 2 pg/mL TPCK-treated trypsin (Sigma-Aldrich, T1426) at 37 °C until cytopathic effects were observed. Subsequently, supernatant was harvested and the virus was isolated by centrifugation for 2 hours at 14000 g. The virus pellet was resuspended in PBS supplemented with 20% glycerol, batched out and frozen at -80°C until use. HEK-293 T, HeLa, and MDCK cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L- glutamine, non-essential amino acids, and 1 mM sodium pyruvate (Gibco) at 37 °C in the presence of 5% carbon dioxide.

Oseltamivir resistant virus. To introduce a mutation at position E117, the NA segment of B/Memphis/12/1997 with E117A was synthetically constructed (IDT Genomics) and inserted in the bidirectional expression vector pHW2000. A co-culture of MDCK and HEK293 cells was transfected with 1 pg of each of the plasmids coding for the segments of mouse-adapted B/Memphis/12/1997 (McCullers et al.2005). The culture medium was recovered after observation of cytopathic effect and the rescued viruses were amplified on MDCK cells to prepare working stocks as described above. The NA gene (with E117A mutation) was sequence verified after isolating viral RNA (Nucleospin viral RNA isolation, Macherey-Nagel), preparation of cDNA (Transcriptor first strand cDNA synthesis, Roche) and PCR amplification with following forward and reverse primers (SEQ. ID NO: 29-30). Microneutralization was performed as described above, in parallel for virus with the NA mutation (E117A), and without (WT), including zanamivir (Sigma-Aldrich, SML0492), peramivir (Merck, SML2486), and laninamivir (MedChemExpress, R125489).

Mice. Specific pathogen-free, female DBA/2J mice were obtained from Janvier (France). The animals were housed in a temperature-controlled environment with 12 h I ight/da rk cycles; food and water were provided ad libitum. The animal facility operates under the Flemish Government License Number LA1400536. All experiments were done under conditions specified by law and authorized by the Institutional Ethical Committee on Experimental Animals (Ethical application EC 2022-112). Intranasal administration of compound or virus dilution was performed under isoflurane sedation in a BL2 graded facility. The reverse genetics B/Washington/02/2019 virus was rescued using a 2:6 combination with B/Memphis/12/1997 internal gene segments. The rescued strain was passed 13 times in DBA/2J mice to increase its pathogenicity, which resulted in an LD₅o corresponding to 25 pfu/mouse. Mice were weighted daily and euthanized when more than 25 % of the bodyweight was lost. Differences in body weight between two groups were tested by two-way ANOVA with Sidak's multiple comparison: *P<0.0332, **P<0.0021, ***P<0.0002, ****P<0.0001 compared to control. Differences in survival were tested with a log-rank (Mantel-Cox), compared to 2.5 mg/kg Palivizumab in Graphpad Prism: * P<0.05, **P<0.01, ***P<0.001. Flow cytometric analysis of cells expressing IBV HA, NA or Ala scan mutants. HEK293T cells were transiently transfected with HA or NA derived from B/Washington/02/2019, or Ala scan mutants, generated after site directed mutagenesis according to the manufacturer's instructions (Agilent) using pcDNA 3.4 expression plasmids (Thermo Scientific). Twenty four hr after co-transfecting with a GFP expression plasmid (Lonza), cells were detached, washed, and blocked with 1% BSA (Sigma-Aldrich) on ice. Cells were stained with hVHH or hVHH-Fc for 1 hr on ice and subsequently washed with 1 % BSA. Binding of hVHHs was detected using a mouse anti-His-tag antibody (Genscript, A00186) and an AF647- conjugated donkey anti-mouse IgG antibody (Invitrogen, A-31571). Binding of hVHH-Fcs or monoclonal antibodies was detected using an AF633-conjugated goat anti-human IgG antibody (Invitrogen). Cells were stained using Live/Dead stain eFluor 506 (Invitrogen, A-21091). Following two subsequent washes, the cells were analysed by flow cytometry using a NovoCyte Quanteon flow cytometer (Agilent). Binding curves were fitted using nonlinear regression in Graphpad Prism.

Biolayer-interferometry. The NA derived from B/Washington/02/2019 binding kinetics of hVHH-525 were assessed using an Octet RED96 system (ForteBio). Streptavidin (SA) biosensors (ForteBio) were soaked in kinetics buffer (PBS supplemented with 0.1 % w/v BSA, and 0.02 % v/v Tween 20) for 20 min. Strepll-tagged NA derived from B/Washington/02/2019 was immobilized on these SA biosensors to an optical shift of 0.3 to 0.5 nm (saturation). Purified P. pastoris produced hVHH-525 was diluted in the assay buffer to 100 nM or 150 nM and a threefold dilution series was prepared. Association was measured for 200 s and dissociation for 600s in kinetics buffer. Between analyses, biosensors were regenerated by three times 20s exposure to 10 mM glycine (pH 1.7) and reloaded. Using ForteBio Data Analysis 9.0 software, data were double-reference-subtracted, and association and dissociation were fit in a global 1:1 model.

Electron Cryo-Microscopy (Cryo-EM). Recombinant protein (either HA or NA) derived from B/Washington/02/2019 was complexed with a 1.3 fold molar excess hVHH (either hVHH-69 or hVHH- 525, respectively) for 1 hr at 4°C. The complex was separated from unbound molecules by size-exclusion chromatography on a Hiload 16/600 superdex 200 column (Cytiva). Four pL of purified Influenza B/Washington/02/2019 Hemagluttinin:hVHH-69 or Neuraminidase:hVHH-525 complex, at a concentration of 0.4 mg/mL and 0.12 mg/mL respectively, were applied to glow-discharged R2/1 300 mesh holey carbon copper grids (Quantifoil Micro Tools GmbH). Grids were blotted and plunge frozen in liquid ethane using a Lecia EM GP2 Plunge Freezer operated at 95 % humidity, and using a blotting time of 5 and 6 s respectively for Hemagluttinin:hVHH-69 and Neuraminidase:hVHH-525 complexes. Grids were screened on a JEOL 1400 Plus TEM operated at 100 keV at the VIB Bio Imaging Core, Ghent Platform (Zwijnaarde, Belgium), and data was acquired on a JEOL cryoARM 300 microscope (BECM, Brussels, Belgium) equipped with an Omega filter (JEOL) and a 6k x 4k K3 direct detector (Gatan) operated in correlated double-sampling (CDS) mode. For both the Hemagluttinin:hVHH-69 and Neuraminidase:hVHH-525 complex, 60-frame movies were collected with a total dose of 60 e“/A², an energy filter slit width of 20 eV and a pixel size of 0.72 A/pixel at the specimen level. For the Hemagluttinin:hVHH-69 complex, 4.352 movies were collected at 40° tilt to overcome preferred orientations, while for the Neuraminidase:hVHH-525 complex 28.057 movies were collected at 0° tilt.

Cryo-EM Image Processing. Data processing of collected movies was performed in CryoSPARC v4.2.1 (Punjani et al., 2017). For both the Influenza B Hemagluttinin:hVHH-69 and Neuraminidase:hVHH-525 complex, motion correction and CTF estimation were performed with Patch motion correction and Patch CTF estimation respectively. For the Hemagluttinin:hVHH-69 complex, particles were first picked from the 40° tilted motion corrected and CTF estimated micrographs using Blob picker with a minimum/maximum particle diameter of 50/150 A, and extracted using a box size of 416 pixels with a pixel size of 0.72 A/pixel. The extracted particle stack was subsequently cleaned using several rounds of iterative 2D classification and 2D class selection to remove junk. Next, the cleaned particle stack was used for Ab Initio model generation using Cl symmetry, followed by homogeneous refinement and Non-Uniform (NU) refinement of this class with enabled C3 symmetry. The resulting particle stack was further cleaned via a round of heterogeneous refinement in C3 starting from the previously obtained NU-refined map, and asking for two 3D classes as output. The generated 3D class corresponding to the highest resolution was further refined via a final round of NU-refinement using particles re-extracted with a box size of 360 pixels, while applying C3 symmetry and optimizing per-particle defocus and pergroup CTF parameters, resulting in a final map with a resolution of 2.9 A based on the 0.143 gold- standard Fourier shell correlation (FSC) criterion (Rosenthal et al., 2003). The final map of the Hemagluttinin:hVHH-69 complex was either sharpened using anisotropic sharpening via the local_aniso_sharpen tool in Phenix 1.19.2-4158 with local sharpening set to false, with the purpose of generating a map for model refinement, or via deepEMhancer⁵⁰ for model building in Coot and visualization in ChimeraX (Pettersen et al., 2021; Liebschner et al. 2019; Emsley & Cowtan, 2004).

For the Neuraminidase:hVHH-525 complex, particles were picked from the untitled motion corrected and CTF estimated micrographs using crYOLO, imported in CryoSPARC and extracted using a box size of 280 pixels down sampled to 140 pixels, corresponding to a pixel size of 1.44 A/pixel (Wagner et al., 2019). The resulting crYOLO picked particle stack was cleaned using several rounds of iterative 2D classification and 2D class selection. A set of selected classes displaying clear secondary structure features, and corresponding to both top and side views of the Neuraminidase:hVHH-525 complex, were used as an input for Topaz (Bepler et al., 2019). Topaz Train and Cross Validation jobs were run in cryoSPARC with the goal of optimizing the amount of Topaz picked particles per micrograph. After cleaning the extracted Topaz picked particle stack via iterative 2D classification and class selection, it became clear that the imaged Neuraminidase:hVHH-525 sample displayed extensive compositional heterogeneity, with 2D classes showing either no, one, two, three or four bound hVHH-525 molecules, which are easily distinguishable as additional bright dots at the corners of Neuraminidase tetramer top views. This compositional heterogeneity necessitated the use of a 3D processing strategy combining symmetry expansion, focused 3D classification without alignment, signal subtraction and localized refinement as follows:

First, an ab initio 3D reconstruction was performed using 3 classes and applying Cl symmetry, followed by 3D heterogeneous refinement of the 3 Ab Initio models with C4 symmetry applied. One obtained 3D class had a significantly higher resolution than the others, and was used for homogeneous refinement and ensuing NU-refinement, both applying C4 symmetry. The obtained NU-refined map displayed clear partial occupancy of bound hVHH-525 molecules as a result of averaging in C4 symmetry. The resulting particle stack was then used as an input for symmetry expansion using C4 as point group symmetry, followed by a focused 3D classification without alignment employing a soft mask around one Neuraminidase monomer (partially) bound by VHH, and asking for two 3D classes as an input. This focused 3D classification resulted in two 3D classes, one without and one with bound hVHH-525 corresponding to roughly 55 % and 45 % of the symmetry expanded particles respectively. Symmetry expanded particles belonging to the second 3D class were then used for a Particle Subtraction job employing a soft mask excluding a Neuraminidase tetramer bound to one hVHH-525 molecule. Signal subtracted particles were then used for a final focused refinement job with Cl symmetry applied, using a soft mask around a Neuraminidase tetramer bound to one hVHH-525 molecule. For local refinement, the rotation and shift search extent were set to 1° and 1 A, respectively, and a pose/shift gaussian prior was enabled with standard deviations of priors over rotation/shifts set to 15° and 7 A, resulting in a 3D reconstruction with a gold-standard FSC 0.143 resolution of 3.6 A. Unbinding of the data to 0.72 A per pixel did not result in an increase in resolution.

The final map of the Neuraminidase:hVHH-525 complex was either sharpened using local filtering in CryoSPARC using a B-factor of -70 A² to generate a map for model refinement in Phenix 1.19.2-4158, or via deepEMhancerfor model building and map visualization.

Cryo-EM Model Building and Refinement. Initial models of Hemagglutinin/Neuraminidase and hVHH- 69/hVHH-525 were generated via AlphaFold2 and ESMfold respectively, and rigid-body fitted in the respective Hemagluttinin:hVHH-69 and Neuraminidase:hVHH-525 maps using ChimeraX (Pettersen et al., 2021; Jumper et al., 2021; Lin et al., 2023).

For the Hemagluttinin:hVHH-69 complex, the rigid-body fitted model was used as an input for automatic molecular dynamics flexible fitting using the NAMDINATOR webserver (Kidmose et al., 2019). Flexible fitting was followed by several rounds of iterative model building guided by the deepEMhancer sharpened map using Coot, and real-space refinement in Phenix 1.19.2-4158 using the anisotropy sharpened map with enabled global minimization, local grid search, ADP refinement, secondary structure restraints and Ramachandran restraints. The final refined model has a map-to-model FSC of 3.20 A at the 0.5 threshold, calculated using the unsharpened, unfiltered full map. For the Neuraminidase:hVHH-525 complex, the rigid-body fitted model was used as a starting point for manual rebuilding of mainly the hVHH-525 CDR loops in Coot using the deepEMhancer sharpened map, followed by several rounds of real-space refinement in Phenix 1.19.2-4158 using the local filtered map with enabled global minimization, local grid search, ADP refinement, secondary structure restraints, Ramachandran restraints and a nonbonded-weight parameter of 300 (Liebschner et al., 2019; Emsley & Cowtan, 2004). A final refinement was performed using REFMAC5 Servalcat as part of the CCP-EM suite vl.6, with automatic weighting applied (Burnley et al., 2017). The resulting final model has a map- to-model FSC of 3.80 A at the 0.5 threshold, calculated using the unsharpened, unfiltered full map.

A summary of all cryo-EM data collection, refinement and validation statistics can be found in Table 4.

Table 4. CryoEM data collection, refinement and validation statistics.

HA/B/Washington/02/2019 + hVHH-69 NA/B/Washington/02/2019 + VHH-525

EMDB-50546 EMDB-50547

PDB: 9FM1 PDB: 9FM2

Data collection and processing

Magnification 60,000 60,000

Voltage (kV) 300 300

Electron exposure (e-/A²) 60 60

Defocus range (pm) 0.5 - 2.5 0.5 - 2.5

Pixel size (A) 0.72 0.72

Symmetry imposed C3 Cl after symmetry expansion in C3

Initial particle images (no.) 4,124,258 409,850

Final particle images (no.) 157,829 158,543 symmetry expanded particles

Map resolution (A) 2.93 3.56

FSC threshold 0.143 0.143

Map resolution range (A) 2.51 - 38.75 3.19 - 58.09

Refinement

Initial model used (PDB code/Alphafold entry)

Model resolution (A) 3.20 3.80

FSC threshold 0.5 0.5

Map sharpening B factor (A²) -98.9 -117.1

Model composition

Non-hydrogen atoms 11,604 11,567

Protein residues 1,533 1,492

Ligands NAG: 6 NAG: 4

B factors (A²)

Protein 54.46 190.30

Ligand 57.52 307.53

R.m.s. deviations

Bond lengths (A) 0.003 0.010

Bond angles (°) 0.704 1.307

Validation

MolProbity score 1.49 1.72

Clashscore 5.96 6.40

Poor rotamers (%) 0.24 0.08

CaBLAM outliers (%) 0.56 2.26

CC (mask/volume) sharpened, 0.81/0.80, 0.78/0.77 0.73/0.73, 0.78/0.78 unsharpened

EMRinger 4.09/4.16 1.61/1.74

(sharpened/unsharpened)

Ramachandran plot

Favored (%) 97.10 94.64

Allowed (%) 2.90 5.36

Disallowed (%) 0.00 0.00

Ramachandran Z-score

Whole 0.04 -1.72

Helix 1.87 N.A.

Sheet 0.38 -0.57

Loop -0.57 -1.55

Conservation analysis. Full-length and non-redundant sequences of the influenza B virus HA (N = 3837) and NA protein (N = 4651) were downloaded from the Influenza Virus Resource at the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/genomes/FLU).

Sequences were aligned to the B/Washington/02/2019 HA (WNG78001, NCBI) or NA (QBP37483, NCBI) reference sequence using the Muscle algorithm in R (version 4.3.2) with msa package (version 1.34.0) and BLOSUM80 substitution matrix. Alignment parameters were set to a gap opening of 12, a gap extension of 3. R package Biostrings 2.70.1 was used to calculate amino acid frequencies per position. Conservation values were calculated compared to reference sequence and ambiguous amino acid frequencies (such as X or B) were disregarded. Lineage annotation of influenza B isolates was downloaded from GISAID (https://platform.epicov.org/). Of 3837 HA sequences, we annotated 1103 B/Victoria and 878 B/Yamagata sequences, which were subsequently used for lineage-specific conservation analysis.

Statistics for in vivo experiments. Differences in body weight between two groups were tested by two- way ANOVA with Sidak's multiple comparison. P<0.0001 for all treatments compared to control. Differences in survival were tested with a log-rank (Mantel-Cox) in graphpad prism. P<0.0001 for all treatments compared to control.

Sequence listing

>SEQ ID N0:l: Amino acid sequence of the NA-specific VHH 504

QVQLQESGGGLVQSGGSLRLSCAASGSIFSTHLMGWYRQAPGKQRELVASVTPGGTTNYADSVKGRFTISRDDAE NAVYLRMNSLKPEDTAVYFCNAEAYYSDYRLPDYWGQGTQ.VTVSS

>SEQ ID NO:2: Amino acid sequence of the NA-specific VHH 525

QVQLQESGGGLVQPGGSLRLSCAASGDIFSTHIMGWYRQAPGKQRVLVATIDTDGSTNYADSVKGRFTISTDNAKN TVYLQMNSLKPEDTAVYYCNAESAYSDYRLPDYWGQGTQ.VTVSS

>SEQ ID NO:3: Amino acid sequence of the NA-specific VHH 508

QVQLQESGGGLVQAGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSCISSSDGSTNFADSVKGRFTISSDDAKN TVYLHM NDLKPEDTAVYYCAAM RYYNDYRGCFIAVDGM DYWGKGTQVTVSS

>SEQ ID NO:4: Amino acid sequence of the HA-specific VHH 69

QVQLQESGGGLVQAGDSLRLSCAASGLVDSANVAWFRQAPGKEREFVAAVKWRSGSTIYADSVEGRFTISRDNAK STVYLQMDSLNPEDTAVYFCAANQWYSGGYYGEKNYDYWGQGTQVTVSS

>SEQ ID NO:5: Amino acid sequence of the HA-specific VHH 75

QVQLQESGGGLVQAGGSLRLSCAASGRAFSGYTMGWFRQAPGKERELVAAISRGGSETYYTVSVKGRFTITRDNIE RTVYLQM DS LKP E DTAVYYCAAG R N YVYS E I YDYWGQGTQVTVSS

>SEQ ID NO:6: Amino acid sequence of the HA-specific VHH 72

QVQLQESGGGLVQPGGSLILSCAASGFSLDYYGIGWFRQAPGKDREAVSCIESSGSAHVVDSVEGRFIISRDNAKNM GYLQMNSLKPEDTALYHCARYWGRRCSMDPSDYDYWGQGTQVTVSS

>SEQ ID NO:7: Amino acid sequence of the humanized NA-specific VHH 504 (hVHH504) dVQLvESGGGLVQpGGSLRLSCAASGSIFSTHLMGWYRQAPGKQRELVASVTPGGTTNYADSVKGRFTISRDnAkNt

VYLqMNSLrPEDTAVYyCNAEAYYSDYRLPDYWGQGTIVTVSS

>SEQ ID N0:8: Amino acid sequence of the humanized NA-specific VHH 525 (hVHH525) dVQLvESGGGLVQPGGSLRLSCAASGDIFSTHIIVIGWYRQAPGKQReLVATIDTDGSTNYADSVKGRFTISrDNAKNT

VYLQM NSLrPEDTAVYYCNAESAYSDYRLPDYWGQGTIVTVSS

>SEQ ID NO:9: Amino acid sequence of the humanized NA-specific VHH 508 (hVHH508) dVQLvESGGGLVQpGGSLRLSCAASGFTLDDYAIGWFRQAPGKEREGVSCISSSDGSTNFADSVKGRFTISrDnAKNT

VYLqMNsLrPEDTAVYYCAAIRYYNDYRGCFIAVDGIDYWGqGTIVTVSS

>SEQ ID NO:10: Amino acid sequence of the humanized HA-specific VHH 69 (hVHH69) dVQLvESGGGLVQpGgSLRLSCAASGLVDSANVAWFRQAPGKEREFVAAVKWRSGSTIYADSVEGRFTISRDNAKST

VYLQMnSLrPEDTAVYyCAANQWYSGGYYGEKNYDYWGQGTIVTVSS

>SEQ ID NO:11: Amino acid sequence of the humanized HA-specific VHH Nb 75 (hVHH75) dVQLvESGGGLVQpGGSLRLSCAASGRAFSGYTMGWFRQAPGKERELVAAISRGGSETYYTVSVKGRFTITRDNskn

TVYLQMDSLrPEDTAVYYCAAGRNYVYSElYDYWGQGTIVTVSS

>SEQ ID NO:12: Amino acid sequence of the humanized HA-specific VHH 72 (hVHH72) dVQLvESGGGLVQPGGSLrLSCAASGFSLDYYGIGWFRQAPGKeREAVSCIESSGSAHVVDSVkGRFtlSRDNAKNtvY

LQMNSLrPEDTALYyCARYWGRRCSIDPSDYDYWGQGTIVTVSS

Table 5. VHH sequence CDR sequences exemplified from annotation disclosed in Figure 18

*CDR1 according to AbM annotation; **CDR2,3 according to Kabat annotation

>SEQ ID NO: 25: amino acid sequence of HA/B/Washington/02/2019 (including N-terminal a leucine zipper (italics underlined} and C-terminal a strep tag (underlined))

EQAATMPMGS .QP .AT.Y. .GM .VASV/.GDRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTET

RGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINAE DAPGRPYEIGTSGSCPNITNGNGFFATMAWAVPKNKTATNPLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGD

SKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGRSKVIK

GSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWE

GMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTI SSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNIT

AASLNDDGLDNHTISGKRMKQIEDKIEEIESKQKKIENEIARIKKLIGERSAWSHPQFEK

>SEQ ID NO:26: Amino acid sequence of the C terminal tag fused to the VHHs of the present application

AAAYPYDVPDYGSHHHHHH

>SEQ ID NO:27: Forward primer used for pMECS-GG vector cloning GGCGGGTATCTCTCGAGAAAAGGCAGGTGCAGCTGCAGGAGTCTGGG

>SEQ ID NO:28: Reverse primer used for pMECS-GG vector cloning

CTAACTAGTCTAGTGATGGTGATGGTGGTGGCTGGAGACGGTGACCTGG

>SEQ ID NO:29: Forward primer

AGCAGAAGCAGAGC >SEQ ID NO:30: Reverse primer

AGTAGTAACAAGAGC

>SEQ ID NO:31: Influenza B/Washington/02/2019 Hemagglutinin ( HA)

>SEQ ID NO:32: Influenza B/Washington/02/2019 Neuraminidase (NA)

REFERENCES

Abed, Y., Fage, C., Checkmahomed, L., Venable, M. C., & Boivin, G. (2020). Characterization of contemporary influenza B recombinant viruses harboring mutations of reduced susceptibility to baloxavir marboxil, in vitro and in mice. Antiviral Research, 179, 104807.

Ambrose, C. S., & Levin, M. J. (2012). The rationale for quadrivalent influenza vaccines. Http://Dx.Doi.Org/10.4161/Hv.8.1.17623, 8(1), 81-88.

Bao, Y., Bolotov, P., Dernovoy, D., Kiryutin, B., Zaslavsky, L., Tatusova, T., Ostell, J., & Lipman, D. (2008). The Influenza Virus Resource at the National Center for Biotechnology Information. Journal of Virology, 82(2), 596-601.

Bepler, T. et al. (2019) Positive-unlabeled convolutional neural networks for particle picking in cryoelectron micrographs. Nature Methods 201916:11 16, 1153-1160.

Brown, S. K., Tseng, Y. Y., Aziz, A., Baz, M., & Barr, I. G. (2022). Characterization of influenza B viruses with reduced susceptibility to influenza neuraminidase inhibitors. Antiviral Research, 200, 105280.

Burnley, T., Palmer, C. M. & Winn, M. (2017) Recent developments in the CCP-EM software suite. Acta Crystallogr D Struct Biol 73, 469-477.

Caini, S., Kusznierz, G., Garate, V. V., Wangchuk, S., Thapa, B., De Paula, F. J., De Almeida, W. A. F., Njouom, R., Fasce, R. A., Bustos, P., Feng, L., Peng, Z., Araya, J. L., Bruno, A., De Mora, D., De Gamez, M. J. B., Pebody, R., Zambon, M., Higueros, R., ... Paget, J. (2019). The epidemiological signature of influenza B virus and its B/Victoria and B/Yamagata lineages in the 21st century. PLoS ONE, 14(9).

Carter, D. M., Bloom, C. E., Nascimento, E. J. M., Marques, E. T. A., Craigo, J. K., Cherry, J. L., Lipman, D. J., & Ross, T. M. (2013). Sequential Seasonal H1N1 Influenza Virus Infections Protect Ferrets against Novel 2009 H1N1 Influenza Virus. Journal of Virology, 87(3), 1400.

Dreyfus et al. (2012). Highly Conserved Protective Epitopes on Influenza B Viruses. Science 337,1343- 1348.

Emsley, P. & Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132.

Glezen, W. P., Schmier, J. K., Kuehn, C. M., Ryan, K. J., & Oxford, J. (2013). The Burden of Influenza B: A Structured Literature Review. American Journal of Public Health, 103(3), e43.

Hannoun, C. (2014). The evolving history of influenza viruses and influenza vaccines. Expert Review of Vaccines, 12(9), 1085-1094. Heikkinen, T., Ikonen, N., & Ziegler, T. (2014). Impact of Influenza B Lineage-Level Mismatch Between Trivalent Seasonal Influenza Vaccines and Circulating Viruses, 1999-2012. Clinical Infectious Diseases, 59(11), 1519-1524.

Hernandez-Davies, J. E., Feigner, J., Strohmeier, S., James Pone, E., Jain, A., Jan, S., Nakajima, R., Jasinskas, A., Strahsburger, E., Krammer, F., Feigner, P. L., Huw Davies, D., Berger, C. T., Poo, H., & Korea Jeffrey Boyington, S. (2021). Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components. 12, 1.

Hernandez-Davies, J. E., Feigner, J., Strohmeier, S., Pone, E. J., Jain, A., Jan, S., Nakajima, R., Jasinskas, A., Strahsburger, E., Krammer, F., Feigner, P. L., & Davies, D. H. (2021). Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components. Frontiers in Immunology, 12, 692151.

Huber, V. C., Thomas, P. G., & McCullers, J. A. (2009). A Multi-valent Vaccine Approach That Elicits Broad Immunity Within an Influenza Subtype. Vaccine, 27(8), 1192.

Hurt, A. C., Hardie, K., Wilson, N. J., Deng, Y.-M., Studies, M. O. B. N. , Grad. Dip. L., Gehrig, N., & Kelso, A. (2011). Community Transmission of Oseltamivir-Resistant A(HlNl)pdmO9 Influenza. New England Journal of Medicine, 365(26), 2541-2542.

Ikematsu, H., Kawai, N., Chong, Y., Bando, T., Iwaki, N., & Kashiwagi, S. (2019). In vitro neuraminidase inhibitory concentration (IC 50 ) of four neuraminidase inhibitors in the Japanese 2017el8 season: Comparison with the 2010ell to 2016el7 seasons *.

Ishiguro, N., Koseki, N., Kaiho, M., Ariga, T., Kikuta, H., Oba, K., Togashi, T., Morita, K., Inagawa, A., Okamura, A., Yamazaki, S., Shida, S., Konno, M., Kawamura, N., Ishizaka, A., Takada, K., Tsubakihara, K., Nagano, N., Shibata, M., ... Yoshioka, M. (2018). Clinical effectiveness of four neuraminidase inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) for children with influenza A and B in the 2014e2015 to 2016e2017 influenza seasons in Japan. J Infect Chemother, 24, 449-457. luliano, A. D., Roguski, K. M., Chang, H. H., Muscatello, D. J., Palekar, R., Tempia, S., Cohen, C., Gran, J. M., Schanzer, D., Cowling, B. J., Wu, P., Kyncl, J., Ang, L. W., Park, M., Redlberger-Fritz, M., Yu, H., Espenhain, L., Krishnan, A., Emukule, G., ... Mustaquim, D. (2018). Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. The Lancet, 391(10127), 1285-1300.

Jumper, J. et al. (2021) Highly accurate protein structure prediction with AlphaFold. Nature 2021 596:7873596, 583-589. Kidmose, R. T. et al. (2019) Namdinator-automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps. doi:10.1107/S2052252519007619. Koutsakos, M., Nguyen, T. H., Barclay, W. S., & Kedzierska, K. (2015). Knowns and unknowns of influenza B viruses. Future Microbiol. 2016;ll(l):119-35.

Krishna, S., Jayaram, A., Shetty, U., Varamballi, P., Mukhopadhyay, C., & Jagadesh, A. (2023). Detection of H275Y oseltamivir resistance gene mutation among Influenza A(HlNl)pdmO9 patients by allelic discrimination real-time RT-PCR. Journal of Medical Virology, 95(5), e28764.

Krissinel E. and Henrick K. (2007). Protein interfaces, surfaces and assemblies' service PISA at the European Bioinformatics Institute. 'Inference of macromolecular assemblies from crystalline state.'. J. Mol. Biol. 372, 774-797.

Kumari, R., Sharma, S. D., Kumar, A., Ende, Z., Mishina, M., Wang, Y., Falls, Z., Samudrala, R., Pohl, J., Knight, P. R, W. T., Hsu, Y. F., Liu, M. T., Chen, C. H., Huang, H. H., Chang, C. H., Chou, J., & Sambhara, S. (2023). Antiviral Approaches against Influenza Virus. Clinical Microbiology Reviews. Vol. 36, No. 1.

Kusi, K. A., Faber, B. W., Thomas, A. W., & Remarque, E. J. (2009). Humoral Immune Response to Mixed PfAMAl Alleles; Multivalent PfAMAl Vaccines Induce Broad Specificity. PLOS ONE, 4(12), e8110.

Laursen et al. (2018) Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science 362,598-602.

Liebschner, D. et al. (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75, 861-877.

Lin, Z. et al. (2023) Evolutionary-scale prediction of atomic-level protein structure with a language model. Science (1979) 379, 1123-1130;

Lo, Y. C., Chuang, J. H., Kuo, H. W., Huang. H., Chang, F. Y., Lin, T. Y., & Chiu, W. T. (2013). Surveillance and Vaccine Effectiveness of an Influenza Epidemic Predominated by Vaccine-Mismatched Influenza B/Yamagata-Lineage Viruses in Taiwan, 2011-12 Season. PLOS ONE, 8(3), e58222.

Mattila, J. M., Vuorinen, T., Waris, M., Antikainen, P., & Heikkinen, T. (2021). Oseltamivir treatment of influenza A and B infections in infants. Influenza and Other Respiratory Viruses, 15( ), 618-624.

McCullers, J. A., Hoffmann, E., Huber, V. C. & Nickerson, A. D. (2005). A single amino acid change in the C -terminal domain of the matrix protein Ml of influenza B virus confers mouse adaptation and virulence. Virology, 336, 318-326.

Momont, C., Dang, H.V., Zatta, F. et al. A pan-influenza antibody inhibiting neuraminidase via receptor mimicry. Nature 618, 590-597 (2023).

Moscona, A. (2009). Global Transmission of Oseltamivir-Resistant Influenza. N Engl J Med, 360(10), 953-956. Nielsen, J., Vestergaard, L. S., Richter, L., Schmid, D., Bustos, N., Asikainen, T., Trebbien, R., Denissov, G., Innos, K., Virtanen, M. J., Fouillet, A., Lytras, T., Gkolfinopoulou, K., Heiden, M. an der, Grabenhenrich, L., Uphoff, H., Paldy, A., Bobvos, J., Domegan, L., ... Krause, T. G. (2019). European allcause excess and influenza-attributable mortality in the 2017/18 season: should the burden of influenza B be reconsidered? Clinical Microbiology and Infection, 25(10), 1266-1276.

Noh, J. Y., Choi, W. S., Song, J. Y., Lee, H. S., Lim, S., Lee, J., Seo, Y. Bin, Lee, J. S., Wie, S. H., Jeong, H. W., Heo, J. Y., Kim, Y. K., Park, K. H., Kim, S. W., Lee, S. H., Lee, J. H., Kim, D. H., Woo, S. II, Lim, C. S., ... Kim, W. J. (2018). Significant circulation of influenza B viruses mismatching the recommended vaccinelineage in South Korea, 2007-2014. Vaccine, 36(35), 5304-5308.

Noshi, T., Kitano, M., Taniguchi, K., Yamamoto, A., Omoto, S., Baba, K., Hashimoto, T., Ishida, K., Kushima, Y., Hattori, K., Kawai, M., Yoshida, R., Kobayashi, M., Yoshinaga, T., Sato, A., Okamatsu, M., Sakoda, Y., Kida, H., Shishido, T., & Naito, A. (2018). In vitro characterization of baloxavir acid, a first-in- class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit. Antiviral Research, 160, 109-117.

Oh, D. Y., Milde, J., Ham, Y., Calderon, J. P. R., Wedde, M., Durrwald, R., & Duwe, S. C. (2023). Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance. Infection and Drug Resistance, 16, 949-959.

Omoto, S., Speranzini, V., Hashimoto, T., Noshi, T., Yamaguchi, H., Kawai, M., Kawaguchi, K., Uehara, T., Shishido, T., Naito, A., & Cusack, S. (2018). Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Scientific Reports 20188:1, 8(1), 1-15.

Paget, J., Caini, S., Del Riccio, M., van Waarden, W., & Meijer, A. (2022). Has influenza B/Yamagata become extinct and what implications might this have for quadrivalent influenza vaccines? Eurosurveillance, 27(39), 1.

Pettersen, E. F. et al. (2021) UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Science 30, 70-82.

Prabakaran, M., Leyrer, S., He, F., Auer, S., Kumar, S. R., Kindsmueller, K., Mytle, N., Schneider, J., Lockhart, S., & Kwang, J. (2014). Progress toward a Universal H5N1 Vaccine: A Recombinant Modified Vaccinia Virus Ankara-Expressing Trivalent Hemagglutinin Vaccine. PLOS ONE, 9(9), el07316.

Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. (2017) CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290-296.

Ramage et al., 2019. Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B HemagglutininAntibodies 2019, 8, 14. Rosenthal, P. B. & Henderson, R. (2003) Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy. J Mol Biol 333, 721-745.

Rota, P. A., Wallis, T. R., Harmon, M. W., Rota, J. S., Kendal, A. P., & Nerome, K. (1990). Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology, 175(1), 59-68.

Saim-Mamoun, A., Abed, Y., Carbonneau, J., & Boivin, G. (2022). Generation and Characterization of Drug-Resistant Influenza B Viruses Selected In Vitro with Baloxavir Acid. Pathogens, 11(9), 1048.

Sanchez-Garcia, R. et al. , (2021) DeepEMhancer: a deep learning solution for cryo-EM volume postprocessing. Common Biol 4.

Schwartzman, L. M., Cathcart, A. L., Pujanauski, L. M., Qi, L., Kash, J. C., & Taubenberger, J. K. (2015). An Intranasal Virus-Like Particle Vaccine Broadly Protects Mice from Multiple Subtypes of Influenza A Virus. MBio, 6(4).

Shang, M., Blanton, L., Brammer, L., Olsen, S. J., & Fry, A. M. (2018). Influenza-Associated Pediatric Deaths in the United States 2010-2016. Pediatrics 141 (4): e20172918.

Schoonooghe et al. (2009). Efficient production of human bivalent and trivalent anti-MUCl Fab-scFv antibodies in Pichia pastoris. BMC Biotechnol. ll;9:70.

Su, S., Chaves, S. S., Perez, A., D'Mello, T., Kirley, P. D., Yousey-Hindes, K., Farley, M. M., Harris, M., Sharangpani, R., Lynfield, R., Morin, C., Hancock, E. B., Zansky, S., Hol lick, G. E., Fowler, B., McDonald- Hamm, C., Thomas, A., Horan, V., Lindegren, M. Lou, ... Fry, A. M. (2014). Comparing Clinical Characteristics Between Hospitalized Adults With Laboratory-Confirmed Influenza A and B Virus Infection. Clinical Infectious Diseases, 59(2), 252-255.

Takashita, E., Kiso, M., Fujisaki, S., Yokoyama, M., Nakamura, K., Shirakura, M., Sato, H., Odagiri, T., Kawaoka, Y., Tashiro, M., Komagome, R., Miyoshi, M., Nagano, H., Ohnishi, A., Tsutsui, R., Takahashi, M., Suzuki, Y., Nakata, A., Aoki, C. S. Y., ... Kyan, H. (2015). Characterization of a large cluster of influenza A(HlNl)pdmO9 viruses cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan. Antimicrobial Agents and Chemotherapy, 59(5), 2607-2617.

Tran, D., Vaudry, W., Moore, D., Bettinger, J. A., Halperin, S. A., Scheifele, D. W., Jadvji, T., Lee, L., Mersereau, T., Bridger, N., Morris, R., Top, K., Thibeaul, R., Dery, P., Lebel, M., Le Saux, N., Ford-Jones, L., Embree, J., Law, B., ... Sauve, L. (2016). Hospitalization for influenza A versus B. Pediatrics, 138(3).

Vanderlinden E, Gbktas F, Cesur Z, Froeyen M, Reed ML, Russell CJ, Cesur N, Naesens L. Novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin. J Virol. 2010 May;84(9):4277-88. Wagner, T. et al. (2019) SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo- EM. Communications Biology 20192:1 2, 1-13.

WHO. (2023). Influenza Laboratory Surveillance Information, https://www.who.int/tools/flunet

Yang, J. R., Huang, Y. P., Chang, F. Y., Hsu, L. C., Lin, Y. C., Huang, H. Y., Wu, F. T., Wu, H. S., & Liu, M. T. (2012). Phylogenetic and Evolutionary History of Influenza B Viruses, which Caused a Large Epidemic in

2011-2012, Taiwan. PLOS ONE, 7(10), e47179.

Yan, S., Weycker, D., & Sokolowski, S. (2017). US healthcare costs attributable to type A and type B influenza. Human Vaccines and Immunotherapeutics, 13(9), 2041-2047.

Zhai, L, Zhang, L, Jiang, Y, et al. (2022). Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus. J Med Virol ; 95:e28106.

Zhang, et al. (2022). Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions. Cell Rep. Med. 3, 100718.

Zhuang, Q„ Wang, S., Liu, S., Hou, G., Li, J., Jiang, W., Wang, K., Peng, C., Liu, D., Guo, A., & Chen, J. (2019). Diversity and distribution of type A influenza viruses: An updated panorama analysis based on protein sequences. Virology Journal, 16(1), 1-38.

Claims

1. An antigen-binding protein specifically binding Influenza B virus (IBV), wherein said antigen-binding protein comprises an immunoglobulin single variable domain (ISVD) specifically binding neuraminidase (NA) and an ISVD specifically binding haemagglutinin (HA).

2. The antigen-binding protein of claim 1, which neutralizes Influenza B Virus from both B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage.

3. The antigen-binding protein of any one of claims 1 or 1, wherein said NA-specific ISVD binds the NA active site, and/or the HA-specific ISVD binds at least partially to the stalk region of the HA protein.

4. The antigen-binding protein of any one of claims 1 to 3, wherein the ISVDs are fused directly or via a linker, and/or are present as part of an ISVD-Fc fusion and/or bispecific antibody.

5. An antigen-binding protein specifically binding Influenza B virus (IBV), wherein said antigen-binding protein comprises an ISVD specifically binding NA which neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage.

6. The antigen-binding protein of claim 5, wherein said ISVD comprises the complementarity determining regions (CDRs) as presented in any of SEQ ID NOs: 1-3, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia, or comprising an ISVD comprising a sequence wherein

CDR1 comprises SEQ ID NO: 19, CDR2 comprises SEQ ID NO: 20, and CDR3 comprises SEQ ID NO: 21;

CDR1 comprises SEQ ID NO: 22, CDR2 comprises SEQ ID NO: 23, and CDR3 comprises SEQ ID NO: 24.

7. The antigen-binding protein of any one of claims 5 or 6, wherein said ISVD comprises a sequence selected from the group of sequences of SEQ ID NO: 1-3, 7-9, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the non-identical amino acids are located in one or more framework residues, or a humanized variant of any one thereof.

8. An antigen-binding protein specifically binding Influenza B virus (IBV), wherein said antigen-binding protein comprises an ISVD specifically binding HA which neutralizes IBV B/Yamagata/16/88-lineage and B/Victoria/2/87-lineage, and wherein said ISVD comprises the CDRs as presented in any of SEQ ID NOs: 4-6, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia, or comprising an ISVD comprising a sequence wherein: CDR1 comprises SEQ ID NO: 13, CDR2 comprises SEQ ID NO: 14, and CDR3 comprises SEQ ID NO: 15;

CDR1 comprises SEQ ID NO: 16, CDR2 comprises SEQ ID NO: 17, and CDR3 comprises SEQ ID NO: 18.

9. The antigen-binding protein of claim 8, wherein the residues at position 44 and 64, according to Kabat numbering, in FR2 are a glutamic acid (E) amino acid residue.

10. The antigen-binding protein of claim 8 or 9, wherein said ISVD comprises a sequence selected from the group of sequences of SEQ ID NO: 4-6 or 10-12, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the non-identical amino acids are located in one or more framework residues, or a humanized variant of any one thereof.

11. The antigen-binding protein of any one of claims 1 to 4, wherein said NA-specific ISVD comprises an ISVD as present in the antigen-binding protein of any one of claims 5 to 7, and/or wherein the HA-specific ISVD comprises an ISVD as present in the antigen-binding protein of any one of claims 8 to 10.

12. The antigen-binding protein of any one of claims 1 to 11, which is labelled, or conjugated to a functional moiety, such as a therapeutic moiety or a half-life extension.

13. A nucleic acid molecule encoding the antigen-binding protein of any one of claims 1 to 12.

14. A pharmaceutical composition comprising an antigen-binding protein of any one of claims 1 to 12, and optionally a further therapeutically active agent, a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

15. The antigen-binding protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, or the pharmaceutical composition of claim 14, for use as a medicament.

16. The antigen-binding protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, or the pharmaceutical composition of claim 14, for use in prevention or treatment of an influenza B infection on a subject.